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Mechanistic investigations of glycosaminoglycan degrading enzymes Jongkees, Seino 2013

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Mechanistic investigations of glycosaminoglycan degrading enzymes by Seino Jongkees  B.Sc.(hons), The University of Otago, 2007 B.A., The University of Otago, 2007 Dip. Grad., The University of Otago, 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Chemistry)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) June 2013 © Seino Jongkees 2013  Abstract Glycosaminoglycans are the main structural polysaccharides of vertebrates, and represent a major barrier to the spread of both bacterial infection and tumours. The enzymes by which mammals and pathogens degrade these polysaccharides use very different mechanisms, and may represent suitable therapeutic targets. In this thesis, work is presented towards an understanding of the mechanisms of Clostridium perfringens unsaturated glucuronyl hydrolase (UGL), the second enzyme in the bacterial pathway for degradation of glycosaminoglycans, and human heparanase, the enzyme by which the abundant glycosaminoglycan heparan sulfate is remodelled. For UGL, evidence was presented for a hydration reaction scheme that had previously been proposed on the basis of crystallographic evidence. This was shown by characterisation of products formed by reaction in D2 O and 10 % methanol, and by demonstrating hydrolysis of three compounds that are only expected to be turned over by the enzyme if this reaction is correct. Investigation of the effects of substituents on the transition state stability, by measurement of a linear free-energy relationship for a series of aryl glycosides, kinetic isotope effects, and rate determination for heteroatom-substituted substrates, led to the proposal of alternate mechanisms. Attempts to verify these mechanisms were made by testing of potential inhibitors, rescue of a catalytic-residue mutant, trapping of a covalent glycosyl-enzyme intermediate, or synthesis of a potential intermediate, but without success. The mechanism that appears most likely proceeds through protonation of the substrate C4-C5  ii  double bond, with the resulting C5 positive charge being quenched by opening of the pyranose ring to give a C5 ketone and a C1-C2 epoxide. Subsequent hydration of the ketone and opening of this epoxide reforms the pyranose ring and gives the same product as direct hydration, but through a lower energy path. For mammalian heparanase, several new potential substrates and a potential inactivator were synthesized and tested. While this work was largely unsuccessful, it indicated that optimisation of sulfation patterns without modification of the aglycone is likely a futile strategy. A redesigned aglycone was proposed, representing a new path towards the goal of studying this enzyme for its eventual use as a therapeutic target in cancer therapy.  iii  Preface Aside from the exceptions noted below, all results presented in this thesis are my own work. Analysis of these results was carried out in consultation with my supervisor, Professor Withers. The work on natural substrate preferences and optimisation of enzyme conditions in Chapter 2, as well as the work on mutagenesis and testing of inhibitors in Chapter 4, was carried out together with Hayoung Yoo, a biochemistry 449 student. Expression and purification of recombinant human heparanase and synthesis of potential substrates and inactivator for this enzyme, presented in Chapter 5, was carried out in collaboration with Professor Jian Liu and co-workers at the University of North Carolina. Material contained in Chapter 2 of this thesis was previously reported in the following publication:  Jongkees, S. A. K.; Withers, S. G. Journal of the American Chemical Society 2011, 133, 19334–19337 “Glycoside Cleavage by a New Mechanism in Unsaturated Glucuronyl Hydrolases”  iv  Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ii  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  iv  Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  v  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  x  List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  xii  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  xvi  . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  xx  Abstract Preface  List of Tables  List of Schemes  List of Abbreviations  Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxii 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  1  1.1  General introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .  1  1.2  Glycosaminoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . .  3  1.2.1  Hyaluronan . . . . . . . . . . . . . . . . . . . . . . . . . . . .  7  1.2.2  Keratan sulfate  . . . . . . . . . . . . . . . . . . . . . . . . .  8  1.2.3  Chondroitin and dermatan sulfate . . . . . . . . . . . . . . .  10  1.2.4  Heparin and heparan sulfate  . . . . . . . . . . . . . . . . . .  12  1.2.5  Other uronic acid-containing polysaccharides . . . . . . . . .  17 v  1.3  Glycoside hydrolases (glycosidases) . . . . . . . . . . . . . . . . . . .  21  1.4  Hydration and elimination mechanisms in glycoside cleavage  . . . .  26  1.4.1  Family GH4 and GH109 glycoside hydrolases . . . . . . . . .  27  1.4.2  Elimination and hydration in sialidases  . . . . . . . . . . . .  33  1.4.3  α-1,4-Glucan lyase . . . . . . . . . . . . . . . . . . . . . . . .  35  1.4.4  Polysaccharide lyases  . . . . . . . . . . . . . . . . . . . . . .  39  1.4.5  Unsaturated glucuronyl and galacturonyl hydrolases . . . . .  43  1.4.6  N -Acetyl-muramic acid 6-phosphate hydrolase (MurQ)  . . .  49  Thesis aims . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  51  . . . . . . . . . . . . . . . .  53  1.5  2 Confirmation of the hydration reaction 2.1  2.2  Cloning and purification of Clostridium perfringens UGL  . . . . . .  54  2.1.1  Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  54  2.1.2  Enzyme expression and purification  . . . . . . . . . . . . . .  54  . . . . . . . . . . . . . . .  57  . . . . . . . . . . . . . . . . . . . . . . . . .  57  Development of a chromogenic substrate 2.2.1  Previous assays  2.2.2  Substrate synthesis  . . . . . . . . . . . . . . . . . . . . . . .  58  2.3  Enzyme optimisation  . . . . . . . . . . . . . . . . . . . . . . . . . .  61  2.4  Natural substrate variation . . . . . . . . . . . . . . . . . . . . . . .  65  2.5  Characterisation of UGL reaction products  . . . . . . . . . . . . . .  74  2.5.1  Reaction in D2 O . . . . . . . . . . . . . . . . . . . . . . . . .  76  2.5.2  Reaction in 10% methanol  . . . . . . . . . . . . . . . . . . .  78  Unusual substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . .  82  2.6.1  Kdn2en . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  83  2.6.2  Axial phenol . . . . . . . . . . . . . . . . . . . . . . . . . . .  87  2.6.3  Thiophenol . . . . . . . . . . . . . . . . . . . . . . . . . . . .  89  Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  90  2.6  2.7  vi  3 Probing the mechanism of UGL . . . . . . . . . . . . . . . . . . . . . 3.1  Detection of initial products by NMR  3.2  Linear free-energy relationship  3.3  3.4  3.5  92  . . . . . . . . . . . . . . . . .  92  . . . . . . . . . . . . . . . . . . . . .  96  3.2.1  Synthesis of aryl glycosides . . . . . . . . . . . . . . . . . . .  96  3.2.2  Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  98  Effects of heteroatoms . . . . . . . . . . . . . . . . . . . . . . . . . . 102 3.3.1  α- and β-ΔGlcA fluorides . . . . . . . . . . . . . . . . . . . . 103  3.3.2  2,4-Dinitrophenyl 2F-ΔGlcA . . . . . . . . . . . . . . . . . . 107  3.3.3  4-F substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . 110  Kinetic isotope effects . . . . . . . . . . . . . . . . . . . . . . . . . . 113 3.4.1  Synthesis of a substrate deuterated at carbon 1 . . . . . . . . 114  3.4.2  Synthesis of a substrate deuterated at carbon 4 . . . . . . . . 115  3.4.3  Kinetic isotope effect measurements . . . . . . . . . . . . . . 116  Conclusions, and possible alternate mechanisms  . . . . . . . . . . . 124  . . . . . . . . . . . . . . . . . . . 130  4 Testing of alternative mechanisms 4.1  Attempted rescue of D113G mutant  . . . . . . . . . . . . . . . . . . 130  4.2  Testing of potential inhibitor leads . . . . . . . . . . . . . . . . . . . 134  4.3  Anticipated trapping reagents for UGL  . . . . . . . . . . . . . . . . 141  4.3.1  2,3-Difluoro Kdn . . . . . . . . . . . . . . . . . . . . . . . . . 141  4.3.2  4-Deoxy-1,5-difluoro-iduronic acid  4.3.3  1-Fluoro-ΔGlcA fluoride  . . . . . . . . . . . . . . . 145  . . . . . . . . . . . . . . . . . . . . 150  4.4  Attempted synthesis of proposed epoxide intermediate . . . . . . . . 153  4.5  Conclusions and future directions  . . . . . . . . . . . . . . . . . . . 159  5 Heparanase substrate and inactivator testing 5.1  Chapter introduction  . . . . . . . . . . . . 161  . . . . . . . . . . . . . . . . . . . . . . . . . . 161  vii  5.2  Synthesis of compounds . . . . . . . . . . . . . . . . . . . . . . . . . 164  5.3  Substrate testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 5.3.1  Confirmation of heparanase activity . . . . . . . . . . . . . . 166  5.3.2  Towards a fluorescent substrate  . . . . . . . . . . . . . . . . 168  5.4  Testing of a potential HPSE inactivator . . . . . . . . . . . . . . . . 173  5.5  Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176  6 Overall conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 7 Materials and methods  . . . . . . . . . . . . . . . . . . . . . . . . . . 185  7.1  Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185  7.2  Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186 7.2.1  General methods . . . . . . . . . . . . . . . . . . . . . . . . . 186  7.2.2  Development of chromogenic substrates . . . . . . . . . . . . 188  7.2.3  Standards for UGL reaction in 10% methanol . . . . . . . . . 194  7.2.4  Unusual substrates for UGL  7.2.5  Substrates for linear free-energy relationship  7.2.6  Substrates for with varied heteroatoms  7.2.7  Substrates for kinetic isotope effects . . . . . . . . . . . . . . 235  7.2.8  Potential inhibitors of UGL . . . . . . . . . . . . . . . . . . . 242  7.2.9  Potential trapping reagents for UGL . . . . . . . . . . . . . . 243  . . . . . . . . . . . . . . . . . . 197 . . . . . . . . . 206  . . . . . . . . . . . . 221  7.2.10 Glucuronides for extension to heparanase substrates. 7.3  Biochemistry.  . . . . 256  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258  7.3.1  Cloning of UGL from Clostridium perfringens.  . . . . . . . . 258  7.3.2  Testing of expression conditions  7.3.3  Heterologous expression of UGL in Escherichia coli. . . . . . 260  7.3.4  Michaelis-Menten kinetics . . . . . . . . . . . . . . . . . . . . 261  . . . . . . . . . . . . . . . . 259  viii  7.3.5  NMR monitoring of UGL-catalysed reaction  7.3.6  Profile of UGL activity at varied pH.  7.3.7  Titration of benzyl ΔGlcA. . . . . . . . . . . . . . . . . . . . 263  7.3.8  Effect of temperature on UGL. . . . . . . . . . . . . . . . . . 263  7.3.9  Reaction in D2 O . . . . . . . . . . . . . . . . . . . . . . . . . 264  7.3.10 Reaction in 10% methanol  . . . . . . . . . 262  . . . . . . . . . . . . . 262  . . . . . . . . . . . . . . . . . . . 264  7.3.11 Testing of competitive inhibitors.  . . . . . . . . . . . . . . . 265  7.3.12 Inactivator testing . . . . . . . . . . . . . . . . . . . . . . . . 266 7.3.13 Kinetic isotope effects.  . . . . . . . . . . . . . . . . . . . . . 266  7.3.14 Attempted Rescue of D113G mutant with nucleophiles  . . . 268  7.3.15 Heparanase kinetics . . . . . . . . . . . . . . . . . . . . . . . 269 References  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272  Appendices A UGL multiple sequence alignment  . . . . . . . . . . . . . . . . . . . 291  B 2D-NMR spectra of UGL products and standards . . . . . . . . . 295 C Kinetic isotope effects . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 D Plots for Michaelis-Menten kinetics  . . . . . . . . . . . . . . . . . . 306  ix  List of Tables 1.1  Michaelis-Menten kinetic parameters for previously characterised Bacillus sp. GL1 UGL mutations . . . . . . . . . . . . . . . . . . . . .  2.1  Michaelis-Menten kinetic parameters for hydrolysis of the two aryl glycoside substrates 6 and 10 by UGL under optimised conditions. .  2.2  97  Michaelis-Menten kinetic parameters for hydrolysis of aryl unsaturated glucuronides by UGL.  3.3  82  Conditions used and yields for the synthesis of unsaturated aryl glucuronides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3.2  68  Michaelis-Menten kinetic parameters for three unusual substrates accepted by UGL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3.1  65  Comparison of kinetic parameters for UGL from different source organisms with GAG-derived natural substrates . . . . . . . . . . . . .  2.3  48  . . . . . . . . . . . . . . . . . . . . . . .  99  Kinetic parameters of UGL substrates with varied heteroatoms at the anomeric carbon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106  3.4  Kinetic isotope effects from deuterium incorporation at carbon 1 and carbon 4 of 2,4,6-trichlorophenyl ΔGlcA, and solvent deuterium effect with 4-nitrophenyl ΔGlcA. . . . . . . . . . . . . . . . . . . . . . . . . 117  x  5.1  Rates of hydrolysis for the commercial pentasaccharide Arixtra and heparin di- and tri-saccharide substrates with fluorescent leaving groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169  xi  List of Figures 1.1  Structures of the simplest repeating unit for each class of glycosaminoglycan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4  1.2  Structures of types I and II keratan sulfate. . . . . . . . . . . . . . .  9  1.3  Sub-types of chondroitin and dermatan sulfate, showing the main sulfation patterns in each . . . . . . . . . . . . . . . . . . . . . . . .  1.4  Known disaccharide units in heparan sulfate and heparin, with the archetypal disaccharides for the N- and S-regions indicated . . . . . .  1.5  11  14  Domain organisation of heparan sulfate and illustration of its domain structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  15  1.6  Minimal structure for heparin anticoagulant activity . . . . . . . . .  16  1.7  Structures of xanthan and gellan. . . . . . . . . . . . . . . . . . . . .  17  1.8  Structure of alginate. . . . . . . . . . . . . . . . . . . . . . . . . . . .  18  1.9  Structures of the pectins homogalacturonan and rhamnogalacturonan I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  19  1.10 Structure of rhamnogalacturonan II . . . . . . . . . . . . . . . . . . .  20  1.11 X-ray crystal structure of the Bacillus sp. GL1 UGL D88N active site from two perspectives, showing an unsaturated hyaluronan disacchar-  2.1  ide substrate bound and all side-chains within 5 Å . . . . . . . . . .  47  Expression of UGL under varying conditions . . . . . . . . . . . . . .  55  xii  2.2  Purification of UGL . . . . . . . . . . . . . . . . . . . . . . . . . . .  2.3  Effect of salt concentration, pH, and temperature on the reaction rate  56  of UGL with 4-nitrophenyl ΔGlcA . . . . . . . . . . . . . . . . . . .  62  2.4  Determination of the pKa of benzyl ΔGlcA . . . . . . . . . . . . . .  63  2.5  Dixon plot showing competitive inhibition of UGL by Mes.NaOH buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  66  2.6  X-ray crystal structure of the S. agalactiae UGL active site . . . . .  71  2.7  Key fragments of a multiple sequence alignment of UGL sequences by the MUSCLE algorithm . . . . . . . . . . . . . . . . . . . . . . . . .  2.8  1 H-NMR  spectrum of 24, the final products of UGL degradation of  4-nitrophenyl ΔGlcA (6) . . . . . . . . . . . . . . . . . . . . . . . . . 2.9  73  75  Overlay of expanded 1 H-NMR spectra of the products from UGLcatalysed hydrolysis of 4-nitrophenyl ΔGlcA (6) in D2 O and H2 O . .  77  2.10 Overlay of expanded 1 H-NMR spectra of the products from UGLcatalysed hydrolysis of phenyl ΔGlcA (10) in 10% MeOH in H2 O (26) and a control in H2 O . . . . . . . . . . . . . . . . . . . . . . . .  78  2.11 Overlay of expanded 1 H-NMR spectra of the anomeric methanol synthetic standard (31), the products from UGL-catalysed hydrolysis of phenyl ΔGlcA (6) in 10% MeOH in H2 O (26) and the carbon 5 methanol epimeric synthetic standard (28) . . . . . . . . . . . . . . . . .  81  2.12 Dixon plot showing competitive inhibition of UGL by Kdn2en . . . .  86  3.1  Offset stack of 1 H-NMR spectra showing reaction of thiophenyl ΔGlcA (46) with a high concentration of UGL . . . . . . . . . . . . . . . . .  3.2  94  Plot of log(kcat ), A, and log(kcat /Km ), B, against leaving group pKa for hydrolysis of aryl unsaturated glucuronides by UGL. . . . . . . . 100  xiii  3.3  Minimal Dixon plot showing competitive inhibition of UGL by axial ΔGlcA fluoride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105  3.4  Inhibition of UGL by equatorial ΔGlcA fluoride . . . . . . . . . . . . 106  3.5  Dixon plot showing competitive inhibition of UGL by 2,4-dinitrophenyl 2-deoxy-2-fluoro ΔGlcA . . . . . . . . . . . . . . . . . . . . . . . . . 110  3.6  Comparison of side-chain carboxyl placement in UGL from Bacillus sp. GL1 and AroA from E. Coli  . . . . . . . . . . . . . . . . . . . . 121  4.1  Elution trace for UGL D113G . . . . . . . . . . . . . . . . . . . . . . 132  4.2  Profile of first order rate for hydrolysis of 4-nitrophenyl ΔGlcA (6) by UGL D113G and wild-type . . . . . . . . . . . . . . . . . . . . . . . 133  4.3  X-ray crystal structure of the Bacillus sp. GL1 UGL active site showing glycine and all side-chains within 5 Å, from two perspectives . . 136  4.4  Dixon plot showing competitive inhibition of UGL by shikimate . . . 138  4.5  Representation of substrate and shikimic acid interaction with D88/113 in the active site of UGL. . . . . . . . . . . . . . . . . . . . . . . . . 139  4.6  Time-dependent inactivation of UGL by 2,3-difluoro Kdn (114) . . . 144  4.7  Time-dependent inactivation of UGL by ΔGlcA fluoride (70) . . . . 144  4.8  19 F-NMR  showing partial hydrolysis of 2,3-difluoro Kdn (114) with  UGL and in a non-enzymatic control . . . . . . . . . . . . . . . . . . 145 4.9  Time-dependent inactivation of UGL by 4-deoxy-1,5-difluoro-iduronic acid (120) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147  4.10 Overnight hydrolysis of 4-deoxy-1,5-difluoro-iduronic acid (120) with UGL and in a non-enzymatic control as monitored by TLC and  19 F-  NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 4.11 Dixon plot showing competitive inhibition of UGL by 4-deoxy-1,5difluoro-iduronic acid (120) . . . . . . . . . . . . . . . . . . . . . . . 149 xiv  4.12 Time-dependent  inactivation  of  UGL  by  1-fluoro-ΔGlcA  fluoride (124) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 4.13 X-ray crystal structure of Haemophilis influenzae sialic acid aldolase (Neu5Ac aldolase) active site . . . . . . . . . . . . . . . . . . . . . . 155 5.1  Sulfation in a substrate oligosaccharide as required for cleavage by HPSE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163  5.2  Overnight reaction of 144 with HPSE, monitored by  19 F-NMR  . . . 171  5.3  Inhibition of HPSE by TFMU substrate trisaccharides . . . . . . . . 172  5.4  Attempted time-dependent inactivation of HPSE by 149 . . . . . . . 175  6.1  Hypothetical energy profile for UGL-catalysed ΔGlcA hydrolysis, compared to the non-enzymatic acid-catalysed reaction . . . . . . . . . . 181  xv  List of Schemes 1.1  The general mechanism of inverting α-glucosidases. . . . . . . . . . .  22  1.2  The general mechanism of retaining β-glucosidases. . . . . . . . . . .  24  1.3  The mechanism for hydration of glucal by a retaining β-glucosidase .  26  1.4  The general mechanism of family GH4 6-phospho-α-glucosidases . . .  28  1.5  The general mechanism of elimination and hydration by sialidases . .  34  1.6  The general mechanism of α-(1,4)-glucan lyases . . . . . . . . . . . .  37  1.7  The anticipated mechanism of inactivation of α-(1,4)-glucan lyases by 1-fluoro-α-d-glucosyl fluoride, if a second residue were to be acting as catalytic base. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  39  1.8  The general mechanism of polysaccharide lyases acting on pectate. .  40  1.9  The general mechanism proposed for unsaturated glucuronyl hydrolases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  44  1.10 Bacterial metabolic pathway for catabolism of free ΔGlcA to the common metabolites pyruvate and d-glyceraldehyde-3-phosphate  . . . .  45  1.11 The general mechanism for N -acetyl-muramic acid 6-phosphate hydrolase (MurQ). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  50  2.1  Final optimised 4-nitrophenyl glycoside substrate synthesis. . . . . .  59  2.2  Final optimised phenyl glycoside substrate synthesis. . . . . . . . . .  59  2.3  Formation of the final product of the UGL-catalysed reaction. . . . .  74  xvi  2.4  Reaction catalysed by UGL, showing the route by which deuterium and methanol are incorporated . . . . . . . . . . . . . . . . . . . . .  2.5  Decomposition of the methanol adduct formed by UGL-catalysed reaction of phenyl ΔGlcA (10) . . . . . . . . . . . . . . . . . . . . . .  2.6  81  Expected reaction pathway for UGL if a mechanism analogous to that for glucal hydration were followed. . . . . . . . . . . . . . . . . . . .  2.9  80  Synthesis of a standard for the product of UGL-catalysed reaction in 10% methanol at carbon 1. . . . . . . . . . . . . . . . . . . . . . . .  2.8  79  Synthesis of a standard for the product of UGL-catalysed reaction in 10% methanol at carbon 5. . . . . . . . . . . . . . . . . . . . . . . .  2.7  77  82  Synthesis of 2,3-dideoxy-d-glycero-d-galacto-non-2-enopyranosonate (Kdn2en, 40). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  84  2.10 Synthesis of an axial phenyl ΔGlcA substrate for UGL. . . . . . . . .  87  2.11 Synthesis of a thiophenyl ΔGlcA substrate for UGL. . . . . . . . . .  89  3.1  Mechanism for UGL catalysis of rearrangement of the hemiketal intermediate 22 to cleave the glycosidic bond. . . . . . . . . . . . . . .  95  3.2  Synthesis of an equatorial ΔGlcA fluoride substrate. . . . . . . . . . 104  3.3  Synthesis of an axial ΔGlcA fluoride substrate. . . . . . . . . . . . . 104  3.4  Synthesis of a 2-deoxy-2-fluoro substrate analogue for UGL. . . . . . 108  3.5  Attempted synthesis of a 4-fluoro substrate, failing at elimination by DBU. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111  3.6  Retrosynthetic analysis of 88 with late oxidation, and test of the first step. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112  3.7  Synthesis of a 1-deuterated subtrate. . . . . . . . . . . . . . . . . . . 114  3.8  Synthesis of a 4-deuterated subtrate. . . . . . . . . . . . . . . . . . . 116  xvii  3.9  Deduced conformation of the transition state that would give rise to the observed KIE on kcat /Km , for the first irreversible step, leading to a hypothetical oxocarbenium ion intermediate . . . . . . . . . . . 119  3.10 Comparison of the reactions catalysed by AroA and UGL. . . . . . . 120 3.11 Illustration of the analogous relationship of deuteriums in unsaturated 1-deutero-glucuronides to 5-deutero-glucosides on formation of an initial oxocarbenium-ion transition state . . . . . . . . . . . . . . 122 3.12 SKIEs for hydration of vinyl ether acetals, proceeding by either initial hydration of the vinyl ether or initial hydrolysis of the acetal. . . . . 123 3.13 Possible mechanisms for UGL to account for KIE and LFER observations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 4.1  Illustration of structural analogies from UGL substrates, intermediates, and putative oxocarbenium ion-like transition states to potential inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135  4.2  Synthesis of Neu2en. . . . . . . . . . . . . . . . . . . . . . . . . . . . 140  4.3  Rationale for attempted trapping of UGL with 2,3-difluoro Kdn (114) 142  4.4  Synthesis of 2,3-difluoro Kdn. . . . . . . . . . . . . . . . . . . . . . . 143  4.5  Synthesis of 4-deoxy-1,5-difluoro-iduronic acid. . . . . . . . . . . . . 146  4.6  Rationale for attempted trapping of UGL with 1-fluoro-ΔGlcA fluoride (124) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151  4.7  Synthesis of 1-fluoro-ΔGlcA fluoride . . . . . . . . . . . . . . . . . . 152  4.8  Epoxide intermediate expected from Kdn2en (40) according to mechanism C of Scheme 3.13 . . . . . . . . . . . . . . . . . . . . . . . . . 154  4.9  Attempted synthesis of the epoxide intermediate expected from Kdn2en (40) under mechanism C of Scheme 3.13 . . . . . . . . . . . . . . . . 156  xviii  4.10 Hafnium tetrachloride and zinc iodide-catalysed rearrangement of dglucal in water. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 4.11 Alternate routes for synthesis of epoxide intermediate 133 from Kdn. 158 5.1  Starting glucuronic acid pseudo-disaccharides and general scheme for chemoenzymatic synthesis of appropriately sulfated substrates and inactivators for HPSE . . . . . . . . . . . . . . . . . . . . . . . . . . 165  5.2  Compounds for the HPSE reducing sugar assay, WST-1 (141) and Arixtra (142). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167  5.3  A potential synthesis for a HPSE substrate with a charged aglycone, and illustration of two possible charge placements by this aglycone to mimic the sulfation of optimal HPSE natural substrates . . . . . . . 173  5.4  Structure of the potential 2-deoxy-2-fluoro inactivator of HPSE. . . . 174  6.1  Conformation of transition states in mechanism C of Scheme 3.13 to account for experimental observations . . . . . . . . . . . . . . . . . 179  6.2  Precedent for mechanism C of Scheme 3.13 from non-enzymatic reactions of glycosides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182  xix  List of Abbreviations BSA  Bovine Serum Albumin  CaZY Carbohydrate active enzymes COSY Correlation spectroscopy DBU  1,8-Diazabicyclo[5.4.0]undec-7-ene  GAGs Glycosaminoglycans Gal  galactose  GalNAc N -acetyl-galactosamine GlcA  glucuronic acid  GlcNAc N -acetyl-glucosamine GlcNTFA N -trifluoroacetyl-glucosamine HPLC High-performance liquid chromatography HPSE human heparanase IdoA  iduronic acid  IPTG isopropyl β-d-thiogalactoside LB  lysogeny broth, also known as Luria broth xx  List of Abbreviations Man  mannose  MU  Methylumbelliferone  NAD+ Nicotinamide adenine dinucleotide (oxidised form) Neu5Ac N -Acetyl-Neuraminic acid O.D.  optical density  PAPS 3 -phosphoadenosine 5 -phosphosulfate PDB  Protein Data Bank  PGs  proteoglycans  Rha  rhamnose  SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SKIE  solvent kinetic isotope effect  TFMU Trifluoromethylumbelliferone TOCSY Total correlation spectroscopy TYP  tryptone-yeast-phosphate growth medium  UDP  Uridine diphosphate  UGH  unsaturated galacturonyl hydrolase  UGL  Unsaturated glucuronyl hydrolase  UV  ultraviolet  xxi  Acknowledgements Thanks first and foremost to Professor Stephen Withers, my supervisor, for all of his help and encouragement over the years, and also for believing in me after a lessthan-stellar first semester. I am grateful to our collaborator Professor Jian Liu and group from the University of North Carolina for their willingness to synthesise the heparanase substrates and inactivator, and provide the enzyme to allow testing of these, as well as troubleshooting when nothing appeared to be working as it should. Thanks to all members of the Withers lab, past and present, for their help, discussions, and also for their distractions. In particular: Dr Hongming Chen for being the best chemistry labmate you could ask for (discussions, advice, keeping everything running, providing intermediates, generally being a fountain of knowledge in the lab); Emily Kwan for her help with molecular biology and orientation within the biochemistry lab; Hayoung Yoo, a biochemistry 449 student, for her hard work on natural substrate preferences, optimisation of conditions, inhibitor testing, and mutagenesis for UGL; Bojana Rakic and Su Hancock for getting me oriented in the NCE lab; Brian Rempel and Tom Morley for getting me oriented in the chemistry lab; Freya Chen for keeping me distracted (and helpful discussions); Ethan GoddardBorger and Jamie Rich for their many helpful suggestions at group meetings (as well as outside of these); Ricardo Resende and Tina Rasmussen for stealing my spectrometer, which forced me to finish my chemistry when I didn’t want to; and Miranda Joyce for her administrative support. The contributions of staff at UBC  xxii  chemistry was also much appreciated, especially but not limited to the NMR and mass spectrometry teams. This document was created using the template for Lyx created by Christopher P. Barrington-Leigh. Thanks to Dr Nancy (Ju-Wei) Liu of the University of Auckland and William Archibald of the University of Victoria for sending me digital versions of the many articles for which the UBC library had only physical copies. Finally, thanks to my parents for all of their support over the years, and Gerritt Lighthart for proofreading several chapters of this thesis, and especially for sticking around these last few years until I finished this business.  xxiii  Chapter 1  Introduction 1.1  General introduction  Carbohydrates are, by mass, the single most abundant class of biomolecule in the world. 1 This abundance arises in large part from the common role of polysaccharides in the structural features of many forms of life, such as glycosaminoglycans in vertebrates, cellulose in plants, chitin in fungi and arthropods, and peptidoglycan in bacteria. These structural polysaccharides are very diverse in their structures across species, and so a great diversity of enzymes is required in order to manipulate them. Structural polysaccharides often play a crucial role in the defence of an organism both from the environment and during pathogenesis and competition, with the rigid layer preventing access to the otherwise vulnerable cell. However, because of the physical limitations of this structural layer, an organism also needs to be able to modify its own structural polysaccharides to be able to grow, either by recycling or replacing the structural layer. The balance between resistance to outside change while maintaining susceptibility to self-modification is an important driving force in carbohydrate-active enzyme evolution. A dramatic example of this is found in the evolutionary history of plants, with the structural components of early trees giving them an enormous evolutionary advantage in competition with other plants, allowing them to literally overshadow those unable to attain the same heights and leading to the formation of vast forests. The  1  1.1. General introduction new structures allowing this, in particular lignin impregnated with cellulose, were so successful at resisting degradation by other species that for millions of years there was a net increase in carbon sequestered as plant biomass (known as the carboniferous era, for this deposition of carbon). This lasted until the eventual evolution of fungal enzymatic systems able to degrade this plant matter that, among other environmental factors, restored carbon balance. 2–4 This co-evolution of new protective features and new methods to bypass them is often referred to as an evolutionary arms race, constantly occurring on a wide variety of time and size scales. Carbohydrates also play a wide variety of roles in living organisms beyond structural features. One of the main energy reservoirs for living organisms is in the form of polysaccharides, such as starch and glycogen. Carbohydrates also comprise one half of the repeating unit of nucleic acids, providing the link between the backbone and the nucleo-bases. In higher organisms, carbohydrates are important signalling molecules, and glycosylation of lipids and proteins can drastically alter their role and fate. Further roles in higher organisms include the formation of mucosal barriers and lubrication of mammalian joints. This diversity of roles is possible in large part because of the vast diversity of structures available to carbohydrates, and the associated differences in physical and chemical properties that arise from those differences. While functionally simple, containing only a few different functional groups, the permutations of branching and the stereo-, and regio-chemistries in carbohydrates gives a class of compounds drastically different from peptides and nucleic acids, the two other main classes of biopolymer. As an example of this difference, a hexamer of nucleic acids allows for 46 (∼ 4 × 103 ) unique structures, a hexamer of the natural amino acids, with all of their different side-chains, allows for 206 (∼ 6 × 107 ) unique structures, while a hexamer of simple reducing d-hexoses allows for a staggering ∼ 1.05 × 1012 structures. 5 Accounting  2  1.2. Glycosaminoglycans for mirror image l-sugars, ketoses as well as aldoses, and variations in monomer size and functional groups (such as pentoses, amino sugars and uronic acids) would vastly increase this figure.  1.2  Glycosaminoglycans  Among primary structural carbohydrates of mammals are the glycosaminoglycans (GAGs). These are composed of a linear repeating unit of two sugars, an amino-sugar and a uronic acid (with one exception, keratan sulfate), with many having extensive sulfation. The simplest repeating units for all GAGs are shown in Figure 1.1. The sulfation, together with the uronic acid carboxylate groups, gives GAGs a high degree of negative charge, with heparin having the highest density of negative charge of any bio-macromolecule. 6 Classification of GAGs is based on the subunits present (GlcNAc or GalNAc, GlcA or IdoA) and the linkages between these (α or β, 1-3 or 14), with further sub-classification based on sulfation pattern and other modifications. The four main classes are hyaluronan, keratan sulfate, chondroitin and dermatan sulfates, and heparin and heparan sulfate. Each of these will be discussed in more depth in the following subsections. Note that, unless specified, all sugars are of the d-absolute stereochemistry, with the exception of idose derivatives, which occur as l-idose. These GAGs primarily exist attached to proteins as proteoglycans (PGs), although one class exists as free polysaccharides. 7 Almost all mammalian cells produce PGs. These are either secreted, attached at the membrane, or stored in granules within the cell. The GAG chains are attached to the protein core by attachment at either serine or threonine (O-glycosylation) or asparagine (N -glycosylation) sidechains. Individual protein cores can have as few as one or as many as >100 GAG chains attached, with some core proteins only being temporarily modified with GAG 3  1.2. Glycosaminoglycans  O  O  HO O OH  O HO 4)-  OH OH  OH  -OOC  O NHAc  O O  Hyaluronan  -  4)-  -O  O  O  O HO GlcA  O O OH NHAc -β-(1,3)- GalNAc -β-(1,  O HO  4)- IdoA  O HO  OH  O HO  O  O AcHN  O 4)- GlcA -β-(1,4)- GlcNAc -α-(1, Heparan sulfate  OH O  OH  O NHAc  O  -α-(1,3)- GalNAc4S -β-(1, Dermatan sulfate  OH  O  3SO  O  -OOC  Chondroitin sulfate  -OOC  O  Keratan sulfate  OH OH  OOC  OH  NHAc HO 3)-GalS-β-(1,4)-GalNAc-β-(1,  -β-(1,3)- GlcNAc -β-(1,  GlcA  O  OH  O HO  -OOC  4)- IdoA  OH  O OH  O HO  O -O  3SHN  O -α-(1,4)- GlcNS -α-(1, Heparin  Figure 1.1: Structures of the simplest repeating unit for each class of glycosaminoglycan. Sulfation is only shown in those cases where it is absolutely conserved. For descriptions of the variable sulfation patterns of each class see the relevant subsection below.  4  1.2. Glycosaminoglycans chains (called part-time PGs). There are no clear defining structural features of the protein component of a PG, with many different types existing. Very broadly, these can be classified 7 into five main types: (1) large extracellular chondroitin sulfate-PGs, including a large number of extracellular matrix components from different tissues, (2) heparan sulfate-PGs of the extracellular membrane/basement membrane, (3) small homologous core proteins with a small number of GAG chains (chondroitin, dermatan or keratan sulfates), (4) membrane-intercalated cell-surface PGs, and (5) intracellular PGs, containing extended sequences of alternating serine and glycine units (see the recognition sequence for xylosylation mentioned above) and heavily substituted with chondroitin sulfate and/or heparin. The first three of these are secreted proteins (e.g. aggrecan, versican, and decorin), the fourth is cell-surface associated (e.g. syndecan), while the last is intracellular (e.g. serglycins). Many of these proteins have domains that contain no GAGs, being involved in other functions. 7 In particular these are important for interactions with other molecules, such as cell membranes, GAGs, or other PGs, in order to create a crosslinked network. This networked structure is important for many of the structural features of GAGs and PGs. The binding of PGs can be tight and specific, but is often loose and general. Amino acids with basic side-chains are often important for these interactions, as many of the interacting partners are poly-anionic. Interactions of PGs, and the functions derived from this, can arise from the GAG chain alone, the protein alone, or both together. The role of the protein can be as simple as providing a simple scaffold to support and space the GAG chains, or it can provide specific interactions such as that of the lectin-like domains of aggrecan and versican, PGs from cartilage and fibroblasts, respectively. Synthesis of the GAG polysaccharide chain takes place in the Golgi apparatus,  5  1.2. Glycosaminoglycans usually starting with xylosylation of serine side-chains within a specific amino acid recognition sequence (broadly aaaGSGaba, where a is D or E and b is G, D, or E, but with many variations) 8 in the endoplasmic reticulum. Not all recognition sites are modified, and protein conformation appears to play a role in determining the extent of this. This initial xylose is subsequently modified with two galactose units before transfer of the first glucuronic acid residue. The subsequent addition of an amino sugar commits the chain to formation of a specific GAG. 9 Following polymerisation to the homopolymeric GAG chain, modification may take place starting with N deacetylation and followed by N -sulfation, O-sulfation, and finally epimerisation of glucuronic acid to iduronic acid. These modification reactions do not occur in all GAGs, or always proceed to completion, and the resulting patterns of modifications define the sub-types of each GAG category, giving rise to different functions. Because of this incomplete modification, GAG chains can exhibit a high degree of both micro(within a chain) and macro- (between chains) heterogeneity. GAG chains from the same tissue but on different core proteins may be different. Given that this process gives a large number of diverse products and is not template driven, the regulation of this synthesis is derived from complex interactions of enzymes and substrates and competing rates between enzymes, and is poorly understood. Glycosaminoglycans have three main roles in tissue. 9 The majority of GAGs function as barriers to diffusion and as adhesion systems for cells and other biomolecules. GAGs are the primary carbohydrate components of the mammalian extracellular matrix, forming the scaffolding between cells. A related role is in providing lubrication and cushioning, with their high density of negative charges occupying a large hydrodynamic volume in solution and resisting compression. The viscosity of GAG solutions is highly dependent on counter-ion nature and concentration. Finally, GAGs function as a reservoir of specific binding sites for proteins, regulating  6  1.2. Glycosaminoglycans or stabilising their activity. Examples 6 include cell-signalling and development, angiogenesis, axonal growth, tumour progression and metastasis, and anticoagulation. GAGs also serve as adhesion points for pathogens, including viruses, protozoa, and bacteria. Organisms defective in GAG metabolism show severe clinical manifestations, while defects in anabolism are embryonic lethal. 9  1.2.1  Hyaluronan  Hyaluronan is the only homopolymeric GAG, being composed of unmodified glucuronic acid and N -acetyl-glucosamine in the sequence [GlcA-β-(1,3)-GlcNAc-β-(1,4)-]n , and is also the only GAG not bound to a protein core as a PG. Synthesis of hyaluronic acid is achieved by three integral-membrane hyaluronan synthases, with subsequent extrusion of the nascent chain into the extracellular space by a transport protein. 10 Chain lengths for hyaluronan vary from 500 to several thousand disaccharide units. The simple repeating unit of hyaluronan belies its complex interactions, with environmental factors such as counter-ions and concentration having dramatic effects on its structure, both in solution and in purified crystalline form. 9 Calcium ions, in particular, are able to bridge the negative charge of carboxylate groups in adjacent chains. At low Ca2+ concentrations hyaluronan forms a viscous, almost gel-like, solution, while higher concentrations of Ca2+ give a much thinner solution. Low concentrations of hyaluronan display non-Newtonian solution behaviour. At higher concentrations, hyaluronan is able to form amphipathic helices, driving selfaggregation into a double helix structure. Hyaluronan also associates with other GAGs, especially keratan and chondroitin sulfate in cartilage. These interactions are enhanced by high salt concentrations to mitigate charge-repulsion. This GAG has roles as lubricant of synovial joints, space filler, wetting agent, flow barrier in synovial fluid, and cartilage surface protector.  7  1.2. Glycosaminoglycans  1.2.2  Keratan sulfate  Keratan sulfate is unique among the GAGs in that it does not contain any uronic acids, being composed of alternating galactose and N -acetyl-glucosamine residues (poly-lactosamine) in the sequence [Gal-β-(1,4)-GlcNAc-β-(1,3)-]n . 11 Carbon 6 of one or both of these residues may be sulfated, with a non-random pattern of monoand di-sulfated subunits. The charged sulfate group on this position can fill many of the roles of the uronic acid carboxylate, but notably does not allow cleavage by polysaccharide lyase by an eliminative mechanism (refer to Subsection 1.4.4 on page 39). Keratan sulfate is broadly classed into three categories, with structures of the two well defined types shown in Figure 1.2. This classification was originally based on the source tissue, but later revised to be based on linkage type to the core protein. 11 Type one, first located in the cornea, is linked by a branched oligosaccharide to an asparagine side-chain. Type two, first located in other tissues and previously referred to as skeletal, is linked by a different branched oligosaccharide to serine or threonine side-chains, and is fucosylated on the carbon 3 hydroxyl of some GlcNAc residues close to the protein core. Type three is poorly characterised, being found in PGs from the brain, and contains a mannose linkage between the keratan sulfate chain and serine or threonine side-chains. Keratan sulfates are synthesised by glycosyl transferases common to glycoproteins and glycolipids, unlike the other glycosaminoglycans, which have specific glycosyl transferases for their synthesis. Keratan sulfate is self-aggregating, 9 and also interacts with collagen fibres. 12 The main role for type I keratan sulfate is in the cornea, in which it is the primary GAG. 12 Its role in this tissue is to establish and maintain the important arrangement of collagen fibrils that allows light to pass through. Type II keratan sulfate is a key component of cartilage, attached to the large protein aggrecan, along 8  1.2. Glycosaminoglycans  Figure 1.2: Structures of types I and II keratan sulfate.  9  1.2. Glycosaminoglycans with the hyaluronan to which it binds. 11  1.2.3  Chondroitin and dermatan sulfate  The disaccharide repeating unit in chondroitin sulfate, as well as the related dermatan sulfate, is composed of glucuronic (or iduronic) acid and N -acetyl-galactosamine in the sequence [GlcA-β-(1,3)-GalNAc-β-(1,4)-]n . These structures can be O-sulfated on carbon 2 and very rarely carbon 3 of the uronic acid and on carbons 4 and 6 of the galactosamine, with the chondroitin sulfates being further sub-classified based on the sulfation patterns (see Figure 1.3). The difference between chondroitin and dermatan sulfates is the presence of iduronic acid, the carbon 5 epimer of GlcA, in the place of some glucuronic acid residues in dermatan sulfate. Dermatan sulfate occurs in small PGs with a low number of short GAG chains (8 to 20 chains of 15 to 55 kDa each), while chondroitin sulfate typically occurs in larger structures composed of longer chains (20 to 100 chains of 15 to 70 kDa each). 9 These chains are attached to the core protein by the same GlcA-β-(1,3)-Galβ-(1,3)-Gal-β-(1,4)-Xyl-β-1-O-(Ser) linker sequence as heparan sulfate and heparin. 8 Synthesis of this linker initiates in the endoplasmic reticulum and is completed in the cis/medial Golgi, followed by addition of the first uronic acid by a specialised glycosyl transferase common to chondroitin/dermatan sulfate and heparin/heparan sulfate. Subsequent chain elongation steps are carried out by GlcA and GalNAc transferases concomitantly with sulfation by sulfotransferases in the medial/trans Golgi. Some sulfation also occurs in the linkage region of chondroitin/dermatan sulfate, which has not been observed in heparin or heparan sulfate, even when present on the same core protein. Chondroitin-6-sulfate and some forms of dermatan sulfate are able to self-associate, but chondroitin-4-sulfate is not. 9 However, chondroitin-4-sulfate is able to associate  10  1.2. Glycosaminoglycans  -OOC  O HO 4)-  -O  3SO  -O  OH O  O  O O OH NHAc GlcA -β-(1,3)- GalNAc4S -β-(1,  Chondroitin-4-sulfate (formerly chondroitin sulfate A)  OH OSO3O O O O O HO OH NHAc 4)- GlcA -β-(1,3)- GalNAc6S -β-(1, -  OOC  Chondroitin-6-sulfate (formerly chondroitin sulfate C)  -  OOC  O HO  O  O3SO  O HO  -OOC  3SO  OH  OH O  O O  O NHAc  4)- IdoA -α-(1,3)- GalNAc4S -β-(1, Dermatan sulfate (formerly chondroitin sulfate B)  OH OSO3O O O O O HO NHAc OSO34)-GlcA2S-β-(1,3)-GalNAc6S-β-(1, -  OOC  Chondroitin-2,6-sulfate (formerly chondroitin sulfate D)  OSO3O  O O OH NHAc 4)- GlcA -β-(1,3)- GalNAc4,6S -β-(1, Chondroitin-4,6-sulfate (formerly chondroitin sulfate E)  Figure 1.3: Sub-types of chondroitin and dermatan sulfate, showing the main sulfation patterns in each.  11  1.2. Glycosaminoglycans with dermatan sulfate. Dermatan sulfate tends to adopt an extended conformation in solution, explained by the presence of iduronic acid units, for which the energy difference between the 4 C1 , 1 C4 , and 2 SO conformers is very small. 6 This flexibility gives rise to poorly defined secondary structures. Chondroitin and dermatan sulfate are synthesised by virtually all vertebrate cells. 8 Their primary function is as a part of the extracellular matrix, providing support and connectivity to cells. They are also found in basement membranes and on cell surfaces, the latter having roles as receptors rather than structural functions.  1.2.4  Heparin and heparan sulfate  Heparan sulfate and heparin are the only glycosaminoglycans with an alpha-linkage at the hexosamine, with the chain composed initially of glucuronic acid and N acetyl-glucosamine in [GlcA-β-(1,4)-GlcNAc-α-(1,4)-]n . This class of GAGs shows the highest degree of heterogeneity. The distinction between heparin and heparan sulfate is one of degree, rather than type, with heparan sulfate showing a higher proportion of acetylated glucosamine, less O-sulfation and a lower proportion of iduronic acid. Counterintuitively, heparin is more heavily sulfated than heparan sulfate. Heparan sulfate molecules, at 10–70 kDa, are also generally larger than heparin, at 10–12 kDa. 6 The other key difference between these molecules is their location, with heparin being stored in granules of mast cells and heparan sulfate being found on cell membranes or extracellularly. Overall, O-sulfation of these GAGs can be found in varying proportions on carbon 2 of the uronic acid and carbons 3 and 6 of the glucosamine, which can also be N -deacetylated and N -sulfated. A list of known disaccharide units found in heparan sulfate and heparin are shown in Figure 1.4. 13–15 Heparan sulfate is composed of long domains containing a low degree of sulfation acting as spacers between shorter sections with high sulfation that resemble heparin,  12  1.2. Glycosaminoglycans with some intermediary mixed domains also present, as depicted in Figure 1.5. These mixed domains have important functional roles as they provide recognition sites for a number of chemokines. Synthesis of heparan sulfate and heparin proceeds in a similar manner to chondroitin and dermatan sulfates.  A core protein is xylosylated on specific serine  residues, the linker region synthesised by specific transferases, then the glycosaminoglycan extended by alternating additions of uronic acid and amino sugar. The divergence of these two classes of GAG is in the addition of the first amino sugar unit. 15 As chain synthesis progresses, an aminosugar deacetylase/sulfotransferase removes acetyl groups from the GlcNAc residues and replaces these with sulfate groups, although some decoupling of these activities does occur, leading to free NH2 groups. 16 The specificity of the subsequent enzyme, an epimerase acting on carbon 5 of the glucuronic acid residues at the non-reducing end of GlcNS residues, is the primary determinant of the various observed sulfation patterns, with less common patterns arising from its low rates of mis-recognition. This enzyme can act in a reversible or irreversible mode, depending on the sequence context. The subsequent and final steps in heparan sulfate and heparin synthesis are sulfation by 2-O-, 6-O-, and 3-O-sulfotransferases. All of these modifying enzymes have been proposed to act as complexes, with the enzyme composition of a complex determining the nature of the final chain. 17 Heparan sulfate adopts a helical conformation in solution, with the dimensions determined in part by the counter ion. Heparan sulfate chains can self-associate under conditions of sufficiently high salt conditions, with chains showing higher affinity for those of the same charge density. Heparin, by contrast, tends to have an extended conformation, again a result of the many conformers of approximately equal energy available to iduronic acid as mentioned for dermatan sulfate. 9 Specific  13  1.2. Glycosaminoglycans  N  -OOC O HO  OH  -OOC O HO  OH  O  O  O HO  AcHN  O 4)- GlcA -β-(1,4)- GlcNAc -α-(1,  -OOC  O HO  OH  O HO  OOC O HO  O OH -O3SO  -OOC O HO  OSO3O  O  -O  3SHN  -OOC  OH  -OOC O HO  OH  O O HO  O -  O3SHN  O HO  -OOC  OSO3O  O OH  O HO  -  -OOC  O O OSO3- O3SO  -  O3SHN  3SHN  -OOC  O OSO3  O  - -O3SO  O  OSO3O H2 N  OOC  O HO  4)-IdoA2S-α-(1,4)GlcNS3,6S-α-(1,  O HO  OH  O HO  -O  OH  O O OH -O3SO  O -O  OH  O O OSO3- HO  O AcHN  OH  O OSO3- HO  O -O  3SHN  O 4)- IdoA2S -α-(1,4)- GlcNS -α-(1,  OSO3O -O  OSO3O  O  O  O HO  O 4)- IdoA -α-(1,4)-GlcNS6S-α-(1,  O HO  3SHN  O 4)- GlcA2S -β-(1,4)- GlcNAc -α-(1,  O IdoA -α-(1,4)- GlcNS -α-(1,  4)-  -O  3SHN O 4)- GlcA -β-(1,4)- GlcNS3S-α-(1,  O 4)- GlcA -β-(1,4)- GlcNS3,6S-α-(1,  O HO  O  3SHN O 4)- GlcA -β-(1,4)- GlcNS6S -α-(1,  AcHN  O 4)- GlcA -β-(1,4)-GlcNAc6S-α-(1,  -OOC O HO  OH  O HO  O 4)- GlcA -β-(1,4)- GlcNS -α-(1,  OSO3O  O  OH  O  -OOC  -OOC  4)-IdoA2S-α-(1,4)GlcNH3,6S-α-(1,  O OSO3- HO  OSO3O -O  S  3SHN  O 4)-IdoA2S-α-(1,4)GlcNS6S-α-(1,  O HO O  O  OH  O OSO3  O  O  - -O3SO  -O  3SHN  O  4)-IdoA2S-α-(1,4)GlcNS3S-α-(1,  Figure 1.4: Known disaccharide units in heparan sulfate and heparin, with the archetypal disaccharides for the N- and S-regions indicated (refer Figure 1.5).  14  1.2. Glycosaminoglycans  S-region  N-region  Figure 1.5: Domain organisation of heparan sulfate (upper) and illustration of its domain structures (lower). The relatively flexible N-regions contain mostly Nacetyl-glucosamine and little sulfation, the stiffer S-regions contain mostly 2-sulfated iduronic acid and 6- as well as N-sulfated glucosamine, while mixed regions represent the transitions between these.  15  1.2. Glycosaminoglycans recognition sequences exist in both heparin and heparan sulfate, with arginine in particular being important for binding interactions with these. 6 An important example of such a sequence is that responsible for the anticoagulant activity of these molecules, a pentasaccharide with the sequence [GlcNS(Ac)6S-α-(1,4)-GlcA-β-(1,4)GlcNS3,(6)S-α-(1,4)-IdoA2S-α-(1,4)-GlcNS6S-α-(1,4)-] as shown in Figure 1.6 (the first glucosamine can be either N -sulfated or N -acetylated, and the second can be with or without 6-O-sulfation). This must be contained within an oligosaccharide of at least 12 to 16 monomers for full activity. The activity of this molecule arises from binding to the blood coagulation factors thrombin and factor Xa, preventing activation of the clotting cascade. 6  O HO  OSO3O -O  -  OOC  3SHN (AcHN)O HO  O O OH -O3SO  (OSO3-) OH O -O  3SHN  O  O HO  -  OOC  O OSO3- HO  OSO3O -O  3SHN  O  -GlcNS(Ac)6S-α-(1,4)-GlcA-β-(1,4)-GlcNS3,(6)S-α-(1,4)-IdoA2S-α-(1,4)-GlcNS6S-α-(1,4)-  Figure 1.6: Minimal structure for heparin anticoagulant activity. Features in parentheses show variants that retain activity. Like all other classes of GAGs discussed, heparan sulfate has important roles in the structure of many vertebrate tissues. However, heparin and heparan sulfate oligosaccharides also have a variety of signalling roles, such as in axonal growth, angiogenesis, and response to growth factors. 6  16  1.2. Glycosaminoglycans  1.2.5  Other uronic acid-containing polysaccharides  While not strictly GAGs, other polysaccharides exist in nature that contain a uronic acid in the repeating unit, making these amenable to degradation by polysaccharide lyases (see Subsection 1.4.4 on page 39) and so of relevance to this thesis. Many of the pathways for depolymerisation of these are thus related to those of GAGs. The microbial gelling agents xanthan 18 and the class of sphingans 19 (for example gellan) both contain a repeating unit with glucuronic acid, among other sugars, in the side-chain and the backbone respectively (Figure 1.7). Xanthan is a polymer of β(1,4)-glucose (the same as cellulose), with every other glucose unit displaying a [Manα-(1,4)-GlcA-α-(1,2)-Man-α-(1,3)-] side-chain. The mannose unit proximal to the backbone can be modified by O-acetylation at carbon 6, while the distal mannose can be modified by pyruvate, forming a (4,6) O-ketal. Gellan is a linear polysaccharide with the sequence [Glc-β-(1,4)-GlcA-β-(1,4)-Glc-β-(1,4)-l-Rha-α-(1,3)]n . Examples of applications include use of both as thickening agents in food, use of gellan to make particularly transparent gels for research, and use of xanthan in concrete to increase its viscosity for use underwater. Gellan  Xanthan 4)-  Glc -β-(1,4)- Glc -β-(1, OH OH O O O O O O OH HO OH OH OH Man-α-(1,3) O OH O OH OH GlcA-α-(1,2) O O COOOH OH O OH Man-α-(1,4) OH  4)-  Glc -β-(1,4)OH  O HO  GlcA  OOC O OH HO  O  -β-(1,4)- Glc -β-(1,4)- L-Rha -α-(1, O  OH  O OH HO  O OH  O O OH  O  OH  Figure 1.7: Structures of xanthan and gellan. Alginate is a polysaccharide from the cell wall of brown algae, 20 and also produced by some bacteria, that is capable of forming a viscous gum. It is composed of 17  1.2. Glycosaminoglycans (1-4)-linked β-d-mannuronate and its C-5 epimer, α-l-guluronate, in discrete blocks of around 20 units, interspersed with short areas of alternating monomers (Figure 1.8). 21 Some bacterially-sourced alginates are modified by acetylation. 22 Like xanthan and gellan, alginate is also used in food as a thickening agent, as well as a number of specialised applications based on its rapid water absorption and physical characteristics. 23 4)- ManA -β-(1, -  OOC O HO  4)- ManA -β-(1,4)- GulA -β-(1,  OH O  -  OOC  O  HO  OH O O HO  4)- GulA -β-(1,  OH O O COO-  OH O O  HO  ~20  COO-  ~20  Figure 1.8: Structure of alginate. Pectins are structural polysaccharides in plant cell walls. They are further divided into three classes, all of which contain uronic acids (Figure 1.9 and Figure 1.10). 24 Homogalacturonan, as the name might suggest, is composed solely of 1,4-linked αd-galacturonic acid. This basic structure may be modified by formation of methyl esters on some carboxyl groups and O-acetylation at carbon 2 or carbon 3. Rhamnogalacturonans make up the other two classes, and are further divided into types I and II. Type I rhamnogalacturonan has a core chain of [GalA-α-(1,2)-Rha-α-(1,4)-]n . The galacturonic acid residues again may be O-acetylated at carbons 2 or 3, while the rhamnose units are heavily modified with linear or branched side-chains on the carbon 4 hydroxyl group (between 20 and 80%). 24 The majority of these side-chains are composed of arabinose and galactose, while fucose and glucuronic acid may also be present. The type II rhamnogalacturonans are not at all similar to the type I, as shown in Figure 1.10. The backbone of this is composed of 1,4-linked α-dgalacturonic acid, as for homogalacturonan, but unlike homogalacturonan it is short 18  Homogalacturonan:  Rhamnogalacturonan I:  1.2. Glycosaminoglycans  19  Figure 1.9: Structures of the pectins homogalacturonan and rhamnogalacturonan I.  1.2. Glycosaminoglycans  Rhamnogalacturonan II:  O HO  COO-  O O  O OR O  O  O B  O  O O  RO  O O O -OOC  OH O  Figure 1.10: Structure of rhamnogalacturonan II core, with side-chain connections represented as blocks (upper), and an illustration of the cross-link formed between adjacent side-chain A moieties (lower), with one unit of the main galacturonic acid shown and the remainder of the side chain indicated by R.  20  1.3. Glycoside hydrolases (glycosidases) and heavily substituted with oligosaccharide side-chains. On four different monomers of this core are attached four different side-chains. Two of these side-chains attach to carbon 2 of a galacturonic acid, an octasaccharide and a nonasaccharide termed side-chains A and B, and two others attach to carbon 3 of a galacturonic acid, two disaccharides termed side-chains C and D. These rhamnogalacturonan II monomers are able to cross-link by formation of borate-diol esters through side-chain A. 25  1.3  Glycoside hydrolases (glycosidases)  The enzymes responsible for hydrolysis of glycosides are termed glycoside hydrolases, or glycosidases. Many of these are very efficient catalysts, with rate enhancements on the order of 1017 fold over the non-enzymatic case. 26 How these enzymes achieve this catalytic feat has been the topic of extensive work over many decades, with the latest insights capably covered in several recent reviews. 27–31 Glycoside hydrolases, along with the related glycosyl transferases, polysaccharide lyases, carbohydrate esterases, and carbohydrate-binding modules, are classified into families on the basis of sequence homology in the Carbohydrate Active Enzymes (CaZY) database (available online at http://www.cazy.org/). 32 For many of these families the key mechanistic details have been elucidated, and representative crystal structures of members have been solved. This database thus provides a framework through which mechanistic similarities between these enzymes can be understood, as well as reflecting structural features and evolutionary relationships. Conservation of mechanistic features has also been found in some cases to extend beyond the family classification, leading to the grouping of related families into clans. Two general mechanisms have been found in most glycoside hydrolases, which result in either a net retention or inversion of the stereochemistry at the anomeric centre, and so the corresponding enzymes are called retaining and inverting glycosi21  1.3. Glycoside hydrolases (glycosidases) dases. The general features leading to these results were outlined in a seminal paper by Koshland, 33 with many more details being added over the subsequent years. For this reason, these are often referred to as Koshland mechanisms. The inverting glycosidases catalyse hydrolysis by a single direct nucleophilic displacement, with a water nucleophile attacking at the anomeric centre while the bond to the nucleofuge is broken. Two key catalytic residues are involved in this mechanism; one acts as a base to deprotonate the water nucleophile and the other acts as an acid to activate the nucleofuge. These are most often aspartate or glutamate residues, one protonated and the other deprotonated in the resting state of the enzyme, with a spacing of approximately 10 Å to allow alignment of the nucleophile, anomeric carbon, and nucleofuge between them in the active site. This reaction proceeds in a dissociative manner, with substantial formal positive charge developing on the anomeric carbon, stabilised by lone-pair electrons of the adjacent endocyclic oxygen in an oxocarbenium-ion like transition state. These key mechanistic features are illustrated in Scheme 1.1.  -O  HO O  HO HO  HO  H O O  H OH  δO  O  H δOH δ+ O  HO HO HO  OR  HO  δ-  δ+  O  HO  HO O  HO HO  OH  OR  H O δO  O  OH HOR  O  -  O  Scheme 1.1: The general mechanism of inverting α-glucosidases. Many of the mechanistic features of the inverting glycoside hydrolases are also present in the retaining glycoside hydrolases. These enzymes, however, catalyse a 22  1.3. Glycoside hydrolases (glycosidases) double inversion in order to achieve a net retention of stereochemistry at the anomeric centre. Two key catalytic residues are again involved in this mechanism, but with slightly different roles, one acting as a nucleophile and the other as an acid/base catalyst, with a spacing of approximately 5.5 Å. The first inversion in the reaction sequence is a replacement of the original nucleofuge in the glycoside by the nucleophile residue side-chain. This forms a covalent glycosyl-enzyme intermediate, with the acid/base residue protonating the nucleofuge to activate it. This first step is termed the glycosylation step. The glycosyl-enzyme intermediate is then hydrolysed, with a water nucleophile being deprotonated by the acid/base residue to activate it for nucleophilic attack, displacing the nucleophile residue and giving a hydrolysed product with the same anomeric configuration as the substrate glycoside. This second step is termed deglycosylation. Both steps pass through oxocarbenium-ion like transition states, similar to that of the inverting mechanism. Either step of this mechanism can be rate-determining, with some enzymes showing evidence of a change in ratedetermining step with increasing activation of the nucleofuge in synthetic substrates. These key mechanistic features are illustrated in Scheme 1.2. Within the context of this general retaining mechanistic scheme, several variations have been reported in both the nucleophile and acid/base residues. 34 In the sialidases and trans-sialidases of clan GH-E the nucleophile residue is a tyrosine. This adaptation has been proposed to minimise charge repulsion between the nucleophile and the carboxylate at carbon 1 of the substrate sialosides, and also to provide a more stable intermediate for the generally more reactive sialosides (a 2-deoxy sugar), making its formation more favourable. Adjacent to the tyrosine nucleophile is a conserved carboxylate side-chain, potentially involved in abstracting the phenolic proton to improve nucleophilicity. Another variation in the nucleophile position is in enzymes that cleave substrates with a 2-acetamido group adjacent to the leaving group,  23  1.3. Glycoside hydrolases (glycosidases)  O H  HO HO HO  O  H HO  O OH  δO  O  HO HO  OR  HO  OO  OR δ+ O δ+ δO  HOR  O H2O -  O  H OH  HO HO HO  O HO  O  O O  O H  HO HO HO  OO  OH  O  H HO  O OH  δO  O  HO HO HO  OH δ+ O δ+ δO O  Scheme 1.2: The general mechanism of retaining β-glucosidases.  24  1.3. Glycoside hydrolases (glycosidases) although not all glycoside hydrolases acting on such substrates show this mechanistic feature. This acetamide can act as an internal nucleophile, forming a non-enzymelinked oxazoline or oxazolinium ion intermediate. An adjacent polarising residue is present in these enzymes, stabilising the intermediate either by charge interactions or by deprotonating it. An exogenous oxazoline analogue of the substrate has been shown to be catalytically competent in these enzymes, and thiazoline analogues have proven to be potent inhibitors. 35 This variation may have evolved as a lowest-energy path for hydrolysis of these already-activated substrates. Variations in the acid/base residue are less common, likely because of the lack of other functional groups in amino acid side chains with an appropriate pKa . One example is found in the unusual inverting endo-sialidase from bacteriophage K1F, for which it has been proposed that the carboxylate at carbon 1 of the substrate sialosides acts as an internal catalytic base, with a glutamate residue acting as catalytic acid. 36 Another example comes from myrosinase, a retaining β-glucosidase that catalyses hydrolysis of plant thioglycosides. 37,38 In this enzyme, the substrate leaving group is already highly activated, meaning the formation of covalent glycosyl enzyme intermediate proceeds without acid catalysis. The subsequent deglycosylation, which is rate-limiting, is catalysed by the carboxylate group of an ascorbate cofactor bound adjacent to the substrate. This may be functioning as a regulatory system, releasing toxic aglycones from their benign glycosides when the plant cell is in a state of poor redox homeostasis through cell lysis, as a defence against herbivores. Finally, in a retaining exo-cellulase from family GH6 a lack of suitable catalytic acid/base residue led to the postulation of a mechanism wherein a group of nearby residues act through a network of water molecules indirectly to provide the required protonation and deprotonation; a so-called Grotthus mechanism. 39 However, this is based on a homology model, so strong conclusions cannot be drawn  25  1.4. Hydration and elimination mechanisms in glycoside cleavage based the placement of residues. A final observation on traditional glycoside hydrolases is warranted in the context of the following section. It has been reported 40–43 that both α- and β-retaining glucosidases are able to catalyse the stereospecific hydration of glucal. This hydration occurs by a syn-addition of the catalytic acid residue across the double bond of the substrate to give a covalently bound 2-deoxy-glycosyl-enzyme intermediate, followed by hydrolysis of this intermediate to give an overall trans-addition of water across the double bond, as illustrated in Scheme 1.3. This hydration proceeds by use of the same catalytic residues and through a similar transition state to that of the hydrolysis reaction of these enzymes.  -  O  O  -  HO HO HO  O  *H O  H OH  HO HO HO O  O  O H* O  O  O H  HO HO HO  O  O H*  OH  OO  O  Scheme 1.3: The mechanism for hydration of glucal by a retaining β-glucosidase. The proton transferred from the catalytic nucleophile is indicated as H* to emphasise the stereochemical result, as determined by NMR studies in D2 O.  1.4  Hydration and elimination mechanisms in glycoside cleavage  In addition to those using the inverting and retaining mechanisms outlined in Section 1.3, a number of enzymes have been reported to cleave glycosides by mechanisms involving elimination, hydration, or both. These represent a set of very different  26  1.4. Hydration and elimination mechanisms in glycoside cleavage mechanisms, some with very different transition states, meaning that any attempts to inhibit or re-purpose these enzymes will likely require a very different set of strategies. Many, but not all, of these mechanisms have been observed in enzymes from bacterial sources.  1.4.1  Family GH4 and GH109 glycoside hydrolases  Both elimination and hydration reactions are found in family GH4 glycoside hydrolases. These employ a fascinating mechanism wherein the hydroxyl group on carbon 3 is transiently oxidised, acidifying the adjacent hydrogen on carbon 2 to allow an elimination across the carbon 2 to carbon 1 bond. In a second half-reaction, water attacks the α,β-conjugated system, followed by reduction of the carbon 3 ketone, giving an overall net hydrolysis of the anomeric bond, as shown in Scheme 1.4. This unique and unusual mechanism, completely different from those of either the inverting or retaining glycoside hydrolase mechanisms outlined in Section 1.3, proceeds through a series of negatively charged intermediates and transition states. This reaction sequence is achieved by the use of two obligate cofactors, a NAD+ to facilitate the transient oxidation and a divalent metal, usually manganese, to stabilise the negative charges developing during the course of the reaction. Evidence for this mechanism, including crystal structures, kinetic isotope effects, and linear free-energy relationships, was capably reviewed by Yip and Withers, 44 with a focus here on work since this publication. The lack of involvement of the anomeric centre in this mechanism has allowed this family to evolve to catalyse hydrolysis of both α- and β-configured substrates. These reactions appear to proceed through the same general mechanism, but with some differences in the residues used to achieve the key catalytic roles. Intriguingly, the 6-phospho-α-glucosidase GlvA from Bacillus subtilis has been demonstrated to  27  O-  O-  H  O  O  H  O  H  O  O  H  2-O  3PO  H* Mn2+  HO-  HO  (slow) (H- transfer)  2-O PO 3  HO  O  OR H O  H2O  Mn2+  O  (slow)  O  O  HO  HOR  -  O  OR H O  H2O  HO OR H O O H O NH2  Mn2+ H*  NH2  N+  H  3PO  H O  H* NH2  O 2-O  HO  HO  H H  H O  HO  HO H O  O  O  N  (fast)  H2O  O-  N O  H O  H H O  2-  O3PO  O  HO O H2O O-  H  O  O-  H  O  O-  O O  H  H O 2-O  3PO  HO-  HO Mn2+  (slow) OH H O  O  28  N+  (H+ transfer)  HO  H O HO  2-O  HO  HO  Mn2+  O  (slow)  NH2  (fast)  H  3PO  Mn H* H O  O  OH H O O  N  O  HO -  OH H O  O  H2O  HO OH H O O H O NH2  Mn2+ H*  NH2 N  H H  H* H O NH2  O  H O 2-O PO 3  O  HO HO  H  O  HO 2+  O N  Scheme 1.4: The general mechanism of family GH4 6-phospho-α-glucosidases, based on experimental results and calculation. Partially rate-limiting steps are indicated as slow, and for those involving redox the relevant half-reaction responsible is indicated. The hydride transferred in redox is denoted by H* .  1.4. Hydration and elimination mechanisms in glycoside cleavage  H O  H  O  1.4. Hydration and elimination mechanisms in glycoside cleavage cleave both α- and β-glucosides within the same active site of the same enzyme, with similar kinetic parameters, 45 provided the substrate contains a sufficiently activated aglycone. This anomerically blind reactivity is possible because the ability to bind substrate adjacent to the NAD+ is the primary determinant of hydrolysis, and aglycone activation is less important. For less activated substrates, where aglycone protonation becomes more important, the enzyme shows more specificity for its natural α-configured substrates. GH4 enzymes have also been shown to cleave thio-linked substrates, for example the unactivated 4-deoxy-4-thio-d-cellobiose-6 -phosphate is cleaved by the β-glucosidase BglT from Thermotoga maritima with kinetic parameters similar to those for the analogous O-glycoside. 46 Incorporation of deuterium at carbon 2 of the glucose-6-phosphate product and determination that the Ki value equalled the Km provided evidence that this hydrolysis proceeded through the same active site and with the same mechanism as for hydrolysis of O-glycosides. Much recent work has focused on the clarification of the rate-determining step(s) of family GH4, through determination of further kinetic isotope effects and linear free-energy relationships, as well as DFT calculations. Kinetic isotope effects in αand β-glycosidases from GH4 45,47–50 generally agree well, and show that, in enzymes acting on both anomeric configurations, oxidation at carbon 3 and deprotonation at carbon 2 are both partially rate limiting. By contrast, glycosidic bond cleavage is shown to be kinetically unimportant by the lack of dependence upon leaving group ability and the lack of a substantial kinetic isotope effect from deuteration at carbon 1. In MelA, an α-galactosidase from Citrobacter freundii, kinetic isotope effects were measured on kcat /Km for substrates with deuterium at carbons 2 and 3, and these were compared to the effect on rate of substitutions on both carbons 2 and 3 simultaneously. This disubstitution was found to show an enhancement of the isotope effect by the same magnitude when compared to either substitution alone,  29  1.4. Hydration and elimination mechanisms in glycoside cleavage suggesting that these isotopes were influencing the same transition state. This result agrees with a concerted oxidation at carbon 3 and deprotonation at carbon 2 with no formation of a discrete ketone intermediate. 49 It is unclear if this is a unique feature of MelA, or represents a more general rate determining step for these enzymes. It is likely that a gradient exists between discrete oxidation/deprotonation and a concerted process, determined by minor variations in the active site. Interestingly, in this same enzyme the kinetic isotope effects on kcat (as opposed to kcat /Km , discussed above) were found to be unity for substitution at carbons 1 and 3 (i.e. kH /kD = 1.0), and inverse for substitution at carbon 2. By comparison, the 6phospho-α-glucosidase GlvA exhibited normal KIEs for deuterium at carbons 2 and 3 on both kcat and kcat /Km , 45 thereby suggesting that the overall rate-determining step and the first irreversible step were one and the same. This suggests a shift in the overall rate-determining step in MelA to one of the steps after cleavage of the glycosidic bond. This step may be re-protonation at carbon 2, on the basis of the calculated reaction coordinate (vide infra), with the change in hybridisation from sp2 to sp3 consistent with the observed inverse effect. An energy profile was calculated 51 for the reaction catalysed by GH4 α-glycosidases, using the product bound crystal structure of the Bacillus subtilis 6-phospho-αglucosidase GlvA 52 as a starting point. Overall, this profile was found to be surprisingly flat, with no single step being clearly rate determining and all intermediates being lower in energy than the enzyme-bound starting material. On the basis of these calculations, the overall rate determining step is predicted to be re-protonation of carbon 2 following the Michael-type addition of water. However, the activation energies for reprotonation of the carbon 3 hydroxyl of the product, deprotonation of carbon 2 in the substrate, abstraction of hydride from the substrate, and reprotonation at carbon 2 of the product are all within 20 kJ.mol-1 . Similarly, the barrier  30  1.4. Hydration and elimination mechanisms in glycoside cleavage height relative to the starting material for reprotonation of the carbon 3 hydroxyl of the product, deprotonation of carbon 2 in the substrate, elimination of the aglycone, and reprotonation at carbon 2 of the product are all within 17 kJ.mol-1 . Given these values, it seems likely that in any given GH4 enzyme there are likely several steps that are at least partially rate-limiting, and subtle factors can easily influence which steps these are. These values also provide an explanation for the difficulty in assigning a single clear rate-determining step for the reaction catalysed by GH4 glycosidases using kinetic isotope effects. Since many steps are very similar in energy, a slight perturbation of any step can influence the overall rate. This calculated reaction path also indicated a role for a manganese-bound hydroxyl group as a catalytic base for the abstraction of a proton from the hydroxyl group on carbon 3 during the oxidation step and the involvement of aspartate 111 in activating tyrosine 265 as a catalytic base for deprotonation of carbon 2, by means of an intermediary water molecule. The oxidation step was found to proceed by an initial fast deprotonation followed by a slower hydride transfer, while reduction was found to proceed in the opposite order and with the opposite relative rates (fast hydride transfer then slow protonation). Unfortunately, the model was built without a phosphorylated substrate, which is the natural substrate of GlvA, so any effect from the phosphate on the reaction coordinate remains unknown. The identity of the catalytic base responsible for deprotonation of carbon 2 in GH4 hydrolases acting on phosphorylated substrates, predicted on the basis of Xray crystal structures, has been experimentally confirmed as tyrosine 241 (BglT numbering) by the careful measurement of kinetic isotope effects on kcat /Km for mutants at this position. 50 In a valiant set of experiments on deuterated substrates with several mutants at varied pH, it was found that removal of this residue ablated the kinetic isotope effect seen from substrates deuterated at carbon 3, while the  31  1.4. Hydration and elimination mechanisms in glycoside cleavage primary kinetic isotope effect from deuterium at carbon 2 is strengthened. This demonstrates that proton abstraction from carbon 2 has become completely rate limiting, a result of the retardation of this step by the removal of its catalytic base. This effect was enhanced at lower pH, where deprotonation is generally more rate limiting. By contrast, at higher pH the isotope effect from deuterium at carbon 2 is decreased while deuterium substitution at carbon 3 remained silent, suggesting that in the presence of adequate exogenous base a later step in the reaction becomes partially rate-limiting, but is silent in these kinetic isotope effects. Again, on the basis of the DFT calculations, this may be the re-protonation at carbon 2. An alternative explanation is that the Michael-type addition of water becomes ratelimiting, on the basis of a small inverse isotope effect that was observed for the Y241A mutant of BglT at its optimal pH. Unfortunately, limited supplies of labelled compound prevented determination of effects at other pH values. Interestingly, the rate-determining nature of proton abstraction from carbon 2 means that during steady state turnover the enzyme has a constitutively bound NADH, rather than the NAD+ of the resting state. The accumulation of this reduced cofactor on-enzyme could be observed spectroscopically for the Y241A mutant, since it binds this cofactor in a particularly tight fashion. GH109, a new family of glycoside hydrolases that appear to follow a very similar mechanism to that of GH4, was recently founded on the basis of an α-GalNAc-ase discovered in Elizabethkingia meningosepticum. 53 This enzyme showed a dependence on NAD+ for its catalytic activity, similar to GH4, but a surprising lack of requirement for a metal cofactor. In GH4 enzymes this metal coordinates to hydroxyls on carbons 2 and 3, one of which is substituted by an N -acetyl group in GalNAc, so another means of activating the hydroxyl proton on carbon 3 is likely being employed in GH109. How this enzyme is able to catalyse a similar reaction, with turnover  32  1.4. Hydration and elimination mechanisms in glycoside cleavage equal to or better than that seen in GH4, is likely to be the subject of much study. This enzyme, in contrast to the 6-phospho-α-glucosidase GlvA, only very slowly hydrolysed activated substrates of non-optimal anomeric configuration or without an N -acetyl moiety at carbon 2. Evidence for a similar redox/elimination/hydration mechanism came from the presence of a tightly-bound NAD+ cofactor appropriately positioned beside the substrate in the crystal structure, incorporation of deuterium at carbon 2 of the product when the reaction was carried out in D2 O, and digestion of thio-linked substrates. A further unusual feature in this enzyme is the lack of a clear candidate in its crystal structure for the acid/base residue that activates the aglycon for departure and water as a nucleophile. Accurate prediction of the substrate preference of GH4 enzymes, of which there are many in both the Archaeal and Bacterial domains, on the basis of sequence identity and phylogenetic relationships was found to be difficult. 54 The presence of a four amino acid domain beginning with the metal-ligating active-site cysteine, was found to correlate with the substrate preference of the enzyme. However, mutation of one such domain to another was not found to change substrate preference but rather to ablate all activity, showing that these motifs are necessary but not sufficient for each type of activity, with the active site context also playing a large role. This finding may prove useful in predicting the substrate preference of newly identified GH4 enzymes, but without further understanding of the effect of context is less useful in any attempts at rationally redesigning the activity of these enzymes.  1.4.2  Elimination and hydration in sialidases  A small number of sialidase enzymes from bacterial and viral sources have been observed to catalyse elimination 55–58 and/or hydration 56–59 reactions of sialosides, forming or degrading the sialidase competitive inhibitor Neu5Ac2en (also known as  33  1.4. Hydration and elimination mechanisms in glycoside cleavage  O HO  O  O-  OH  H O O  AcHN HO HO  HOR H2O COO-  OR  HO  O  OH  O  AcHN HO HO  H O  O  H COO-  O H O  O H H O-  O  HO  O-  OH  H  O  HO  O  OH O  OH  O AcHN HO HO  HO COO-  OH O  H O  O  OH  AcHN HO HO  COOH  O  AcHN HO HO  O  O  H O  H O COO-  O H O  O H H O-  Scheme 1.5: The general mechanism of elimination and hydration by sialidases. A given sialidase may catalyse either half reaction in isolation, both half reactions, or neither (giving only hydrolysis).  34  1.4. Hydration and elimination mechanisms in glycoside cleavage 2,3-dehydro-2-deoxy-N -acetylneuraminic acid, DANA). The mechanism for this is outlined in Scheme 1.5. Elimination proceeds through the same glycosyl-enzyme intermediate as the hydration reactions catalysed by these enzymes. However, proton abstraction from carbon 3, to trigger elimination, competes with hydrolysis at carbon 2 to varying degrees. A similar mechanism is also responsible for hydration of Neu5Ac2en, with formation of the same glycosyl-enzyme intermediate preceding hydrolysis to give overall hydration. This reaction is similar to the hydration of glucal by retaining glycosidases (refer to the end of Section 1.3), although notably tyrosine is unable to undergo a concerted syn-addition across the double bond as seen for aspartate/glutamate. The balance of elimination and hydration is thus determined by access of the water nucleophile to the anomeric carbon and the proton on carbon 3, as well as competition between substrates (Neu5Ac2en for hydration and sialosides for the formation of hydrolysed product and/or Neu5Ac2en). In most cases these elimination and hydration reactions represent minor pathways for hydrolases, and are correspondingly slow, but in one case it is the primary reaction catalysed and is reasonably efficient (kcat of 19 min-1 in NanC from Streptococcus pneumoniae). 58 Evidence for this mechanism came from reaction in D2 O, showing an overall transaddition of water, catalytic competence of synthetic Neu5Ac2en for hydration, and a lack of reaction with thio-sialosides, arguing against direct elimination without a covalent intermediate. 58 Given that Neu5Ac2en is a reasonably potent inhibitor of sialidases, its formation and decomposition has been proposed to be a regulatory system for sialidase activity. 58,60  1.4.3  α-1,4-Glucan lyase  An elimination mechanism, but no hydration, has also been found to be acting in some enzymes of family GH31, the α-1,4-glucan lyases. As the name suggests,  35  1.4. Hydration and elimination mechanisms in glycoside cleavage these cleave the glycosidic bond of α-(1,4)-linked polymers through an elimination mechanism, and are predominantly involved in the metabolism of energy storage polysaccharides such as glycogen and starch. 61 The first step in this mechanism is the displacement of the α-linked anomeric group by a catalytic nucleophile, in much the same manner as the first step in the mechanism of a retaining α-glycosidase. However, the glycosyl-enzyme intermediate thus formed then undergoes a syn-elimination of an adjacent proton and the nucleophile, rather than a second displacement by water, to form anhydrofructose. On release from the enzyme active site, this tautomerises to its keto form, and then hydrates to give a geminal alcohol. 62 The product of these lyases can be used directly in energy metabolism, 61 or alternatively can be hydrated by α-glucosidases from GH13 or GH31 to yield glucose in what is proposed to be an anhydrofructose scavenging system. 63 The mechanism for this hydration has not been investigated, but presumably mimics that of glucal hydration (refer to the end of Section 1.3. The mechanism of α-(1,4)-glucan lyases and the subsequent product rearrangements are shown in Scheme 1.6. This mechanism uses the same basic catalytic machinery, and indeed the same first step, as the α-glucosidases of the same family. The formation of the covalent glycosyl-enzyme intermediate was observed by trapping with 5-fluoro-β-l-idosyl fluoride and detection of the labelled peptide by mass spectrometry. 64 Interestingly, trapping could not be observed with 1-, 2- or 5-fluoro-α-d-glucosyl fluorides, which instead acted as slow substrates. 65 The formation of this covalent glycosyl-enzyme intermediate was seen to be rate limiting, with a strong α-secondary kinetic isotope effect from 1-[2 H] substrates showing a dissociative transition state, and a shallow negative slope in the linear free energy plot of both kcat and kcat /Km showing substantial proton donation to the leaving group. Consistent with this, substitution with deuterium at carbon 2 revealed only a small β-secondary effect. For the partic-  36  1.4. Hydration and elimination mechanisms in glycoside cleavage  O  HO  O-  O  HO HO  HO  HO  O  O  O  HO HO  OR  H O  δ+  δ+  HO δ- H O  O  δ-  OR  HOR  O  HO  O HO  HO HO  O  OH OO  O H  HO  O δ- O HO  O  HO HO  H  HO HO  O-  δ-  O  δ+  δ+  HO  OH  O-  O  O  HO HO HO  O  GH13/ GH31  O HO  H2O OH  HO HO HO  O  +/- H+  HO HO HO  O O  H2O H2O  HO HO HO  HO O OH  OH  Scheme 1.6: The general mechanism of α-(1,4)-glucan lyases (upper), with rearrangements and further reaction of the product in water also shown (lower).  37  1.4. Hydration and elimination mechanisms in glycoside cleavage ularly slow substrate 5-fluoro-α-d-glucosyl fluoride the deglycosylation/elimination step was found to be rate limiting, and a small primary kinetic isotope effect was seen from a 2-[2 H] substrate, while a 1-[2 H] substitution showed a fairly large α-secondary effect. Together these suggest that the proton abstraction step occurs in a concerted but asynchronous process. In other words, proton abstraction only occurs after substantial bond cleavage at the anomeric carbon to generate an oxocarbenium ion-like transition state, the charge of which increases the acidity of the proton on carbon 2. The catalytic base for this proton abstraction has been suggested to be the catalytic nucleophile itself. The asynchronous nature of this elimination makes this plausible, as at the transition state the glycosyl-enzyme bond has largely broken to give a proton, acidified by an adjacent carbocation, with a carboxylate in close proximity. Indeed, the placement of the carbonyl oxygen in GH31 α-glucosidases is over the proton on carbon 2 of glucose. 66 Such a syn-elimination is the reverse of the syn-addition of the catalytic nucleophile in glucal hydration (Section 1.3). A similar elimination has been observed in the formation and subsequent hydration of a minor product d-ribal in the 2-deoxy-ribosyltransferase from Lactobacillus leishmanii, 67 and also in the observation of 1,5-anhydro-d-arabino-hex-1-enitol, an isomer of anhydrofructose, in the active site of an X-ray crystal structure of glycogen synthase from Escherichia coli, 68 both formed by glycoside-synthesising enzymes in the absence of an appropriate acceptor. In the first case the same enzyme is responsible for both the elimination and hydration reactions, emphasising the implied relationship between the mechanisms of α-(1,4)-glucan lyases and glucal hydration by glycoside hydrolases. In the latter case the observed 1,5-anhydro-d-arabino-hex1-enitol did not seem to be catalytically competent, suggesting this is an off-path intermediate formed from the highly activated glycosyl donor in the absence of any suitable acceptor. If this assignment of the nucleophile residue as the catalytic  38  1.4. Hydration and elimination mechanisms in glycoside cleavage base for its own elimination is correct, this provides a convenient explanation for the lack of trapping observed with 1-fluoro-α-d-glucosyl fluoride, which is instead a slow substrate. 65 These were anticipated to undergo a trans-elimination of HF from the glycosyl-enzyme intermediate to form a stable covalent species, as illustrated in Scheme 1.7. However, if the departing nucleophile itself is the catalytic base for this step then the catalytic base would never be present at the same time as the covalent glycosyl-enzyme intermediate, rendering such a trapping scheme impossible. While this explanation is appealing, other anticipated trapping reagents for this enzyme also did not provide trapped covalent glycosyl-enzyme intermediates, including the C5 epimer of the successful 5-fluoro-α-d-idosyl fluoride, indicating that other explanations are also plausible.  HO  B:  HO HO  O O  O-  HF F  HO  HO  B: O HO  HO HO  HO  F  H O O  F  HF O  HO  BH+ O O  HO HO  O  +/- H+  BH+ O O  HO HO  OO  O  O  HO  OO  HO  OO  Scheme 1.7: The anticipated mechanism of inactivation of α-(1,4)-glucan lyases by 1-fluoro-α-d-glucosyl fluoride, if a second residue were to be acting as catalytic base.  1.4.4  Polysaccharide lyases  Polysaccharide lyases are a class of enzymes that catalyse an elimination reaction that is superficially similar to that of the α-(1,4)-glucan lyases, but the mechanism by which this is achieved is very different. These enzymes are classified separately in the CaZY database, and substantially fewer of these families are known compared to the glycoside hydrolases (22 compared to 131, respectively, as of March 2013). The general mechanism of this class of enzymes involves three key components. 69 The 39  1.4. Hydration and elimination mechanisms in glycoside cleavage  OO  OO  H O  O  δO  O  RO HO  OH  H  OR'  H O O  RO HO H  -O  OH  OR'  O  OO -  O  RO HO H O  H O O OH  OR'  OOO O  H O O OR' OH  HO -O  O δO RO  H O O  HO  H  OH  OR'  O  Scheme 1.8: The general mechanism of polysaccharide lyases acting on pectate.  40  1.4. Hydration and elimination mechanisms in glycoside cleavage first is the neutralisation of charge from the carboxylate at carbon 6, presumably occurring immediately on binding of substrate to the enzyme active site, followed by abstraction of the proton at carbon 5 to form a carbanion intermediate with resonance stabilisation by the adjacent carboxylate. Finally, the free electron pair forms a double bond between carbons 4 and 5, expelling the leaving group at carbon 4 in the process and giving an unsaturated glucuronide as the product. This overall E1 cb reaction mechanism is outlined in Scheme 1.8. Because of the mechanistic involvement of the carboxylate group, all substrates for polysaccharide lyases must contain this moiety. Indeed for some polyanionic saccharides, degradation by lyases is the only known path for catabolism. 70 This mechanism is very similar to that of the C5-epimerases acting on the same substrates, differing in the nature of the final step — protonation either occurring on the carbon 4 oxygen to give elimination or on carbon 5 from the opposite face to give epimerisation. 69 On the basis of a set of experiments using chemically defined short substrates with chondroitin AC lyase from Flavobacterium heparinum, 71–74 the rate determining step for this mechanism was found to be abstraction of the proton from carbon 5 of the substrate. 71 When this proton was substituted with deuterium a small primary kinetic isotope effect was observed, while variation of the leaving group at carbon 4 was found to have no effect on the rate of reaction (for a set of activated aryl substrates). Consistent with this, a deuterium substitution at carbon 4 resulted in no significant kinetic isotope effect (kH /kD = 1.0). Deuterium was not observed to have exchanged at carbon 5 in a partially-cleaved sample, suggesting that deprotonation is irreversible, but may also indicate solvent inaccessibility of the active site during catalysis (elimination is faster than reprotonation or solvent exchange). A corollary of this E1 cb mechanism is that unactivated thio-linked substrates are turned over by these enzymes, but only very poorly. 75  41  1.4. Hydration and elimination mechanisms in glycoside cleavage The various structural features of polysaccharide lyases have recently been very thoroughly reviewed by Garron and Cygler. 76 These enzymes can generally be grouped into one of two types, depending on the residues responsible for the catalytic functions of charge neutralisation, base catalysis, and acid catalysis. One group, containing almost exclusively pectate and pectin lyases, employs a divalent metal for charge neutralisation, an arginine or a lysine as a catalytic base, and water as a catalytic acid. An example is the pectin lyase from Cellvibrio japonicus, which contains a single calcium bridging the carboxylates of the +1 and -1 subsites, an arginine catalytic base, and no clear acid residue, although a water ligand of the calcium ion or an aspartate acting via the carbon 3 hydroxyl were proposed as candidates for this role. 77 Because of the high charge density of pectate, a poly-galacturonide, these enzymes may contain as many as four divalent metals. 78 By contrast, in enzymes acting on pectins with methyl esters, many sites homologous to these metal binding sites are poorly conserved, reflecting the lack of a requirement for charge neutralisation in these substrates. The other group of polysaccharide lyases is more diverse, but generally employs an amide or acid side chain to protonate the substrate carboxylate, a histidine or tyrosine as the catalytic base, and a tyrosine as the catalytic acid. In syn-eliminating enzymes, a single tyrosine can act as both acid and base. 74,79 An interesting case within this class is exemplified by the chondroitin ABC lyases from Bacteriodes thetaiotaomicron and Proteus vulgaris. 80 These enzymes are able to catalyse both syn- and anti -elimination in the same active site. It appears that binding of each substrate type (glucuronides or iduronides) recruits the appropriate catalytic machinery for its degradation through conformational shifts, with a single substrate binding domain forming a part of two partially overlapping active sites. For syn-elimination, a single tyrosine acts as both base and acid, as has been seen in the related chondroitin AC lyases. 74 For the anti -elimination the same  42  1.4. Hydration and elimination mechanisms in glycoside cleavage tyrosine still acts as a catalytic acid, but the role of catalytic base is filled by a pair of histidine residues that are located 12 Å away in the enzyme’s resting state. A metal ion is also involved in the active site of this enzyme, and appears to mostly be important for turnover of dermatan sulfate, which contains iduronic acid residues and thus requires the catalytic residues for anti -elimination .  1.4.5  Unsaturated glucuronyl and galacturonyl hydrolases  Unsaturated glucuronyl and galacturonyl hydrolases (UGL and UGH, respectively) degrade the products released by polysaccharide lyases by use of a hydration reaction. UGL are the primary family of enzyme studied in this thesis. These were identified in Bacillus sp. GL1 in a pathway for total degradation of xanthan (for the structure of this substrate, see Subsection 1.2.5), 81 and have since been cloned and expressed from a variety of bacteria. 82–85 These were initially thought to be a specialised group of hydrolases operating by a Koshland mechanism, 86 but structural and limited biochemical evidence has suggested otherwise. 87 The mechanism based on this evidence comprises an initial hydration of the double bond between carbons 4 and 5, followed by rearrangement of the hemiketal product, through an intermediate hemiacetal, to cleave the glycosidic bond and afford a free unsaturated uronic acid (a α-keto acid) as shown in Scheme 1.9. The free unsaturated glucuronic acid thus liberated is further catabolised in a pathway in which it is reduced and tautomerised at carbon 2 over two steps, phosphorylated at what was carbon 1 (now 6, from a change in numbering), and finally cleaved by an aldolase to afford two common metabolites, pyruvate and d-glyceraldehyde-3-phosphate, as shown in Scheme 1.10. 88 The expression of an unsaturated glucuronyl hydrolase and polysaccharide lyase pair, along with a phosphoenolpyruvate-dependent phosphotransferase system for import of unsaturated glucuronic acid disaccharides, are necessary for  43  1.4. Hydration and elimination mechanisms in glycoside cleavage growth of Streptococcus pneumoniae on hyaluronic acid as a sole carbon source. 89 HN  O O  COO-  H  H O H  O OR OH  HO O  O-  HN  O N  O-  COO+  O  HO  H2N  O  H O H  N  O OR OH  O HN  O  H2 N  O-  N  OH -OOC  O  HO O  O  O  HN N  OH -OOC  ROH  O O  HO  O  HO O  O-  COOHO  O OR OH  (glycosidase)  OH  O OR NH2  O-  N H OO  OH O  H  HN  OH -OOC  NH2  O  OH  O OR NH2  O-  H+ COO O B: HO O OH H  O  OH  -OOC  O OH  Scheme 1.9: The general mechanism proposed for unsaturated glucuronyl hydrolases (upper). Also shown is a rearrangement to form the same final product following the hypothetical cleavage of a ΔGlcA derivative by a glycoside hydrolase mechanism (lower). Two related GH families have been identified that operate by this same general mechanism; GH88, 83 containing unsaturated glucuronyl hydrolases (UGL), and GH105, 84 containing unsaturated galacturonyl hydrolases (UGH, also known as URH for unsaturated rhamnogalacturonyl hydrolase). These names refer to the source polysaccharide of the substrates for each enzyme, with UGL substrates being derived from polymers containing β-glucuronide monomers in the repeating unit and UGH substrates being derived from polymers containing α-galacturonide monomers in the repeating unit. 90 These polymers are primarily glycosaminoglycans for UGL 44  1.4. Hydration and elimination mechanisms in glycoside cleavage  O  4-deoxy-5keto-uronate isomerase  OH  HOOC  O  OH  OH  HOOC  O  2-dehydro-3deoxy-D-gluconate 5-dehydrogenase  OH  O  OH OH  HOOC  O  OH 2-keto-3-deoxy -D-gluconate kinase  O  2-keto-3-deoxy6-phosphogluconate aldolase  O +  OPO32-  HOOC OH pyruvate  O  OH OPO32-  HOOC OH  D-glyceraldehyde-  3-phosphate  Scheme 1.10: Bacterial metabolic pathway for catabolism of free ΔGlcA to the common metabolites pyruvate and d-glyceraldehyde-3-phosphate. The site of modification for each step is in red for emphasis. and pectins for UGH. Because the 4,5-unsaturated glucuronide and galacturonide products are the same compound, with the distinction at carbon 4 being lost on formation of the double bond, the actual distinction in the substrate specificities of these enzyme families is the stereochemistry of the anomeric bond. UGL are thus αΔGlcAses, with pseudo-equatorial anomeric constituents, and UGH are β-ΔGlcAses, with pseudo-axial anomeric constituents (the reference stereo-centre for α/β nomenclature also being affected by the double bond between carbons 4 and 5 — to avoid confusion, the anomeric configuration of unsaturated glucuronides will often be referred to as axial or equatorial in this thesis, derived from α- and β-glucuronides respectively). Because of this specificity, enzymes from mammalian pathogens and symbiotes predominate in GH88 and enzymes from plant pathogens and symbiotes predominate in GH105, but many cases of both are found in soil bacteria for decomposition of dead organic matter. 90 A large proportion of the work carried out on UGL and UGH has focused on X-ray crystallography, with an excellent series of structures being determined by  45  1.4. Hydration and elimination mechanisms in glycoside cleavage Hashimoto, Mikami, Murata and co-workers at Kyoto University. These have been determined for UGL from a non-pathogenic Bacillus strain with an inhibitor bound (glycine), 91,92 with substrates bound, 87,93 and in apo-form, 93 and also from a pathogenic Streptococcus species both in apo-form 85 and with substrate bound. 94 Similarly, structures for UGH have been determined in apo-form, with an inhibitor bound (UGL substrate in the +1 subsite), 84 and with substrate bound. 95 The active sites of these are very similar, with catalytic residues overlaying well. In the resting state, the two catalytic aspartate residues hydrogen bond to one another, likely accounting for the high pKa of the second residue. On substrate binding, the catalytic acid/base residue rotates through 70° to position itself adjacent to carbon 4 of the substrate. No residue is positioned close to the anomeric oxygen. Strong interactions are seen with the ΔGlcA in the -1 subsite, while less interactions take place in the +1 subsite. This gives UGL and UGH fairly wide substrate specificities, the strongest requirement being for a ΔGlcA moiety. An example active site structure of UGL with a hyaluronan substrate disaccharide bound is shown in Figure 1.11. Aside from crystallography, little mechanistic work has been reported for GH88 or GH105. Two experiments were performed when the mechanism was first proposed, showing a solvent kinetic isotope effect of 2.1 and 2.2 on kcat and kcat /Km , respectively, and incorporation of  18 O  at carbon 5 of the product by GC/MS following  reaction in H2 18 O. However, these SKIE values are consistent with many alternative mechanisms, and incorporation of  18 O  at carbon 5 of the product is possible by  non-enzymatic means, for example by hydration/dehydration of the product carbon 5 ketone in water (see Scheme 1.9, lower panel). Some mutagenesis of conserved residues in the active site of the Bacillus sp. GL1 UGL has also been carried out, as summarised in Table 1.1. Conservative mutation of two aspartate residues (D88 and D149, all residue numbers in this section from Bacillus sp GL1 UGL) 92 was found  46  1.4. Hydration and elimination mechanisms in glycoside cleavage  Figure 1.11: X-ray crystal structure of the Bacillus sp. GL1 UGL D88N active site from two perspectives (top and looking at the ΔGlcA anomeric bond), showing an unsaturated hyaluronan disaccharide substrate bound and all side-chains within 5 Å (glycine and alanine omitted). The substrate is coloured by atom (dark grey, carbon; red, oxygen; blue, nitrogen), while amino acid side-chains are coloured by type: light grey, non-polar; yellow, polar; red, negatively charged; blue, positively charged. (PDB: 2FV1) 47  1.4. Hydration and elimination mechanisms in glycoside cleavage to have a drastic effect on turnover, with little associated change in binding to the enzyme. Mutation of an arginine (R221) 87 adjacent to the ΔGlcA carboxylate, initially thought to be responsible for neutralisation of its charge, was found to have no effect on substrate binding. A histidine residue (H193) 87 appears to be much more important for binding, but the main source of charge neutralisation for the substrate carboxylate was instead proposed to be the positive end of an inner α-helix dipole. Finally, a glutamine residue (Q211) 87 was also found to be somewhat important for turnover. Table 1.1: Michaelis-Menten kinetic parameters for previously characterised Bacillus sp. GL1 UGL mutations, using unsaturated gellan tetrasaccharide as substrate. Enzyme Wild type 92 D88N 92 D149N 92 R221A 87 H193A 87 Q211A 87  kcat (s-1 ) 7.3 0.00057 0.0059 4.2 0.42 0.20  Km (µM) 90 200 60 69 980 170  kcat /Km (rel., %) 100 0.0036 0.12 75 0.53 1.4  Based on the structures published, and the placement of residues within them, one aspartate (D149) has been proposed to act as the proton donor in the initial hydration step, and as the base in the subsequent addition of water. 85,87,94,95 Another catalytically important aspartate, D88, was proposed to hydrogen bond to the hydroxyl groups on carbons 2 and 3, preventing their interference with the role of D149 by hydrogen bonding, and also to stabilise the oxocarbenium ion-like transition state for the hydration reaction. D88 was also proposed to modulate the acidity of D149, ensuring it is protonated through the hydrogen bond seen in the apo-form. 93 However, this role seems inadequate in explaining the large effect seen on turnover when this residue is mutated to asparagine. If the role of D88 is in aiding D149, its mutation to an amide should not have a larger effect on turnover than the same 48  1.4. Hydration and elimination mechanisms in glycoside cleavage mutation in the acid itself. Moreover, an amide at this position should still be able to form hydrogen bonds, and D88 is located on the opposite side of the hexose ring to the site of charge development, making any direct transition state stabilisation of this charge implausible. The role of this residue thus remains unclear.  1.4.6  N -Acetyl-muramic acid 6-phosphate hydrolase (MurQ)  While not strictly a glycoside hydrolase as it is cleaving an ether, the enzyme N acetyl-muramic acid 6-phosphate hydrolase (MurQ) catalyses an elimination and hydration reaction in a monosaccharide, as shown in Scheme 1.11, and so warrants mention here. This enzyme is involved in the scavenging and recycling of bacterial cell wall components, and is required by Escherichia coli for growth on N -acetylmuramic acid as a sole carbon source, 96,97 forming GlcNAc-6-phosphate by cleavage of the lactate ether at carbon 3 of MurNAc-6-phosphate. Interestingly, this enzyme appears to have undergone duplication and fusion with itself, along with loss of most but not all etherase activity, to become a mammalian glucokinase regulatory protein that responds to glucose- and fructose-6-phosphate, using the original etherase active site. 98 In the mechanism of this enzyme, 99 the aldehydic tautomer of the monosaccharide substrate is used to activate the proton at carbon 2 for abstraction by a catalytic base residue. The enolate product of this is then able to rearrange to cleave the ether bond at carbon 3 with assistance from a catalytic acid residue, in a similar manner to the mechanism of the polysaccharide lyases outlined in Subsection 1.4.4. Following exchange of the leaving group for water, a Michael type addition to the α/β unsaturated intermediate then occurs, with deprotonation by the former catalytic acid residue. In a final step the proton at carbon 2 is then replaced by the former catalytic base residue to give the hydrolysed product, which is then able to  49  1.4. Hydration and elimination mechanisms in glycoside cleavage  +  HB  :B 2-O  3PO  HO  O  2-O  O OH NHAc  3PO  HO  H OH  O  H CO2- A  CO2-  O NHAc  2-O  3PO  HO  OH ONHAc  O  CO2-  H  lactate H2O  A  2-O  +HB  3PO  OH  HO  O H :B 2-O  3PO  HO HO  2-O  O OH NHAc  3PO  H OH  HO HO H  O NHAc A  2-O  H  3PO  O  H  NHAc A-  B+  OH  HO HO  ONHAc H  A  Scheme 1.11: The general mechanism for N -acetyl-muramic acid 6-phosphate hydrolase (MurQ). re-tautomerise to its cyclic form on release from the enzyme. This mechanism is very similar to that established for the rapid non-enzymatic thermal decomposition of the disaccharides Gal-(1,3)-GalNAc and Gal-(1,3)-GlcNAc at neutral pH, with a half-life on the order of minutes. This proceeds through an elimination half reaction to the same α/β unsaturated intermediate, but instead of re-hydrating this forms intramolecular adducts. 100 MurQ may have an important role in accelerating the hydration reaction to prevent formation of such off-path products. Evidence for this mechanism comes from an excellent set of isotope exchange, NMR, and mutagenesis experiments. 99 Abstraction of the proton on carbon 2 was shown through the observation of its exchange with deuterium when the MurQcatalysed reaction is carried out in D2 O, while reaction in H2 18 O gave incorporation of  18 O  at carbon 3, but not carbon 1. This second observation indicated that a  Schiff’s base, a common enzymatic strategy for activating aldehydes and ketones,  50  1.5. Thesis aims likely does not form. A small primary kinetic isotope effect was also observed using a substrate deuterated at carbon 2, indicating that this step is rate-limiting for the first half-reaction. The α/β unsaturated intermediate was observed to accumulate in solution during the course of the reaction, showing that hydration is the slower of the two half-reactions. Finally, a homology model of MurQ built using the structure of a distantly related protein of unknown function, solved in a structural genomics project but never published, indicated two aspartate residues that could fill the roles of the two acid/base catalysts. Mutants of each of these were indeed found to be substantially reduced in activity. For one of these, D83, deuterium exchange at carbon 2 persisted in the mutant, suggesting that this is the catalytic acid residue responsible for the elimination reaction, and the other, D114, is thus likely the initial catalytic base residue.  1.5  Thesis aims  Given that unsaturated glucuronyl hydrolases are involved in the degradation of the extracellular matrix of mammalian tissue, and the role of this degradation both in providing an energy source and in removing a physical barrier to bacterial motility, they are clear pathogenicity factors. This family of enzymes represents a particularly promising target for therapeutic agents with minimal side-effects as they are not produced by vertebrates, and appear to be acting through a completely novel mechanism for glycoside hydrolysis. An eventual long-term goal of this work is to furnish either an inhibitor or inactivator of UGLs, allowing the selective removal of this activity in infected tissue, thus slowing or even halting its spread. In order to facilitate design of such a bacteriostatic agent, a clear understanding is required of the exact reaction catalysed and the mechanism by which this is achieved. Furthermore, the vast majority of glycoside hydrolysing enzymes that have been characterised op51  1.5. Thesis aims erate through either the inverting or retaining mechanisms of Koshland, 33 with very few variations on this. The only other completely novel mechanism for glycoside hydrolysis that has been established to date is that of the related families GH4 and GH109. If the proposed hydration mechanism is shown to be correct it will represent a second, although with much more limited scope given its requirement for an unsaturated uronide. The first goal of this thesis is thus to clearly show that UGL does indeed catalyse a hydration reaction. While some evidence has been published for this reaction, 87 alternate interpretations of the data remained possible. Following confirmation of the general reaction catalysed, a more in-depth study will be undertaken, aiming to characterise the rate determining step of the reaction and the structure of its associated transition state. Knowledge of the charge and geometry of the transition state is essential for guiding design of a strongly binding competitive inhibitor that mimics these features, as enzymes are known to bind very strongly to the transition state in order to catalyse their reactions. This will be achieved by careful characterisation of UGL reaction products, measurement of a linear free-energy relationship, determining the effects of heteroatom substitution in substrates, kinetic isotope effects, and finally the testing of some potential inhibitors and inactivators of UGL. A further goal of this thesis, related to the work on UGL, is to investigate the mechanism of mammalian heparanase (HPSE). This is an enzyme responsible for degradation of heparan sulfate and heparin during tissue remodelling and growth, and has been strongly implicated in cancer metastasis. This thesis presents work towards the development of a convenient fluorogenic assay of HPSE, and an attempt at development of a 2-deoxy-2-fluoro-glucuronide inactivator both as a lead compound for treatement and in order to allow confirmation of the catalytic nucleophile.  52  Chapter 2  Confirmation of the hydration reaction In the simple hydration-initiated mechanism presented in Scheme 1.9 on page 44, UGL is proposed to catalyse hydration of the carbon 4 to carbon 5 double bond. This hypothesis needs to be tested before any further investigation of the details of this mechanism can be carried out. Following cloning, expression, and optimisation of UGL, and investigation of the natural substrate preferences, the UGL-catalysed reaction of unsaturated glucuronides in D2 O, to give an isotopic label for the site of protonation, and in methanol, as an alternate nucleophile to give a stable intermediate, were envisaged as first tests of this mechanism. Further confirmation was sought in compounds that would only be anticipated to act as substrates under a hydration-initiated mechanism. Clostridium perfringens was chosen as a source organism for UGL as it is an organism of very high disease relevance, with infections often having very serious consequences — it is a common cause of food poisoning and post-surgical infection, with this infection necessarily involving degradation of the extracellular matrix and its GAGs. 101–103 Because of this strong infectivity this organism was expected to have a suitable copy of UGL, which was confirmed by homology searching of the genomic DNA. The commercial availability of genomic DNA for C. perfringens was also an advantage of using this organism, with DNA for the ATCC13124 strain 53  2.1. Cloning and purification of Clostridium perfringens UGL already being present in the laboratory at the time of commencing this study.  2.1  Cloning and purification of Clostridium perfringens UGL  In order to investigate the reaction carried out by UGL, a reliable source of pure enzyme is required. To achieve this, the enzyme was cloned from C. perfringens and heterologously expressed in E. coli using the pET28a vector to provide a hexahistidine tagged enzyme, and was purified by metal ion affinity chromatography.  2.1.1  Cloning  Primers for cloning of the gene for UGL from C. perfringens were designed to amplify the gene and to allow insertion with two of the restriction sites present in the multiple cloning site of the commercial pET28a(+) expression plasmid — XhoI and NheI. The insertion site was selected in order to generate a fusion peptide with an Nterminal hexa-histidine tag. PCR from C. perfringens genomic DNA proved difficult initially, but longer primers to overcome the low GC content and optimisation of temperature and magnesium concentration allowed isolation of a small amount of product. Gel purification of this product and its use as a template for a second round of amplification yielded sufficient DNA to insert the gene into the desired plasmid and, following direct transformation into BL21(DE3) cells by electroporation, several colonies were picked and sequenced to confirm the insert.  2.1.2  Enzyme expression and purification  Expression was first tested at small scale in a variety of growth conditions, looking for a protein of the correct size by SDS-PAGE in the supernatant or pellet of lysed  54  2.1. Cloning and purification of Clostridium perfringens UGL cells (Figure 2.1). The effects of growth temperature, growth medium, cell density at induction, and concentration of IPTG used for induction on protein levels were assessed. All conditions gave acceptable growth of cells and high levels of soluble protein, with some variability, so the choice of expression conditions was based largely on ease of workflow, opting for overnight expression following induction with a low level of IPTG at low cell density in TYP medium.  Figure 2.1: Expression of UGL under varying conditions. Calculated mass for UGL is 48 kDa. The red arrow indicates selected conditions for large-scale expression. Protein expression was then scaled up to a half litre culture for purification of the enzyme. Cells were grown to mid log phase (OD600 of 0.5–1), induced, and the protein expressed overnight before harvesting. Lysing of cells and pelleting of cellular debris gave a clarified lysate that was purified using a nickel-affinity column. Stepwise elution with increasing concentrations of imidazole gave protein that was deemed to be sufficiently pure for enzymatic work, as assessed by SDS-PAGE (see Figure 2.2). The imidazole and salt from the purification were removed by repeated spin filtration in a 30 000 kDa cut off spin filter, with a final estimated dilution of 15 000 fold, bringing the imidazole to low micromolar concentrations. The concentration of enzyme was determined from a calculated extinction coefficient at 280 nm, 55  2.1. Cloning and purification of Clostridium perfringens UGL  Figure 2.2: Purification of UGL, with sample elution trace for wild type UGL (upper), showing A280 in blue, conductivity in brown, % buffer B in green (up to 100% at its maximum), and fraction numbers in red. Axes are eluted volume in mL and absorbance in mAU, with axes not shown for other traces. (Lower) Sample SDS-PAGE for purification of UGL, with fractions and marker masses as labelled.  56  2.2. Development of a chromogenic substrate with this process yielding around 45 mg of protein per litre of culture.  2.2 2.2.1  Development of a chromogenic substrate Previous assays  In earlier work, 82,84–87,93 kinetic parameters for cleavage of natural substrate by UGL were determined by monitoring the decreasing absorbance at 240 nm from the double bond in the starting material. A phenyl unsaturated glucuronide has also been reported in a study of UGL anomeric specificity. 104 Monitoring the substrate double bond, while successful for the limited characterisation carried out previously, has many restrictions. Because of the short wavelength of this absorption peak, there are many compounds that may interfere with these readings. Related to this, this method requires the measurement of a decrease in an initially high substrate concentration, meaning that there are practical limitations on how high the starting substrate concentration can be before this method becomes unsuitable. The assay has a useful range from high tens of micromolar, where signal is low, to low millimolar concentrations, where the initial absorption becomes too high. Finally, because this method monitors the consumption of starting material, and not the formation of product, this means that if the enzyme catalysed a fast reaction followed by a slower second step, which is a plausible scenario given the initially proposed hydration mechanism, this second step would not be observable using a method that only monitors reactants. In a mechanistic study it is useful to be able to monitor both substrates and products. Access to natural substrates is also very limited, coming from the degradation of glycosaminoglycans by polysaccharide lyases, and commercial sources are very expensive for small amounts. Available quantities are sufficient for characterisation of basic kinetic parameters, but insufficient for in-depth study.  57  2.2. Development of a chromogenic substrate While this older method remained useful for characterisation of natural substrates (Section 2.4 on page 65), most kinetic measurements in this work were performed using aryl unsaturated glucuronide substrates. These provided a product that could be easily and accurately detected at wavelengths where there is minimal background interference, are able to be synthesised in large quantities, and are easily variable at key positions with heteroatoms or isotopes, all while avoiding perturbation of the natural mechanism.  2.2.2  Substrate synthesis  Several sources in the literature had previously reported the synthesis of simple unsaturated glucuronides, for use either as substrates in early work on UGL 104 or investigations of the bleaching process of paper from the Kraft process, where such unsaturated glucuronides are an important side product of the strong bases used and are responsible for brightness reversion of the paper and excess consumption of bleaching chemicals. 105,106 Synthetic compounds reported were predominantly methyl or phenyl glycosides, and are largely unsuitable for in depth mechanistic studies because of difficulties in varying the anomeric group, low yields, or requiring an inconveniently large number of steps. A route was envisaged, based largely on that reported by Azoulay et al., 107 which comprised four key steps: global protection of the starting material, glycosylation of the desired anomeric alcohol, elimination across carbons 4 and 5, and global deprotection of the product. The final optimised routes for 4-nitrophenyl ΔGlcA (6) and phenyl ΔGlcA (10) are given in Scheme 2.1 and Scheme 2.2, respectively. Global protection was achievable through several routes, with either methyl ester formation or acetylation taking place first. The most convenient was found to be from glucuronic acid-γ-lactone (1), opening the lactone with sodium methoxide in  58  2.2. Development of a chromogenic substrate 1. NaOMe, MeOH 2. Ac2O, HClO4  HO O O  MeOOC AcO AcO  OH O  1. NaOMe, MeOH 2. LiOH, THF/H2O  COOH O O OH  HO  Br  97%  Ag2O, 4-nitrophenol ACN DBU, DCM  MeOOC AcO AcO  O O  OAc  NO2  NO2  5  80%  AcO  58%  O O OAc  NO2  O  3  COOMe  AcO  6  OAc  OAc  MeOOC AcO AcO  2  OH  1  HBr, AcOH  O  4  92%  54%  Scheme 2.1: Final optimised 4-nitrophenyl glycoside substrate synthesis. 1. NaOMe, MeOH 2. Ac2O, HClO4  HO O O  OH O  O OAc  58%  COOH O O OH  H2NNH2.AcOH OAc DMF  MeOOC AcO AcO  2  OH  1  HO  MeOOC AcO AcO  NaOH, acetone/H2O AcO  COOMe O O OAc  O AcO  OH  7 65% 1. Trichloroacetonitrile/DBU, DCM 2. BF3.OEt2/phenol, DCM DBU, DCM  MeOOC AcO AcO  O OAc  10  9  8  77%  51%  30%  O  Scheme 2.2: Final optimised phenyl glycoside substrate synthesis. methanol then acetylating with acetic anhydride under acidic conditions (to avoid elimination side-products) to give a mixture of anomeric forms of per-O-acetylated methyl glucuronate (2). 108 Purification of this product on the large scale proved to be problematic. The reactions appeared to go cleanly by TLC, but on workup and removal of solvent a thick slightly yellow syrup was formed. While this product could be used without further purification for many reactions, for long term storage and for reactions requiring more controlled stoichiometry a solid was more convenient. The most effective means found to achieve this was trituration from a small volume of methanol, yielding a white powder suspended in a residual amount of solvent 59  2.2. Development of a chromogenic substrate that could be conveniently filtered off and washed with a small additional portion of methanol then dried and stored. This process could be repeated several times with the filtrate to increase recovery, with no apparent loss in purity of the solid recovered. Crystallisation from ethanol, isopropanol, or toluene with petroleum ether gave a lower recovery, and the product remained sticky and difficult to handle. The overall yield for this protection was 58%, mostly limited by the number of times the evaporation from methanol was carried out, with each iteration giving diminishing recovery. The method used for glycosylation by Azoulay, using tin tetrachloride and 4nitrophenol directly with globally protected glucuronic acid (2), was relatively lowyielding. Improvement was sought through activation of the glycosyl donor using two alternate methods. Deprotection of the anomeric position to give the free hemiacetal (7) and formation of the Schmidt donor was somewhat successful, but the activated donor was found to be unstable and it was difficult to avoid significant hydrolysis when reacting with BF3 diethyl etherate and either phenol or 4nitrophenol (Scheme 2.2). By contrast, formation of the glucuronyl bromide (3) using hydrobromic acid in acetic acid proceeded smoothly and gave a very stable product that could be purified by flash column chromatography and stored for an extended period at -20 °C, although for routine preparations this was used without purification. Koenigs-Knorr glycosylation using this was also found to proceed smoothly, giving the desired product in moderate to good yields for most phenol derivatives (Scheme 2.1), although notably not for phenol itself, with purification often achievable by crystallisation (see Subsection 3.2.1 on page 96 for further discussion). Elimination of acetic acid from the protected aryl glucuronides (4 and 8) was achieved using DBU in dichloromethane overnight, and the reaction mixture could be loaded directly onto a flash column for purification (where inconvenient to purify  60  2.3. Enzyme optimisation immediately, the reaction was stopped by filtering through a small plug of silica then evaporating the solvent in vacuo). The success of this reaction was found to be highly dependent on the purity of the starting material, with crystalline starting materials often giving significantly better yields, but was otherwise found to be a reliable process with moderate to good yields. Deprotection of the resultant final products (5 and 9) was attempted by several methods, with the most reliable found to be deacetylation using sodium methoxide in mixed methanol and dichloromethane followed by hydrolysis of the methyl ester by a slight excess of lithium hydroxide in tetrahydrofuran and water. This method produced very clean products, although a C18 Sep-pak was used to remove salts and trace non-polar contaminants such as partially deprotected intermediates. An alternative using excess NaOH in 1:1 acetone and water then quenched with a slight excess of aqueous HCl was used in some cases, including phenyl ΔGlcA (9), but generated a large amount of salt that required subsequent removal. This method was found to be faster but more likely to give hydrolysis of the product. The overall yield for the 4-nitrophenyl ΔGlcA substrate (6) was 22% over 7 steps. Synthesis of the phenyl substrate (10) was much less efficient at 4.4%. This synthesis of 6 could be carried out with only one flash column — using the crude products of the first three steps to achieve 4, which can be crystallised from ethanol, then a single column after the elimination step yields the protected final product 5, which is deprotected in two steps with a final Sep-pak clean up.  2.3  Enzyme optimisation  With enzyme and substrate in hand, the enzymatic reaction conditions needed to be optimised before careful kinetic measurements could be made. BSA was found to be important for stabilising the enzyme, with around 0.1 % w/v proving sufficient and 61  2.3. Enzyme optimisation giving approximately twice the rate compared to a control without BSA. Inclusion of EDTA at 10 mM was found to have no effect on the rate of reaction, suggesting no metal ions are important in the reaction. A previous report 82 had suggested that sodium chloride may be important for the reaction, with 50–100 mM being required in that case for maximal rate, but any concentration of added salt was found to be detrimental to C. perfringens UGL (Figure 2.3A).  Figure 2.3: Effect of A, salt concentration; B, pH; and C, temperature, on the reaction rate of UGL with 4-nitrophenyl ΔGlcA (6). Y axes are a percentage of maximal kcat /Km for A and B, and of kcat /Km , kcat , and Km as noted for C. Enzyme stability was confirmed across the pH and temperature ranges tested (not shown). Measurement of kcat /Km for hydrolysis of 4-nitrophenyl ΔGlcA (6) over a range of pH values yielded the curve shown in Figure 2.3B. This showed an optimal pH of 6.6 and two pKa values of 6.1 and 7.0. Since the pH of dependence of kcat /Km reflects ionisation in the free enzyme or free substrate it was necessary to determine whether these could reflect the substrate ionisation state. The pKa of a representative substrate benzyl ΔGlcA (53 from Section 3.2 on page 96) was determined to be 4.5 ± 0.2 (Figure 2.4). This substrate was chosen as it did not show interference from the aryl group at low wavelengths, unlike the phenyl substrates. However, as  62  2.3. Enzyme optimisation measurements in this spectral region are inherently noisy, an average of the 200 to 220 nm spectral region with adjustment for the substrate double bond peak height at 235 nm was required to give a reasonable fit. This shows that ionisation of the substrate carboxylate group is not responsible for either of these inflection points. Given that the enzyme active site contains two aspartate residues that have been shown by mutagenesis to be critical for activity, it is parsimonious to attribute these pKa values to those two residues. The optimal pH of this enzyme is close to physiological pH, particularly that of new wounds, where the pH is slightly decreased relative to normal tissue. 109  Figure 2.4: Determination of the pKa of benzyl ΔGlcA (53). A, spectra of 53 from 260–200 nm at the following pH values: 2.7( ), 3.0( ), 3.5( ), 4.1( ), 4.6( ), 5.7( ), 6.5( ), and 7.3( ); B, fit of ratio (average A220 to 200 )/A235 against pH. Temperature was found to have an unusual effect on the rate of UGL hydrolysis, since the optimal temperatures determined for kcat /Km and kcat were not the same as each other, and together these show an increase in Km as temperature increases (Figure 2.3C). The increase in kcat with temperature was close to linear, rather than 63  2.3. Enzyme optimisation exponential as would be predicted by the Arrhenius equation. The enzyme was stable at all temperatures measured for longer than the assay required, showing no sign of denaturation. This is somewhat consistent with what was observed with F. heparinum UGL, 82 where activity peaked at 30 °C using a substrate concentration slightly above Km , despite the enzyme being stable to much higher temperatures. By contrast, UGL from Bacillus Sp GL1 86 showed a more conventional temperature profile with an activity peak at around 50 °C but decreasing stability above 30 °C, with a sharp drop in both above the activity maximum. A similar temperature effect to that observed for C. perfringens UGL has been observed for other enzymes, such as 3-phosphoglycerate kinases, 110 where enzymes sourced from mesophiles and thermophiles both showed this effect to varying extents, but at higher temperatures than for C. perfringens UGL (optima around 50–70 °C). In that case, the observation was explained by invoking an inactive intermediate I on the enzyme unfolding pathway in equilibrium with the native active form N, from which the enzyme then unfolds irreversibly to the denatured state D according to the equation N −− −− I −−→ D. The proportion of the I form increases with temperature, accounting for the apparent increase in Km , with saturating substrate ’S’ stabilising the enzyme in the N.S form, accounting for the trend in kcat readings. This reversible instability could be on a global or local scale, with potentially only the active site reversibly denaturing in a stable global background to give this folded but inactive form. 111 Given that an enzyme active site is generally optimised for increased flexibility over the protein as a whole in order to allow for catalysis, 112,113 this is an appealing explanation. The final reaction conditions determined to be used for further study involve use of 40 mM Mes.NaOH buffer at pH 6.6 with 0.1% BSA w/v at 37 °C, to balance optimisation of kcat and kcat /Km , or phosphate buffer at pH 7.0 for experiments involving NMR spectroscopy. The Michaelis-Menten kinetic parameters for hydrolysis  64  2.4. Natural substrate variation of the two aryl glycoside substrates discussed in section 2.2.2 (10 and 6) by UGL under these optimised conditions are given in Table 2.1. This buffer was much later found to act as a competitive inhibitor (Figure 2.5), with a Ki of 57 ± 5 mM. Given its use in the majority of experiments, use of this buffer was continued for all further experiments unless otherwise stated, with care taken to not vary its concentration from 40 mM to allow direct comparison of results. Table 2.1: Michaelis-Menten kinetic parameters for hydrolysis of the two aryl glycoside substrates 6 and 10 by UGL under optimised conditions. Structure  kcat (s-1 )  Km (mM)  kcat /Km (s-1 .mM-1 )  2.05 ± 0.06  0.26 ± 0.02  7.9 ± 0.8  4.3 ± 0.2  3.2 ± 0.4  1.3 ± 0.2  COOH HO  O O OH  NO2  (6)  COOH HO  O O OH  2.4  (10)  Natural substrate variation  The natural substrates for C. perfringens UGL are the products of reactions of polysaccharide lyases acting on the GAGs of mammalian tissue, and as such they can vary significantly in their glycosidic linkage and sulfation pattern, depending on the source GAG. Other UGLs that have previously been studied, from both pathogenic and non-pathogenic source organisms, have shown variability in their preferred substrates. 82,85,86 In order to probe the natural substrate preferences of C. perfringens UGL, a variety of unsaturated disaccharides of GAG origin were purchased and the Michaelis-Menten kinetic parameters for their cleavage determined. Activity was highly variable with these substrates, ranging from the highest kcat detected with any substrate in this work at 112 s-1 (11) to no detectable activity at all (16), while Km was less variable among the better substrates — see Table 2.2. For one sub-  65  2.4. Natural substrate variation  Figure 2.5: Dixon plot showing competitive inhibition of UGL by Mes.NaOH buffer with a Ki of 57 ± 5 mM. Substrate (6) was at the following concentrations: 25( ), 50( ), 170( ), and 600( ) µM.  66  2.4. Natural substrate variation strate (14), Km could not be determined as no sign of substrate saturation could be achieved within the limitations of both the assay and availability of compound. Substrates derived from both chondroitin and heparin sulfate were accepted by C. perfringens UGL, with no clear a priori determinant of the level of activity. By far the best substrate was ΔGlcA-β-1,3-GalNAc-6-sulfate (11), for which C. perfringens UGL showed a kcat an order of magnitude higher than the next best, while the equivalently sulfated substrate from heparin sulfate (14) was very poorly acted on by C. perfringens UGL. By contrast, the N-sulfated compound derived from heparin sulfate (12) was the second best of the natural substrates tested with C. perfringens UGL. For the other chondroitin-derived substrates, removing the sulfate still gives a reasonable level of activity, while when on the carbon 4 hydroxyl it gives very low activity, while moving it onto the 2 -position of the ΔGlcA completely ablates this activity. This lack of activity with a 2 -sulfated substrate is not particularly surprising on an intuitive level, as it places this negatively charged sulfate in close proximity to Asp113, and the interaction of these two negative charges could very plausibly interfere with that residue’s catalytic function. However, it is notable that some strains were able to cleave this substrate, as the Streptococcal strains all showed a small level of activity and for Bacillus this is a close third best substrate. Clearly, it is possible for UGL to accommodate a sulfate in this position, but how this occurs and what this means for the role of this residue is unclear. It is possible that the sulfate group itself takes over the role of Asp113 in these cases, since it has a similar charge and likely occupies a similar location. The pattern of relative activity with these substrates is fairly similar across C. perfringens, F. heparinum, Streptococcus pneumoniae, S. pyogenes, and S. agalactiae, while the Bacillus data are notably different. This likely reflects the different environments each organism lives in and the metabolic requirements of this environ-  67  Table 2.2: Comparison of kinetic parameters for UGL from different source organisms with GAG-derived natural substrates (Ch., chondroitin; He., heparin: Hy., hyaluronan; nd, not detected; blank, no data). Data from C. perfringens, F. heparinum, S. agalactiae, S. pyogenes, S. pneumoniae, and Bacillus sp. GL1. F. heparinum 82  C. perfringens Source GAG HO  COOH OH OSO3-Na+ O O O OH NHAc OH COOH  He. NS  Km (mM)  kcat /Km (rel.)  kcat (s-1 )  Km (mM)  kcat /Km (rel.)  (11)  112  3.7  1.00  11.7  0.107  1.00  HO  (12)  14  1.2  0.39  4.9  0.251  0.18  (13)  8.5  3.0  0.09  15.3  0.283  0.49  OH  O O OH HO  O OH NHSO3-Na+  COOH OH OH  Ch. 0S  HO  O  O O  COOH  He. 6S  OH NHAc  OH  HO  O O OH HO  -Na+  OSO3 O  OH NHAc  (14)  0.0009  -Na+  Ch. 4S  HO  COOH OSO3 OH O O O OH NHAc OH  (15)  0.09  4.5  0.0007  COOH OH OH  Ch. 2 S  HO  O  O O OSO3-Na+  OH NHAc  (16)  OH NHSO3-Na+  (17)  nd  COOH OH OH  Ch. 2 SNS Ch. 2 S6S Ch. NS6S 68  Ch. NH2 6S Hy. 0S  HO  HO  HO  HO  HO  O O OSO3-Na+  O  COOH OH OSO3-Na+ O O O OH NHAc OSO3-Na+ COOH OH OSO3-Na+ O O O OH NHSO3-Na+ OH COOH OH OSO3-Na+ O O O OH NH2 OH COOH O HO O OH  (18) (19)  8.8  0.334  0.24  (20)  2.4  0.235  0.09  OH O OH NHAc  nd  (21) {continued on the following page}  2.4. Natural substrate variation  Ch. 6S  kcat (s-1 )  Structure  Bacillus 86  Streptococcus 85 Source GAG Ch. 6S  Structure HO  COOH OH OSO3-Na+ O O O OH NHAc OH COOH  He. NS  HO  Km (mM)  kcat /Km (rel.)  1.3, 4.0, 24(10.2) 94  0.18, 0.39, 0.54(0.10) 94  1.00  2.7  1.27  0.003– 0.01  kcat (11)  (s-1 )  O O OH HO  O OH NHSO3-Na+  COOH  He. 6S  OH NHAc  O O OH HO  OSO3-Na+ O OH NHAc -  Ch. 4S  HO  0.22  14.1  0.38  0.98  (12)  O O  OH  HO  19  (13) (14)  nd– 0.0008  +  COOH OSO3 Na OH O O O OH NHAc OH  (15)  nd  COOH OH OH  Ch. 2 S  HO  O  O  O OSO3-Na+  OH NHAc  (16)  OH NHSO3-Na+  (17)  0.0001– 0.003  0.20  COOH OH OH  Ch. 2 SNS Ch. 2 S6S Ch. NS6S Ch. NH2 6S Hy. 0S  HO  HO  HO  HO  HO  O O OSO3-Na+  O  COOH OH OSO3-Na+ O O O OH NHAc OSO3-Na+ COOH OH OSO3-Na+ O O O OH NHSO3-Na+ OH COOH OH OSO3-Na+ O O O OH NH2 OH COOH O HO O OH  (18)  nd  (19) (20)  OH O OH NHAc  (21)  1.00  2.4. Natural substrate variation  Ch. 0S  O  50  kcat  OH  COOH OH OH HO  Km (mM)  kcat /Km (rel.)  (s-1 )  69  2.4. Natural substrate variation ment. Interestingly, F. heparinum, a soil-isolated bacterial strain, shows a substrate preference pattern in many ways similar to those of the pathogenic Streptococcus and Clostridium species, with high activity on sulfated substrates. The Bacillus species, which is also a soil isolate, has a quite different pattern of activity, however, but both do seem to have high activity on the unsulfated substrate. Since the UGL from Bacillus was initially isolated by growth on the unsulfated bacterial polysaccharide gellan, while the F. heparinum UGL was isolated from growth on heparin sulfate and chondroitin sulfate, this may reflect diverse diets within the different soil-isolated samples, decomposing organic matter from different organisms. Interestingly, in these other species the Km values for the GAG-derived substrates were lower than for C. perfringens UGL, with correspondingly lower kcat values to give similar kcat /Km values. The X-ray crystal structure of the Streptococcus agalactiae UGL with substrate bound revealed a binding motif, R-//-SXX(S)XK (where -//- is a large break in the sequence, and X is any amino acid), conserved in pathogenic bacteria-sourced UGL that is important for binding of the 6-sulfate of chondroitin-derived substrate 11, as shown in Figure 2.6. 94 This motif is located on a dynamic loop that moves upon substrate binding, and mutation of three key residues in this motif was observed to reverse the preference for 6-sulfated substrate over unsulfated substrate. Alignment of UGL sequences for the four species shown in Table 2.2 (with S. agalactiae chosen as a representative Streptococcal species) proved difficult, with different methods giving very different alignments of the F. heparinum sequence. These often showed no conservation of the otherwise very well conserved D113 and D173 catalytic residues (C. perfringens numbering). The result deemed the best was returned by the MUSCLE algorithm, 114 and the key results are given in Figure 2.7. Even in this best alignment, no residue is  70  2.4. Natural substrate variation  Figure 2.6: X-ray crystal structure of the S. agalactiae UGL active site showing the 6-sulfated chondroitin disaccharide substrate (11), the catalytic residues (D115 and D(N)175), and the sulfate binding domain (S365, S368, and K370), from two perspectives (PDB: 3ANK). 71  2.4. Natural substrate variation aligned with D113, although there are several other residues close in sequence (if not necessarily space) that could potentially be filling the same role. The putative catalytic acid residue, D173, is conserved, although the sequence context that is preserved in the other species is changed for F. heparinum. A BLAST search using the F. heparinum sequence returned GH88 sequences wherein the aspartate of the ’QNTRDART’ motif aligning with D113 is conserved in all but 4 of the top 99 hits, suggesting this may be filling the same role in these species. The 6-sulfate binding domain is present in its entirety in C. perfringens and S. agalactiae and completely absent in Bacillus, while F. heparinum contains one of the key residues and has a conservative mutation for a second. This provides some explanation for the apparent mixture of activities displayed by F. heparinum UGL, showing no strong preference for sulfated or unsulfated substrates. Interestingly, the histidine immediately preceding D113 also appears to be highly conserved, and in the substrate-bound crystal structure it is within hydrogen bonding distance, suggesting that these two residues may be acting as a catalytic diad, although no mutagenesis of this histidine has been reported. In F. heparinum the sequence aligning with D113 also has two basic residues (arginine) that appear to be well conserved in one or both positions (arginine or lysine). In two of the species presented, C. perfringens and Bacillus, the 6-sulfated substrate derived from chondroitin is turned over much more efficiently than all other substrates tested, as shown by their substantially higher kcat values. Given that the 6-sulfate binding motif has been identified by crystallography for only one of these two species, this suggests that the sulfate group may also be influencing the reaction in some other way, perhaps through long-range interactions that optimise the positioning of the catalytic residues. Alternatively, direct intramolecular catalysis of the reaction may be taking place, with the negatively charged sulfate group somehow  72  2.4. Natural substrate variation  Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  catalytic residue ↓ KDIELDHHD....LGFLY QFIWVEQNTRDARTGLLY NRIALDHHD....LGFLY RFENLDHHD....IGFLY  catalytic residue ↓ Clostridium_perfringens YRFIID.......CLLN Flavobacterium_heparinum WYDILDQPNRKGNYFES Streptococcus_agalactiae YRLIID.......CLLN GRIIID.......CLLN Bacillus_sp_GL_1  Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  118 204 120 93  177 293 179 153  4- and 6-sulfate binding ↓ RGVTRQG YSWHSGKGV 370 RDGSYEY EIFKTAKQL 560 KGVTRQG YSWHSGKGV 372 RGGTHQG YHVRGGISP 346  Figure 2.7: Key fragments of a multiple sequence alignment of UGL sequences by the MUSCLE algorithm. Identical residues are coloured yellow on a purple background, conserved residues are coloured white on a blue background, the putative catalytic residues D113 and D173 (C. perfringens numbering) are indicated with arrows, the 4- and 6-sulfate binding domains are indicated with an arrow and a brace, and the vertical red line indicates a break in the sequence. The full alignment is in Appendix A on page 291.  73  2.5. Characterisation of UGL reaction products stabilising a partial positive charge in the transition state of the rate-determining step. The arginine residue identified in the crystal structure of S. agalactiae UGL to bind 4-sulfated substrate 85,94 appears to be a poor predictor of high activity with these substrates for UGLs from other species. A homologous residue is present in C. perfringens UGL, but the chondroitin 4-sulfate derived substrate is only poorly hydrolysed. While no kinetic parameters for the chondroitin 4-sulfate derived substrate were provided for S. agalactiae to allow quantitative comparison, TLC images in the above reference show complete hydrolysis of the compound by S. agalactiae UGL while Bacillus shows no conversion, suggesting a relatively high level of activity.  2.5  Characterisation of UGL reaction products COOH  HO  UGL O OR OH  HO HO HOOC  22  O OH  OR  O  (UGL?)  OH  HOOC +  HOOC ROH  H2O O (+/- H )  23  OH  HO  O OH OH  OH  24 + isomers  Scheme 2.3: Formation of the final product of the UGL-catalysed reaction. The final products of UGL (GH88) and UGH (GH105) catalysed degradation of unsaturated glycuronides via the hydrated intermediate 22 have previously been described as the leaving group alcohol and free unsaturated glucuronic acid as the open chain 5-keto form (23), 82,84,87,92,93,95 as demonstrated by mass spectrometry. 87 However, on observing the products of this reaction by 1 H-NMR, the characteristic peaks from protons on carbon 1 and 4 of an unsaturated uronic acid are not visible. Instead, a mixture of products is clearly visible, resulting from addition of water to and cyclisation of this open chain as shown in Scheme 2.3, with the hemiketal and hemiacetal both existing as an equilibrium mixture to give four compounds with both 74  2.5. Characterisation of UGL reaction products  Figure 2.8: 1 H-NMR spectrum of 24, the final products of UGL degradation of 4-nitrophenyl ΔGlcA (6).  75  2.5. Characterisation of UGL reaction products stereochemistries at each of carbons 1 and 5. The peaks arising from the dominant form of this product, being that shown in Scheme 2.3 (24), are labelled in the 1 HNMR spectrum given in Figure 2.8. These assignments were further confirmed by direct correlation (COSY, Appendix B on page 295) and total correlation (TOCSY, Appendix B) spectra.  2.5.1  Reaction in D2 O  The same UGL-catalysed reaction was carried out with D2 O as a solvent. This allowed any accessible exchangeable protons in the enzyme to become deuterated, in particular the catalytic acid. This means that, on catalysing the hydration reaction, a deuterium would be incorporated into the products at the site of protonation. If a simple glycoside hydrolysis reaction were taking place, this catalytic acid-sourced deuterium would be on the leaving group oxygen and exchangeable with bulk solvent, while subsequent keto-enol tautomerisation at carbon 4 is unlikely to effect stereospecific protonation. However, in a hydration reaction, this deuterium would be stably incorporated at carbon 4 of the sugar product, and this would occur in a stereospecific manner determined by the placement of the catalytic residue. Such an incorporation is detectable by 1 H-NMR. The 1 H-NMR spectrum of the products of this enzymatic reaction in D2 O, overlaid on the spectrum from a control reaction in H2 O, is shown in Figure 2.9. This clearly shows that all peaks are the same, except for those affected by exchange of the axial proton at carbon 4 with deuterium. This signal itself is completely ablated, the equatorial proton on the same carbon no longer shows geminal coupling, and the signal from the proton on carbon 3 has lost a diaxial coupling partner to become a doublet of doublets. Together these show stereospecific incorporation of deuterium by the enzyme to the re face of the double bond at carbon 4 to form 25.  76  2.5. Characterisation of UGL reaction products  Figure 2.9: Overlay of expanded 1 H-NMR spectra of the products from UGLcatalysed hydrolysis of 4-nitrophenyl ΔGlcA (6) in D2 O (upper) and H2 O (lower).  The spatial location of this deuterium correlates well with the aspartate residue 173 (149 in Bacillus numbering), which was proposed to be the principal catalytic acid residue based on the crystal structure of UGL with substrate bound, 87 as shown in Figure 1.11 on page 47 and represented in Scheme 2.4. D173 DO O  OD  D  COOD O OPh OD  DO  D  (-PhOD) OD  H DO DOOC  O OD  OPh  10 D173 HO O  OH HO  D H DO  COOD  O  OD OD  OD  25  Me  COOH O OPh OH  10  H  OMe O H OPh HO HOOC OH  26  Scheme 2.4: Reaction catalysed by UGL, showing the route by which deuterium (upper) and methanol (lower) are incorporated.  77  2.5. Characterisation of UGL reaction products  2.5.2  Reaction in 10% methanol  To show that the solvent nucleophilic attack catalysed by the enzyme occurs at carbon 5, the UGL catalysed reaction was carried out in 10% methanol in H2 O. This was the highest proportion of methanol in which the enzyme was stable. Methanol can act as an alternate nucleophile, substituting for water in the active site and attacking in its place. This forms a product which has a methyl ketal in place of the hemiketal in 22, and so cannot undergo the same rearrangement steps to cleave the glycosidic bond shown in Scheme 2.3 on page 74. In practice, the product of this reaction was found to not be very stable, and attempts to purify it by HPLC or C-18 Sep-pak were unsuccessful. However, the product was clearly visible as an additional set of small peaks in the 1 H-NMR spectra of the crude reaction mixture following removal of enzyme, as shown in Figure 2.10. These peaks were further analysed by correlation spectroscopy (COSY45 and NOESY, Appendix B on page 295).  Figure 2.10: Overlay of expanded 1 H-NMR spectra of the products from UGLcatalysed hydrolysis of phenyl ΔGlcA (10) in 10% MeOH in H2 O (26, lower) and a control in H2 O (upper). The final structure determined for this product (26) results from addition of methanol to the si face of the double bond at carbon 5, as shown in Scheme 2.4. Thus an overall syn addition of methanol occurs, when taken together with the result 78  2.5. Characterisation of UGL reaction products of the reaction with D2 O detailed in Subsection 2.5.1. This product stereochemistry was deduced from the NOESY correlation of the methyl singlet peak, which showed a cross peak with the axial proton on carbon 4, but not the equatorial proton at the same position. This product conformation is likely the reason for the compound’s instability, either through ground state destabilisation or intramolecular catalysis by the C-6 carboxylate, which is now in close proximity to the anomeric carbon. It is worth noting that the product formed from hydrolytic degradation of 26 initiated at carbon 1 or carbon 5 is the same, as both will form the same hydrated product seen in the enzymatic reaction (24), as illustrated in Scheme 2.5. OMe O OPh  HO COOHO  OH O(rotamers)  O  O OPh OH  10  OMe O  UGL, 10% MeOH -  OOC  OH O  HO  HO  -  HO O  O  OOC  OOC  O OH  OH  (rotamers)  H2O -  OMe OH OPh  OMe  OH O+  -  OOC  OMe OH  OH O  OMe OH OH  26 OMe O HO O  OMe O HO O  OH O-  26  OH  OH  -  O  COO-  OPh  HO OH HO  O OH  O  OPh  O  OH OH  OH  24  O-  Scheme 2.5: Decomposition of the methanol adduct formed by UGL-catalysed reaction of phenyl ΔGlcA (10), showing possible mechansims by which decomposition is accelerated (upper) and formation of the same product from decomposition initiated at either carbon 1 or carbon 5 (lower). To further confirm the structure of this methanol adduct, a synthetic standard was sought. Protracted treatment of protected phenyl ΔGlcA (9) in methanol and HCl, generated in situ using acetyl chloride, followed by purification by HPLC af79  2.5. Characterisation of UGL reaction products forded the axial methyl acetal 27 (Scheme 2.6). This was then deprotected with sodium hydroxide in acetone/water to give the deprotected standard 28 in 49% yield over 2 steps. This compound was found to be the carbon 5 epimer of the enzymatic product 26, and was correspondingly much more stable. This structure was determined again by 1 H (Figure 2.11), TOCSY (not shown), COSY45, and NOESY (Appendix B) NMR spectroscopy. The stereochemistry at carbon 5 is inferred from NOE correlations between the methyl singlet and the protons on carbons 1 and 3 as well as the equatorial proton on carbon 4 — the opposite to that with which the methyl singlet correlates in the enzymatic reaction product. COOMe AcO  O O OAc  9  AcCl, MeOH  MeOOC HO  NaOH, acetone/H2O HOOC  O OMeOH  O  27  HO  O OMeOH  O  28 49% over 2 steps  Scheme 2.6: Synthesis of a standard for the product of UGL-catalysed reaction in 10% methanol at carbon 5. A further standard for methanol addition to the anomeric carbon, the product expected from a Koshland glycoside hydrolase mechanism, was also synthesised as detailed in Scheme 2.7. Glucuronyl bromide 3 was reacted with silver carbonate in methanol and acetone to give the methyl β-glucuronide 29 in low yield. This was then subjected to DBU-mediated elimination in good yield to 30, followed by deprotection by NaOH in acetone and water to give 31 in 16% yield over 4 steps, without optimisation. An overlay of 1 H-NMR spectra for both 31 and 28 with the enzymatic products is presented in Figure 2.11. Finally, the observation of the syn adduct allows the exclusion of a mechanism analogous to that acting in the hydration reaction of glucal by glycoside hydrolases, wherein the catalytic acid residue itself is added in a syn- fashion across the double bond to form a glycosyl enzyme intermediate, followed by attack of the eventual 80  2.5. Characterisation of UGL reaction products MeOOC AcO AcO  3  O AcO  Ag2CO3, methanol/ Br acteone  MeOOC AcO AcO  29 56%  O OAc  OMe  DBU DCM  AcO  COOMe  COOH  NaOH, O OMe acetone/ HO OAc H2O  O OMe OH  30  31  78%  36%  Scheme 2.7: Synthesis of a standard for the product of UGL-catalysed reaction in 10% methanol at carbon 1.  Figure 2.11: Overlay of expanded 1 H-NMR spectra of the anomeric methanol synthetic standard (31, upper), the products from UGL-catalysed hydrolysis of phenyl ΔGlcA (6) in 10% MeOH in H2 O (26, middle) and the carbon 5 methanol epimeric synthetic standard (28, lower). nucleophile from the opposite face, to give an overall anti-addition (see the end of Section 1.3 on page 21). If such a mechanism were acting in UGL, one of two possible outcomes would be observed, depending on where the methanol nucleophile attacks. In the case where methanol attacks the hypothetical glycosyl-enzyme intermediate at carbon 5, the product formed would reflect the overall anti addition, and would thus be the same as the synthetic standard 28. The alternative would be for methanol to attack at the acyl carbon of the catalytic acid/nucleophile residue. This would give an appropriate product stereochemistry, a syn addition product of water (not methanol) across the double bond, but would give a methyl ester of this catalytic  81  2.6. Unusual substrates D173 D173  O  O  O H  COOH O OR  HO  OH  (A) D173 Me (B) OH O O O OR HO HOOC OH (A) (B)  OH HOOC HO  O OMeOH  OR  D173 O  OMe HO  HO HOOC  O OH  OR  Scheme 2.8: Expected reaction pathway for UGL if a mechanism analogous to that for glucal hydration were followed. residue, thereby preventing further turnover of substrate by the enzyme (paths A and B in Scheme 2.8, respectively).  2.6  Unusual substrates  Because UGL cleaves glycosidic bonds indirectly through hydration of the carbon 4carbon 5 double bond, there are several compounds that are expected to be accepted by this enzyme as substrates that otherwise would not undergo reaction with a standard hydrolase. Kinetic parameters for hydration of each of these are summarised in Table 2.3, with discussion of each following. Table 2.3: Michaelis-Menten kinetic parameters for three unusual substrates accepted by UGL (*, estimate based on Ki ). Structure  kcat (s-1 )  Km (mM)  kcat /Km (s-1 .mM-1 )  (40)  0.036 ± 0.0067  2.7 ± 0.4*  0.0133 ± 0.0002  (43)  0.6 ± 0.1  38 ± 9  0.015 ± 0.006  (46)  9.3 ± 0.2  4.8 ± 0.3  1.9 ± 0.2  COOH OH O  HO  OH HO  OH  COOH O  HO  HOO COOH HO  O S OH  82  2.6. Unusual substrates  2.6.1  Kdn2en  The first such unusual substrate, 2,3-dideoxy-d-glycero-d-galacto-non-2-enopyranosonate (Kdn2en, 40), is an unsaturated C-glucuronide analogue with a glycerol chain in place of a leaving group. The relationship between sialic acid and ΔGlcA derivatives has previously been exploited to design inhibitors for sialidases based on a ΔGlcA scaffold, 115,116 while in this case a sialic acid is being used for ease of access to a C-glycoside, a substrate that could only be acted on if a hydration mechanism were employed by UGL. A small amount of 40 could be purchased for initial testing, which confirmed that this compound was hydrated by UGL, with the formation of a hydration product being confirmed by mass spectrometry. In order to determine the Michaelis-Menten kinetic parameters for this substrate a larger quantity was required, so synthesis was attempted based on that of Schreiner and Zbiral 117 , as outlined in Scheme 2.9. Kdn (32) was formed enzymatically from mannose and pyruvate by Neu5Ac aldolase using a method based on that reported for synthesis of sialic acid, 118 then protected by methanol/TFA followed by acetic anhydride/pyridine. For the elimination of 33 by trimethylsilyl trifluoromethanesulfonate, a more recent method from Chang et al. 119 was attempted first, with reaction at ambient temperature in ethyl acetate instead of at 0 °C in acetonitrile. This appeared to proceed smoothly by TLC, but gave a second set of NMR very similar to the published product characterisation and of a similar abundance to the product. This was initially assumed to be a rotamer of the product, given the apparent purity of the product by TLC analysis in several different solvent systems, so deprotection was undertaken. However, these additional peaks persisted. Further investigation of the literature 120 revealed a propensity for this product to racemise at carbon 4 under these conditions, dependent on temperature, solvent, and reagent quantities, with the reaction proceeding through an allylic carbocation 83  2.6. Unusual substrates intermediate formed by the Lewis acid reagent. OH HO HO  OH  OH O  OH  Neu5Ac aldolase HO HO  O  OH  COONa  HO  O  1. MeOH, TFA 2. Ac2O, pyridine  COOH  HO  32  OAc  AcOAcO AcO AcO  OAc O  COOMe  33 60%  48%  TMSOTf EtOAc HO Ph  OH COOMe  O O O O Ph  O PhCH(OMe)2  38 23%  OH  36  COOMe HO HO  39  OAc O  AcOAcO NaOMe, MeOH AcO  37  COOMe  34  COOMe  80% overall  18%  COOMe  OAc  + O  HO HO  O HO  Ph  HO  pTSA, ACN  O  O HO  HO HO  OH  +  OAc OH O  AcOAcO AcOAcO  +  COOMe  O  35  77% overall  1. LiOH, THF/H2O 2. IEX H+ OH COOH HO HO HO  O  HO  COOH OH O OH  HO  40  HO  OH  81%  Scheme 2.9: Synthesis of 2,3-dideoxy-d-glycero-d-galacto-non-2-enopyranosonate (Kdn2en, 40). While it was possible to repeat the reaction where the racemisation had occurred to try to avoid this side-product, this reaction had already been scaled up and little starting material remained. It was decided to attempt separation of these epimers, despite literature claims that they are inseparable. Chromatography and crystallisation attempts using various solvent systems, on both the acetylated (35 and 34) and de-acetylated forms (37 and 36), largely confirmed this claim, although some enrichment was possible with diligent flash column chromatography. A solution to the lack of resolution was then sought on the basis of the differing reactivity of 84  2.6. Unusual substrates cis- and trans-oriented vicinal diols in the formation of cyclic protecting groups, such as a benzylidene. Because the carbon 4 and 5 alcohols in the undesired sideproduct are in a cis-oriented arrangement, it was anticipated that these could form a benzylidene where the trans-oriented groups in the desired product could not, giving easily resolvable products. This proved to indeed be the case, although the yield of each product (39 and 38) was low, owing to the formation of various side products that were not characterised. These could then be deprotected by methyl ester hydrolysis using lithium hydroxide followed by protracted stirring under acidic conditions to hydrolyse the benzylidene and protonate the carboxylate at carbon 1. The yield for this deprotection likely could have been improved by a two step deprotection removing the benzylidene first, as some hydration of the double bond occurred, but with sufficient product in hand optimisation was unwarranted. Testing of this substrate indicated a high Km , which was not surprising given the nature of the group at the position equivalent to the anomeric position of ΔGlcA (a glycerol chain rather than an aryl group or sugar). This meant that substrate binding could not be saturated within the limits of the assay used (as discussed in Subsection 2.2.1 on page 57). Data from direct assay of this substrate at low concentrations were fit to a linear equation to determine kcat /Km , and Ki (see Figure 2.12) was then approximated as a surrogate for Km to allow estimation of kcat (see Table 2.3 on page 82). This estimation of Km derived from inhibition testing (2.7 ± 0.4 mM) agreed well with the value calculated from non-linear regression of data from reactions below 1 mM of 40 (2.4 ± 1.3 mM), despite this showing only slight curvature.  85  2.6. Unusual substrates  Figure 2.12: Dixon plot showing competitive inhibition of UGL by Kdn2en (40) with a Ki of 2.7 mM. Substrate (6) was at the following concentrations: 25( ), 100( ), 200( ), 400( ), and 600 ( ) µM.  86  2.6. Unusual substrates  2.6.2  Axial phenol  Glycoside hydrolases are very specific for a particular anomeric configuration as the placement of catalytic residues needs to be very precise to effect catalysis. With a hydration-initiated mechanism, such as that in UGL, the anomeric configuration is only important in determining binding. As long as the enzyme active site can accommodate the compound it should catalyse hydration by virtue of the catalytic residues around the double bond, the placement of which should be unaffected. Hydrolysis of both anomers of a substrate has previously been observed in family GH4 enzymes, 45,121 the elimination/hydration mechanism of which also acts largely independent of this configuration. To test if this is also possible in UGL, 43, the axial analogue of phenyl ΔGlcA (10) was synthesised (see Scheme 2.10) and subjected to UGL-catalysed hydrolysis. 1. NaOMe, MeOH 2. Ac2O, HClO4  HO O O  OH O  1  MeOOC AcO AcO  OH  ZnCl2  O OAc  OAc  phenol, 80 °C  MeOOC AcO AcO  O AcO O  2  41  58%  6% DBU, DCM  COOH O  HO  HOO  NaOH, acetone/H2O  COOMe O  AcO  AcOO  43  42  64%  44%  Scheme 2.10: Synthesis of an axial phenyl ΔGlcA substrate for UGL. The phenyl leaving group was attached in an axial configuration by reaction of acetylated methyl glucuronate (2) in melted neat phenol as solvent with zinc (II) chloride as a Lewis acid catalyst. The yield of pure alpha product was very low, as a mixture of anomers formed and resolution of the pure desired product from this  87  2.6. Unusual substrates as well as the excess phenol proved difficult. However, as only a small amount of this compound was required for testing and a sufficient amount of the intermediate 41 was in hand to achieve this, it was not deemed important to repeat or improve on this method. Subsequent elimination by DBU to 42 and deprotection by NaOH in acetone/water proceeded smoothly, if with slightly lower yields than with the equatorial phenyl analogue, to afford 43. While other syntheses of this product have been published, 104,106,122 with better yields, this route represented the smallest number of discrete steps required to achieve a compound for testing from available starting materials. Testing of this compound (43) as a substrate with UGL revealed a lower kcat and higher Km than for the equatorial phenyl substrate 10 (see Table 2.3 on page 82, and Table 2.1 on page 65). The observation of any activity clearly demonstrates a reaction that is largely independent of anomeric configuration and consistent with a hydration-initiated hydrolytic mechanism, with the kcat of the two phenyl anomers differing only by a single order of magnitude. The Km is increased by approximately an order of magnitude also, which is unsurprising since the active site is likely optimised for binding of a group with an equatorial configuration at the anomeric carbon. Hydrolysis of this substrate was found to deviate significantly from the classical Michaelis-Menten kinetics at higher substrate concentrations, possibly as a result of either substrate inhibition or non-specific enzyme inactivation (as discussed in Section 4.3 on page 141). Hydrolysis of both anomers of phenyl ΔGlcA by UGL from F. heparinum may have previously been observed, 104 but the extent to which this occurred and whether or not this activity came from a single enzyme was not clear as the authors pursued an enzyme with activity specific for one isomer from one source GAG. These authors dismissed this observation as the activity was low by comparison.  88  2.6. Unusual substrates  2.6.3  Thiophenol  Thioglycosides are generally very poor substrates for glycoside hydrolases, and are in common use as inert substrate analogues. However, carbohydrate-active enzymes that use eliminative mechanisms, such as family GH4 enzymes 46 and polysaccharide lyases, 75 have been shown to be able to hydrolyse these substrates. Because the hydration-initiated hydrolytic mechanism of UGL acts distal to the thioglycosidic bond, UGL was anticipated to be able to hydrolyse a suitable ΔGlcA thioglycoside only if such a mechanism were acting. Thiophenyl ΔGlcA was thus synthesised and tested as a substrate for UGL. 1. NaOMe, MeOH 2. Ac2O, HClO4  HO O O  OH O  MeOOC AcO AcO  OAc  OAc  MeOOC AcO AcO  2  OH  1  HBr, AcOH  O  Na2HCO3, thiophenol, TBAB EtOAc/H2O  HO  O S OH  NaOH, acetone/H2O AcO  COOMe O S OAc  AcO Br  3  58%  COOH  O  DBU, DCM  MeOOC AcO AcO  97%  O OAc  46  45  44  86%  56%  78%  S  Scheme 2.11: Synthesis of a thiophenyl ΔGlcA substrate for UGL. Synthesis of this substrate proceeded along a similar route to that for the 4nitrophenyl substrate 6, as shown in Scheme 2.11. Glycosylation was accomplished in good yield under phase transfer conditions with base catalyst, taking advantage of the good nucleophilicity of thiophenol to afford 44 without requiring expensive silver reagents. This was then subjected to DBU-mediated elimination with a modest yield. The protected thioglycoside 45 was deprotected in one step using NaOH in acetone/water as for the other phenyl glycosides (10 and 43), in good yield, to afford 46. 89  2.7. Conclusions Hydrolysis of 46 by UGL proceeded very efficiently, with both higher kcat and Km compared to its phenyl analogue 10 (see Table 2.3 on page 82, and Table 2.1 on page 65), giving a slightly higher kcat /Km as a result.  2.7  Conclusions  These results demonstrate conclusively that UGL catalyses the hydrolysis of unsaturated glucuronides through hydration of the carbon 4–carbon 5 double bond to generate a hemiketal. It remains unclear whether or not the subsequent rearrangement of this hemiketal to give the final products is catalysed by the enzyme or occurs spontaneously in solution, and what, if any, intermediate steps are involved in this hydration process. Optimal conditions for UGL-catalysed hydrolysis were determined to be pH 6.6, 37 °C and with 0.1 % w/v BSA. The optimal temperature was seen be different for kcat and kcat /Km , and not determined by irreversible global thermal instability, but likely rather by local reversible instability. UGL from C perfringens appears to show similar substrate preferences to most of the species previously profiled in the literature when presented with substrates derived from natural GAGs, and is also able to hydrolyse aryl substrates with similar efficiency. One substrate in particular, the chondroitin-6-sulfate derived 11, showed a higher kcat than any other substrates assayed in this work or reported elsewhere. The products of the UGL-catalysed hydrolysis of aryl glucuronides in D2 O and 10% methanol are completely consistent with the mechanism proposed, as well as the tentative assignment of D173 as a catalytic acid, based on crystallography. Proton addition takes place stereoselectively from the face where this residue is located, while nucleophilic attack takes place from the same face. Indeed, a well resolved water molecule is also located here in the structure and is presumed to be the nucleophile. 90  2.7. Conclusions These results also rule out a syn addition of this residue over the double bond. As a further test of the hydration mechanism, several compounds were synthesised and tested, each of which was predicted to be accepted as a substrate by UGL only if this novel mechanism were acting — Kdn2en (40), axial phenyl ΔGlcA (43), and thiophenyl ΔGlcA (46). All of these were indeed seen to be turned over, confirming the results of the above experiments.  91  Chapter 3  Probing the mechanism of UGL With the overall reaction catalysed by UGL established in the previous chapter, attention next turned to probing of the details of how this reaction was catalysed. Specific goals were identifying the nature of the transition state for the hydrolysis reaction, establishing whether or not the rearrangements of the hemiketal were catalysed on-enzyme, and determining the role of D113, the second residue identified previously as catalytically important by mutagenesis experiments in the literature. 87,92  3.1  Detection of initial products by NMR  In their publication on the crystal structure of UGL from Bacillus sp. GL1 with bound substrate, 87 Itoh et al. claim that the rearrangements of the initial hemiketal product (22 in Scheme 2.3 on page 74) occur on-enzyme, but provide no evidence of this. While that would be a plausible mechanism, it remains possible that this unstable intermediate degrades non-enzymatically. Since the initial hydration reaction is likely the slowest step the system gains nothing by accelerating this subsequent step. One means by which this hypothesis can be tested is by monitoring the reaction using NMR as it progresses, looking for short-lived intermediates in solution. By using a high concentration of enzyme in the reaction any enzymatic steps should be accelerated, while rates for non-enzymatic steps should be zero order in enzyme concentration. Thus by maximising the difference between the enzymatic and any  92  3.1. Detection of initial products by NMR non-enzymatic rates the likelihood of detecting intermediates is also maximised, if such intermediates are present. This experiment was carried out using thiophenyl ΔGlcA (46) as a substrate because of its high kcat , giving a high rate of enzymatic reaction under the saturating substrate conditions used, and also because sufficiently large quantities were available to allow monitoring of the reaction by 1 H-NMR. The substrate/buffer/BSA reaction mixture was used to tune the spectrometer and to give a baseline for the unreacted substrate, then the enzyme in D2 O was added, the solution mixed, and monitoring initiated as quickly as possible. The resultant spectra are shown in Figure 3.1. New peaks derived from the products are already apparent in the t = 0 min scan, from reaction in the time taken to lower the sample into the spectrometer and re-establish the lock. These peaks increase in intensity over the next 15 minutes, at which point the starting material is almost completely consumed. Allowing the reaction to continue for longer, up to 266 min, does not appear to change the spectrum of the product. At no stage are there any peaks formed that are no longer present in the final scan, as would be expected if an intermediate were being formed and released into solution — the only peaks visible at any stage are those from the starting material and those from the final products. While not observing an intermediate is not evidence for no intermediate, these results do show that either no intermediate is released into solution or that it is extremely short-lived. Since this rearrangement proceeds in a similar fashion to mutarotation of free sugars, it could be expected that the kinetics would be similar. Half-life values for uncatalysed mutarotation of simple sugars range from around 5 minutes to an hour, and are highly dependent on temperature and pH, 123 but the process can generally be observed using 1 H-NMR. The best model system for the rearrangement in the UGL reaction is likely that of mutarotation in sialic acid, for  93  3.1. Detection of initial products by NMR  94  Figure 3.1: Offset stack of 1 H-NMR spectra showing reaction of thiophenyl ΔGlcA (46) with a high concentration of UGL. Lowest trace, initial spectrum before addition of UGL; highest trace, thiophenol in the reaction buffer. Numbers in italics are integrals for the spectral regions indicated by the horizontal lines below the lowest trace.  3.1. Detection of initial products by NMR which the half-life at pD 6.7 is 25 min, 124 and appears to be similar for Kdn. 125 This suggests that the rearrangement of the hemiketal intermediate 22 in the UGLcatalysed reaction occurs on-enzyme. Examination of the enzyme active site and associated mutagenesis 87 shows a potential pair of residues that may be involved in general base catalysis of this reaction, H193 and Q211 (Bacillus numbering), and likely proceeding through a mechanism as shown in Scheme 3.1, by analogy to basecatalysed mutarotation. 126 In this possible mechanism, these two residues act to deprotonate the newly-introduced hydroxyl group at carbon 5 in a fast equilibrium, the transient product of which then undergoes a rate-limiting rearrangement to the products. This rearrangement may occur in either a concerted manner or in two steps. However, in the substrate-bound crystal structure of UGL from S. agalactiae the placement of the histidine invoked in this mechanism is not conserved, despite strong sequence conservation, and has no obvious replacement in its suggested possible role as base catalyst, casting some doubt on this role. Q211  H193 N O  C  NH O  H  HN  NH2  R OH C  NH O  +  NH2 O  -  O  O  HO HOOC  Q211  H193  OH  OR  (fast)  HO HOOC  OH  O OH  OR  HOOC  (slow)  O OH  22  23 O via  HO HOOC  O- ?  OH OR  Scheme 3.1: Mechanism for UGL catalysis of rearrangement of the hemiketal intermediate 22 to cleave the glycosidic bond.  95  3.2. Linear free-energy relationship  3.2  Linear free-energy relationship  Measurement of a free energy relationship for the UGL-catalysed hydrolysis of aryl glycosides was undertaken as a way of probing the transition state of the hydration reaction. Free energy relationships show the effects of electron-withdrawing and/or -donating groups on the activation energy of a reaction (ΔG‡ ), through measurement of the effect on the rate of a reaction. 127 For example, a partial negative charge developing on a phenolic oxygen at the transition state will be stabilised by electron-withrdawing substituents on the phenol ring, decreasing the free energy of this transition state relative to that of the unsubstituted phenol, and thus giving faster reaction. In addition to informing on the polarity of charge development, the degree to which different electron-withdrawing or -donating substituents effect the rate of reaction can also provide information on the magnitude of charge development. In measurement of free energy relationships for glycoside hydrolases, the pKa of a phenyl group is commonly used as a measure of its electron withdrawing ability, with a more withdrawing substituent stabilising a negative charge on the phenolic oxygen and thus lowering the pKa .  3.2.1  Synthesis of aryl glycosides  To measure such a free energy relationship for UGL, a series of aryl glycosides was required with leaving groups of varying pKa . These were synthesised chemically using the same methods as for the phenyl and 4-nitrophenyl ΔGlcA substrates (6 and 10) outlined in Scheme 2.1 and Scheme 2.2 on page 59, with a summary of the reaction conditions used and yields for each substrate presented in Table 3.1. Glycosylation of aryl groups was performed using three different methods, depending on the pKa . Low pKa phenols, up to 3-nitrophenol at 8.39, were attached using Koenigs-Knorr glycosylation in moderate to good yields (as exemplified in 96  3.2. Linear free-energy relationship  Table 3.1: Conditions used and yields for the synthesis of unsaturated aryl glucuronides. TCA, trichloroacetonitrile (Schmidt donor); BnBr, acylation of free hemiacetal 7 (refer Scheme 2.2 on page 59) with benzyl bromide. Numbering for intermediates in parentheses. Substrate  Elimination  Deprotection  55% (55)  HCl(aq.) , 32%  50% (57)  HCl(aq.) , 56%  72% (59)  NaOH, 63%  92% (5)  NaOMe then LiOH, 80%  8.39  Glycosylation KoenigsKnorr, 79% (54) KoenigsKnorr, 85% (56) KoenigsKnorr, 65% (58) KoenigsKnorr, 54% (4) KoenigsKnorr, 53% (60)  72% (61)  NaOMe then H2 O, 85%  9.38  TCA, 20% (62)  72% (63)  NaOH, 46%  9.99  TCA, 30% (8)  51% (9)  NaOH, 77%  10.37  TCA, 28% (64)  4% (65)  NaOH, 54%  15.40  BnBr, 31% (66)  73% (67)  NaOH, 96%  pKa  COOH HO  O O OH  NO2  O2N  3.96  47 COOH HO  NO2  O O OH O2N  5.15  48 COOH  Cl  O O  HO  OH  Cl Cl  6.39  49 COOH HO  O O OH  NO2  7.18  6 COOH HO  O O  NO2  OH  50 COOH HO  O O OH  Cl  51 COOH O O  HO  OH  10 COOH HO  O O OH  52 COOH HO  O O OH  53  97  3.2. Linear free-energy relationship Scheme 2.1), while for all other phenols the free hemiacetal 7 was activated to the Schmidt donor using trichloroacetonitrile and catalytic DBU, then glycosylation of the appropriate phenol using BF3 diethyl etherate (as exemplified in Scheme 2.2). The leaving group for the benzyl substrate was attached by alkylation of the same free hemiacetal 7 using benzyl bromide and silver carbonate, as the other methods were found to not be effective. Eliminations were all performed using DBU, also with moderate to good yield. Deprotection was more varied, but in general also depended on the pKa of the aryl group — most were deprotected under basic conditions, in either one or two steps, while for the substrates with the most activated leaving groups aqueous acid was used as they were not stable under basic conditions. Overall yields ranged from 2.5% for 4-chlorophenyl ΔGlcA to 22% for 4-nitrophenol. The halide-substituted phenols in particular were found to be more difficult to manage, with lower solubilities, less visibility on TLC, and smaller Rf changes during reactions, which made monitoring difficult.  3.2.2  Kinetics  Kinetic parameters for the hydrolysis of these substrates by UGL are summarised in Table 3.2. The best substrates are those with a higher pKa , largely as a result of changes in kcat rather than kcat /Km . Log plots of kcat and kcat /Km against pKa are given in Figure 3.2, revealing slopes of βlg = 0.16 ± 0.04 and βlg = 0.04 ± 0.05, respectively. The error range for the plot of log(kcat /Km ) contains zero, and so is considered to be a flat line, showing negligible effect from the leaving group, while the slope of log(kcat ) is slightly positive and excludes zero, showing that more electron withdrawing groups make this step slower. Such a relationship would make sense in the context of a transition state involving a a partial positive charge that is destabilised by electron withdrawing groups, either a small amount of charge close  98  3.2. Linear free-energy relationship  Table 3.2: Michaelis-Menten kinetic parameters for hydrolysis of aryl unsaturated glucuronides by UGL. Substrate  pKa  kcat (s-1 )  Km (mM)  kcat /Km (mM-1 .s-1 )  3.96  0.140 ± 0.005  0.16 ± 0.02  0.9 ± 0.1  5.15  0.64 ± 0.04  0.59 ± 0.06  1.1 ± 0.2  6.39  0.312 ± 0.007  0.57 ± 0.04  0.55 ± 0.05  7.18  2.05 ± 0.06  0.26 ± 0.02  7.9 ± 0.8  8.39  14.7 ± 0.5  0.88 ± 0.03  16 ± 1  9.38  3.9 ± 0.1  0.84 ± 0.09  4.6 ± 0.6  9.99  4.3 ± 0.2  3.2 ± 0.4  1.3 ± 0.2  10.37  3.27 ± 0.02  0.88 ± 0.02  3.7 ± 0.1  15.40  18.6 ± 0.6  9.0 ± 0.5  2.1 ± 0.2  COOH HO  O O OH  NO2  O2N  47 COOH HO  NO2  O O OH O2N  48 COOH  Cl  O O  HO  OH  Cl Cl  49 COOH HO  O O OH  NO2  6 COOH HO  O O  NO2  OH  50 COOH HO  O O OH  Cl  51 COOH O O  HO  OH  10 COOH HO  O O OH  52 COOH HO  O O OH  53  99  3.2. Linear free-energy relationship to the leaving group or a larger charge at a greater distance. This second situation matches what would be expected from the proposed hydration mechanism, with the addition of a proton to the double bond between carbon 4 and carbon 5, generating significant carbocation character at carbon 5, which is then quenched by attack of the nucleophile. However, the observation of two different slopes for the plots of log(kcat ) and log(kcat /Km ) suggests that these parameters represent two different steps, meaning any mechanism must involve at least two distinct steps. Further discussion of this will be taken up in Section 3.5 on page 124 after all experimental data have been presented.  Figure 3.2: Plot of log(kcat ), A, and log(kcat /Km ), B, against leaving group pKa for hydrolysis of aryl unsaturated glucuronides by UGL. An apparent outlier in these is 3-nitrophenyl ΔGlcA, which has an unusually high kcat , several fold higher than most other aryl substrates, although still not as high as the best natural substrates. Given that there are several other nitrophenyl substrates presented in Table 3.2, it is unlikely that this is a specific effect of the nitro group, and 2,5-dinitrophenol has one of its two nitro groups in the same position on the phenyl ring as 3-nitrophenol. This higher kcat may arise from a specific interaction  100  3.2. Linear free-energy relationship of the 3-nitro group with a subdomain that interacts with and improves turnover of some natural substrates, but given that there is no improvement in binding over any of the other substrates, a simple binding sub-domain interaction seems unlikely. It is also possible that this group is involved in some fortuitous interaction that results in optimisation of the catalytic residue placements, as suggested for the 6-sulfated natural substrates in Section 2.4 on page 65, but again it is difficult to conceive of a mechanism whereby the 3-nitrophenol group achieves this but the 2,5-dinitrophenol does not. This source of the high activity with this substrate remains unclear. Free energy relationships for other glycoside hydrolases generally follow one of two patterns, depending on the mechanism employed and which step within that mechanism is rate determining. For inverting hydrolases and retaining hydrolases with rate-limiting glycosylation 128 the slope of log(kcat /Km ) and log(kcat ) against pKa is negative, as the leaving groups with a more stabilised negative charge are activated and accelerate the reaction. For retaining hydrolases with rate-limiting deglycosylation the plot is usually flat, reflecting the lack of effect of charge stabilisation on transition state energy as the charged group is no longer present in this step. In some cases, such as with many retaining β-glycosidases, the plot will show a shift between these two cases as the rate-limiting step changes with leaving group ability. 129–131 It is also possible to observe a flat plot for enzymes with ratelimiting glycosylation if there is substantial proton donation to the leaving group at the transition state 132 as this will minimise the developing charge that needs to be stabilised. Polysaccharide lyases 71 and glycoside hydrolases from family 4 48 as well as some members of family 31 65 (those operating with eliminative mechanisms) also follow a similar pattern, showing either a negative slope or a flat plot, depending on the extent of charge developed and whether this occurs in the rate-limiting step. Flat plots can also be indicative of a non-chemical step being rate-limiting, such as  101  3.3. Effects of heteroatoms domain movement in the enzyme or substrate conformational changes, 133 but kinetic isotope effects (see Subsection 3.4 on page 113) show this to not be the case for UGL. These data thus show that UGL exhibits a different trend to all of these cases, reflecting the different charge development pattern in its unusual mechanism.  3.3  Effects of heteroatoms  To further investigate this trend, a substrate with an even more activated leaving group, and thus even stronger electron withdrawing effect, was sought in (4-deoxyα-l-threo-hex-4-enopyranosyl fluoride)uronic acid (70). The high electronegativity of fluorine generally makes glycosyl fluorides very good substrates for carbohydrateactive enzymes, and they are in fact considered good substrates for essentially all previous glycoside hydrolases, 134 including those with eliminative mechanisms such as glycoside hydrolases from family GH31, 65 while 4-fluoro glucuronides are good substrates for polysaccharide lyases. 71 In the hydration mechanism, however, this is expected to be a very poor substrate. Because fluorine is an almost isosteric substitution with hydrogen and activity had already been observed with both anomers of phenyl ΔGlcA (10 and 43), the alternate anomer of (70), (4-deoxy-β-l-threo-hex-4-enopyranosyl fluoride)uronic acid (73), was also synthesised to test as a substrate. In order to investigate the effect of the proximity of this highly electronegative group on the transition state energy, a substrate with fluorine substitution at carbon 2 (78) was also synthesised and tested, while synthesis of a substrate containing fluorine at carbon 4 (88) was attempted but unsuccessful.  102  3.3. Effects of heteroatoms  3.3.1  α- and β-ΔGlcA fluorides  Previous syntheses of glucuronyl fluorides reported in the literature were accomplished by selective oxidation of carbon 6 of glucosyl fluoride using TEMPO catalysis. 135 However, to achieve the desired unsaturated glucuronides it was deemed more convenient to form the protected glucuronyl fluorides directly to allow the same elimination reaction by DBU used for all other compounds reported in this work, rather than trying to re-protect the reactive glucuronyl fluoride. The two anomers of ΔGlcA fluoride were synthesised by similar routes, as shown in Scheme 3.2 and Scheme 3.3, differing only in the means by which the fluorine was incorporated. The equatorial fluorine in 68 was incorporated using silver fluoride displacement of the axial bromine in 3, overall a net double displacement at the anomeric carbon from acetylated methyl glucuronate (2), first with HBr then with AgF. This reaction sequence was much higher yielding than for the axial anomer as the axial glucuronyl bromide appears to be a more stable product than the axial glucuronyl fluoride under their respective work-up conditions, and the equatorial fluoride is not worked up in the presence of excess acid. The axial fluoride in 71 was incorporated directly from acetylated methyl glucuronate (2) by use of HF in pyridine overnight at 4 °C. The yield for this reaction was low both because the reaction was slow and the product was unstable during work-up; extending the reaction to drive complete product formation resulted in increased hydrolysis, likely by atmospheric water even in the sealed reaction vessel. However, this reaction generated sufficient product to continue with the synthesis, and the hydrolysis by-product and recovered starting material were both useful intermediates in other syntheses. The elimination reaction of both these products was lower yielding than for other substrates (see Table 3.1 on page 97 for comparison), which may be a result of the electron-withdrawing fluoride accelerating formation of over-eliminated side-products, as was also seen for the 103  3.3. Effects of heteroatoms 4-fluoro unsaturated glucuronide 87 in Scheme 3.5 on page 111. Deprotection over two steps by sodium methoxide then lithium hydroxide proceeded smoothly, while acetyl chloride in methanol unsurprisingly led to decomposition of the product. 1. NaOMe, MeOH 2. Ac2O, HClO4  HO O O  OH O  MeOOC AcO AcO  OAc  OAc  MeOOC AcO AcO  2  OH  1  HBr, AcOH  O  O AcO Br  3  58%  97% AgF, ACN  COOH O F OH  HO  1. NaOMe, MeOH 2. LiOH, THF/H2O  COOMe  DBU, DCM  O F OAc  AcO  MeOOC AcO AcO  O OAc  70  69  68  70%  33%  85%  F  Scheme 3.2: Synthesis of an equatorial ΔGlcA fluoride substrate.  1. NaOMe, MeOH 2. Ac2O, HClO4  HO O O  OH O  1  MeOOC AcO AcO  OH  O OAc  HF, pyridine OAc  4 °C  MeOOC AcO AcO  O AcO F  2  71  58%  20% DBU, DCM  COOH O  HO  COOMe  1. NaOMe, MeOH 2. LiOH, THF/H2O  O  AcO  HO F  AcO F  73  72  79%  34%  Scheme 3.3: Synthesis of an axial ΔGlcA fluoride substrate. Testing of these two substrates revealed that the equatorial fluorine compound 70 was a very poor substrate, while the axial anomer 73 showed no detectable hydrolysis by UGL. This lack of any activity with 73 is somewhat surprising given that 70 shows detectable hydrolysis and both phenyl isomers are hydrolysed by this enzyme, but 104  3.3. Effects of heteroatoms it is possible that the equatorial anomer is able to make a weak interaction that the axial cannot, and a small decrease under the already low activity of 70 renders this substrate’s hydrolysis undetectable above background. It seems unlikely that steric arguments are appropriate here, given both the size of the fluorine atom and the observation that much larger axial phenyl ΔGlcA (43) can be accommodated by the enzyme active site, and 73 was also shown to bind to the UGL active site as a competitive inhibitor with a Ki of 6.4 ± 1.2 mM Figure 3.3). The low activity detectable with 70 could not be saturated within the limits of the assay used (as discussed in Subsection 2.2.1). Data from reaction progress curves were fit by linear regression to determine kcat /Km , and Ki was then approximated as a surrogate for Km (Figure 3.4), as was done for Kdn2en (40), to allow estimation of the kcat given in Table 3.3.  Figure 3.3: Minimal Dixon plot showing competitive inhibition of UGL by axial ΔGlcA fluoride (73) with a Ki of 6.4 mM: 1/Vmax is shown as a dashed line. Substrate (6) was at the following concentrations: 250( ), and 400( ) µM. Taken in the context of the other heteroatom-substituted substrates already  105  3.3. Effects of heteroatoms  Figure 3.4: Inhibition of UGL by equatorial ΔGlcA fluoride (70) showing a Ki of 10.7 mM, determined from the intercept with 1/Vmax (dashed line). Substrate (6) was at 125 µM.  Table 3.3: Kinetic parameters of UGL substrates with varied heteroatoms at the anomeric carbon (*, estimate based on Ki ).  Substrate  Electronegativity (Pauling scale)  kcat (s-1 )  Km (mM)  kcat /Km (mM-1 .s-1 )  3.98  0.00047 ±0.00007  10.7 ± 0.9*  0.000044 ±0.000002  3.44  4.3 ± 0.2  3.2 ± 0.4  1.3 ± 0.2  2.58  9.3 ± 0.2  4.8 ± 0.3  1.9 ± 0.2  2.55  0.036 ± 0.006  2.7 ± 0.4*  0.0133 ± 0.0002  COOH O F  HO  OH  70 COOH HO  O O OH  10 COOH HO  O S OH  46 HO  COOH OH O OH HO  40  OH  106  3.3. Effects of heteroatoms presented, namely thiophenyl ΔGlcA with sulfur at the anomeric position (46, Subsection 2.6.3 on page 89) and the reference case of phenyl ΔGlcA with oxygen at the anomeric position (10, Section 2.3 on page 61), a trend is seen wherein more electronegative atoms at the anomeric position destabilise the transition state of the rate-determining step. However, Kdn2en, with carbon at the analogous position to the anomeric carbon of ΔGlcA (40, Subsection 2.6.1 on page 83), is a clear exception to this trend. Because these compounds all have quite different groups at the anomeric position, ranging from a single atom through a glycerol chain to an aromatic ring, it is difficult to draw any strong conclusions from these trends, especially since binding of these substrates to the enzyme (as reflected in Km ) is different in each case, and only 4 datapoints are available. However, these results are in broad agreement with the previous section. Assuming that UGL does catalyse the rearrangement of the intermediate hydrate 22, as suggested by the NMR-monitoring results in Subsection 3.1 on page 92, this low kcat with Kdn2en may reflect the slower release of the intermediate hydrate, as this substrate is unique in not being able to undergo this rearrangement.  3.3.2  2,4-Dinitrophenyl 2F-ΔGlcA  The class of 2-deoxy-2-fluoroglycosides are known mechanism-based inactivators of glycosidases operating via retaining Koshland mechanisms. 136 The fluorine on carbon 2 inductively destabilises the positive charge that develops on carbon 1 in the transition state of both the glycosylation and deglycosylation steps, slowing both steps. By also including a highly activated leaving group such as fluoride or 2,4dinitrophenol the glycosylation step is accelerated, and if the balance of these two rates is appropriate this gives rise to a trapped intermediate that only turns over slowly. Given that UGL has been shown in Chapter 2 to work through a hydra-  107  3.3. Effects of heteroatoms tion reaction, and that the positive charge in that reaction is understood to develop on carbon 5 rather than carbon 1, it was expected that an unsaturated 2-deoxy-2fluoroglucuronide would be a substrate for these enzymes. MeOOC AcO AcO  3  NaH2PO4/Zn,  O  acetone  AcO  MeOOC AcO AcO  Br  74  O  Selectfluor, nitromethane/ AcOH  MeOOC AcO AcO  O F  OAc  75  88%  16% (+23% manno) 1. HBr/AcOH 2. 2,4-dinitrophenol/Ag2O, ACN  COOH O O  HO F  78 55%  O2N  COOMe LiOH, THF/H2O NO2  O O  AcO F  77 74%  DBU, DCM NO2  O2N  MeOOC AcO AcO  O F  76 37%  O O2N  NO2  Scheme 3.4: Synthesis of a 2-deoxy-2-fluoro substrate analogue for UGL. Synthesis of 2,4-dinitrophenyl 2-deoxy-2-fluoro ΔGlcA (78) proceeded as shown in Scheme 3.4. Glycal 74 was formed from glucuronyl bromide 3 (see Scheme 2.1 on page 59) by reductive elimination with zinc dust, then fluorinated with the electrophilic fluorinating reagent Selectfluor (1-chloromethyl-4-fluoro-1,4-diazoniabicyclo [2.2.2]octane bis(tetrafluoroborate)) in 5:1 acetic acid/nitromethane to give protected 2-deoxy-2-fluoroglucuronate 75 as well as its manno-configured epimer, in modest overall yield. Generation of the substrate analogue then proceeded in a similar manner to synthesis of 2,4-dinitrophenyl ΔGlcA (47) as outlined in Subsection 3.2.1 on page 96, with activation by HBr in acetic acid then glycosylation of 2,4-dinitrophenol under Koenigs-Knorr conditions to 76, elimination by DBU to 77 and finally deprotection by LiOH in THF/water, instead of by aqueous HCl, to 78. Yields were different from those of the non-fluorinated compound as might be expected. Glycosylation proceeded much more slowly and in lower yield as both stages of this process involve development of positive charge in the transition states, which the fluorine destabilises. The anomeric stereochemistry in this reaction may be set by 108  3.3. Effects of heteroatoms the influence of the 2-fluoro group on the oxonium ion conformation, giving preferential attack from one face, 137–139 or the destabilising effect of the fluorine on charged intermediates may push the reaction towards a more SN 2-like mechanism. Elimination gave a better yield in a shorter reaction time, perhaps suggesting that the carbon 2 fluorine’s inductive effect extends to carbon 5, on the opposite side of the pyranose ring, increasing the acidity of the proton at this position. Finally, 78 was overall more stable than 47, allowing deprotection in one step by aqueous base rather than the slow acid hydrolysis used for the most activated leaving groups in Subsection 3.2.1. Surprisingly, no detectable hydrolysis of 78 by UGL was observed under standard assay conditions. Upon addition of a much larger amount of enzyme a very small amount of activity was observed over that in a control with no enzyme, with the rate estimated at 0.006% of that with the 2-hydroxy analogue 47 (an approximately 15 000 fold decrease). In order to determine if this lack of activity arises from poor binding or poor turnover, 78 was tested as an inhibitor of UGL-catalysed hydrolysis of 4-nitrophenyl ΔGlcA (5), and was found to be a competitive inhibitor as shown by the Dixon plot in Figure 3.5, with a Ki determined by non-linear regression to be 150 ± 10 µM. This is within the error range of the Km of the analogous nonfluorinated substrate 47, at 160 ± 20 µM (Section 3.2 on page 96). This indicates that binding to the enzyme is essentially unchanged, and that the large decrease in activity is a result of very poor turnover. While it is possible that this low activity is a cumulative effect of the two electron-withdrawing substituents on the pyranose ring (2,4-dinitrophenol and fluorine) and loss of potential hydrogen bonding by the hydroxyl group on carbon 2, these would not be expected to have such a large effect. Substitution by fluorine at carbon 4 reduced rates of glycoside hydrolysis only 8–500 fold (i.e. 0.2% activity at least, with most substantially more than this), 140–142 yet  109  3.3. Effects of heteroatoms this is an analogous substitution relative to the site of charge development. Another possible explanation for this effect is proposed in Section 3.5 on page 124.  Figure 3.5: Dixon plot showing competitive inhibition of UGL by 2,4-dinitrophenyl 2-deoxy-2-fluoro ΔGlcA (78) with a Ki of 150 µM. Substrate (6) was at the following concentrations: 52( ), 86( ), 260( ), 780( ), and 1300( ) µM  3.3.3  4-F substrate  Given the apparent positive charge developing at carbon 5 in the transition state of the rate-determining step, and the strong effect of a fluorine at carbon 1 on this, it was of interest to synthesise a substrate with fluorine at carbon 4 (88). This fluorine would be directly adjacent to the developing charge, and so would be expected to drastically reduce, if not completely ablate, hydrolytic activity. Previous syntheses of 4-fluoro glucuronides all report oxidation to the uronic acid after incorporation of fluorine, 71,74,75,143–145 so a route to the product was designed with this in mind, given in Scheme 3.5. 110  3.3. Effects of heteroatoms  OH OBz  Me-DAST  O BzO  79  BzO  O  F DCM, -30 °C BzO OBz  1. HBr, AcOH 2. Ag2O/pNP, ACN  OBz  BzO  80  O OAc  84  1. MeOH, AcCl 2. Ac2O, IEX H+ HOOC F O HO NO2  73%  83  85  Br F AcO  81  OBz  O NO2  63%  NaOMe, DCM/MeOH  O OH  TEMPO/NaOCl/ NaBr, H2O F O HO NO2  95%  Br2/CaCO3, hν, CCl4 DBU, DCM MeOOC F O F 0 °C O AcO AcO Br OAc NO2  O  F BzO  OBz  96%  MeOOC F AcO  OBz  COOMe O O OAc  87  OH O  82  OH  NO2  44%  1. NaOMe 2. LiOH  F HO  NO2  O  COOH O O OH  88  NO2  + DABCO, hν, ACN  O OAc COOMe  O NO2  86 85% (total)  Scheme 3.5: Attempted synthesis of a 4-fluoro substrate, failing at elimination by DBU. Selectively benzoylated galactose 79 was fluorinated using Me-DAST to afford 80 in very good yield, followed by activation to the glycosyl bromide by HBr then glycosylation of 4-nitrophenol under Koenigs-Knorr conditions (81) with a more moderate yield to give the desired leaving group. Deprotection of this under Zemplén conditions to 82 proceeded poorly, for unknown reasons, but subsequent oxidation to the acid 83 by TEMPO-catalysed oxidation with sodium hypochlorite proceeded in very good yield, and because of the aryl aglycone this product could be easily purified by Sep-pak. Protection of 83 under mild acid conditions afforded 84 in good yield. In order to allow a convenient elimination reaction, bromine was incorporated at carbon 5 by radical photobromination in good overall yield, with a mixture of the intended  111  3.3. Effects of heteroatoms axial brominated product (85) and a small proportion of the equatorial brominated product (86). Attempted elimination of this mixture using DBU proceeded very rapidly at 0 °C, with a significant proportion over-eliminating to form side-products in the time taken to run TLC. A small amount of starting material was recovered from this reaction, which was exclusively the equatorially brominated 86. Elimination of this recovered starting material was attempted, using a radical-initiated synelimination, 146,147 but also without success. This disappointing result, one step away from the desired product, indicated that the protected product (87) was much too reactive under the standard DBU-mediated elimination conditions to allow isolation. If a synthetic route is to have any hope of achieving 87 oxidation to the acid likely would need to occur as the last step. Control of the elimination reaction without the influence of the carboxyl group at carbon 6 then becomes a more significant challenge. F  OH  COOH F  O O  HO  Br2, hν or NBS/(BzO)2, Δ  O OBz  81  OH  NO2  OBz F BzO  O O  HO  OH  O  F PGO  CCl4 or DCM  NO2  Br OPG  O NO2  NO2  OBz F BzO  OPG O  O Br OBz  89  F PGO  O NO2  OPG O OPG  O NO2  Scheme 3.6: Retrosynthetic analysis of 88 with late oxidation, and test of the first step. Retrosynthetic analysis of a route to 88 with oxidation as a last step is presented in Scheme 3.6. This was envisaged as proceeding with introduction of bromine to carbon 5 of a 4-deoxy-4-fluoroglucoside under radical conditions followed by elimination as a means of giving the desired double bond cleanly without a 6-carboxyl group, then deprotection and TEMPO-mediated oxidation to the desired compound. 112  3.4. Kinetic isotope effects As a test of this, radical bromination of glucoside 81 to 89 was attempted, with both thermal and photochemical initiation, but without any success. The adjacent fluorine likely has too much of a destabilising effect on the radical intermediate, which was overcome by the capto-dative stabilisation in 84. While it remains possible that this reaction could proceed with different protecting groups, the choice of group(s) is not immediately apparent. Determining this empirically was decided to be more work than this compound is worth given that it was only sought as a confirmation of other results already obtained, and even if the desired result were obtained (no activity as a substrate) other possible explanations for this would also be plausible. Synthesis of this compound was thus abandoned.  3.4  Kinetic isotope effects  Further investigation of the transition state and potential steps of the UGL-catalysed hydrolysis of unsaturated glucuronides can be achieved through measurement of kinetic isotope effects, wherein a specific atom in a substrate is exchanged for one of its isotopes and the effect of this on the rate of reaction measured. Generally speaking, such differences in rate arise from the effect of the isotopic substitution on the relative stability of the transition state compared to the ground state for the two isotopomers, and can arise from events such as breaking or forming of bonds to the isotope, changes in hybridisation or charge at or near the isotopic substitution, and occasionally steric effects. A full discussion of the theory behind kinetic isotope effects is presented in Appendix C on page 300. In this work, hydrogen was replaced by deuterium on carbons 1 and 4 independently in an aryl unsaturated glucuronide, and rates of reaction for each compared to those for the non-deuterated control. The reaction rate in D2 O compared to that in H2 O for a non-deuterated substrate was also measured. 113  3.4. Kinetic isotope effects  3.4.1  Synthesis of a substrate deuterated at carbon 1  MeOOC AcO AcO  O OH  AcO  DMP, DCM  MeOOC AcO AcO  7  O  NaBD4  AcO  O  90  D2O/THF  MeOOC AcO AcO  O D  91  83%  OH  AcO  58% Ac2O/TCA, DCM  MeOOC AcO AcO  2,4,6-trichlorophenol, MeOOC O Ag2O AcO D AcO ACN AcO Br Cl 93 86%  Cl  O O  AcO D  94 50%  Cl  HBr/AcOH DCM  MeOOC AcO AcO  92  O OAc  AcO D  91%  DBU, DCM  AcO  COOMe Cl O O AcO D Cl  COOH NaOH Cl  H2O/acetone  Cl  O O  HO  Cl  HO D  95  96  84%  67%  Cl  Scheme 3.7: Synthesis of a 1-deuterated subtrate. A substrate with deuterium incorporated at carbon 1 of an aryl unsaturated glucuronide was synthesised as outlined in Scheme 3.7. Starting from methyl glucuronate hemiacetal (7), the deuterium was first incorporated in two steps by oxidation to a 1,5-lactone (90) using Dess-Martin periodinane then reduction of this lactone with sodium borodeuteride, selectively at the lactone in the presence of the ester protecting groups by virtue of its higher activity due to the strain of a planar centre in a 6-membered ring, giving the deuterated version of the hemiacetal (91) in moderate yield. Attempts at oxidation using Moffatt conditions were unsatisfactory as a large proportion of acetylation was found to occur, and this product was inseparable from the lactone. The deuterated hemiacetal was then acetylated (92) and brominated (93) in good yields to activate it for glycosylation of 2,4,6-trichlorophenol.  114  3.4. Kinetic isotope effects This was chosen as it absorbs light at a wavelength with a low background and also allows for a hydrogenation reaction required for synthesis of the substrate deuterated at carbon 4 (see Scheme 3.8 on the next page), which renders nitrophenyl groups unsuitable despite their superior optical properties. Glycosylation was achieved using Koenigs-Knorr conditions as before (58 in Table 3.1 on page 97) to give the deuterated 2,4,6-trichlorophenyl glucuronide 94 in slightly lower yield than for the non-deuterated isotopomer. This was then subjected to DBU-catalysed elimination to afford 95 in good yield, which was subsequently deprotected by NaOH in 1:1 water/acetone to give the desired compound (96) with an overall yield of 11% over 7 steps.  3.4.2  Synthesis of a substrate deuterated at carbon 4  Synthesis of a substrate with deuterium incorporated at carbon 4 was less straightforward. Oxidation of carbon 4 of a selectively protected galacturonide or glucuronide was found to give a very unstable product, likely because of the 1,3-dicarbonyl moiety formed. Use of a glucoside or galactoside may have overcome this, but without a carboxyl group at carbon 6 to activate the proton at carbon 5 this would have necessitated more extensive protecting group manipulations to achieve the double bond between carbons 4 and 5. Together these discouraged incorporation of deuterium by sodium borodeuteride as for 96, and an alternative method was sought. The route eventually employed is that shown in Scheme 3.8. Deuterium was incorporated by hydrogenation of 59 using deuterium gas with palladium on carbon as a catalyst to afford the 4-deoxy-4,5-dideutero-glucuronide 97 in near-stoichiometric overall yield but of two isomers, arising from syn-addition of D2 to either face of the double bond. These isomers were easily separable by flash column chromatography, and the more abundant desired isomer was then brominated under radical conditions  115  3.4. Kinetic isotope effects  AcO  COOMe Cl O O OAc  59  D2, Pd/C EtOAc  MeOOC D AcO  OAc  D  Cl  O  CCl4  MeOOC D AcO  COOH  OH  100  O Cl  Cl  65% DBU, DCM  NaOH, acetone/H2O  Cl  O O  HO  OAc  98  63% (+ 37% 4,5-isomer)  D  Cl  O Br  Cl  Cl  97  Cl  NBS, (BzO)2, Δ  Cl  O  D AcO  Cl Cl  54%  COOMe Cl O O OAc  99  Cl  Cl  72%  Scheme 3.8: Synthesis of a 4-deuterated subtrate. in moderate yield to give a bromine at carbon 5 anti -periplanar to the remaining hydrogen at carbon 4 in 98. Elimination of HBr using DBU proceeded in similar yield to elimination reactions for previous compounds to give the protected product 99, which was then deprotected in modest yield using NaOH in 1:1 water/acetone as for the 1- and non-deuterated isotopomers 49 and 96, to give 100 in 16% yield over 4 steps.  3.4.3  Kinetic isotope effect measurements  Using these two deuterated compounds (96 and 100) the effect of the isotope substitutions on kcat and kcat /Km were measured. For effects on kcat substrate was used at a concentration well in excess of Km in order to saturate the enzyme, and initial linear rates were determined. For effects on kcat /Km substrate was used at a concentration well below Km , and first order rate constants were determined (spectrophotometrically, by depletion) to remove any effect from variations in substrate concentration between samples. Averages of multiple reactions were then calculated, along with standard errors, and used to determine the ratio of activity with hydrogen over that with deuterium. The results are presented in Table 3.4. For the  116  3.4. Kinetic isotope effects 4-deuterated substrate the result on kcat /Km was verified by competition of equal amounts of the two substrates in one reaction, using integrals of H-1, H-4, and aryl signals in 1 H-NMR as an alternate method to follow the reaction. This method gave the same result within error (1.08 ± 0.03). The SKIE on UGL has previously been reported by Itoh et al. 87 to be 2.1 and 2.2 for kcat and kcat /Km , respectively (errors not given, but estimated from errors on individual kinetic parameters to give SKIE ranges that overlap), which agrees well with the results reported here. No directly equivalent experiments to these kinetic isotope effects from deuterium at carbons 1 and 4 have previously been reported, but analogies can be made to several other cases (vide infra). Table 3.4: Kinetic isotope effects from deuterium incorporation at carbon 1 and carbon 4 of 2,4,6-trichlorophenyl ΔGlcA, and solvent deuterium effect with 4nitrophenyl ΔGlcA. Substrate COOH  kH /kD for kcat /Km  kH /kD for kcat  (96)  1.03 ± 0.02  0.93 ± 0.02  (100)  1.06 ± 0.02  1.46 ± 0.09  2.51 ± 0.05  2.69 ± 0.12  Cl  O O  HO  Cl  HO D Cl D  COOH  Cl  O O  HO  OH  Cl Cl  COOH HO  O O OH  NO2  (6, in D2 O)  The kinetic isotope effects on kcat and on kcat /Km are clearly different from one another, for both the 1- and 4-deuterated substrates, and the error ranges exclude a unity effect. This supports the conclusion drawn from the linear free-energy relationship in Section 3.2 on page 96, that these parameters represent two independent and kinetically important chemical steps in the mechanism, with different charge densities and/or distributions. If the first step was the overall rate-limiting step these parameters would be expected to be the same, for example as was the case for  117  3.4. Kinetic isotope effects α-1,4-glucan lyase. 65 What these two steps might be are discussed in more detail in Section 3.5 on page 124. Kinetic isotope effects on kcat /Km represent the effect of isotope substitution on the energy of the transition state for the first irreversible step. 148 Deuterium substitutions at carbons 1 and 4 both give small normal effects on this step. In the accepted mechanism for hydration of a vinyl ether the initial addition of a proton to the carbon-carbon double bond is indeed an irreversible process. 149–151 If, for the sake of discussion, the first irreversible step in UGL-catalysed hydration is assumed to be protonation of the substrate double bond between carbons 4 and 5, a plausible explanation for both of these effects can be given in terms of hyperconjugation and hybridisation changes as follows. The effect from deuterium at carbon 4 can be seen as arising from two competing effects. Because the orbitals at this centre change in hybridisation from sp2 to sp3 on going to the transition state, an inverse KIE would be expected on this basis. However, substantial carbocation character develops at carbon 5, which is stabilised by hyperconjugation from the hydrogen/deuterium on carbon 4, and this will give rise to a normal KIE. 152,153 Because the overall net KIE observed is a small normal effect, the effect from hyperconjugation can be seen to be larger than that from hybridisation. From this it can be deduced that the deuterium at carbon 4 assumes a pseudo-axial geometry at the transition state, being close to the optimal geometry to give a high degree of overlap of the C−D σ-orbital with the empty p-orbital of the carbocation at carbon 5. An example of the importance of this geometry in determining the magnitude of a KIE can be seen in the effect from substitution of both hydrogens with deuteriums at carbon 3 of a substrate sialoside for Micromonospora viridifaciens sialidase; as only one of the hydrogens is appropriately aligned for hyperconjugation, the KIE for this hydrogen substitution alone is almost identical to that observed for both substitutions. 154 The small to  118  3.4. Kinetic isotope effects insignificant effect on kcat /Km from deuterium at carbon 1 can be attributed to the inability of the carbon-hydrogen bond at carbon one to stabilise positive charge on the endocyclic oxygen as there is no vacant orbital to allow hyperconjugation. A possible transition state geometry to account for these effects is given in Scheme 3.9. D  COOH  Cl Cl  O O  HO HO  D  H D  Cl  δ+  Cl  OH δ+  Cl Cl  Cl  OH O  O  OH  D  Cl  δ+ O  H D  δ+  O  D  Cl  OH  Scheme 3.9: Deduced conformation of the transition state that would give rise to the observed KIE on kcat /Km , for the first irreversible step, leading to a hypothetical oxocarbenium ion intermediate. Deuteriums at both carbon 1 and carbon 4 are represented in one molecule for simplicity, but each substitution was made independently. The transition state for this first irreversible step is assumed to strongly resemble the oxocarbenium ion product, but whether this oxocarbenium ion actually forms as a stable species is unclear. The magnitude of charge thought to develop at each centre is qualitatively represented by font size. Catalysis by the enzyme enolpyruvylshikimate 3-phosphate synthase (AroA) provides an interesting comparison with UGL, as shown in Scheme 3.10. Extensive calculations of the reaction coordinate for both the non-enzymatic (acid-catalysed) 155 and AroA-catalysed 156 cases showed a difference in the transition states, with experimental KIE data in good agreement. Both reactions proceed through an irreversible initial protonation followed by fast attack of the nucleophile on the carbocation, but the enzymatic case exhibited a much earlier transition state in formation of the carbocation (C−H bond order of 0.24 vs. 0.6 in the non-enzymatic case). This earlier transition state arose from substantial stabilisation of the carbocation intermediate, as well as the transition state leading to it, by an “electrostatic sandwich” of two carboxyl-containing sidechains. While UGL also contains two such sidechains of importance in the reaction, the arrangement in the active site is very different (see Figure 3.6). The KIE observed in AroA for two deuteriums in the equivalent 119  3.4. Kinetic isotope effects position to carbon 4 in UGL was 0.990 in the enzymatic case and 1.04 in the nonenzymatic case, reflecting a similar balancing of hyperconjugation and hybridisation effects, with the lower contribution from hyperconjugation in the earlier transition state of the enzymatic case perhaps reflecting a more substrate-like than product-like conformation, which limits orbital overlap. In this case there are two deuteriums in the substrate giving an effect from hybridisation, while only one can have substantial orbital overlap to give an effect from hyperconjugation, explaining the net inverse effect. AroA: COO-  COO(slow)  COO-  COO-  (fast)  (fast) OH  2-O  3PO  O  COO-  2-O  3PO  OH  O  COO-  2-O  3PO  COO-  O  OH  OH  2-O  3PO  OH OH2 + O COO-  UGL: COOHO  O OR OH  HO  ?? HO  -  OOC  OH  OH  O  O OR  -OOC  O +  OH  R OH  Scheme 3.10: Comparison of the reactions catalysed by AroA and UGL. Kinetic isotope effects on kcat represent the effect of isotope substitution on the energy of the highest transition state, the overall rate-determining step. 148 The nature of this second step remains unclear, but several observations about the kinetic isotope effects observed can still be made. The effect from deuterium incorporation at carbon 4 is very large, around the limit for a secondary isotope effect. 157 This suggests substantial positive charge at the transition state and a conformation that gives rise to a large degree of overlap of the C−D σ-orbital with the empty p-orbital  120  3.4. Kinetic isotope effects  Figure 3.6: Comparison of side-chain carboxyl placement in UGL from Bacillus sp. GL1 (A, PDB: 2FV1) and AroA from E. Coli (B, PDB: 4EGR), showing their distance to the carbon at which charge develops. (PEP, phosphoenolpyruvate). at carbon 5, as discussed for the effect on kcat /Km , but without a compensatory effect from change in hybridisation. The effect from deuterium at carbon 1 is much harder to explain. Common sources of inverse isotope effects include changes in hybridisation from sp2 to sp3 , as mentioned above for the effect on kcat /Km , decreases in hyperconjugation on going from the ground state to the transition state, crowding of associative transition states in nucleophilic attack, and small effects arising from induction. None of these effects are expected to be present in a simple one-step mechanism for the hydration reaction. Previous examples of the effect of deuterium substitution at carbon 5 in hydrolysis of a glycoside, in many ways analogous to carbon 1 in unsaturated glucuronide hydration (as illustrated in Scheme 3.11), include small inverse effects in acid-catalysed hydrolysis of methyl 5-{2 H}-α- and β-glucosides (0.987 and 0.971, respectively) attributed to induction, 158 and no effect in a retaining glycosyltransferase, despite substantial charge from a highly dissociative transition state. 159 An induction effect of this magnitude seems particularly unlikely given the KIE arising from deuterium on carbon 4 that suggests the majority of the charge 121  3.4. Kinetic isotope effects resides on the carbon rather than the oxygen at the transition states. The KIE from deuterium at carbon 1 on kcat thus seems to be arising from a very different transition state from that of most other glycoside hydrolases. COOH  HOOC  O OR  HO  HO  HO D  OH OR D  HO  OR  HO  D  OH O  HO HO  HOOC  O  OH  D  HO  OR D  OH O  HO HO  O  OH  O  HO HO D  OH  Scheme 3.11: Illustration of the analogous relationship of deuteriums in unsaturated 1-deutero-glucuronides (upper) to 5-deutero-glucosides (lower) on formation of an initial oxocarbenium-ion transition state. The effect from deuterium in the solvent (solvent kinetic isotope effect, SKIE) is also difficult to interpret, as there are many possible effects in play at the same time, some of which could be having opposite effects to others. Since the enzyme is incubated in D2 O the majority of exchangeable protons are assumed to be replaced with deuterium, and this will have effects on hydrogen-bonding, acid-base reactions, sterics to some extent, and all properties derived from interactions of these. This means that the effect of isotope substitution on transition state energies is unlikely to arise from a single such factor. Given this caveat, the most common interpretation of a solvent kinetic isotope effect in a reaction where proton transfer is expected to occur in the rate-determining step(s) is based on pKa effects and the ease of A−H bond breaking and formation (where A is any atom but hydrogen). The SKIE values for UGL, 2.51 on kcat /Km and 2.69 on kcat , are reasonable for a rate-determining hydrogen transfer in each step. These do not, however, inform on which hydrogen is being transferred, or even if it is the same hydrogen in both kinetically relevant steps. For non-enzymatic hydration of vinyl ethers SKIE values of 1.56–7.1 have been 122  3.4. Kinetic isotope effects reported, 43,150,155,160–162 while for the hydration of maltal by β-amylase a SKIE value of 8 was reported, 41 with the values for UGL falling toward the lower end of this range. SKIE = 1.56 - 7.1 R' R  O  R' O  R''  R  O  OH2 R' O  R''  H+  O  O  O  R  O  R''  +/- H+  R' R''  O  H 2O  R' R  R  R'  O R  +  O  + HO  R''  R' O H  R''  R  O  O H + HO  H R''  SKIE = 0.37 - 0.55  Scheme 3.12: SKIEs for hydration of vinyl ether acetals, proceeding by either initial hydration of the vinyl ether or initial hydrolysis of the acetal. In the UGL-catalysed hydrolysis of unsaturated glucuronides, the most likely proton transfer that this isotope substitution will affect is the initial hydrogen donation by the catalytic acid residue to the double bond between carbons 4 and 5. As noted earlier for the other kinetic isotope effects, a single step cannot explain the different isotope effects on kcat and on kcat /Km as observed here, meaning a second kinetically important proton transfer is taking place to account for the SKIE on kcat . It is worth noting that SKIEs reported for the few cases of hydrolysis of vinyl ether acetals determined to proceed through initial hydrolysis of the acetal, rather than initial protonation of the vinyl group, gave inverse effects characteristic of acid-catalysed acetal hydrolysis in the order of 0.37–0.55, 150,161,163 arising from a pre-equilibrium protonation then rate-determining reaction of the protonated state (see Scheme 3.12). This is clearly not the case for UGL, providing further evidence for reaction at the vinyl group rather than the acetal.  123  3.5. Conclusions, and possible alternate mechanisms  3.5  Conclusions, and possible alternate mechanisms  While the simple hydration-initiated mechanism presented in Scheme 1.9 on page 44 is plausible, several of the experimental results outlined in this chapter appear to indicate that this picture is not complete. The linear free energy relationship, showing a flat line for kcat /Km but a positive slope for kcat , and the kinetic isotope effects observed, showing different effects from deuterium substitutions at carbons 1 and 4 on kcat /Km and on kcat , together suggest that there are two kinetically relevant steps — an initial irreversible step and an overall rate-limiting step. The electron withdrawing effect of the anomeric group appears to have a bigger effect on the latter, and KIE data suggest that both involve substantial increases in positive charge at the transition state over that at the relevant ground states. An exception to this is the inverse effect at carbon 1 on kcat , which cannot be easily explained by any direct effect on a positive charge in the transition state of that step. Although a formal oxocarbenium ion intermediate could be invoked between the transition states for these two steps, such an explanation would not be in agreement with the kinetic isotope effects observed. A full positive charge at the intermediate would be expected to give no KIE from hyperconjugation in the second step, as the degree of charge would not change substantially on going from the intermediate to the transition state (or would decrease, giving an inverse effect from deuterium at carbon 4). Quenching of this oxocarbenium ion is also not likely to involve ratelimiting proton transfer as indicated by the SKIE on kcat . However, given the potential complexity of such effects some mitigating factor, as discussed at the end of Subsection 3.4.3 on page 116, could potentially allay this concern. Glycopyranosyl cations are very short-lived species in water. 164–167 While 2-deoxy glucopyranosyl cations are slightly longer lived 168,169 and the enzyme active site would no doubt be optimised to stabilise this hypothetical intermediate, it seems unlikely that the 124  3.5. Conclusions, and possible alternate mechanisms lifetime is increased in the UGL active site anywhere near enough for its hydration to become rate-limiting. Rate-limiting rearrangement of the hydrated intermediate 22 also cannot account for these results, as the breakdown of this intermediate is unlikely to proceed through a transition state with the substantial positive charge indicated by the KIE results. Also unexplained in the simple hydration-initiated mechanism is the role of the second catalytically important aspartate residue (D88 in Bacillus sp. GL1, D113 in C. perfringens, refer to Table 1.1 on page 48). The crucial catalytic importance of this residue, even more so than the putative catalytic acid (D149 in Bacillus sp. GL1, D173 in C. perfringens), suggests it may play a role in stabilisation of the transition state of the rate determining step over that in the uncatalysed reaction. It seems likely that this residue is involved in formation of the intermediate species implied by the kinetic studies. Given this evidence for an uncharged semi-stable intermediate in the hydration reaction, several possible alternative mechanisms were considered. These mechanisms are outlined in Scheme 3.13, each involving a clear role for D88/D113 in stabilising the transition state, either as nucleophile at carbon 5 (A), a nucleophile at carbon 1 (B), or as an acid/base residue to activate the substrate carbon 2 hydroxyl as a nucleophile (C). These potential mechanisms will be assessed in the following paragraphs in the context of the experimental evidence presented in this work as well as earlier publications. The simplest role for this residue in stabilising the positive charge at carbon 5 is through direct nucleophilic attack at this carbon, shown in mechanism A of Scheme 3.13. This provides a neutral covalent on-enzyme intermediate that can then be degraded by nucleophilic attack of water at carbon 5 to give the hydrated product. If this attack by water proceeds through a dissociative transition state, this scheme can account for the KIE on kcat /Km and kcat from deuterium at carbon 4.  125  3.5. Conclusions, and possible alternate mechanisms A D173  O O HO  COOH  H  O OR OH  O D113  D173  H OH O HOOC O O OR HO O OH D113  O  O  D173  OH HO  O HO  OH COOH  O D113  O OR  22  O  B D173  O O HO  COOH  H  O OR OH  O D113 C  D173  H OH O HOOC O O OR HO HO O O  O  O O HO  O OR O H O  D173  H OH O HOOC O O HO O O OH D113  D173  OH HO  O HO  D173  OR  22  O OH HO  O OR  O  OH COOH  O D113  D113 COOH  H  O D113  D173  HO  OH COOH  O D113  O  O  OR  22  Scheme 3.13: Possible mechanisms for UGL to account for KIE and LFER observations. However, this mechanism suffers from an inability to explain the inverse KIE on kcat from deuterium at carbon 1, as this effect would be expected to be similar to that on kcat /Km , both arising as a result of hyperconjugation to stabilise charge developing on the ring oxygen. A further significant objection to this mechanism arises from the distance and orientation of the D88/113 carboxylate group relative to carbon 5 of the substrate. In the substrate-bound crystal structure of UGL from Bacillus sp. GL1 this distance is 5.33 Å, or 5.71 Å in the structure from S. agalactiae, and furthermore is positioned on the same plane as the pyranose ring but on the opposite side from carbon 5, which makes this residue too far away and poorly placed for a  126  3.5. Conclusions, and possible alternate mechanisms nucleophile (see Figure 2.6 on page 71 and Figure 3.6 on page 121). While it is possible that it moves during catalysis, to invoke this explanation is a further ad hoc adjustment to the mechanism for which there is currently no evidence. A second potential role for this residue, presented in mechanism B in Scheme 3.13, better accounts for the placement of residue D88/D113. This mechanism proceeds through a similar type of nucleophilic catalysis, but with the nucleophile adding at carbon 1, rather than carbon 5, of the pyranose ring. Concomitant breaking of the bond from carbon 1 to the endocyclic oxygen gives an intermediate with a ketone instead of a positive charge at carbon 5. In a subsequent step this ketone is then hydrated by water as a nucleophile while the endocyclic oxygen attacks at carbon 1 to reform the pyranose ring, expelling the D88/D113 nucleophile to give the hydrated product 22. This mechanism accounts for the isotope effects on kcat /Km in the same manner as mechanism A and, assuming bond formation is sufficiently early on the reaction coordinate for the second step, it can provide a better explanation for the kinetic isotope effects on kcat . This early attack in the reaction coordinate would be expected to generate substantial positive charge at carbon 5, explaining the KIE on kcat from deuterium at carbon 4 by hyperconjugation to stabilise this charge, and gives steric crowding of carbon 1 in the transition state, accounting for the inverse effect of deuterium at this position, while also allowing for a weak destabilisation of the transition state by electron-withdrawing leaving groups as shown in the LFER and heteroatom substitutions. If the carbon 1 to endocyclic oxygen bond breaking is sufficiently advanced in the transition state of the first step this provides an explanation for the lack of leaving group effect on this step, as seen in the flat LFER plot on kcat /Km . The distance and alignment of D88/D113, while better in mechanism B than in mechanism A, remains a potential problem for this mechanism as the residue is 4.01 Å from carbon 1 in the substrate-bound  127  3.5. Conclusions, and possible alternate mechanisms crystal structure of UGL from Bacillus sp. GL1, or 4.48 Å in the structure from S. agalactiae, and is not optimally aligned for nucleophilic attack to displace the endocyclic oxygen (see Figure 2.6 and Figure 3.6). A final mechanism, labelled C in Scheme 3.13, involves D88/D113 acting as an acid/base catalyst for activation of the carbon 2 hydroxyl group as a nucleophile. The distance of 2.34 Å from the oxygen on carbon 2 in the crystal structure of UGL from Bacillus sp. GL1 or 2.62 Å in that from S. agalactiae (see Figure 2.6 and Figure 3.6) means that no movement of residues is required during catalysis. In this mechanism, similar to in mechanism B, formation of a ketone at carbon 5 stabilises the positive charge developing there as a result of protonation at carbon 4, in this case with formation of an epoxide across carbons 1 and 2 by the hydroxyl from carbon 2 stabilising the resulting charge on carbon 1. In the second step, nucleophilic attack of water at carbon 5 re-forms the pyranose ring with opening of the epoxide, further aided by re-protonation by D88/D113. This mechanism accounts for the lack of reactivity of carbon 2-substituted substrate analogues (see 3.3.2), as these are unable to form the required epoxide. While epoxides such as that in the intermediate shown for mechanism C are known to have a short half-life, and thus are not very stable, 170,171 they are much more stable than would be a carbocation or oxocarbenium ion. This instability may in fact be beneficial, as an overly stable intermediate can act as a kinetic trap to decrease catalysis by slowing the subsequent step. The formation of an unstable ido-configured hydrated intermediate (22) may similarly promote fast rearrangement, as seen in the poor stability of the methyl ketal analogue to this intermediate (26) formed by UGL-catalysed reaction in dilute methanol (refer to Subsection 2.5.2) compared to its gluco-epimer (28). These three mechanisms present possible explanations for the data presented in this chapter, but further work is required to discriminate between them. Attempts  128  3.5. Conclusions, and possible alternate mechanisms at such a discrimination are presented in the following chapter.  129  Chapter 4  Testing of alternative mechanisms In Section 3.5 at the end of the previous chapter, a set of alternative mechanisms for UGL was proposed and discussed . Several experiments were carried out in an attempt to discriminate between them and these form the basis of this chapter. Based on the apparent importance of, and lack of clear role for, the catalytic residue D88/113, a mutant in which this residue had been replaced by glycine was generated, and rescue of the largely catalytically inactive mutant was attempted with various small molecules. Further testing of these mechanisms was sought in the analysis of various compounds as potential competitive inhibitors, matching the geometry and postulated sites of charge development in these mechanisms. For mechanisms invoking nucleophilic catalysis by the enzyme, A and B of Scheme 3.13 on page 126, attempts were made to kinetically trap these covalent glycosyl-enzyme intermediates. Finally, an attempt was made to synthesise a small molecule intermediate as postulated in mechanism C.  4.1  Attempted rescue of D113G mutant  Alongside their publication of a crystal structure of UGL, Itoh et al. 92 presented kinetic data on two mutants in which active site aspartate residues had been replaced by asparagine. These mutations were seen to have a dramatic effect on kcat , but little effect on Km . One of these residues (aspartate 149 in Bacillus sp. GL1 or aspartate  130  4.1. Attempted rescue of D113G mutant 173 in C. perfringens — D149/173) was later proposed to act as a catalytic acid, 87 donating a proton to the double bond between carbons 4 and 5. The other of these aspartate residues (D88 in Bacillus sp. GL1 or D113 in C. perfringens — D88/113) had no role assigned beyond hydrogen bonding to hydroxyl 2 and 3 for substrate binding and stabilising the transition state by some unknown mechanism. Several possible roles for this residue that would account for its great importance in catalysis are proposed in the concluding section of Chapter 3. Testing of these roles, as either a nucleophile or acid/base residue, was attempted by mutation of this residue to glycine in C. perfringens UGL. This is a much less conservative mutant than the asparagine introduced into Bacillus sp. GL1 UGL, creating a relatively large space in the UGL active site. The ability of exogenous small molecules to occupy this space and rescue activity of the D113G mutant was investigated. The C. perfringens UGL D113G mutant was generated using the Quikchange method. 172 Primers were designed to cover this region of the UGL gene, with a single mis-match introduced to codon 113 to make it encode glycine instead of aspartate. These primers were used for PCR of the pET28a::UGL plasmid in Subsection 2.1.1 on page 54, followed by selective digestion of the template on the basis of methylation by the restriction enzyme Dpn1. The resulting mutant plasmid was transformed into TOP10 E. coli cells by heat shock, the cells allowed to recover, and the plasmid then isolated and transformed into BL21(DE3) E. coli cells for expression. The mutant UGL protein was expressed and purified in the same way as the wild-type (see Subsection 2.1.2 on page 54), using a new column to avoid trace contamination with wild-type enzyme (see Figure 4.1). The yield of the mutant enzyme was similar to that of the wild-type. Initial characterisation of this mutant showed very low activity at pH 6.6, with kcat = 0.0033 ± 0.0001 s-1 and Km = 0.8 ± 0.1 mM for hydrolysis of 3-nitrophenyl  131  4.1. Attempted rescue of D113G mutant  Figure 4.1: Elution trace for UGL D113G, showing A280 in blue, conductivity in brown, % buffer B in green (up to 100% at its maximum), and fraction numbers in red. Axes are eluted volume in mL and absorbance in mAU, with axes not shown for other traces. ΔGlcA (50), one of the best substrates with wild-type UGL ( Table 3.2 on page 99). These parameters indicate little to no change in Km and a drastic reduction in kcat to only 0.02% of wild-type. This is similar to the 0.008% residual activity reported by Itoh et al. for a conservative asparagine substitution at the analogous position. 92 This D113G mutant enzyme also exhibited an altered profile of activity with pH. The mutant UGL showed greater relative activity in more acidic solutions, with a single point of inflection in the pH profile at 4.99 ± 0.05, as shown in Figure 4.2. Given that this profile was determined by measuring kcat /Km , which reports on reaction of free enzyme with free substrate, it is important to note that this is above the pKa measured for the substrate, at 4.5 ± 0.2 ( Figure 2.4 on page 63), and so likely represents an intrinsic ionisation of the enzyme itself, and not the substrate. This shift in pKa arising from mutagenesis of one aspartate to glycine is dramatic, and represents a large change in the local environment of the remaining ionisable residue 132  4.1. Attempted rescue of D113G mutant — assumed to be the catalytic acid residue D149/173. Such a shift in pKa is similar to that observed for the nucleophile residue of a retaining glycosidase on removal of the adjacent charge on the acid/base residue. 173 While this indicates that the low residual activity reported at pH 6.6 can partially be overcome by reaction at a more acidic pH, the highest activity observed with this mutant is still around two orders of magnitude less than that of the wild-type at its optimum pH.  Figure 4.2: Profile of first order rate for hydrolysis of 4-nitrophenyl ΔGlcA (6) by UGL D113G ( , right scale) and wild-type ( , left scale), plotting kcat /Km (µM-1 .min-1 ) against pH. Rescue of catalytic activity in this mutant was attempted with a variety of small molecules at pH 6.6, but with no success. Addition of sodium azide or sodium formate buffer at pH 6.6 to the UGL-catalysed hydrolysis of 4-nitrophenyl ΔGlcA and monitoring of the reaction by spectrophotometry (detection of released phenol) showed no enhancement of hydrolysis at 100 mM of either small molecule. Further testing of a wider range of nucleophiles also showed no rescue, with UGL, 4-nitrophenyl ΔGlcA (6), and the small molecules (at molar concentrations) incubated at 25 °C and monitored daily by TLC. The potential nucleophiles tested were sodium formate, 133  4.2. Testing of potential inhibitor leads sodium acetate (both adjusted to pH 6.6 with NaOH), methanol, β-mercaptoethanol, sodium azide, sodium cyanate, sodium thiocyanate, and sodium cyanide. While this shows no rescue with any exogenous nucleophile, formate and acetate could also be expected to rescue activity if the mutated residue functions as an acid/base residue, and so this experiment provides a disappointing lack of information on the role of this residue.  4.2  Testing of potential inhibitor leads  Compounds that mimic the transition state of a reaction are often very potent competitive inhibitors, taking advantage of the enzyme’s strong binding to the transition state, which stabilises it and thereby catalyses the reaction. 174 The mechanisms proposed in Section 3.5 on page 124 involve different transition states, with charge developing in different locations and in different magnitudes during the course of the reaction. In particular, both the simple hydration-initiated mechanism in Scheme 1.9 on page 44 and direct nucleophilic catalysis at carbon 1 in mechanism A in Scheme 3.13 on page 126 invoke development of positive charge on the endocyclic oxygen of the substrate. By contrast, the ring-opening mechanisms B and C of Scheme 3.13 may involve development of negative charge on this same oxygen, depending on the timing of bond breaking and bond formation. Thus, inhibition of the reaction by a compound with charge matching that which develops in the transition state would provide evidence for that aspect of the mechanism. Scheme 4.1 shows a representation of the first step in a direct hydration mechanism and potential inhibitors mimicking structures in this mechanism. Inhibition of Streptococcal UGLs has previously been reported for the monosaccharides d-glucuronic acid and d-galacturonic acid, with 0.4–3.3 % maximal activity remaining in reactions with substrate at or around Km (0.2 mM) and inhibitor at 1 mM (no Ki value reported), 85 while glycine 134  4.2. Testing of potential inhibitor leads has been reported to competitively inhibit Bacillus sp. GL1 UGL with a Ki of 6.5 mM. 93 COOH  COOH (UGL)  O HO  H2O  OR  HOOC  OH O  O+ HO  OH  OR  HO  OR OH  OH  22 COOH  COOH  HOOC  OH  NH2+ HO  HO  OH  101  OH  102  O OH  103  COOH OH  O  R= R  HO NH3+ .TFA-  OH OH  108 Scheme 4.1: Illustration of structural analogies from UGL substrates, intermediates, and putative oxocarbenium ion-like transition states to potential inhibitors: d/lproline (101), shikimate (102), shikimate’s biosynthetic precursor 3-dehydroquinate (103), and deacetylated DANA (Neu2en, 108). Proline (101) was selected as a a simple lead compound exhibiting positive charge at a centre analogous to the endocyclic oxygen at the assay pH. Both isomers of proline were tested as competitive inhibitors, with no inhibition observed up to 10 mM, clearly different from the case of glycine (vide supra). The inhibition by glycine is presumably the result of binding at the active site as seen in the X-ray crystal structure of wild-type UGL from Bacillus sp. GL1 with glycine and DTT bound (Figure 4.3). 92 In this structure glycine binds with its nitrogen situated between D88 and D149, but substantially closer to the latter, presumably taking advantage  135  4.2. Testing of potential inhibitor leads  Figure 4.3: X-ray crystal structure of the Bacillus sp. GL1 UGL active site showing glycine (centre) and all side-chains within 5 Å, from two perspectives (PDB: 1VD5). 136  4.2. Testing of potential inhibitor leads of ionic interactions to enhance binding, and the positioning of the positive charge is not analogous to the position of the substrate endocyclic oxygen. This binding mode is perhaps not available to the sterically larger proline, making the latter a better mimic of the postulated charge. These results are consistent with a positively charged nitrogen residue at a position analogous to the endocyclic oxygen of UGL substrates not giving inhibition, suggesting that such a positive charge may not develop on the endocyclic oxygen during catalysis. However, other possible explanations for this lack of inhibition are also possible, including the displacement of tightly bound water molecules by the alkyl chain of proline, which is unable to form hydrogen bonds. Designing of a transition state mimic with positive charge at an analogous position to carbon 5 or negative charge in an analogous position to the endocyclic oxygen is more difficult, because of steric limitations of this centre and the adjacent negative charge on the carbon 6 carboxylate group. The natural product shikimic acid (102) is a carbocyclic analogue of UGL substrates, albeit with different stereochemistries for two hydroxyl groups. This compound was found to exhibit competitive inhibition of UGL with a Ki of 3.0 ± 0.4 mM (Figure 4.4), and was not hydrated by UGL. This affinity is slightly better than anticipated for a substrate mimic, given the Km of substrates with a small anomeric group such as ΔGlcA fluoride (10.7 mM, refer Table 3.3 on page 106), and similar to the binding affinity of Kdn2en (40) and most of the natural substrates tested (1–3 mM, Table 2.2 on page 68). While the stereochemical mismatch would be expected to decrease binding affinity, examination of the substrate-bound crystal structure of UGL suggests that such differences in stereochemistry could be sterically accommodated at the active site. 102 may also be able to bind in a 1 H2 conformation to allow the hydroxyls on carbons 2 and 3 to interact with D88/113 in a similar manner to the substrate in its 2 H1 conformation, placing the remaining hydroxyl in a pseudo-axial  137  4.2. Testing of potential inhibitor leads configuration as illustrated in Figure 4.5. Derivatisation of a suitable hydroxyl group in 102 (on carbon 1 or perhaps carbon 3) with an aryl group would likely improve upon the binding observed, while its biosynthetic precursor 3-dehydroquinate (103) or suitable derivatives may also furnish improved binding as a result of the hydroxyl and carboxylate groups at carbon 5 acting as mimics of the hydrated intermediate 22 (see Scheme 4.1).  Figure 4.4: Dixon plot showing competitive inhibition of UGL by shikimate with a Ki of 3.0 mM. Substrate (6) was at the following concentrations: 52( ), 86( ), 260( ), 780( ), and 1300( ) µM. A substrate mimic intended to take advantage of the interactions of the substrate hydroxyl on carbon 2 and the enzyme D88/113 residue was sought in the deacetylated form of sialidase inhibitor DANA, Neu2en (108). 175 The amine group in 108 was predicted to form a strong ionic interaction with D88/113, so this compound was expected to bind much more tightly than the related substrate Kdn2en (40). Synthesis of 108 proceeded from acetylated Neu5Ac2en (DANA, 104) by the method of Gervay et al., 176,177 through an N -acetyl-N -Boc intermediate (105) with 79% yield over 138  4.2. Testing of potential inhibitor leads  B  A COOH O HO  O OR  D88/113  .  COOH O OR  O H O H O-  2  H1  OH substrate (ΔGlcA) OH COOH OH  OH  COOH  2H 1  HO  OH COOH  OH OH O  shikimic acid D88/113  H O O H O-  1  H2  Figure 4.5: (A) Representation of substrate (upper) and shikimic acid (lower) interaction with D88/113 in the active site of UGL. (B) X-ray crystal structure of the Bacillus sp. GL1 UGL active site showing ΔGlcA and side-chains of all residues within 3 Å (N(D)88, D143, and R221), from two different perspectives (PDB file 2FV0).  139  4.2. Testing of potential inhibitor leads AcO  OAc  AcHN AcOAcO  O  OAc Boc2O/ AcO COOMe DMAP, DCM Ac N Boc AcOAcO  104  COOMe O  HO NaOMe, MeOH  OH COOMe O  BocHN HO HO  105  106 NaOH, MeOH/H2O  HO  OH COOH  TFA-.+H3N HO HO  O  TFA, H2O  108  HO  OH COOH  BocHN HO HO  O  107  79% (over 4 steps)  Scheme 4.2: Synthesis of Neu2en. four steps, as shown in Scheme 4.2. Surprisingly, 108 did not show any inhibition of UGL when tested in the micromolar concentration range. Given the anticipated proximity of the 108 amine group and D113, and the strong ionic interaction this would be anticipated to give, this lack of binding is perplexing. However, while 1-amino glycosides are reasonably good inhibitors of glycosidases (low micromolar affinity), 178 2-deoxy-2-fluoro glycosides are not, 179 despite the similar proximity of complementary charges on the amino group and catalytic carboxylates, weakening any conclusions that can be drawn from this. Incubation of 108 with UGL and subsequent spectrophotometric monitoring of the reaction showed that this compound also was not hydrated by this enzyme, in agreement with the lack of activity seen with a fluorine substituent on this same carbon (78, Subsection 3.3.2). One potential argument against the relevance of the inactivity of the 2-deoxy-2-fluoro ΔGlcA substrate is that it has lost the hydrogen bonds made by the 2-hydroxy group, which may be crucial for activity. The amine in Neu2en (108) should be able to make more similar hydrogen bonds to the analogous 2-hydroxy substrate Kdn2en (40, Subsection 2.6.1), and so potentially provides a control for this mitigating factor. However, 140  4.3. Anticipated trapping reagents for UGL 2-deoxy-2-amino glucosides are poor substrates for glucosidases, showing that this substitution is clearly not able to compensate completely. 179  4.3  Anticipated trapping reagents for UGL  Mechanisms A and B in Scheme 3.13 on page 126, which involve nucleophilic catalysis, invoke covalent glycosyl-enzyme intermediates. Under appropriate conditions these may be expected to be trapped as stable species. In order for these species to have an appreciable lifetime, their rate of formation needs to be accelerated relative to the rate of decomposition. Because the reactivity of the UGL substrate (ΔGlcA) carbon-carbon double bond is difficult to modulate, this was attempted by placement of fluorine substituents in product mimics, in the hope that the reverse half-reaction could be observed.  4.3.1  2,3-Difluoro Kdn  Given the activity of Kdn2en (40) as a substrate for UGL (see Subsection 2.5.2 on page 78), it was hoped that a sufficiently activated leaving group at carbon 5 of a compound resembling the hydration product of this reaction (22) would be able to drive the reverse of the normal reaction, as illustrated in Scheme 4.3 for the example case of mechanism A in Scheme 3.13. Addition of a second fluorine at the adjacent position, carbon 3 in Kdn numbering or carbon 4 in ΔGlcA numbering, might inductively destabilise transition states leading to and from this intermediate, in an analogous strategy to the use of 2-deoxy-2-fluoro glycosides, giving a stabilised glycosyl-enzyme intermediate. Placement of this additional fluorine at carbon 3 in an axial stereochemistry would also prevent abstraction of this proton by the enzyme, preventing elimination of the intermediate to give an unsaturated uronide as a product, and thereby preventing completion of the reverse reaction. 141  4.3. Anticipated trapping reagents for UGL A D173  O O HO  H  D173  O R OH  O D113  COOH  H OH O HOOC O O R HO O OH D113  O  O  D173  OH HO  O HO  OH COOH  O D113  O R  22  O  B D173  O O HO  D173 H  O R OH  O D113  O  O  COOH O  D173 F  COOH  HO O D113  O  O OH  OH O  R  F F  HO O D113  O OH COOH  R  O  Scheme 4.3: Rationale for attempted trapping of UGL with 2,3-difluoro Kdn (114), comparing one proposed mechanism involving nucleophilic catalysis (A) and the corresponding anticipated mechanism of trapping (B). Synthesis of 2,3-difluoro Kdn is presented in Scheme 4.4, following the method of Watts et al. 180 Briefly, 3-fluoro Kdn (109) was synthesised from d-mannose and 3-fluoropyruvate by means of Neu5Ac aldolase, then protected in two steps by trifluoroacetic acid in methanol followed by acetic anhydride in pyridine to afford 110 in 46% over 3 steps, with a 4.35:1 ratio of axial to equatorial fluorine at carbon 3. The anomeric position was subsequently deprotected using hydrazine acetate in methanol to give 111 (differing from the dichloromethane solvent used in the reference cited) in good yield, then the anomeric position fluorinated by means of methyl-DAST (differing from the DAST used in the reference cited) in low yield to the protected product 112. This was then deprotected in two steps, deacetylating to give 113 by sodium methoxide in dichloromethane/methanol then saponifying the methyl ester to afford the desired compound 114 in 5.5% total yield over 6 steps. On testing, 114 showed unusual inactivation behaviour with UGL. At a high concentration of inactivator (20 mM; well below the buffer concentration of 40 mM), rapid time-dependent inactivation was observed, but with a poor fit to first order kinetics. However, at lower concentrations no inactivation was observed, as shown 142  4.3. Anticipated trapping reagents for UGL OH HO HO  OH  OH O  F  HO HO  O  OH  O  HO HO  109  COONa  OAc  1. MeOH, TFA 2. Ac2O, pyridine  OH  Neu5Ac aldolase  COOH  OAc  AcOAcO AcOAcO  F  O  110  COOMe F  58%  79%  H2NNH2.HOAc, MeOH  OH  OAc  COOMe O  HO HO  F  NaOMe, DCM/MeOH  HO HO  113  F  OAc  COOMe  AcOAcO AcOAcO  O  F  DCM  112 F  65%  OH  Me-DAST, AcOAcO AcOAcO  O  111  34%  COOMe F  70%  NaOH, THF/H2O  OH  COOH O  HO HO  HO HO  114  F F  F F HO HOOC  O  OH  OH  OH HO  78%  Scheme 4.4: Synthesis of 2,3-difluoro Kdn. in Figure 4.6. The possibility that inactivation was due to pH shifts in the inactivation mixture was discounted by checking the pH of reaction mixtures before and after inactivation. No change in pH was observed.  The results in Figure 2.3 on  page 62 also showed no inactivation of the enzyme as a result of ionic strength. An assay for time-dependent inactivation was subsequently carried out using the equatorial ΔGlcA fluoride substrate 70 at the same concentration, and a similar rate of inactivation was seen (Figure 4.7). This compound is not predicted to inactivate based on any mechanism proposed, suggesting that the inactivation seen for 114 is not mechanism-dependent. Indeed the axial phenyl substrate 43 (2.6.2) showed decreasing activity when testing hydrolysis of substrate at 20 mM or above, which  143  4.3. Anticipated trapping reagents for UGL  Figure 4.6: Time-dependent inactivation of UGL by 2,3-difluoro Kdn (114) at 0( ), 0.2( ), 2( ), and 20( ) mM. Fits are to first order decay.  Figure 4.7: Time-dependent inactivation of UGL by ΔGlcA fluoride (70) at 20 mM. Fit is to first order decay.  144  4.3. Anticipated trapping reagents for UGL may have arisen from the same non-specific inactivation phenomenon. The reason for this inactivation by 70 or 114 at high concentrations remains unclear. Further testing also showed 114 to not be a substrate of UGL. Monitoring of the reaction over a period of days at 25 °C by TLC and  19 F-NMR  showed slow degradation of  the compound to 3-fluoro Kdn 109 and fluoride at the same rate with or without enzyme, as illustrated in Figure 4.8.  Figure 4.8: 19 F-NMR showing partial hydrolysis of 2,3-difluoro Kdn (114) with UGL (lower) and in a non-enzymatic control (upper) after 1 week. Labelled are fluorines 2 and 3 of 114 as well as fluorine 3 of 109 (labelled 3 ) and free fluoride.  4.3.2  4-Deoxy-1,5-difluoro-iduronic acid  Given the lack of inactivation by 2,3-difluoro Kdn, a better product mimic for use as a mechanism-based inactivator was sought in a compound with fluorine at the anomeric carbon, to stabilise the molecule, while retaining the activated leaving group fluorine at carbon 5. This compound, 4-deoxy-1,5-difluoro-iduronic acid (120), was anticipated to be a potential mechanism-based inactivator for the same reasons outlined in Scheme 4.3 of Subsection 4.3.1, although abstraction of a proton from 145  4.3. Anticipated trapping reagents for UGL carbon 4 of a covalent glycosyl-enzyme to give elimination to an unsaturated uronide product might be possible in this case. O O O AcO AcO  O DBU DCM  O OAc  O  O  Ph  F  115  O  O  O 2. PhCOCH2Br/ AcO TEA, EtOAc  O F OAc  AcO  Ph  1. H2, Pd/C, Ph EtOAc  O OAc  116  117  84%  48%  F  1. NBS, hν, CCl4 2. AgF, ACN F  O  HO HOOC  NH3, MeOH  OH  F  F AcO HOOC  120  119  12%  69%  O OAc  F  H2, Pd/C MeOH/H2O  F  O  AcO O  O  118  F OAc O Ph  39%  Scheme 4.5: Synthesis of 4-deoxy-1,5-difluoro-iduronic acid. Synthesis of this compound proceeded by the same route as that used for synthesis of 1,5-difluoro iduronic acid by Wong et al., 181 with additional steps to generate the 4-deoxy analogue as shown in Scheme 4.5. Starting from the protected glucuronyl fluoride intermediate 115 of this same reference, carbon 4 was deoxygenated by DBU-mediated elimination (116) followed by hydrogenation and reprotection of the phenacyl ester at carbon 6 (117) in 40% yield over 3 steps. Fluorine was subsequently incorporated at carbon 5 by radical bromination with photochemical initiation followed by replacement of bromine by fluorine using silver (I) fluoride to afford the protected final product (118) in low yield. This was deprotected over 2 steps, removing the phenacyl ester by hydrogenation (119) then deacetylating by ammonia in methanol to give the desired 120. While the deprotection appeared to proceed smoothly by TLC, this final product was unstable, decomposing during both Sep-pak and flash column chromatographic purification, but a small sample of 146  4.3. Anticipated trapping reagents for UGL pure product was isolated for testing. Unsurprisingly, an attempt at synthesis of an analogous compound with a phenyl group at the anomeric position by a similar pathway failed when the product decomposed completely on attempting deprotection by sodium methoxide followed by lithium hydroxide.  Figure 4.9: Time-dependent inactivation of UGL by 4-deoxy-1,5-difluoro-iduronic acid (120) at 0( ) and 5( ) mM. Fits are to first order decay. Incubation of UGL with 5mM 120 resulted in no inactivation, as shown in Figure 4.9. Nor was there any sign of hydrolysis or elimination, as determined by TLC and  19 F-NMR  (Figure 4.10). However relatively rapid non-enzymatic decomposi-  tion was observed, as might be expected from its behaviour on deprotection, to give the same final product as UGL-catalysed hydrolysis (24 and free fluoride, shown in Scheme 2.3 on page 74). As with 114, pH was tested before and after reaction at higher concentrations and confirmed to not change. This non-reactivity with UGL was determined to not be a result of poor binding, since 120 was seen to be a competitive inhibitor of UGL with a Ki of 7.5 ± 0.8 mM (see Figure 4.11 on page 149), demonstrating a slightly higher binding affinity than the analogous ΔGlcA fluoride substrate (70 at 10.7 mM, refer 3.3.1 on page 103). The lack of inactivation or turnover of 114 and 120 by UGL appears to be 147  4.3. Anticipated trapping reagents for UGL  A  .  B  .  Figure 4.10: Overnight hydrolysis of 4-deoxy-1,5-difluoro-iduronic acid (120) with UGL (right lanes of TLC, upper spectrum of NMR) and in a non-enzymatic control (left lanes of TLC, middle spectrum of NMR) as monitored by TLC (A) and 19 FNMR (B), with starting material in the lower spectrum.  148  4.3. Anticipated trapping reagents for UGL  Figure 4.11: Dixon plot showing competitive inhibition of UGL by 4-deoxy-1,5difluoro-iduronic acid (120) with a Ki of 7.5 mM. Substrate (6) was used at the following concentrations: 25( ), 100( ), 300( ), and 600( ) µM. a result of inappropriate reactivity or placement of reactive moieties. Given that fluoride is a very good leaving group, and would certainly be expected to react if a suitably nucleophilic side-chain is appropriately placed to displace it, this suggests that mechanism A in Scheme 3.13 (in which a nucleophilic side-chain attacks at carbon 5) is not correct. Mechanism B (in which a nucleophilic side-chain attacks at carbon 1) and mechanism C (in which an acid/base side-chain activates the carbon 2 hydroxyl as a nucleophile to attack at carbon 1) both remain plausible as the immediate leaving group for both cases is the endocyclic oxygen, which is expected to be only slightly activated by the inductive effect of the adjacent fluorine. In these mechanisms the driving force for nucleophilic attack is postulated to be donation of a proton to carbon 4 and the development of positive charge at carbon 5 (refer to discussion in Section 3.5), and thus would not be substantially influenced by a fluorine at carbon 5 unless a suitable residue were present to accelerate its departure, 149  4.3. Anticipated trapping reagents for UGL such as by hydrogen bonding. The D88/D113 catalytic acid residue would possibly fill this role, since the D88/D113 carboxylate to carbon 5 distance is 3.28 Å. However, the distance to a heteroatom substituent on this carbon in the hydration product is presumably slightly greater (compared with 2.98 Å to carbon 4). This increased distance, combined with any difference in side-chain placement of this residue when the product is bound, rather than the substrate, may be sufficient to explain the lack of efficient proton donation to this fluorine.  4.3.3  1-Fluoro-ΔGlcA fluoride  The potential UGL inactivator 1-fluoro-ΔGlcA fluoride (124) was designed as a further test for rearrangement of the initial hydrate product (22) in the UGL active site. Inactivation of UGL by this compound was expected to arise from hydrolysis of one of the glycosyl fluorides to give an acyl fluoride at the anomeric carbon, which can subsequently react with any nucleophile at or close to the active site in a non-specific manner, giving inactivation of the enzyme as shown in Scheme 4.6. Although 124 was expected to only be very slowly hydrolysed by UGL, based on the results with the unsaturated glucuronyl fluorides 73 and 70 in Subsection 3.3.1 on page 103, each enzyme only needs to catalyse a single turnover to inactivate, and so reactivity does not need to be high for this probe to provide an answer to this question. If inactivation were to be seen with this molecule it would be indicative of rearrangement taking place in the active site, while no inactivation would be uninformative, as the acyl fluoride may be sufficiently long-lived to leave the active site, the probe may be too deactivated to form the acyl fluoride, or the rearrangement may be taking place outside of the enzyme active site. Synthesis of this probe was accomplished over three steps from the glucuronyl 1,5 lactone 90 in 11% overall yield, as shown in Scheme 4.7. Both fluorines were  150  4.3. Anticipated trapping reagents for UGL COOH HO  O UGL F +H2O HO F  124  HO HO  O HO COOH F  HOOC  O F UGL? HO -HF  O OH  O UGL-Nu HO COOH F  O  -FHO  Nu UGL  Scheme 4.6: Rationale for attempted trapping of UGL with 1-fluoro-ΔGlcA fluoride (124), showing reaction to form the hydrated product and subsequent rearrangement to form an acyl fluoride followed by trapping of an active site nucleophile (Nu). introduced in one step by protracted treatment with methyl-DAST to afford 121 in a low yield, but a sufficient amount to proceed with synthesis. A contaminant arising from elimination of a fluorinated intermediate was also observed (122). The desired di-fluorinated intermediate was then subjected to DBU-mediated elimination in good yield to give 123, followed by deprotection over two steps in very good yield by acid-catalysed trans-esterification in methanol then hydrolysis of the methyl ester by lithium hydroxide in a mixture of water and tetrahydrofuran to give the desired probe 124. This scheme represents a novel route for introduction of two fluorines at the anomeric position, with previous routes typically using a sequential scheme of halogenations, such as chlorination by hydrochloric acid, then radical bromination at the anomeric position, followed by replacement of these halogens with fluorine using silver fluoride. 182–186 The previously employed reaction sequence was unsuitable for a uronic acid because of the high reactivity of carbon 5 under radical bromination conditions. Previously, 1-fluoroglycosyl fluorides have been observed to act as slow substrates for inverting and retaining α- and retaining β-glycosidases, as well as trehalase, 187 and α-1,4-glucan lyases of glycoside hydrolase family 31, 65 despite being anticipated to give mechanism-based inactivation similar to 2-deoxy-2-fluoroglycosides. This was attributed to a stabilising back-bonding effect from the fluorine p-orbitals compensating for the destabilising inductive effects on transition state energies. 134 Compounds  151  4.3. Anticipated trapping reagents for UGL MeOOC AcO AcO  90  O AcO  Me-DAST DCM O  MeOOC AcO AcO  O  DBU F DCM  AcO F  121 16% MeOOC AcO AcO +  AcO  COOMe 1. AcCl, MeOH O HO F 2. LiOH, H2O/THF AcO F  COOH O F HO F  123  124  73%  98%  O F AcO  122 10%  Scheme 4.7: Synthesis of 1-fluoro-ΔGlcA fluoride with additional fluorides at either carbon 2 or carbon 5 in addition to the geminal anomeric fluorines were observed to not react as either inactivators or substrates, despite evidence of their binding to the enzyme active site. 185 Unfortunately, 124 was seen to give non-specific inactivation of UGL, as shown in Figure 4.12, similar to that seen with 2,3-difluoro Kdn (114) and ΔGlcA fluoride (70). The rate of this inactivation did not appear to vary in any meaningful way with changes in concentration above 20 mM, and fits to first order kinetics for this inactivation were relatively poor, while concentrations below this did not appear to give any inactivation. Given the apparent binding of both anomers of ΔGlcA fluoride (70 and 73, see Subsection 3.3.1 on page 103), there is no reason why 124 should not bind. This inactivation is thus unlikely to be mechanism-based, and is more likely to either arise from some contaminant (no contaminant was detected, but it would not necessarily be expected to be, as the concentration of enzyme in an inactivation reaction was at around 0.02% that of the inactivator in a 20 mM reaction) or from non-specific effects such as an increase in surface adsorption as seen with yeast α-glucosidase with 1-fluoro-d-glucopyranosyl fluoride. 187  152  4.4. Attempted synthesis of proposed epoxide intermediate  Figure 4.12: Time-dependent inactivation of UGL by 1-fluoro-ΔGlcA fluoride (124) at 0( ), 5.5( ), 11( ), 22.1( ), 36.8( ), and 51.5( ) mM. Fits are to first order decay.  4.4  Attempted synthesis of proposed epoxide intermediate  As a direct test of the epoxide-based mechanism C in Scheme 3.13 on page 126, a stable form of the proposed epoxide intermediate was sought to assay for catalytic competence. Reports of alkoxy oxiranes in the literature, 170,171 which would be the intermediates formed from O-glycosidic substrates if this mechanism were acting, indicate half-lives in water on the order of minutes. While this would be consistent with enzymatic intermediacy, it would be difficult to synthesise these for testing. However, the epoxide formed from the C -glycoside analogue Kdn2en (40), as illustrated in Scheme 4.8, should be much more stable, and thus presents a viable target for synthesis. Given that synthesis of Kdn (32, Scheme 2.9 on page 84) was achieved using 153  4.4. Attempted synthesis of proposed epoxide intermediate COOH OH O OH  HO  OH  40  HO  UGL?  HOOC O HO  OH  O  133  HO  OH  HO  UGL? HO  O  OH COOH  OH  OH  HO  Scheme 4.8: Epoxide intermediate expected from Kdn2en (40) according to mechanism C of Scheme 3.13 the enzyme Neu5Ac aldolase and mannose, as opposed to the natural substrate N acetyl-mannosamine, the active site of this enzyme was investigated to determine if unsaturated substrates could be expected to be accommodated. A published set of X-ray crystal structures of Neu5Ac aldolase with several product-like inhibitors bound 188 showed that the product and Schiff’s base intermediate of this enzyme, and presumably its substrate also, bind in the active site in an open chain extended conformation, as illustrated in Figure 4.13. While the 4-hydroxy group of Neu5Ac (Neu5Ac numbering), derived from the anomeric aldehyde of N -acetyl-mannosamine, was found in the above publication to be important for the formation of the Schiff’s base intermediate, the carbon 5 and 6 hydroxyl groups (Neu5Ac numbering) appear to make few contacts with the enzyme and thus are plausibly less important for binding and catalysis. Based on these observations, it was hoped that aldehydo2,3-dideoxy-erythro-trans-hex-2-enose (131, an α,β-unsaturated hexose enal) would also be accepted as a substrate by Neu5Ac aldolase and provide a facile means of constructing the desired carbon skeleton. A simple synthesis of 133 was envisaged in extension of the α,β-unsaturated hexose enal 131 (also known as a Perlin aldehyde) to an appropriate non-5-enulosic acid by use of Neu5Ac aldolase followed by epoxidation to the desired compound, as shown in Scheme 4.9. Rearrangement of acetyl and benzyl protected glucal (125 and 126) by hafnium tetrachloride and zinc iodide under reflux, following the method of Saquib et al., 189 proceeded in lower selectivity than reported to give a mixture of  154  4.4. Attempted synthesis of proposed epoxide intermediate  Figure 4.13: X-ray crystal structure of Haemophilis influenzae sialic acid aldolase (Neu5Ac aldolase) active site 188 with the inhibitor 4-oxo-sialic acid bound as a stable Schiff’s base derivative of Lys164, showing all amino-acid side-chains within 3.5 Å (PDB: 1F7B). cis (127 and 128) and trans (129 and 130) α,β-unsaturated hexose enal products, while acetate migration from hydroxyl 4 to hydroxyl 5 was also observed. These products proved difficult to separate, especially those with benzyl protecting groups, so deprotection was undertaken on the crude product mixture. Unfortunately, the α,β-unsaturated hexose enals were found to be very difficult to deprotect and purify (vide infra). An attempted rearrangement of glucal (134) in water to the unprotected α,β-unsaturated hexose enal was unsuccessful, forming predominantly the overeliminated product 135 and a small amount of the hydrated product 2-deoxy-glucose (136) shown in Scheme 4.10, as reported for similar reactions catalysed by indium trichloride, iron trichloride, and acidified mercuric salts. 190–193 For the acetyl-protected 127 and 129, deprotection was attempted under a variety of standard conditions. Trans-esterification with methanol, catalysed by either hydrochloric acid or sodium methoxide, gave rise to Michael-type addition to the 155  4.4. Attempted synthesis of proposed epoxide intermediate OR O  RO  OH OR O  RO RO  127 128  HfCl4/ZnI2  +  ACN/H2O, Δ  125 126  OR O  RO  R = Ac R = Bn  OBn: H2, Pd/C or AlCl3  R = Ac R = Bn  H  OH  129 130  OH  OAc: NaOH/H2O/acetone or NH3/MeOH or NaOMe/MeOH or HCl/MeOH  O  HO H  OH  131 COOH  R = Ac R = Bn  O Neu5Ac aldolase  O HO  COOH mCPBA or  OH HO OH  O  O  OH COOH  HO  O  OH  OH O  133  132  Scheme 4.9: Attempted synthesis of the epoxide intermediate expected from Kdn2en (40) under mechanism C of Scheme 3.13 OH O  HO HO  H2O  134  HO  HfCl4/ZnI2  O  HO  135  OH  + HO HO  O OH  136  Scheme 4.10: Hafnium tetrachloride and zinc iodide-catalysed rearrangement of dglucal in water. α,β-unsaturated moiety. Saponification of the ester with sodium hydroxide showed signs of forming the desired product, but on quenching of the reaction and evaporation of solvent (either by rotary evaporator or lyophilisation) a black insoluble compound was seen to form, presumed to be a result of self-polymerisation of the poly-ol with the α,β-unsaturated moiety, again by Michael-type addition. Finally, methanol saturated with ammonia gave a new product with mass consistent with further elimination of acetate, with no sign of the desired deprotection product 131. Deprotection of the benzyl-protected 128 and 130 was slightly more success-  156  4.4. Attempted synthesis of proposed epoxide intermediate ful. Lewis acid-catalysed (AlCl3 ) benzyl ether hydrolysis and palladium on carboncatalysed hydrogenation were attempted, the former giving no reaction but the latter showing a mixture of several products, with indications of the desired compound being seen in a mass spectrum of the crude product mixture. However, this reaction proved not to be repeatable, and analysis of crude product mixtures was complicated by the formation of a hydrate from the aldehyde when dissolving the product in water, removing its characteristic peak from 1 H-NMR spectra at around 9–10 ppm. TLC analysis of a small-scale reaction of Neu5Ac aldolase and pyruvate over a period of days showed complete consumption of most starting material spots. Further, a small peak of appropriate mass was detected by MS (along with several other peaks), suggesting that these unsaturated and/or 2,3-dideoxy hexoses can indeed be accommodated in the Neu5Ac aldolase active site, but synthesis of a pure starting material for this reaction proved to be problematic. Two possible alternate synthetic routes to 133 are presented in Scheme 4.11. Following selective protection of Kdn, 194 the first route (A in Scheme 4.11) takes advantage of an activated leaving group equatorially at the anomeric position to drive a similar epoxidation to that proposed in mechanism C of Scheme 3.13, with catalysis by a suitable base such as DBU. If this attempted epoxidation reaction is found to give elimination, despite the poor overlap of the leaving group σ* with adjacent proton σ orbitals, an alternate route (B in Scheme 4.11) may also be possible by reducing the anomeric ketone 195 then selectively protecting the resultant alcohol to allow chemistry at the endocyclic oxygen. This oxygen could subsequently be activated as a nucleofuge for direct nucleophilic epoxidation with the adjacent hydroxyl on carbon 5. However, this is clearly a more involved route compared to that in Scheme 4.9 on the previous page, and elimination rather than epoxidation remains a risk.  157  4.5. Conclusions and future directions  OH HO HO HO HO  O  OBn  BaO, Ba(OH)2, BnBr  SPh COOBn  PMB-Br NaH  SPh O  BnO HO BnO BnO  COOBn  OBn  BnO  SPh  PMBO BnO BnO  O  COOBn  NBS (A) OBn  BnO  HO BnO BnO  COOBn O  OBn  BnO  DDQ  F  COOBn  PMBO BnO BnO  O  DAST  F  OBn  BnO  OH  PMBO BnO BnO NaBH4  BnO  OBn  OTBDMS OH  PMBO BnOBnO  COOBn  imidazole  COOBn  (B)  OBn  BnO  TBDMSCl  O  OH  PMBO BnOBnO  OH  COOBn  Tf2O  DBU  BnO  OBn  PMBO BnO BnO  DDQ  OTBDMS OTf  OBn  BnO  COOBn  OTBDMS  HO BnO BnO  OTf  COOBn  DBU BnO  BnO  BnO  TBAF  BnO  OH OBn O  OTBDMS OBn O  COOBn BnO  COOBn BnO  DMP  BnO  HO  BnO  HO H2, Pd/C  O OBn O  COOBn BnO  HOOC OH  O  O  O  HO COOH  HO  OH  OH  O  133  HO  Scheme 4.11: Alternate routes for synthesis of epoxide intermediate 133 from Kdn.  158  4.5. Conclusions and future directions  4.5  Conclusions and future directions  Unfortunately, none of the attempted means of testing the mechanisms proposed in Section 3.5 on page 124 provided clear answers. Substitution of D113, the residue proposed to either act as a nucleophile directly or as an acid/base to activate the carbon 2 hydroxyl group to act as a nucleophile resulted in a dramatic decrease in hydrolytic activity at the optimal pH of wild-type UGL, but a proportion of this activity could be recovered in sufficiently acidic solution. This clearly shows a shift in the pKa of the remaining carboxylate and a stronger requirement for acid catalysis. The activity of the D113G mutant could not be rescued by addition of exogenous small molecules such as azide or formate. Further circumstantial evidence against the simple hydration-initiated mechanism, presented in Scheme 1.9 on page 44, was found in the lack of inhibition by proline. This was intended to be a mimic of the transition state charge of this mechanism, with the proline amino group occupying the same position as the substrate endocyclic oxygen, although other explanations are possible for this lack of inhibition. Surprisingly, strong inhibition was also not seen from a compound with a positively charged substituent on carbon 2 (Neu2en, 108), which should be located very close to the negatively charged residue D88/113 (but similar charge placement has previously been seen to provide only poor inhibition of glycosidases). Inhibition was however observed with the substrate mimic shikimic acid, with a higher affinity than expected given that the hydroxyl group stereochemistries did not match that of the substrate. It is postulated that binding of this compound in a slightly different conformation from that adopted by the substrate allows similar interactions of this compound with the adjacent D88/113. The inactivation seen with all predicted mechanism-based inactivators was concluded to be a non-specific effect, and thus not mechanism based, as clearly demon159  4.5. Conclusions and future directions strated by the control in which UGL is inactivated by 20 mM equatorial ΔGlcA fluoride substrate (70). What the nature of this non-specific effect is remains unknown. Undesired variation in pH by the substrate/inactivator carboxylate group was ruled out by quenching each compound stock to pH 6.5 using sodium hydroxide, and by monitoring the pH of the inactivation reactions. In all cases where inactivation was observed it occurred at or above 20 mM, with no clear concentration dependence above this. This suggests that the same non-specific effect is responsible for the inactivation by all of the compounds tested, and not a contaminant, as it is unlikely that the same contaminant is present at the same concentration in all of these independent samples. Below this threshold no inactivation was observed for any compound, despite all of these compounds being seen to bind as competitive inhibitors. Thus, no evidence was found for any mechanism involving direct nucleophilic catalysis. Finally, an attempt at synthesis of a stable epoxide intermediate, as required by mechanism C of Scheme 3.13 on page 126, was unsuccessful, as deprotection of the key α,β-unsaturated hexose enal was found to be problematic. While a preliminary test reaction of Neu5Ac aldolase with this α,β-unsaturated hexose enal in impure form suggests that this synthetic route may still be feasible, an alternative method of synthesising the α,β-unsaturated hexose enal is required if mechanism C is to be tested directly. Discussion of these results, together with those of other chapters, will be taken up in Chapter 6.  160  Chapter 5  Heparanase substrate and inactivator testing 5.1  Chapter introduction  Mammalian degradation of heparan sulfate structures is catalysed by the enzyme heparanase (HPSE) from family GH71. 196 This enzyme is expressed as a single chain, pre-pro-heparanase, which is then truncated N-terminally to remove a signalling peptide, glycosylated, and finally cleaved in two locations to give the mature heterodimer (8 and 50 kDa subunits, excising a 6 kDa fragment). Proheparanase is typically stored in an intracellular pool in lysosomes until required, at which point it is mobilised to either the cell surface or secreted. 197 HPSE is a retaining endo-βglucuronidase that cleaves only at specific sites within heparin and heparan sulfate, typically producing products of around 5–10 kDa (10–20 sugars). By homology modelling, the catalytic residues of HPSE have been proposed to be Glu225 and Glu343 for the acid/base and nucleophile, respectively, 196 but experimental evidence of these assignments is still lacking. Consistent with its internal cleavage mode, HPSE has an extended active site with recognition subsites for several sugar units. 198 The substrate specificity of HPSE has been the subject of much recent study. While earlier work was based on selective digestion of heparan sulfate with other enzymes to form defined sets of oligosaccharides for testing, 199,200 and thus neces161  5.1. Chapter introduction sarily used heterogeneous substrates, more recent work has benefitted from advances in chemoenzymatic synthesis of very specific oligosaccharides as substrates for testing. 201,202 Further insights have been provided by docking of potential substrate structures into the active site of homology models of HPSE. 198,203 Together, these works show that HPSE has a complicated substrate specificity, and it is difficult to arrive at a single model to explain this. While all works agree that a sulfated trisaccharide domain within a larger oligosaccharide is important (predominantly found in the mixed N/S regions of heparan sulfate, as outlined in Figure 1.5 on page 15), the exact effects of specific sulfations remain difficult to predict, and results are occasionally contradictory. Sulfation of the hydroxyl group on carbon 2 of a glucuronic acid residue (but not iduronic acid) in either the -3 or +2 subsites is beneficial for cleavage, 199 although not essential, 200 while sulfation of the hydroxyl group on carbon 6 of glucosamine in the +1 subsite is also beneficial for cleavage. Sulfation of the hydroxyl group on carbon 3 of the glucosamine in the +1 subsite appears to be beneficial in the absence of other secondary sulfations 201 (those coloured blue in Figure 5.1), but it is inhibitory in their presence. 200 Recalcitrant substrates, such as those with an N -acetyl group on the glucosamine in the -2 subsite, can be activated by multiple adjacent sulfates, especially of the hydroxyl group on carbon 6 of the N -acetyl-glucosamine in the +1 and -2 subsites. 200,202 A simple recognition sequence for heparanase cleavage based on these works is presented in Figure 5.1. Whether or not a substrate is cleaved by HPSE appears to be determined by the way in which the substrate binds to the active site, with appropriate substrates binding with the scissile bond close to the two catalytic residues and inappropriate substrates binding in such a way that these residues are slightly further away, by as little as 1–2 Å. 203 In its normal function, HPSE has roles in growth and remodelling, especially in foetal tissue and sites of injury. However, it is also highly expressed and active in  162  5.1. Chapter introduction -OOC  O HO  OSO3O  O O OSO3- HO  -3 -2  -  O3SHN  -  OOC  O HO  O OH -O3SO -1  OSO3O  O  -O SHN 3  +1  -OOC  O HO  O O OSO3-  +2  Figure 5.1: Sulfation in a substrate oligosaccharide as required for cleavage by HPSE. Sulfates in boxes are vital for substrate recognition, sulfates underlined are beneficial for substrate recognition, although not all are necessary together, while the sulfate coloured in red can be beneficial or inhibitory, depending on other sulfates present. The scissile bond is indicated by an arrow. Subsites relative to this cleavage site are numbered. many cancers, where it is seen as an important factor in determining malignancy. This arises because the action of heparanase allows the tissue surrounding a growing tumour to expand, and also because some oligosaccharide products of HPSE are angiogenesis stimulants. 204 HPSE also has a direct role in signal transduction, mediated by a C-terminal domain separate from its catalytic site, increasing gene transcription associated with tumour progression. 205 There is only one known mammalian heparanase enzyme, and activity is typically low in healthy adult tissue, so inhibition of its activity presents an appealing target for cancer treatment. One of the largest current problems for HPSE research is the development of a simple and sensitive assay system for the activity of this enzyme on a short timescale (minutes) to allow testing of inhibitors and inactivators. While much progress has been made in this area in recent years, an assay comparable to the use of aryl glycosides for other glycosidases has yet to be achieved. Presented here is work towards designing and testing of a short oligosaccharide substrate with a fluorescent aglycone, and also a potential active-site trapping reagent intended to allow unam163  5.2. Synthesis of compounds biguous identification of the catalytic nucleophile residue, the development of the former being intended to facilitate testing of the latter as well as further mechanistic work on this enzyme.  5.2  Synthesis of compounds  Given the size and complexity of heparan sulfate oligosaccharides, much previous synthesis of substrates, inhibitors, or inactivators has involved very laborious chemical synthetic routes (see, for example, Chen et al. 206 and Hu et al. 207 ). In this work, substrates and an inactivator for HPSE were synthesised by a collaborative chemo-enzymatic scheme, as represented in Scheme 5.1, 208 allowing much easier access to these compounds. The starting pseudo-disaccharides methylumbelliferyl βd-glucopyranosiduronic acid (137, commercially available), trifluoromethylumbelliferyl β-d-glucopyranosiduronic acid (138, synthesised from 3 by Koenigs-Knorr glycosylation to 140 and deprotected by HCl in methanol then lithium hydroxide) and 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-d-glucopyranosiduronic acid (139, deprotected from 76 by HCl in methanol then lithium hydroxide) were sent to a collaborator for extension to appropriately sulfated trisaccharides, as outlined below. Briefly, the pseudodisaccharide is first extended by addition of a glucosamine residue using the N -acetylglucosamine transferase KfiA from E. coli, with UDPGlcNTFA as glycosyl donor. If the desired product requires an N -acetyl group then UDP-GlcNAc is used instead. The oligosaccharide chain is subsequently elongated by addition of a glucuronic acid residue using the glucuronic acid transferase heparosan synthase 2 from Pasteurella multocida, with UDP-GlcA as glycosyl donor. These two steps can be repeated to give longer chains. The completed oligosaccharide chain can then undergo a series of modifications, depending on the desired final sulfation pattern. Trifluoroacetamide-protected glucosamine can be deprotected by 164  5.2. Synthesis of compounds HOOC HO HO  O OH  OR  UDP-GlcNTFA, α-(1,4)-GlcNAc MnCl2, pH 7.2 transferase (E. coli KfiA) OH HOOC HO HO  HO HO  O OH  O  O  O  -OOC  CF3  HO HO  O OH  O  -OOC TFAHN (AcHN)O HO  O  OH  O HO  O  -OOC TFAHN (AcHN)O HO  O OH  OR  MeOH/H2O/TEA 2:2:1, 37 °C, 16 hr  NO2  OOC HO HO  OH  O OH  O  O HO  -OOC H2 N O (AcHN) HO  O F  OR  O  -  HOOC HO HO  OH  OH  O  138  O2N  O  UDP-GlcA, β-(1,4)-GlcA MnCl2, pH 7.2 transferase (Pasteurella multocida heparosan synthase 2)  137  HOOC HO HO  O  O  O  OH PAPS N-sulfotransferase  139 -OOC  HO HO  OH  O OH  O HO  O -  OOC O3SHN O (AcHN) HO  O  OH PAPS 6-O-sulfotransferase OOC HO HO  O HO  OR  OSO3O  O OH  OR  -  -OOC O3SHN (AcHN)O HO  O OH  OR  Scheme 5.1: Starting glucuronic acid pseudo-disaccharides (left) and general scheme for chemoenzymatic synthesis of appropriately sulfated substrates and inactivators for HPSE (right). Positions of sulfation are indicated in red, and N-acetylated glucosamine nitrogen derivatives are indicated in parentheses. "R" must be an aryl group or N -acetyl-glucosamine analogue, to occupy the GlcNAc transferase -2 subsite, for efficient transfer in the first step. 165  5.3. Substrate testing triethylamine in methanol and water at 37 °C for 16 hours, followed by treatment with an N -sulfotransferase using 3 -phosphoadenosine 5 -phosphosulfate (PAPS) as a source of activated sulfate, to give N -sulfated glucosamine. Further sulfation, of the oxygen on carbon 6 of glucosamine, is possible with a 6-O-sulfotransferase, again using PAPS as a source of activated sulfate. Finally, epimerisation of glucuronic acid to iduronic acid and subsequent sulfation of the oxygen on carbon 2 of iduronic acid is possible using a C5-epimerase and 2-O-sulfotransferase, but was not carried out for the compounds presented in this work. Overall, this pathway largely mimics the natural synthesis of heparan sulfate (see Subsection 1.2.4), but additional control is available by variation of the glucosamine donor nitrogen derivative and omission of specific steps where desired. To obtain disaccharide substrates, a trisaccharide substrate was initially synthesised, with the additional sugar being required for the activity of the sulfotransferase enzymes, then the terminal glucuronic acid residue was removed by treatment with bovine β-glucuronidase. Unfortunately, a large amount of enzyme and long treatment was required to obtain satisfactory cleavage of this recalcitrant substrate. Heparanase enzyme as a single constitutively active chain (fusing the 50 and 8 kDa subunits), expressed by insect cells using a baculovirus vector, was sourced from the same collaborators. 202  5.3 5.3.1  Substrate testing Confirmation of heparanase activity  In order to provide a positive control for the activity of HPSE following shipment as frozen aliquots on dry ice, the assay system of Hammond et al. was used. 209 This employs a synthetic pentasaccharide heparan sulfate fragment capped as its methyl anomer (Fondaparinux, 142, traded as Arixtra by GlaxoSmithKline, U.K.,  166  5.3. Substrate testing O2N -  N  O3S  N SO3-  N+ N  HO HO  I  141  O  OSO3O  OSO3O -O  3SHN  -OOC  O HO  O OH -O3SO  -O  3SHN  O HO  -  142  O OOC  O OSO3- HO  OSO3O -O  3SHN  OMe  Scheme 5.2: Compounds for the HPSE reducing sugar assay, WST-1 (141) and Arixtra (142). for use as an anticoagulant) as substrate in a reducing sugar assay, as detected by the dye WST-1 (water soluble tetrazole, 141) shown in Scheme 5.2. While this pentasaccharide can be cleaved by heparanase, it is not an ideal substrate as it is shorter than the optimal case (an octasaccharide), and contains a 2-sulfated iduronic acid in the +2 subsite, as opposed to the optimal 2-sulfated glucuronic acid, and 3sulfation on the N -sulfo-glucosamine in the +1 subsite, which is inhibitory in the presence of other sulfates (see Subsection 5.1). Overnight incubation of up to 500 µM Arixtra with HPSE resulted in complete hydrolysis. A time course for cleavage of 100 µM substrate over a shorter time frame showed a slow linear rate, from which a kcat of 0.31 s-1 can be derived, which is lower than the value of 3.5 s-1 reported by Hammond et al. 209 These results clearly demonstrate that the heparanase enzyme received had a detectable but relatively low level of activity. This discrepancy may, at least in part, arise from the differences in the enzyme preparation, as the enzyme 167  5.3. Substrate testing used by Hammond et al. is a native heterodimer rather than a single constitutively active chain as used here.  5.3.2  Towards a fluorescent substrate  Based on the publication by Pearson et al. 210 showing activity of HPSE on the fluorescent and chromogenic substrates GlcNS-α-1,4-GlcA-β-DNP (17 nmol.hr-1 .mg-1 ) or -MU (48 nmol.hr-1 .mg-1 ), the first compounds to be designed and tested for improved HPSE substrates were the trisaccharides GlcA-β-1,4-GlcNS6S-α-1,4-GlcA-β-TFMU (143) and GlcA-β-1,4-GlcNAc6S-α-1,4-GlcA-β-TFMU (144), as shown in Table 5.1. The additional glucuronic acid subunit, sulfation of the hydroxyl on carbon 6 of the glucosamine, and a more sensitive fluorophore were together anticipated to give a substrate that allows rapid quantification of HPSE activity. Unfortunately, very little hydrolysis of these compounds was detected when 100 µM substrate was digested overnight by HPSE. Indeed, quantification of this overnight digestion was only possible by virtue of the extremely sensitive fluorophore. Activity was at an insignificant level when compared to Arixtra, with 5 orders of magnitude less substrate cleaved, and also substantially less than that reported by Pearson et al. for their disaccharides, although this may partially reflect the low level of activity seen for this source of HPSE. Cleavage of these trisaccharides to disaccharides, by use of bovine liver β-glucuronidase, gave GlcNS6S-α-1,4-GlcA-β-TFMU (145) and GlcNAc6S-α1,4-GlcA-β-TFMU (146). These shorter substrates were, surprisingly, found to be slightly better substrates for HPSE, particularly 146, but again still hydrolysed at much lower rates than required for a convenient assay. As Pearson et al. also reported an effect from the nature of the aglycone in their disaccharide substrates, further trisaccharides were designed that employed a methylumbelliferyl aglycone, as this was the best leaving group reported in that  168  5.3. Substrate testing  Table 5.1: Rates of hydrolysis for the commercial pentasaccharide Arixtra and heparin di- and tri-saccharide substrates with fluorescent leaving groups. Key features of substrates are emphasised in red and the intended scissile bond is indicated by an arrow. Reactions were performed in acetate buffer at pH 5.5 with 100 µM substrate. Rate (nmol.hr-1 .mg-1 HPSE)  Substrate HO HO  OSO3O -O SHN 3  -OOC O HO  OSO3O  O O  OH -O3SO -O SHN 3  O HO  -OOC  -  OOC HO HO  -OOC  HO HO  O HO  OSO3 O -  O HO  HO HO  OOC  HO HO  -  OOC  HO HO  -O SHN 3  -O SHN 3  (142)  1.3 × 104  O  (143)  0.35  O  (144)  0.28  O  (145)  0.58  OMe  O OH  O  AcHN  O  CF3 -OOC O HO  O OH  O  O  CF3 -OOC  O HO  O OH  O  OSO3O  O  CF3 -OOC  O HO  O OH  O  O  O  (146)  3.3  O  O  O  (147)  5.9  O  O  O  (148)  2.3  -  OSO3 O  O HO  -O SHN 3  -OOC O HO  O  -OOC O HO  O  OH  OH  O OH  O HO  OSO3O  O OH  O3SHN  AcHN  HO HO  -  OSO3O  CF3 -OOC  OSO3O  O OH  O OSO3- HO  -  O OH  O  O HO  O -  O3SHN  OH  169  5.3. Substrate testing work. The trisaccharides GlcA-β-1,4-GlcNS6S-α-1,4-GlcA-β-MU (147) and GlcA-β1,4-GlcNS-α-1,4-GlcA-β-MU (148) showed slightly improved activity, but still vastly less than than seen with Arixtra, itself a sub-optimal substrate. While the hydrolysis of these MU trisaccharides is sufficient for detection of activity following overnight incubation, again by virtue of the sensitive fluorophore released, the level of signal is still nowhere near sufficient for an assay on a timescale of minutes as desired. Repeating the assays for 147 and 148 under identical conditions to those reported by Pearson et al. (5 mM substrate) gave only marginal increases over the rates reported above for 100 µM substrate concentrations (6.4 and 3.5 nmol.hr-1 .mg-1 , respectively). Several experiments were performed in order to test whether the enzyme is cleaving at a site other than that between the fluorophore and the oligosaccharide. Overnight HPSE digestion of GlcA-β-1,4-GlcNS6S-α-1,4-GlcA-β-TFMU (143), the anticipated best substrate, at 100 µM was assayed with WST-1 (141), but no signs of free reducing sugars were detected above a background control. To test for modification of the aglycone, particularly of the ester in TFMU, the reaction of 144 with HPSE was monitored by  19 F-NMR.  No change was detected in the spectrum  after overnight incubation, as shown in Figure 5.2. Finally, in order to test if the substrates are able to bind to HPSE, 143 and 144 were tested as inhibitors of HPSE hydrolysis of Arixtra. Figure 5.3 shows the measurement of initial hydrolysis rates for a single Arixtra concentration and a range of TFMU trisaccharide concentrations. Inhibition was calculated from these to be at 120 and 690 µM, respectively. An assumption of competitive inhibition was made, as would be expected to arise from binding of a substrate to the enzyme active site, but insufficient compound was available to test this assumption. These Ki values are similar to the Km of the Arixtra pentasaccharide at 46 µM, and indicates that the lack of activity with these  170  5.3. Substrate testing substrates is not a result of inability to bind at the active site.  Figure 5.2: Overnight reaction of 144 with HPSE, monitored by  19 F-NMR  Together these results clearly demonstrate that, in order to obtain a fluorescent or chromogenic substrate with sufficiently high activity to allow convenient assaying of HPSE on a minute timescale, much further optimisation of the substrate is required. Given the requirement for sulfation of the glucosamine in the +1 subsite for appropriate positioning of a substrate in the HPSE active site (as discussed in Subsection 5.1), the nature of the leaving groups used here appears to be inappropriate. Future efforts likely need to focus on design of an activated leaving group with appropriately placed charges, while sulfation of the hydroxyl on carbon 2 of the GlcA in the -3 subsite may also help activity. Although two charged aglycones were used by Pearson et al., the placement of these charges did not appear to be matched to that in an ideal HPSE substrate, and no hydrolysis of these compounds by HPSE was detected. One potential substrate incorporating these changes is illustrated in Scheme 5.3.  171  5.3. Substrate testing  Figure 5.3: Inhibition of HPSE by TFMU substrate trisaccharides ( : 143 and : 144) showing Ki values of 120 and 690 µM, respectively, determined from the intercept with 1/Vmax (dashed line). Substrate (142) was at 100 µM.  172  5.4. Testing of a potential HPSE inactivator HO  COOMe +  MeOOC AcO AcO  COOMe O  Br HO  MeOOC AcO AcO  O  O OAc  O  O O COOMe  COOMe -  OOC  HO HO  O O OSO3- HO  -  O3SHN  -  OOC O HO  OH  COO-  O O  COO-  O  O  O  O  HO HO  OSO3O  -  OOC  COOMe  COOMe  + HO  AcO Br  HO  Cl  O  COO-  O  OH  O COO-  -O  O  O RO  O-  O-  O O  O  RO =  O O  OO S O O O  RO HO  O -  O  -  O  S  NH O O  OO  O  Scheme 5.3: A potential synthesis for a HPSE substrate with a charged aglycone (upper), and illustration of two possible charge placements by this aglycone to mimic the sulfation of optimal HPSE natural substrates (lower).  5.4  Testing of a potential HPSE inactivator  A potential 2-deoxy-2-fluoro-glucuronide inactivator of HPSE was also designed, synthesised and tested. Although the hydrolysis rates of the di- and tri-saccharide substrates in the previous section were disappointingly low, an inactivator needs only to catalyse half a turnover, the glycosylation half-reaction, to give a trapped intermediate. The potential inactivator synthesised was a trisaccharide, GlcA-β-1,4GlcNAc6S-α-1,4-2FGlcA-β-DNP (149, Scheme 5.4), with N -acetyl-glucosamine being used in the -2 subsite despite it giving poorer hydrolysis as the conditions for deprotection of N -trifluoroacetyl-glucosamine were found to cleave off the dinitrophenyl  173  5.4. Testing of a potential HPSE inactivator leaving group. Time-dependent inactivation of HPSE by 149 was monitored by reaction of an aliquot from the inactivation reaction with Arixtra at 250 µM for 30 min at each timepoint, with the results shown in Figure 5.4. The initial loss and subsequent recovery of HPSE activity seen with inactivator present over the first 4 hours was deemed to be an artefact, as the activity in the control varies by a similar magnitude. The inactivator was also present in a large excess over enzyme, so reactivation is not expected without substantial amounts of dinitrophenol being released, which would give an associated observable colour change. Insufficient Arixtra remained to repeat this experiment to control for this noise. While the assay was much more noisy than desired, these results are still able to show that no inactivation of HPSE by 149 is taking place on this timeframe. If, for the sake of comparison, the rate of inactivation with this compound were assumed to be the same as that of the related substrate GlcA-β-1,4-GlcNAc6S-α-1,4-GlcA-β-TFMU (144), inactivation would be expected to have a half life of around 40 hours. Given that 2-deoxy-2-fluoro compounds react more slowly than their parent substrates, it is not surprising that no inactivation was detected over 20 hours. Any future design of such inactivators for HPSE will likely be dependent on the successful design of small molecule substrates, particularly the effect of charge in the leaving group. -OOC  HO HO  O OH  O HO  OSO3O AcHN  149  -  OOC  O HO  O2N  NO2  O F  O  Scheme 5.4: Structure of the potential 2-deoxy-2-fluoro inactivator of HPSE.  174  5.4. Testing of a potential HPSE inactivator  Figure 5.4: Attempted time-dependent inactivation of HPSE by 149 at 0( ) and 1( ) mM.  175  5.5. Conclusions  5.5  Conclusions  Presented here was an attempt at design and synthesis of small molecule substrates and an inactivator for HPSE. While literature precedent from Pearson et al. 210 had suggested that substantial acivity from such compounds may be possible, the relevance of the level of activity detected in that work may have been overestimated by selection of a less appropriate native substrate for comparison, and by comparison to rates reported in other work rather than a control carried out under the same assay conditions. The Arixtra pentasaccharide substrate 209 was used here to confirm activity of the HPSE enzyme, and as a native-like substrate to provide a baseline comparison for the activity seen with other substrates. This clearly indicated that the activity of HPSE with the six potential fluorogenic small molecule substrates tested here, which were anticipated to be better substrates than those of Pearson et al., was negligible. Similarly, no inactivation of HPSE was detected from a 2deoxy-2-fluoro reagent based on these. Progress in this area is likely dependent on the design of a chromophore that can make suitable binding interactions with the +1 subsite of HPSE, and one potential candidate based on the placement of sulfates in optimal substrates was suggested here.  176  Chapter 6  Overall conclusions Overall conclusions In this thesis, three chapters were presented on the mechanism of unsaturated glucuronyl hydrolases. The first chapter presented evidence for a hydration reaction that had previously been proposed on the basis of crystallographic evidence. 87 Careful characterisation of the products of reaction in D2 O and 10 % methanol led to the conclusion that this hydration is indeed the reaction catalysed by UGL, with supporting evidence from reaction of UGL with three compounds that are only expected to be turned over by the enzyme if reaction occurs through such a hydration mechanism. The subsequent chapter presented a series of experiments designed to probe the steps of this reaction, and the transition states of these steps. Evidence for rearrangement of the hydrated product outside of the enzyme active site was sought, but not found, leading to the tentative conclusion that the enzyme actively catalyses this step. The effect of activated leaving groups and heteroatoms on the rate of UGL hydrolysis supported the hypothesis that the transition state of the rate-determining step involved development of positive charge at carbon 5, with the unexpected observation that a 2-deoxy-2-fluoro substrate was almost completely inert to hydrolysis by UGL. Subsequent measurement of kinetic isotope effects showed that two kinetically important steps must be present in the mechanism, from observation of distinct  177  Overall conclusions effects on kcat and on kcat /Km . The effects on kcat /Km are consistent with an initial irreversible protonation step, while those on kcat clearly show that the intermediate formed is not an oxocarbenium ion. Clear solvent kinetic isotope effects were observed on both parameters, showing a likely importance of solvent-derived proton transfer in each step. On the basis of these results, three alternate mechanisms were proposed for UGL. The third chapter presented work towards testing each of these hypotheses, but clear, incontrovertible evidence was not obtained for any one. Mutagenesis of D113, the aspartate residue without a clear role in the simple hydration mechanism, to glycine followed by subsequent unsuccessful attempts at rescue with exogenous nucleophiles confirmed the importance of this residue for turnover, but provided no clues as to its role. Inhibitors intended to mimic the charge distribution of transition states in some of the candidate mechanisms were tested, but none were found to bind strongly to the enzyme. Neu2en, a further compound modified at carbon 2 (numbering based on ΔGlcA), was also found not to be turned over by UGL. To test the mechanisms that involved nucleophilic catalysis, two potential trapping reagents were tested but neither inactivated UGL in a concentration-dependent manner, and indeed nor were they turned over by the enzyme. A further potential trapping reagent, intended to test the hypothesis that the hydrated product rearranges in the active site, also did not inactivate the enzyme, and neither was it converted, thereby providing no clear answers. Finally, synthesis of a small molecule intermediate proposed for mechanism C was attempted, but unsuccessful. Despite the lack of conclusive evidence, mechanism C from Scheme 3.13 on page 126 best accounts for all of the experimental observations presented in this work. Scheme 6.1 shows potential transition states that illustrate this. In the first irreversible step, bond formation is advanced between carbon 4 and its second proton  178  Overall conclusions D173  O O HO O  D113  H  D173  COOH O OR O  O-  H  O- H OH HOOC O O HO O O OH  D173  OR  D173  OO  δ+  D113  D O  O δD113  D173 O OR  H  Oδ+ H  HO HO O  D113 HOOC OH  OH O  OO  O  OH COOH O- 22  OR  H OH HOOC OH δ+  O  H  D  D O  OR  Oδ+  D  H  OD113  Scheme 6.1: Conformation of transition states in mechanism C of Scheme 3.13 to account for experimental observations. Dashed lines represent partially formed or broken bonds at the transition state. Deuteriums for which kinetic isotope effects were measured are both shown in the one structure for simplicity, but each substitution was made independently. and between carbon 1 and the carbon 2 hydroxyl oxygen. Bond breaking is largely complete in the double bond between carbons 4 and 5, between the hydroxyl on carbon 2 and its proton, and between carbon 1 and the endocyclic oxygen, but formation of the double bond between this same oxygen and carbon 5 is less developed. This lag in the formation of the ketone double bond gives the substantial positive charge at carbon 5 and large change in hybridisation at carbon 4 required to account for the small normal overall KIE on kcat /Km from deuterium at carbon 4, and may arise from the requirement for a change in hybridisation at the endocyclic oxygen atom to give appropriate orbital overlap with the adjacent carbon. The low bond order between carbon 1 and the endocyclic oxygen isolates the deuterium at carbon 1 from this charge, explaining its small kinetic isotope effect on this first step, and also the lack of detectable effect from electron withdrawing groups in the LFER. In the second step, this situation is largely reversed. Bond formation is early between the epoxide oxygen and its proton, which provides a driving force for attack  179  Overall conclusions of the endocyclic oxygen (ketone) at carbon one. The double bond from the endocyclic oxygen to carbon 5 is also broken early in the reaction coordinate, while attack of the water nucleophile at carbon 5 occurs later in the reaction coordinate. This again gives a substantial positive charge at carbon 5 and, with no accompanying change in hybridisation at carbon 4 in this step, leads to a large normal KIE on kcat from deuterium at carbon 4. The higher bond order between the endocyclic oxygen and carbon 1 in this transition state means that the anomeric substituent is more able to influence its stability through electron withdrawing effects on the positive charge. Attack of the endocyclic oxygen (ketone) at carbon one proceeds through an SN 2-like mechanism, and the associative nature of this transition state at carbon 1 leads to steric crowding and limitation of the out-of-plane bending modes of the hydrogen at this position. This leads to an inverse KIE, with the lower energy vibration of the carbon-deuterium bond in this mode resulting in less destabilisation from crowding at the transition state than a carbon-hydrogen bond. Kinetic isotope effects on acidcatalysed epoxide opening 211–214 are similar to the effect from deuterium at carbon 1 on kcat , lending further credibility to this interpretation. Protonation of the epoxide likely provides the main driving force for this step, as the ketone at carbon 5 is otherwise a poor nucleophile, and this proton transfer accounts for the SKIE on kcat . A hypothetical energy profile for reaction by mechanism C compared to the non-enzymatic acid-catalysed case is given in Figure 6.1. It is worth noting that some precedent exists for this mechanism in non-enzymatic reactions of glycosides, as shown in Scheme 6.2. 215 In non-enzymatic acid-catalysed hydrolysis of glycosides an initial protonation can occur on either the anomeric oxygen, giving a cyclic oxocarbenium ion intermediate stabilised by the endocyclic oxygen, or on the endocyclic oxygen, giving a linear oxocarbenium ion stabilised by the anomeric oxygen. Ring-opening mechanisms for glycoside hydrolysis have previously  180  I+ COOH O OR OH  HO  E.I  HO  HOOC  E+S COOH HO  O OR OH  E.S  O  O  OH COOH  OR  E.J  O HO  Overall conclusions  Energy  HO  O  OR  OH  HOOC  HOOC  J'  HO OH  E+P  O OH  OR  O OH  E.P  181  Reaction coordinate Figure 6.1: Hypothetical energy profile for UGL-catalysed ΔGlcA hydrolysis (in black), compared to the non-enzymatic acid-catalysed reaction (in red).  Overall conclusions  O  HO  SMe  HO  OH +  O HO  SMe  OH  H+  OH  HO  S+Me OH  OH  O  -H+  SMe  HO  OH  OH  OH + O  HO  SMe  HO  OH  OH O  OR  OH  OH  -ROH  OH O  -  OH  O  MeOH  OMe  O HO  HO  HO  OH  OH  O O O  OH  O H+  O OMe OBn  OH  O  O H2O  O  HO  OMe OBn  HO  O O  HO  OMe OBn  Scheme 6.2: Precedent for mechanism C of Scheme 3.13 from non-enzymatic reactions of glycosides.  182  Overall conclusions been suggested, and distinguishing between these two mechanisms was the topic of much work. Indeed some rearrangements of thioglycosides in aqueous acid do appear to proceed through such linear intermediates. Furthermore, an epoxide intermediate has been proposed to accelerate non-enzymatic decomposition under basic conditions of activated glycosides with a 1,2-trans arrangement, while non-enzymatic opening of an epoxide at carbons 3 and 4 of a hexose is accelerated by participation of an ester substituent at carbon 6. Further evidence for this mechanism comes from the importance of the hydroxyl group at carbon 2, shown by the dramatically low hydrolysis rate for the 2-deoxy-2fluoro glucuronide (78, see Subsection 3.3.2) and the lack of any hydrolysis detected for Neu2en (108, see Subsection 4.2). These compounds are unable to form the epoxide required to stabilise the positive charge in the transition state, and so their hydrolysis proceed at a much lower rate than for their oxygen-bearing analogues DNP ΔGlcA (47) and Kdn2en (40). The very low level of residual activity seen with 78 may arise by an alternate mechanism, as the acid catalyst and water nucleophile are still appropriately placed but substantially less stabilisation is available for the transition state. One likely candidate for this mechanism is direct hydration through an oxocarbenium ion-like transition state. The chondroitin-derived 2 -sulfated natural substrate 16, which is hydrolysed by UGL from Bacillus sp. GL1 but not from any of the other organisms for which data have been reported (see Section 2.4), clearly cannot proceed through the epoxide mechanism. However, the sulfate group on the 2 -hydroxyl may be able to fill the same stabilisation role with no need for acid-base catalysis as a result of the already low pKa of the sulfate group. This explanation could be tested by determining the effect of the D88N mutant in Bacillus sp. GL1 UGL on hydrolysis of this 2 -sulfated substrate, which would be expected to not show the same dramatic decrease in activity seen for other substrates with this mutant.  183  Overall conclusions One potential advantage for the remote charge stabilisation proposed in this mechanism is that the identical charges of the substrate and enzyme carboxylates do not need to be in close proximity, so charge repulsion does not need to be overcome. A similar explanation has been invoked to explain the action of a neutral tyrosine residue as a catalytic nucleophile in sialidases, where a carboxylate is normally found in other glycoside hydrolases (refer to Section 1.3). Finally, work towards simple substrates and an inactivator of mammalian heparanase was presented in the preceding chapter. While this work was not successful in achieving the desired assay sensitivity, it indicated that optimisation of sulfation patterns without modification of the aglycone is likely a futile strategy. A potential aglycone that takes advantage of knowledge of optimal HPSE sulfation patterns was presented, and represents a new path towards the goal of studying this enzyme for its eventual use as a therapeutic target in cancer therapy.  184  Chapter 7  Materials and methods 7.1  Materials  Chemicals were purchased from Sigma-Aldrich unless otherwise stated, and used without further purification. Natural substrates 11, 12, 13, 14, 15, and 16 as well as a small sample of Kdn2en (40) were purchased from Carbosynth, UK (http://www. carbosynth.com/). TLC was performed on pre-coated 60F254 silica plates (Merck, Germany), with visualisation by UV light followed by charring with 10% ammonium molybdate in 2 M H2 SO4 . Flash column chromatography was performed using 230– 400 mesh silica gel and an in-house compressed air system. For anhydrous reactions solvents were freshly distilled (CH2 Cl2 over CaH2 , MeOH over Mg) and glassware dried in an oven. NMR spectra were recorded on Bruker Avance 300 and 400 (with either an inverse or a direct probe) spectrometers at 300 and 400 MHz, respectively. Chemical shifts are reported in δ scale in parts per million from tetramethylsilane (TMS) with internal reference to solvent for 1 H and  13 C  shifts, while  19 F  shifts are  reported relative to an external standard of CFCl3 at 0 ppm. Low resolution mass spectra were recorded on a Waters ZQ equipped with ESCI ion source and Waters 2695 HPLC for sample delivery, while high resolution mass spectra were submitted to the University of British Columbia mass spectrometry facility for analysis on a Waters/Micromass LCT with electrospray ionisation and time of flight detection in either positive or negative mode. HPLC was carried out using an Agilent eclipse  185  7.2. Synthesis XD-C18 column (5 µm pore size, 9.4 x 250 mm) on a Waters 600 at 4 mL.min-1 with a Waters 2996 photodiode array detector. Kinetic measurements were performed in matched reduced-volume quartz cuvettes using a Varian Cary 4000 spectrophotometer with automatic cell changer and Peltier temperature controller, at 37 °C unless otherwise specified, and reaction mixtures were allowed to pre-incubate for 5 min before adding enzyme to start the reaction. All non-linear regression was performed using GraFit 5.0 (Erithacus software limited; www.erithacus.com/grafit).  7.2 7.2.1  Synthesis General methods  General method for Koenigs-Knorr glycosylation. The acceptor alcohol (1.3 eq.) was dried over acetonitrile where necessary, then dissolved along with the relevant glycosyl bromide in dry acetonitrile (to give approximately 0.1 M of sugar). Ag2 O (2-3 eq.) was added, and the reaction mixture stirred vigorously overnight at ambient temperature in the dark. The reaction mixture was filtered through a plug of Celite and concentrated before being dissolved in ethyl acetate and washed extensively with sat. NaHCO3 , water and brine . The organic phase was dried over MgSO4 then the solvent evaporated. General method for DBU-catalysed elimination. Protected aryl glucuronide was dissolved in dichloromethane (to give approximately 0.1 M of sugar), DBU (1.3 eq.) was added slowly through a septum and the reaction mixture stirred at room temperature overnight. Where found to be necessary, 4 Å molecular sieves were used and the solution flushed with argon or nitrogen to dry before addition of reagent. Once finished, the reaction mixture was concentrated and 186  7.2. Synthesis filtered through a plug of silica (washing with 2:1 ethyl acetate/petroleum ether). General method for ester saponification. The protected final compound was dissolved in acetone to approximately 0.1 M, cooled to 0 °C, and an equal volume of NaOH added (1 M). The reaction mixture was stirred 5 min then quenched with a slight excess of HCl (1 M). General method for Zemplén deprotection. The acetyl- or benzoyl-protected compound was dissolved in 1:1 dichloromethane/methanol to approximately 0.1 M, cooled to 0 °C, and either sodium methoxide in methanol (stock at 5.4 M) or a small piece of sodium metal added to give a final concentration of between 5 and 50 mM. Deacetylation was monitored by TLC and, upon completion, the reaction was quenched with Sephadex ion exchange resin (H+ form) then filtered. General method for acidic trans-esterification. The acetyl- or benzoyl-protected compound was dissolved in 1:1 dichloromethane/methanol to approximately 0.1 M, cooled to 0 °C, and acetyl chloride added (approximately 1-5% v/v). Longer reactions were transferred to a cold cabinet at 4 °C. Deacetylation was monitored by TLC and, upon completion, the solvent was evaporated in vacuo for an extended period to remove HCl traces. General method for hydrolysis by aqueous lithium hydroxide. The methyl ester-protected compound was dissolved in tetrahydrofuran and water (1.75:1, to approximately 0.1 M), cooled to 0 °C, and 2 eq. of 1 M lithium hydroxide added. The reaction was allowed to proceed for 5 min before quenching with  187  7.2. Synthesis Sephadex ion exchange resin (H+ form) then filtered.  7.2.2  Development of chromogenic substrates  Methyl (1,2,3,4-tetra-O-acetyl-α/β-d-glucopyranosid)uronate (2) MeOOC AcO AcO  O OAc OAc  Glucuronic acid gamma lactone (9.936 g, 56.4 mmol) was suspended in MeOH (500 mL), a sodium methoxide solution was added (1 mL at 5.4 M) at 0 °C, and the reaction mixture stirred until TLC indicated completion. The methoxide was then quenched with 500 µL acetic acid, and the solvents evaporated in vacuo. The intermediate was subsequently dissolved in Ac2 O (100 mL), cooled to 0 °C, perchloric acid (0.5 mL) added in small portions, and the reaction mixture allowed to warm to ambient temperature with stirring overnight. Upon completion, the reaction was quenched with ice water, extracted with 1 L ethyl acetate and the organic phase then washed extensively with water and saturated sodium bicarbonate, washed once with brine and dried with magnesium sulfate. The solvent was then evaporated to a syrup, which was allowed to stand at 4 °C until a slurry formed, the solid collected and washed with cold methanol, and the process repeated several times (with diminishing returns) to give a white powder (12.32 g, 58 %, α/β ratio 1.73:1). 1H  NMR (300 MHz; CDCl3 ) δ 6.36 (d, J1,2 = 3.6 Hz, 1H, H-1α), 5.74 (d, J1,2 =  7.7 Hz, 1H, H-1β), 5.49 (t, J2,3 = J3,4 = 9.9 Hz, 1H, H-3β), 5.32-5.06 (m, 3H, H-2,3,4), 4.38 (d, J4,5 = 10.2 Hz, 1H, H-5α), 4.16 (d, J4,5 = 9.3 Hz, 1H, H-5β), 3.72 (s, 3H, OMeα) , 3.71 (s, 3H, OMeβ), 2.16 (s, 3H, OAc), 2.09 (s, 3H, OAc), 2.01 (s, 6H, OAc), 2.00 (s, 4H, OAc), 1.98 (s, 3H, OAc); MS : Calcd. for C15 H20 NaO11 : 399.1; found: 399.3.  188  7.2. Synthesis Methyl (2,3,4-tri-O-acetyl-α-d-glucopyranosyl bromide)uronate (3) MeOOC AcO AcO  O AcO Br  Globally protected glucuronic acid (2, 1.18 g, 3.13 mmol) was dissolved in dichloromethane (4.5 mL) then acetic anhydride (0.5 mL) and 33% HBr in acetic acid (20 mL) were added at 0 °C. This was stirred at 4 °C until the reaction was complete as determined by TLC (2:1 petroleum ether/ethyl acetate). The reaction was quenched in ice/water, the aqueous layer extracted thrice with dichloromethane and this pooled organic phase then extracted quickly with cold water, cold sat. NaHCO3 twice and brine. After drying over MgSO4 , concentrating and co-evaporating from toluene the colourless syrup (1.20 g, 3.02 mmol, 97%) was used without further purification. A sample for analysis was purified by flash column chromatography (3:1 petroleum ether/ethyl acetate), yielding a white powder. 1 H-NMR (300 MHz; CDCl3 ): δ 6.67 (d, J1,2 = 4.0 Hz, 1H, H-1), 5.64 (t, J2,3 = J3,4 = 9.8 Hz, 1H, H-3), 5.27 (dd, J4,5 = 10.3 Hz, 1H, H-4), 4.88 (dd, 1H, H-2), 4.61 (d, 1H, H-5), 3.79 (s, 3H, OMe), 2.13 (s, 3H, OAc), 2.09 (s, 3H, OAc), 2.08 (s, 3H, OAc) ppm.  13 C-NMR  (75 MHz; CDCl3 ):  δ 169.63 (OAc), 169.43 (OAc), 166.6 (C-6), 85.5 (C-1), 72.1 (C-3), 70.3 (C-2), 69.3 (C-4), 68.5 (C-5), 53.1 (OMe), 20.60 (2xOAc), 20.45 (OAc) ppm. MS : Calcd. for C13 H17 BrNaO9 : 419.0/421.0; found: 419.2/421.2 Methyl (4-nitrophenyl 2,3,4-tri-O-acetyl-β-d-glucopyranosid)uronate (4) MeOOC AcO AcO  O OAc  O NO2  4-Nitrophenol (1.14 g, 7.98 mmol) was reacted with globally protected glucuronyl bromide (3, 2.51 g, 6.32 mmol) by the general method for Koenigs-Knorr glyc189  7.2. Synthesis osylation (page 186). Purification by flash column chromatography (1:1 petroleum ether/ethyl acetate) yielded a white powder (1.54 g, 3.38 mmol, 54%).  1 H-NMR  (300 MHz; DMSO-d6): δ 8.24 (d, J2 ,3 = J5 ,6 = 9.3 Hz, 2H, H-2 ,6 ), 7.22 (d, 2H, H-3 ,5 ), 5.88 (d, J1,2 = 7.8 Hz, 1H, H-1), 5.48 (t, J2,3 = J3,4 = 9.6 Hz, 1H, H-3), 5.16 (dd, 1H, H-2), 5.09 (br. t, J4,5 = 9.9 Hz, 1H, H-4), 4.76 (d, 1H, H-5), 3.62 (s, 3H, OMe), 2.01 (s, 3H, OAc), 2.01 (s, 3H, OAc), 2.00 (s, 3H, OAc) ppm. MS : Calcd. for C19 H21 NNaO12 : 478.1; found: 478.2 Methyl (4-nitrophenyl 2,3-di-O-acetyl-4-deoxy-α-l-threo-hex-4enopyranosid)uronate (5) COOMe AcO  O O OAc  NO2  Globally protected 4-nitrophenyl β-d-glucuronide (4, 0.521 g, 1.14 mmol) was subjected to the general method for DBU-catalysed elimination (page 186). Purification by flash column chromatography (2:1 petroleum ether/ethyl acetate) gave a white powder (0.416 g, 1.05 mmol, 92%).  1 H-NMR  (300 MHz; CDCl3 ): δ 8.23 (d,  J2 ,3 = J5 ,6 = 9.2 Hz, 2H, H-2 ,6 ), 7.21 (d, 2H, H-3 ,5 ), 6.34 (dd, J3,4 = 4.5, J2,4 = 1.6 Hz, 1H, H-4), 5.96 (dd, J1,2 = 2.5, J1,3 = 0.9 Hz, 1H, H-1), 5.32-5.29 (m, 2H, H-2,3), 3.81 (s, 3H, OMe), 2.16 (s, 3H, OAc), 2.15 (s, 3H, OAc) ppm. MS : Calcd. for C17 H17 NNaO10 : 418.1; found: 418.2 4-Nitrophenyl 4-deoxy-α-l-threo-hex-4-enopyranosiduronic acid (6) COOH HO  O O OH  NO2  Globally protected 4-nitrophenyl unsaturated β-d-glucuronide (5, 60 mg, 0.152 190  7.2. Synthesis mmol) was subjected to the general method for Zemplén deprotection (page 187) followed by the general method for hydrolysis by lithium hydroxide (page 187). The reaction was quenched with Sephadex ion exchange resin (H+ form) then filtered. Purification by flash column chromatography (7:2:1 ethyl acetate/methanol/water) then 5 g C-18 Sep-pak, washed with water, 40% acetonitrile in water and 100% acetonitrile, gave a white powder following lyophilisation (36.1 mg, 0.122 mmol, 80%).  1 H-NMR  (D2 O, 400 MHz): δ 8.09 (d, J2 ,3 = J5 ,6 = 9.2 Hz, 2H, H-2 ,6 ),  7.17 (d, 2H, H-3 ,5 ), 6.22 (d, J3,4 = 4.1 Hz, 1H, H-4), 5.84 (d, J1,2 = 4.8 Hz, 1H, H-1), 4.29 (br. t, J2,3 = 4.4 Hz, 1H, H-3), 4.07 (br. t, 1H, H-2) ppm.  13 C-NMR  (D2 O, 101 MHz): δ 176.8 (C-6), 165.1 (C-5), 161.1 (Ar), 142.7 (Ar), 126.1 (Ar), 116.9 (Ar), 112.5 (C-4), 97.3 (C-1), 69.3 (C-3), 65.7 (C-2) ppm. HRMS : Calcd. for C12 H10 NO8 : 296.0406; found: 296.0411 Methyl (2,3,4-tri-O-acetyl-α-d-glucopyran)uronate (7) MeOOC AcO AcO  O AcO OH  Globally protected glucuronic acid (2, 1.902 g, 5.05 mmol) was dissolved in 1:1 dichloromethane/methanol (90 mL) and hydrazine acetate added (0.719 g, 1.6 eq.), and the reaction mixture then stirred at 0 °C followed by ambient temperature for 2 hours each. The solvent was subsequently evaporated in vacuo, the reaction mixture dissolved in ethyl acetate and the product washed with water, 1 M HCl and brine, then dried over MgSO4 . The product was purified by flash column chromatography (3:2 to 1:1 petroleum ether/ethyl acetate) to give a white foam (1.09 g, 3.26 mmol, 65%). 1 H-NMR (300 MHz; CDCl3 ) δ 5.50 (t, J2,3 = J3,4 = 9.4 Hz, 1H, H-3), 5.46 (d, J1,2 = 3.0 Hz, 1H, H-1), 5.09 (t, J4,5 = 10.1 Hz, 1H, H-4), 4.82 (dd, 1H, H-2), 4.52 (d, 1H, H-5), 3.66 (s, 3H, OMe), 2.01 (s, 3H, OAc), 1.96 (s, 3H, OAc), 1.95 (s, 3H, 191  7.2. Synthesis OAc) ppm.  13 C-NMR  (75 MHz; CDCl3 ) δ 170.2 (OAc), 170.0 (OAc), 169.7 (OAc),  168.5 (C-6), 89.9 (C-1), 70.6 (C-3), 69.4 (C-2), 69.0 (C-4), 67.7 (C-5), 52.7 (OMe), 20.5 (2xOAc), 20.3 (OAc) ppm. MS : Calcd. for C13 H18 NaO10 : 357.1; found: 357.3 Methyl (phenyl 2,3,4-tri-O-acetyl-β-d-glucopyranosid)uronate (8) MeOOC AcO AcO  O OAc  O  Globally protected glucuronyl hemiacetal (7, 1.82 g, 5.45 mmol) was dissolved in dichloromethane (30 mL), 4 Å molecular sieves were added and the solution flushed with argon, then cooled in a dry ice/acetone bath. Trichloroacetonitrile (10 mL, 10 eq.) then DBU (0.5 mL, 0.3 eq.) were added and the reaction mixture stirred for 30 min at -78 °C. The reaction mixture was concentrated and filtered through a plug of silica gel (eluting with 2:1 petroleum ether/ethyl acetate). The resultant crude trichloroacetimidate intermediate and phenol (0.66 g, 1.3 eq.) were dissolved in dichloromethane (30 mL) and dried over 4 Å molecular sieves under argon, then cooled in a dry ice/acetone bath. Boron trifluoride diethyl etherate (0.22 mL, 0.3 eq.) was added and the reaction mixture stirred 30 min at -78 °C before allowing to warm to ambient temperature while stirring overnight. The reaction mixture was diluted with an equal volume of dichloromethane and washed with sat. NaHCO3 thrice then brine. The organic phase was dried over MgSO4 then purified by flash column chromatography (1:1 hexanes/ethyl acetate) to give a white powder (0.68 g, 1.65 mmol, 30%). 1 H-NMR (300 MHz; DMSO-d6): δ 7.33 (t, J2 ,3 = J3 ,4 = J4 ,5 = J5 ,6 = 7.7 Hz, 2H, H-3 ,5 ), 7.06 (t, 1H, H-4 ), 6.99 (d, 2H, H-2 ,6 ), 5.65 (d, J1,2 = 7.9 Hz, 1H, H-1), 5.47 (t, J2,3 = J3,4 = 9.6 Hz, 1H, H-3), 5.12-5.03 (m, 2H, H-2,4), 4.70 (d, J4,5 = 9.9 Hz, 1H, H-5), 3.63 (s, 3H, OMe), 2.01 (s, 3H, OAc), 2.00 (s, 3H, OAc), 1.99 (s, 3H, OAc) ppm. MS : Calcd. for C19 H22 NaO10 : 433.1; found: 433.1 192  7.2. Synthesis Methyl (phenyl 2,3-di-O-acetyl-4-deoxy-α-l-threo-hex-4enopyranosid)uronate (9) COOMe AcO  O O OAc  Globally protected phenyl β-d-glucuronide (8, 678 mg, 1.64 mmol) was subjected to the general method for DBU-catalysed elimination (page 186). Purification by flash column chromatography (3:2 hexanes/ethyl acetate) gave a white powder (295 mg, 0.84 mmol, 51%).  1 H-NMR  (300 MHz; CDCl3 ): δ 7.33-7.27 (m, 2H, H-3 ,5 ),  7.11-7.03 (m, 3H, H-2 ,4 ,6 ), 6.29 (d, J3,4 = 5.9 Hz, 1H, H-4), 5.82 (dd, J1,2 = 2.5, J1,3 = 1.3 Hz, 1H, H-1), 5.31-5.29 (m, 2H, H-2,3), 3.79 (s, 3H, OMe), 2.13 (s, 3H, OAc), 2.10 (s, 3H, OAc) ppm.  13 C-NMR  (75 MHz; CD3 OD): δ 170.4 (OAc), 169.8  (OAc), 162.1 (C-6), 156.4 (C-5), 142.3 (Ar), 129.5 (Ar), 123.2 (Ar), 116.9 (Ar), 107.6 (C-4), 95.2 (C-1), 68.4 (C-3), 65.0 (C-2), 51.9 (OMe), 19.6 (OAc), 19.5 (OAc) ppm. Phenyl 4-deoxy-α-l-threo-hex-4-enopyranosiduronic acid (10) COOH HO  O O OH  Globally protected phenyl unsaturated β-d-glucuronide (9, 84 mg, 0.24 mmol) was deprotected by the general method for ester saponification (page 187). Purification was by 5 g C-18 Sep-pak, washed with water, 10% acetonitrile in water, 40% acetonitrile in water and 100% acetonitrile. All fractions determined by TLC (3:3:1:1 toluene/ethyl acetate/methanol/acetic acid) to contain pure product were pooled and lyophilised, while those containing impure product were pooled, lyophilised and purified again to give a white powder (46 mg, 0.19 mmol, 77%). 1 H-NMR  193  7.2. Synthesis (400 MHz; CD3 OD) δ 7.29 (t, J2 ,3 = J3 ,4 = J4 ,5 = J5 ,6 = 7.9 Hz, 2H, H-3 ,5 ), 7.08 (d, 2H, H-2 ,6 ), 7.05 (t, 1H, H-4 ), 5.81 (d, J3,4 = 3.7 Hz, H-4), 5.61 (d, J1,2 = 5.2 Hz, H-1), 4.18 (br. t, J2,3 = 4.5 Hz, H-3), 3.93 (br. t, H-2) ppm.  13 C-NMR  (101 MHz; CD3 OD) δ 157.3  (C-6), 129.3 (Ar), 122.8 (Ar), 117.2 (Ar), 117.1 (Ar), 112.4 (C-4), 99.5 (C-1), 70.8 (C-3), 67.0 (C-2) ppm (carbon 5 signal too weak to detect, but seen in protected form). HRMS : Calcd. for C12 H11 O6 : 251.0550; found: 251.0556  7.2.3  Standards for UGL reaction in 10% methanol  Methyl (phenyl 4-deoxy-5-C-methoxy-β-d-glucopyranosid)uronate (27) MeOOC HO  O OMeOH  O  Globally protected phenyl unsaturated β-d-glucuronide (9, 10 mg) was dissolved in dry methanol (1 mL) under argon and acetyl chloride added (50 µL). The reaction mixture was left at room temperature for 8 days then quenched with saturated NaHCO3 , and a precipitate was seen to form. This was redissolved by addition of glacial acetic acid, then the organic solvents evaporated and the product purified by HPLC, eluting with H2 O (5 min), 5% acetonitrile (linear gradient over 40 min), 20% acetonitrile (linear gradient over 20 min) and finally 100% acetonitrile (linear gradient over 10 min). Product fractions were identified by UV-vis absorbance online and confirmed by TLC (3:3:1:1 toluene/ethyl acetate/methanol/acetic acid). The title compound was seen to elute with a retention time of approximately 74 min.  1 H-NMR  (400 MHz; CD3 OD): δ 7.31 (t, J2 ,3 = J3 ,4 = J4 ,5 = J5 ,6 = 7.7  Hz, 2H, H-3 ,5 ), 7.18 (d, 2H, H-3 ,5 ), 7.06 (t, 1H, H-4 ), 5.05 (d, J1,2 = 7.8 Hz, 1H, H-1), 3.94 (ddd, J3,4ax = 11.5, J2,3 = 9.3, J3,4eq = 5.1 Hz, 1H, H-3), 3.79 (s,  194  7.2. Synthesis 3H, OMe), 3.45 (dd, 1H, H-2), 3.25 (s, 3H, OMe), 2.32 (dd, J4ax ,4eq = 13.1 Hz, 1H, H-4eq ), 1.75 (dd, 1H, H-4ax ) ppm. MS : Calcd. for C14 H18 NaO7 : 321.1; found: 321.2 Phenyl 4-deoxy-5-C-methoxy-β-d-glucopyranosiduronic acid (28) HOOC HO  O OMeOH  O  Methyl (phenyl 4-deoxy-5-C-methoxy-β-D-glucopyranosid)uronate (27) was deprotected by the general method for ester saponification (page 187). The product was purified by 5 g C-18 Sep-pak washed with H2 O, 40% acetonitrile in water and 100% acetonitrile. The product was identified by TLC (3:2:2 1-butanol/acetic acid/water) to be in the flowthrough and water washes, so these were pooled and lyophilised before dissolving in ethanol to desalt, then filtering and removing the solvent to give a white powder (4 mg, 14 µmol, 49% over two steps). 1 H-NMR (600 MHz; D2 O): δ 7.29 (t, J2 ,3 = J3 ,4 = J4 ,5 = J5 ,6 = 7.9 Hz, 2H, H-3 ,5 ), 7.11 (d, 2H, H-3 ,5 ), 7.05 (t, 1H, H-4 ), 5.06 (d, J1,2 = 8.0 Hz, 1H, H-1), 3.90 (ddd, J3,4ax = 11.8, J2,3 = 9.4, J3,4eq = 5.1 Hz, 1H, H-3), 3.44 (dd, Hz, 1H, H-2), 3.07 (s, 3H, OMe), 2.22 (dd, J4ax ,4eq = 13.1 Hz, 1H, H-4eq ), 1.62 (dd, 1H, H-4ax ) ppm. HRMS : Calcd. for C13 H15 O7 : 283.0818; found: 283.0813 Methyl (methyl 2,3,4-tri-O-acetyl-β-d-glucopyranosid)uronate (29) MeOOC AcO AcO  O OAc  OMe  Globally protected glucuronyl bromide(3, 0.564 g, 1.42 mmol) was dissolved in acetone (12 mL) and methanol (5 mL), Ag2 CO3 (0.907 g, 2.3 eq.) was added, and the reaction mixture stirred overnight at room temperature in the dark. The solid was then removed by filtration through Celite, the solvents evaporated in vacuo, and 195  7.2. Synthesis the product purified by flash column chromatography (3:2 petroleum ether/ethyl acetate) to yield a colourless syrup (277 mg, 0.795 mmol, 56%).  1 H-NMR  (300  MHz, CDCl3 ): δ 5.16 (t, J2,3 = J3,4 = 9.3 Hz, 1H, H-3), 5.10 (t, 1H, H-4), 4.89 (dd, J1,2 = 7.8 Hz, 1H, H-2), 4.41 (d, 1H, H-1), 3.98 (d, J4,5 = 9.3 Hz, 1H, H-5), 3.66 (s, 3H, COOMe), 3.42 (s, 3H, OMe), 1.95 (s, 3H, OAc), 1.92 (s, 3H, OAc), 1.91 (s, 3H, OAc) ppm.  13 C-NMR  (75 MHz, CDCl3 ): δ 169.9 (OAc), 169.26 (OAc), 169.18  (OAc), 167.2 (C-6), 101.5 (C-1), 72.4 (C-3), 72.0 (C-2), 71.0 (C-4), 69.4 (C-5), 57.2 (OMe), 52.8 (OMe), 20.53 (OAc), 20.45 (OAc), 20.36 (OAc) ppm. Methyl (methyl 2,3-di-O-acetyl-4-deoxy-α-l-threo-hex-4-enopyranosid) uronate (30) COOMe AcO  O OMe OAc  Globally protected methyl glucuronide (29, 277 mg, 0.80 mmol) was subjected to the general method for DBU-catalysed elimination (page 186). Purification by flash column chromatography (5:2 petroleum ether/ethyl acetate) gave a pale yellow syrup (179 mg, 0.62 mmol, 78%). 1 H-NMR (300 MHz, CDCl3 ): δ 6.09 (dd, J3,4 = 4.5, J2,4 = 1.1 Hz, 1H, H-4), 5.07 (dd, J1,2 = 4.3, J1,3 = 1.9 Hz, 1H, H-1), 5.01 (dd, 1H, H-3), 4.97-4.95 (dd, 1H, H-2), 3.72 (s, 3H, OMe), 3.40 (s, 3H, COOMe), 1.98 (s, 3H, OAc), 1.97 (s, 3H, OAc) ppm. MS : Calcd. for C14 H20 NaO10 : 371.1; found: 371.3  196  7.2. Synthesis Methyl 4-deoxy-α-l-threo-hex-4-enopyranosiduronic acid (31) COOH O OMe OH  HO  Globally protected methyl glucuronate (30, 20 mg, 70 µmol) was deprotected as per the general method for ester saponification (page 187). Purification was by 5 g C-18 Sep-pak, washed with H2 O in 5 mL fractions. All fractions containing product as determined by TLC (3:3:1:1 toluene/ethyl acetate/methanol/acetic acid) were pooled and lyophilised to give a white powder (4.5 mg, 24 µmol, 36%).  1 H-NMR  (400 MHz, D2 O): δ 6.05 (d, J3,4 = 4.0 Hz, 1H, H-4), 4.99 (d, J1,2 = 4.8 Hz, 1H, H-1), 4.10 (br. t, J2,3 = 4.3 Hz, 1H, H-3), 3.76 (br. t, 1H, H-2), 3.44 (s, 3H, OMe) ppm. MS : Calcd. for C7 H9 O6 : 189.0; found: 189.3  7.2.4  Unusual substrates for UGL  3-Deoxy-d-glycero-d-galacto-2-nonulopyranosonic acid( 32) OH HO HO  OH O  COOH  HO HO  Pyruvic acid (2.55 g, 28.95 mmol, adjusted to pH 7 with 1 M NaOH) and Dmannose (10.161 g, 56.4 mmol) were dissolved in distilled water (200 mL), Neuraminic acid aldolase (260 mg) was added, and the reaction allowed to proceed at ambient temperature until TLC indicated consumption of the pyruvic acid (3:2:2 ethyl acetate/methanol/water). The product was then purified by Dowex 1XB ion exchange resin (pre-equilibrated with 6 M formic acid then water) eluting with water then 1 M formic acid. Fractions containing product were pooled and the solvent evaporated under reduced pressure until the volume was sufficiently low for lyophilisation,  197  7.2. Synthesis yielding a white foam (3.75 g, 14.0 mmol, 48 %). MS : Calcd. for C9 H15 O9 : 267.2; found: 267.3 Methyl 2,4,5,7,8,9-hexa-O-acetyl-3-deoxy-d-glycero-d-galacto-2nonulopyranosonate (33) OAc AcOAcO AcOAcO  OAc O  COOMe  3-Deoxy-d-glycero-d-galacto-2-nonulopyranosonic acid (32, 3.75 g, 14.0 mmol) was dissolved in methanol (100 mL) and trifluoroacetic acid (0.5 mL), and stirred overnight at ambient temperature. The solvents were then evaporated in vacuo, and acetic anhydride (15 mL) and pyridine (75 mL) added and again stirred overnight at ambient temperature. The solvents were again evaporated in vacuo and the crude product dissolved in ethyl acetate before washing with water, saturated NaHCO3 , 1 N HCl and brine before drying over MgSO4 . Filtering through a plug of silica (eluting with 1:1 ethyl acetate/petrol) gave a pale yellow syrup of sufficient purity for the subsequent reaction (4.47 g, 8.36 mmol, 60 %). 1 H-NMR (300 MHz; CDCl3 ): δ 5.39 (dd, J7,8 = 6.2, J6,7 = 2.3 Hz, 1H, H-7), 5.25 (ddd, J3ax ,4 = 11.6, J4,5 = 10.0, J3eq ,4 = 5.2 Hz, 1H, H-4), 5.14 (td, J8,9b = 6.2, J8,9a = 2.6 Hz, 1H, H-8), 4.97 (t, J5,6 = 10.0 Hz, 1H, H-5), 4.43 (dd, J9a ,9b = 12.5 Hz, 1H, H-9a ), 4.18 (dd, Hz, 1H, H-6), 4.11 (dd, Hz, 1H, H-9b ), 3.78 (s, 3H, OMe), 2.62 (dd, J3ax ,3eq = 13.6 Hz, 1H, H-3eq ), 2.15 (s, 3H, OAc), 2.11 (s, 3H, OAc), 2.06 (s, 3H, OAc), 2.06 (dd, 1H, H-3ax ), 2.03 (s, 3H, OAc), 2.01 (s, 3H, OAc), 2.00 (s, 3H, OAc) ppm. MS : Calcd. for C22 H30 NaO15 : 557.15; found: 557.3  198  7.2. Synthesis Methyl 4,5,7,8,9-penta-O-acetyl-2,3-dideoxy-d-glycero-d-galacto-non-2enopyranosonate (35) OAc COOMe AcOAcO AcOAcO  O  and Methyl 4,5,7,8,9-penta-O-acetyl-2,3-dideoxy-d-glycero-d-talo-non-2enopyranosonate (34) OAc AcOAcO AcO  OAc O  COOMe  To a solution of Methyl 2,4,5,7,8,9-hexa-O-acetyl-3-deoxy-d-glycero-d-galacto-2-nonulopyranosonate (33, 4.47 g, 8.36 mmol) in ethyl acetate (40 mL) at 0 °C under nitrogen was added Trimethylsilyl trifluoromethanesulfonate (3.36 mL, 2.2 eq.) dropwise, and allowed to react for 5 hours while warming to ambient temperature. The reaction was subsequently quenched with triethylamine (6 mL) and water (60 mL) at 0 °C, then the aqueous phase extracted thrice with ethyl acetate. The pooled organic phases were then washed with brine and dried over MgSO4 . Purification by flash column chromatography (2:1 petroleum ether/ethyl acetate) yielded a pale yellow syrup, composed of the desired product and its C4 epimer in a 1.2:1 ratio, which proved to not be separable by further chromatography (3.07 g, 6.47 mmol, 77%). 1 H-NMR  (300 MHz; CDCl3 ): δ 6.04 (d, J3,4 = 5.9 Hz, 1H, H-3 34), 5.95 (d, J3,4 =  2.9 Hz, 1H, H-3 35), 5.57-5.46 (m, 4H, H-4,7 35 & 34), 5.42-5.34 (m, 2H, H-8 35 & 34), 5.20 (dd, J5,6 = 9.4, J4,5 = 7.0 Hz, 1H, H-5 35), 5.00 (dd, J5,6 = 11.0, J4,5 = 4.0 Hz, 1H, H-5 34), 4.67 (dd, J9a ,9b = 12.5, J8,9a = 2.3 Hz, 1H, H-9a 34), 4.55 (dd, 199  7.2. Synthesis J9a ,9b = 12.5, J8,9a = 2.5 Hz, 1H, H-9a 35), 4.38 (dd, J6,7 = 1.7 Hz, 1H, H-6 34), 4.32 (dd, J6,7 = 3.0 Hz, 1H, H-6 35), 4.17 (dd, J8,9b = 6.2 Hz, 2H, H-9b 35 & 34), 3.80 (s, 3H, OMe 34), 3.79 (s, 3H, OMe 35), 2.09 (s, 3H, OAc), 2.08 (s, 6H, 2xOAc), 2.07 (s, 3H, OAc), 2.06 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.04 (s, 3H, OAc), 2.03 (s, 6H, 2xOAc), 1.99 (s, 3H, OAc) ppm. MS : Calcd. for C20 H26 NaO13 : 497.13; found: 497.2 Methyl 2,3-dideoxy-d-glycero-d-galacto-non-2-enopyranosonate (37) OH COOMe HO HO  O  HO HO  and Methyl 2,3-dideoxy-d-glycero-d-talo-non-2-enopyranosonate (36) OH HO HO  OH O  COOMe  HO  Methyl 4,5,7,8,9-penta-O-acetyl-2,3-dideoxy-d-glycero-d-galacto-non-2-enopyranosonate (35, 2.08 g, 4.38 mmol), contaminated with its C4 epimer (34), was subjected to the general method for Zemplén deprotection (page 187). Purification by flash column chromatography (18:1 ethyl acetate/methanol) yielded a white powder, composed of the desired product and its C4 epimer in a 1.5:1 ratio, which proved to not be separable by further chromatography or crystallisation (0.925 g, 3.50 mmol, 80%). 1 H-NMR (300 MHz; CD3 OD): δ 6.05 (d, J3,4 = 5.9 Hz, 1H, H-3 36), 5.87 (d, J3,4 = 2.6 Hz, 1H, H-3 37), 4.27 (dd, J4,5 = 7.7 Hz, 1H, H-4 37), 4.21 (dd, J4,5 = 3.9 Hz, 1H, H-4 36), 4.13 (d, J5,6 = 11.5 Hz, 1H, H-6 37), 4.10 (d, J5,6 = 7.3 Hz, 1H, 200  7.2. Synthesis H-6 36), 3.89-3.82 (m, 6H, H-7,8,9a 37 & 36), 3.78 (s, 3H, OMe 36), 3.78 (s, 3H, OMe 37), 3.75-3.65 (m, 4H, H-5,9b 37 & 36) ppm. MS : Calcd. for C10 H16 NaO8 : 287.07; found: 287.2 Methyl 6,8-O-phenylmethylene-2,3-dideoxy-d-glycero-d-galacto-non-2enopyranosonate (39) OH COOMe O  O HO  O HO  Ph  and Methyl 4,5:6,8-bis-O-phenylmethylene-2,3-dideoxy-d-glycero-d-talo-non2-enopyranosonate (38) HO Ph COOMe  O O O O  O  Ph  Methyl 2,3-dideoxy-d-glycero-d-galacto-non-2-enopyranosonate (37, 0.570 g, 2.16 mmol), contaminated with its C4 epimer (36), was dissolved in acetonitrile (40 mL), then benzaldehyde dimethyl acetal and p-toluenesulfonic acid (40 mg, 0.1 eq., monohydrate) were added and the reaction allowed to proceed at ambient temperature for 3.5 hours. The reaction was quenched with triethylamine and the solvent evaporated in vacuo before purification by flash column chromatography (3:1 petroleum ether/ethyl acetate followed by neat ethyl acetate). Compound 38 was crystallised from ethanol to yield white plates (222 mg, 0.504 mmol, 23%), while the desired product 39 was purified on a second flash column (6% methanol in dichlorometh201  7.2. Synthesis ane) to yield a white powder (138 mg, 0.392, 18%). 38: 1 H-NMR (300 MHz; CDCl3 ): δ 7.51-7.45 (m, 4H, Ph), 7.41-7.34 (m, 6H, Ph), 6.37 (d, J3,4 = 4.3 Hz, 1H, H-3), 5.89 (s, 1H, CHPh), 5.56 (s, 1H, CHPh), 4.66 (dd, J4,5 = 6.3 Hz, 1H, H-4), 4.55 (dd, J5,6 = 9.5 Hz, 1H, H-5), 4.37 (dd, J9a ,9b = 10.3, J8,9a = 5.4 Hz, 1H, H-9a ), 4.27 (dd, J6,7 = 5.3 Hz, 1H, H-6), 4.04-3.98 (m, 2H, H-7,8), 3.81 (s, 3H, Me), 3.67 (t, J8,9b = 10.3 Hz, 1H, H-9b ) ppm. MS : Calcd. for C24 H24 NaO8 : 463.1; found: 463.2 39: 1 H-NMR (400 MHz; acetone-d6): δ 7.51-7.46 (m, 2H, Ph), 7.38-7.31 (m, 3H, Ph), 5.85 (d, J3,4 = 2.5 Hz, 1H, H-3), 5.56 (s, 1H, CHPh), 4.32 (dd, J4,5 = 7.7 Hz, 1H, H-4), 4.28 (dd, J9a ,9b = 10.4, J8,9a = 4.8 Hz, 1H, H-9a ), 4.21 (dd, J6,7 = 0.9 Hz, 1H, H-6), 4.16-4.08 (m, 1H, H-7,8), 3.88 (dd, J5,6 = 10.4 Hz, 1H, H-5), 3.72 (s, 3H, OMe), 3.64 (t, J8,9b = 10.4 Hz, 1H, H-9b ) ppm. MS : Calcd. for C17 H20 NaO8 : 375.1; found: 375.3 2,3-Dideoxy-d-glycero-d-galacto-non-2-enopyranosonate (40) OH COOH HO HO  O  HO HO  Benzylidene-protected Kdn2en (39, 45 mg, 0.128 mmol) was subjected to the general method for hydrolysis by lithium hydroxide (page 187), at ambient temperature. The reaction was then quenched with Sephadex ion exchange resin (H+ form) and stirred until the benzylidene was cleaved (7 days), stopping short of completion to mitigate hydration of the final product under the acidic conditions. The reaction mixture was then filtered, the organic solvent evaporated in vacuo, and the product purified by 5 g C-18 Sep-pak eluted with water. Lyophilisation yielded a white powder (26 mg, 0.104 mmol, 81%). 1 H-NMR (400 MHz; D2 O): δ 5.97 (d, J3,4 = 2.4 Hz, 1H, H-3), 4.44 (dd, J4,5 = 7.9 Hz, 1H, H-4), 4.19 (d, 1H, H-6), 3.94-3.90  202  7.2. Synthesis (m, 3H, H-7,8,9a ), 3.80 (dd, J5,6 = 10.4 Hz, 1H, H-5), 3.70 (dd, J9a ,9b = 13.3, J8,9b = 4.0 Hz, 1H, H-9b ) ppm. HRMS : Calcd. for C9 H13 O8 : 249.0610; found: 249.0607 Methyl (phenyl 2,3,4-tri-O-acetyl-α-d-glucopyranosid)uronate (41) MeOOC AcO AcO  O AcO O  Globally protected glucuronic acid (2, 2.013 g, 5.35 mmol) was dissolved in phenol (4.17 g) at 80 °C, ZnCl2 added (1 g) and the reaction mixture stirred under vacuum (aspirator) for two hours. The reaction mixture was allowed to cool, dissolved in ethyl acetate and then extracted with 10% Na2 SO4 , 1M NaCl, and 10% Na2 SO4 again. After drying over MgSO4 and concentrating, the residue was purified by flash column chromatography (2:1 hexanes/ethyl acetate) to give a white foam of the title compound (0.135 g, 0.33 mmol, 6%), as well as the beta anomer (0.329 g, 0.80 mmol, 15%).  1 H-NMR  (300 MHz; DMSO-d6): δ 7.33 (m, 2H, H-3 ,5 ), 7.13-7.04 (m, 3H,  H-2 ,4 ,6 ), 5.93 (d, J1,2 = 3.6 Hz, 1H, H-1), 5.53 (t, J2,3 = J3,4 = 9.8 Hz, 1H, H-3), 5.17-5.08 (m, 2H, H-2,4), 4.39 (d, J4,5 = 9.8 Hz, 1H, H-5), 3.59 (s, 3H, OMe), 2.01 (s, 3H, OAc), 2.00 (s, 3H, OAc), 1.98 (s, 3H, OAc) ppm. Methyl (phenyl 2,3-di-O-acetyl-4-deoxy-β-l-threo-hex-4enopyranosid)uronate (42) COOMe AcO  O AcOO  Globally protected phenyl α-d-glucuronide (41, 135 mg, 0.33 mmol) was subjected to DBU-catalysed elimination by the general method . Purification by flash column chromatography (5:2 petroleum ether/ethyl acetate) gave a white foam (50 203  7.2. Synthesis mg, 0.14 mmol, 44%). 1 H-NMR (300 MHz; CDCl3 ): δ 7.31 (dd, J2 ,3 = J5 ,6 = 8.7, J3 ,4 = J4 ,5 = 7.3 Hz, 2H, H-3 ,5 ), 7.11-7.05 (m, 3H, H-2 ,4 ,6 ), 6.14 (d, J2,4 = 2.8 Hz, 1H, H-4), 5.84 (dd, J1,2 = 8.2, J1,3 = 2.8 Hz, 2H, H-1), 5.82 (d, 1H, H-3), 5.30 (dd, 1H, H-2), 3.78 (s, 3H), 2.11 (s, 6H) ppm.  13 C-NMR  (101 MHz; CD3 OD):  δ 170.6 (OAc), 170.3 (OAc), 161.9 (C-6), 156.6 (C-5), 141.8 (Ar), 129.5 (Ar), 123.3 (Ar), 116.9 (Ar), 108.4 (C-4), 95.9 (C-1), 68.6 (C-3), 66.4 (C-2), 51.8 (OMe), 19.5 (OAc), 19.3 (OAc) ppm. Phenyl 4-deoxy-β-l-threo-hex-4-enopyranosiduronic acid (43) COOH HO  O HOO  Globally protected phenyl unsaturated α-d-glucuronide (42, 25 mg, 0.071 mmol) was deprotected by the general method for ester saponification (page 187). Purification was by 5 g C-18 Sep-pak, washed with water, 10% acetonitrile in water, 40% acetonitrile in water, 60% acetonitrile in water and 100% acetonitrile. All fractions containing product as determined by TLC (3:3:1:1 toluene/ethyl acetate/methanol/acetic acid) were pooled and lyophilised to give a white powder (11.5 mg, 0.046 mmol, 64%).  1 H-NMR  (400 MHz; D2 O): δ 7.22 - 7.36 (m, 2H, H-3 ,5 ),  7.00 - 7.11 (m, 3H, H-2 ,4 ,6 ), 6.00 (d, J3,4 = 3.1 Hz, 1H, H-4), 5.70 (d, J1,2 = 2.5 Hz, 1H, H-1), 4.47 (dd, J2,3 = 7.8 Hz, 1H, H-3), 3.90 (dd, 1H, H-2) ppm.  13 C-NMR  (151 MHz; D2 O): δ 165.9 (C-6), 155.5 (C-5), 141.3 (Ar), 129.5 (Ar), 123.2 (Ar), 117.0 (Ar), 110.9 (C-4), 97.4 (C-1), 69.2 (C-3), 65.3 (C-2) ppm. MS : Calcd. for C12 H12 NaO6 : 275.0; found: 275.4  204  7.2. Synthesis Methyl (phenyl 2,3,4-tri-O-acetyl-1-thio-β-dglucopyranosid)uronate (44) MeOOC AcO AcO  O OAc  S  Globally protected glucuronyl bromide (3, 0.482 g, 1.21 mmol) was dissolved in ethyl acetate (10 mL), then tetra-n-butylammonium bromide (0.315 g), thiophenol (200 µL, 1.6 eq.) and 1 M Na2 CO3 (10 mL) were added. The reaction mixture was stirred vigorously at ambient temperature for 2 hours then diluted with ethyl acetate and washed with 1 M NaOH, sat. NaHCO3 , water and brine before drying over MgSO4 . Purification by flash column chromatography (3:1 petroleum ether/ethyl acetate) gave a white foam (405 mg, 0.95 mmol, 78%). 1 H-NMR (300 MHz; DMSOd6): δ 7.45-7.33 (m, 5H, H-2 ,3 ,4 ,5 ,6 ), 5.43 (t, J2,3 = J3,4 = 9.8 Hz, 1H, H-3), 5.40 (d, J1,2 = 9.8 Hz, 1H, H-1), 4.96 (t, 1H, H-2), 4.86 (dd, J4,5 = 10.0 Hz, 1H, H-4), 4.58 (d, 1H, H-5), 3.65 (s, 3H), 2.03 (s, 3H), 1.98 (s, 3H), 1.95 (s, 3H) ppm. Methyl (phenyl 2,3-di-O-acetyl-4-deoxy-1-thio-α-l-threo-hex-4enopyranosid)uronate (45) COOMe AcO  O S OAc  Globally protected phenyl thio-β-d-glucuronide (44, 163 mg, 0.38 mmol) was subjected to the DBU-catalysed elimination as per the general method. Purification by flash column chromatography (5:2 petroleum ether/ethyl acetate) gave a white powder (78 mg, 0.21 mmol, 56%). 1 H-NMR (300 MHz; CDCl3 ): δ 7.60-7.57 (m, 2H, Ar), 7.37-7.32 (m, 3H, Ar), 6.35 (dd, J3,4 = 4.8, J2,4 = 1.4 Hz, 1H, H-4), 5.71 (dd, J1,2 = 2.3, J1,3 = 1.5 Hz, 1H, H-1), 5.32 (br. q, J2,3 = 1.5 Hz, 1H, H-2), 5.20 (dt, 205  7.2. Synthesis Hz, 1H, H-3), 3.87 (s, 3H, OMe), 2.17 (s, 3H, OAc), 2.08 (s, 3H, OAc) ppm. Phenyl 4-deoxy-1-thio-α-l-threo-hex-4-enopyranosiduronic acid (46) COOH O S OH  HO  Globally protected phenyl unsaturated thio-β-d-glucuronide (45, 65 mg, 0.177 mmol) was deprotected by the general method for ester saponification (page 187). Purification was by 5 g C-18 Sep-pak, washed with water, 10%, 20%, 30%, 40%, 60% and 100% acetonitrile in water. All fractions containing pure product as determined by TLC (3:3:1:1 toluene/ethyl acetate/methanol/acetic acid) were pooled and lyophilised to give a white powder (41 mg, 0.153 mmol, 86%).  1 H-NMR  (400  MHz; D2 O): δ 7.58 (dd, J2,3 = J5,6 = 6.5, J2,4 = J4,6 = 3.2 Hz, 2H, H-2 ,6 ), 7.34 7.44 (m, 3H, H-3 ,4 ,5 ), 6.23 (dd, J3,4 = 4.0, J2,4 = 0.9 Hz, 1H, H-4), 5.56 (dd, J1,2 = 4.9, J1,3 = 0.9 Hz, 1H, H-1), 4.21 (td, J2,3 = 4.0 Hz, 1H, H-3), 3.98 (ddd, 1H, H-2) ppm.  13 C-NMR  (101 MHz; D2 O): δ 165.5 (C-6), 141.7 (C-5), 133.1 (Ar), 132.2  (Ar), 129.5 (Ar), 128.8 (Ar), 111.7 (C-4), 86.3 (C-1), 69.7 (C-3), 65.7 (C-2) ppm. HRMS : Calcd. for C12 H11 NaO5 S: 291.0303; found: 291.0304  7.2.5  Substrates for linear free-energy relationship  Methyl (2,4-dinitrophenyl 2,3,4-tri-O-acetyl-β-dglucopyranosid)uronate (54) MeOOC AcO AcO  O OAc  O  O2N  NO2  2,4-Dinitrophenol (0.983 g, 5.34 mmol, 3.75 eq.) was reacted with globally protec206  7.2. Synthesis ted glucuronyl bromide (3, 0.585 g, 1.47 mmol) by the general method for KoenigsKnorr glycosylation (page 186). The resulting off-white powder (0.580 g, 1.16 mmol, 79%) was used without further purification. 1 H-NMR (300 MHz; DMSO-d6): δ 8.79 (d, J3 ,5 = 2.8 Hz, 1H, H-5 ), 8.54 (dd, J2 ,3 = 9.3 Hz, 1H, H-3 ), 7.65 (d, 1H, H-2 ), 5.97 (d, J1,2 = 7.4 Hz, 1H, H-1), 5.45 (t, J2,3 = J3,4 = 9.3 Hz, 1H, H-3), 5.16 (t, 1H, H-2), 5.13 (t, J4,5 = 9.9 Hz, 1H, H-4), 4.80 (d, 1H, H-5), 3.62 (s, 3H, OMe), 2.02 (s, 3H, OAc), 2.01 (s, 3H, OAc), 2.00 (s, 3H, OAc) ppm. Methyl (2,5-dinitrophenyl 2,3,4-tri-O-acetyl-β-dglucopyranosid)uronate (56) MeOOC AcO AcO  O OAc  O  NO2  O2N  2,5-Dinitrophenol (0.245 g, 1.33 mmol, 1.3 eq.) was reacted with globally protected glucuronyl bromide (3, 0.403 g, 1.02 mmol) by the general method for KoenigsKnorr glycosylation (page 186). The resulting off-white powder (0.442 g, 0.883 mmol, 85%) was used without further purification. 1 H-NMR (300 MHz; DMSO-d6): δ 8.22-8.11 (m, 3H, H-3 ,4 ,6 ), 5.98 (d, J1,2 = 7.4 Hz, 1H, H-1), 5.41 (t, J2,3 = J3,4 = 9.2 Hz, 1H, H-3), 5.16 (t, J4,5 = 9.5 Hz, 1H, H-4), 5.14 (dd, 1H, H-2), 4.83 (d, 1H, H-5), 3.64 (s, 3H, OMe), 2.02 (s, 6H, 2xOAc), 1.99 (s, 3H, OAc) ppm.  207  7.2. Synthesis Methyl (2,4,6-trichlorophenyl 2,3,4-tri-O-acetyl-β-dglucopyranosid)uronate (58) MeOOC AcO AcO  Cl  O OAc  O Cl  Cl  2,4,6-Trichlorophenol (0.34 g, 1.72 mmol) was reacted with globally protected glucuronyl bromide (3, 0.527 g, 1.33 mmol) by the general method for KoenigsKnorr glycosylation (page 186). Purification by flash column chromatography (3:1 petroleum ether/ethyl acetate) yielded a white powder (0.441 g, 0.86 mmol, 65%). 1 H-NMR  (300 MHz; DMSO-d6): δ 7.73 (s, 2H, H-3 ,5 ), 5.52 (d, J1,2 = 7.9 Hz, 1H,  H-1), 5.46 (t, J2,3 = J3,4 = 9.6 Hz, 1H, H-3), 5.13 (dd, 1H, H-2), 5.02 (t, J4,5 = 10.0 Hz, 1H, H-4), 4.51 (d, 1H, H-5), 3.59 (s, 3H, OMe), 2.05 (s, 3H, OAc), 1.99 (s, 3H, OAc), 1.97 (s, 3H, OAc) ppm. MS : Calcd. for C19 H19 Cl3 NaO10 : 535.0/537.0; found: 535.1/537.1 Methyl (3-nitrophenyl 2,3,4-tri-O-acetyl-β-dglucopyranosid)uronate (60) MeOOC AcO AcO  O OAc  O  NO2  3-Nitrophenol (0.220 g, 1.58 mmol, 1.25 eq.) was reacted with globally protected glucuronyl bromide (3, 0.505 g, 1.27 mmol) by the general method for KoenigsKnorr glycosylation (page 186). Purification by flash column chromatography (2:1 petroleum ether/ethyl acetate) yielded an off-white powder (0.304 g, 0.668 mmol, 53%). 1 H-NMR (300 MHz; DMSO-d6): δ 7.95 (ddd, J4 ,5 = 8.2, J2 ,4 = 2.2, J4 ,6 = 0.8 Hz, 1H, H-4 ), 7.80 (t, J2 ,6 = 2.2 Hz, 1H, H-2 ), 7.65 (t, J5 ,6 = 8.2 Hz, 1H,  208  7.2. Synthesis H-5 ), 7.48 (ddd, 1H, H-6 ), 5.87 (d, J1,2 = 7.8 Hz, 1H, H-1), 5.46 (t, J2,3 = J3,4 = 9.6 Hz, 1H, H-3), 5.14 (dd, 1H, H-2), 5.09 (t, J4,5 = 9.9 Hz, 1H, H-4), 4.76 (d, 1H, H-5), 3.62 (s, 3H, OMe), 2.02 (s, 3H, OAc), 2.01 (s, 3H, OAc), 1.99 (s, 3H, OAc) ppm. Methyl (4-chlorophenyl 2,3,4-tri-O-acetyl-β-dglucopyranosid)uronate (62) MeOOC AcO AcO  O OAc  O Cl  Globally protected glucuronyl hemiacetal (7, 0.4 g, 1.20 mmol) was dissolved in dichloromethane (12 mL), 4 Å molecular sieves were added and the solution flushed with argon, then cooled in a dry ice/acetone bath. Trichloroacetonitrile (1.2 mL, 10 eq.) then DBU (0.25 mL, 1.5 eq.) were added and the reaction mixture stirred for 30 min at -78 °C, then allowed to warm to ambient temperature. The reaction mixture was concentrated and filtered through a plug of silica gel (eluting with 2:1 petroleum ether/ethyl acetate). The resultant crude trichloroacetimidate intermediate and 4-chlorophenol (0.19 g, 1.48 mmol, 1.2 eq.) were dissolved in dichloromethane (25 mL) and dried over 4 Å molecular sieves under argon, then cooled in a dry ice/acetone bath. Boron trifluoride diethyl etherate (0.05 mL, 0.3 eq.) was added and the reaction mixture stirred 30 min at -78 °C before allowing to warm to ambient temperature while stirring overnight. The reaction mixture was diluted with an equal volume of dichloromethane and washed with sat. NaHCO3 thrice then with brine. The organic phase was dried over MgSO4 then purified by flash column chromatography (3:1 petroleum ether/ethyl acetate) to give a white powder (0.103 g, 0.232 mmol, 20%). 1 H-NMR (300 MHz; CDCl3 ): δ 7.25 (d, J2 ,3 = J5 ,6 = 9.0 Hz, 2H, H-3 ,5 ), 6.94 (d, 2H, H-2 ,6 ), 5.36-5.23 (m, 3H, H-2,3,4), 5.11 209  7.2. Synthesis (d, J1,2 = 7.2 Hz, 1H, H-1), 4.19 (d, J4,5 = 9.5 Hz, 1H, H-5), 3.73 (s, 3H, OMe), 2.06 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.04 (s, 3H, OAc) ppm. Methyl (4-tert-butylphenyl 2,3,4-tri-O-acetyl-β-dglucopyranosid)uronate (64) MeOOC AcO AcO  O OAc  O  Globally protected glucuronyl hemiacetal (7, 0.4 g, 1.20 mmol) was dissolved in dichloromethane (12 mL), 4 Å molecular sieves were added and the solution flushed with argon, then cooled in a dry ice/acetone bath. Trichloroacetonitrile (1.2 mL, 10 eq.) then DBU (0.25 mL, 1.5 eq.) were added and the reaction mixture stirred for 30 min at -78 °C, then allowed to warm to ambient temperature. The reaction mixture was concentrated and filtered through a plug of silica gel (eluting with 2:1 petroleum ether/ethyl acetate). The resultant crude trichloroacetimidate intermediate and 4-tert-butylphenol (0.25 g, 1.66 mmol, 1.4 eq.) were dissolved in dichloromethane (25 mL) and dried over 4 Å molecular sieves under argon, then cooled in a dry ice/acetone bath. Boron trifluoride diethyl etherate (0.025 mL, 0.3 eq.) was added and the reaction mixture stirred 30 min at -78 °C before allowing to warm to ambient temperature while stirring overnight. The reaction mixture was diluted with an equal volume of dichloromethane and washed with sat. NaHCO3 thrice then brine. The organic phase was dried over MgSO4 then purified twice by flash column chromatography (2:1 petroleum ether/ethyl acetate then 5:2 hexanes/ethyl acetate) to give an amorphous solid (0.158 g, 0.339 mmol, 28%). 1 H-NMR (300 MHz; CDCl3 ): δ 7.31 (d, J2 ,3 = J5 ,6 = 8.9 Hz, 2H, H-3 ,5 ), 6.93 (d, 2H, H-2 ,6 ), 5.36-5.25 (m, 3H, H-2,3,4), 5.14 (d, J1,2 = 7.2 Hz, 1H, H-1), 4.19 (d, J4,5 = 9.5 Hz, 1H, H-5), 3.74  210  7.2. Synthesis (s, 3H, OMe), 2.06 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.05 (s, 3H, OAc) ppm. Methyl (1-O-benzyl-2,3,4-tri-O-acetyl-β-d-glucopyranosid)uronate (66) MeOOC AcO AcO  O OAc  O  Globally protected glucuronyl hemiacetal (7, 0.343 g, 1.03 mmol) was dissolved in toluene (2 mL) and the solution flushed with nitrogen. Benzyl bromide (0.245 mL, 2 eq.) and silver(I) carbonate (0.885g, 3 eq.) were added and the reaction mixture stirred overnight at ambient temperature in the dark. The reaction was quenched with triethylamine, diluted with dichloromethane and filtered through Celite. The filtrate was then washed with 1 M HCl, saturated NaHCO3 , water and brine before drying over MgSO4 . Purification by flash column chromatography (7:2 petroleum ether/ethyl acetate) followed by recrystallisation from toluene/petroleum ether yielded white plates (0.135 g, 0.318 mmol, 31%).  1 H-NMR  (300 MHz; CDCl3 ): δ  7.36-7.29 (m, 5H, H-2 ,3 ,4 ,5 ,6 ), 5.28-5.18 (m, 2H, H-7 a ,7 b ), 5.08 (br. t, J3,4 = 8.3, J4,5 = 9.2 Hz, 1H, H-4), 4.92 (d, J1,2 = 12.3 Hz, 1H, H-1), 4.64-4.58 (m, 2H, H-2,3), 4.02 (d, 1H, H-5), 3.76 (s, 3H, OMe), 2.01 (s, 3H, OAc), 2.01 (s, 3H, OAc), 1.99 (s, 3H, OAc) ppm.  13 C-NMR  (75 MHz; CDCl3 ): δ 170.3 (OAc), 169.51 (OAc),  169.35 (OAc), 167.4 (C-6), 136.6 (Ar), 128.6 (Ar), 128.2 (Ar), 127.9 (Ar), 99.4 (C-1), 72.8 (C-3), 72.2 (C-2), 71.3 (C-4), 71.0 (OCH2 Ar), 69.6 (C-5), 53.1 (OMe), 20.74 (2xOAc), 20.64 (OAc) ppm. MS : Calcd. for C20 H24 NaO10 :447.1; found: 447.4  211  7.2. Synthesis Methyl (2,4-dinitrophenyl 2,3-di-O-acetyl-4-deoxy-α-l-threo-hex-4enopyranosid)uronate (55) COOMe AcO  O O OAc  NO2  O2N  Globally protected 2,4-dinitrophenyl β-d-glucuronide (54, 0.275 g, 0.550 mmol) was subjected to the general method for DBU-catalysed elimination (page 186). Purification by flash column chromatography (5:2 petroleum ether/ethyl acetate) gave a white powder (0.133 g, 0.302 mmol, 55%). 1 H-NMR (300 MHz; DMSO-d6): δ 8.80 (d, J3 ,5 = 2.8 Hz, 1H, H-5 ), 8.60 (dd, J2 ,3 = 9.3 Hz, 1H, H-3 ), 7.83 (d, 1H, H-2 ), 6.57 (dd, J3,4 = 2.1, J2,4 = 0.9 Hz, 1H, H-4), 6.25 (dd, J1,2 = 4.8, J1,3 = 1.3 Hz, 1H, H-1), 5.27-5.25 (td, J2,3 = 2.1 Hz, 1H, H-3), 5.20 (ddd, 1H, H-2), 3.73 (s, 3H, OMe), 2.10 (s, 3H, OAc), 2.09 (s, 3H, OAc) ppm. Methyl (2,5-dinitrophenyl 2,3-di-O-acetyl-4-deoxy-α-l-threo-hex-4enopyranosid)uronate (57) COOMe AcO  O O OAc  NO2  O2N  Globally protected 2,5-dinitrophenyl β-d-glucuronide (56, 0.222 g, 0.444 mmol) was subjected to the general method for DBU-catalysed elimination (page 186). Purification by flash column chromatography (3:1 petroleum ether/ethyl acetate) gave a white powder (0.098 g, 0.223 mmol, 50%).  1 H-NMR  (300 MHz; CDCl3 ): δ  8.40 (d, J4 ,6 = 2.2 Hz, 1H, H-6 ), 8.04 (dd, J3 ,4 = 8.8, 1H, H-4 ), 7.90 (d, 1H, H-3 ), 6.35 (dd, J3,4 = 4.9, J2,4 = 1.4 Hz, 1H, H-4), 6.05 (dd, J1,2 = 1.9, J1,3 = 1.1 Hz, 1H,  212  7.2. Synthesis H-1), 5.28 (ddd, J2,3 = 1.1 Hz, 1H, H-2), 5.24 (dt, 1H, H-3), 3.80 (s, 3H, OMe), 2.14 (s, 3H, OAc), 2.13 (s, 3H, OAc) ppm. Methyl (2,4,6-trichlorophenyl 2,3-di-O-acetyl-4-deoxy-α-l-threo-hex-4enopyranosid)uronate (59)  AcO  COOMe Cl O O OAc  Cl  Cl  Globally protected 2,4,6-trichlorophenyl β-d-glucuronide(58, 0.441 g, 0.858 mmol) was subjected to the general method for DBU-catalysed elimination (page 186). Purification by flash column chromatography (3:1 petroleum ether/ethyl acetate) gave a white powder (0.279 g, 0.61 mmol, 72%).  1 H-NMR  (300 MHz; CDCl3 ): δ  7.28 (s, 2H, H-3 ,5 ), 6.36 (dd, J3,4 = 5.1, J2,4 = 1.4 Hz, 1H, H-4), 5.79 (dd, J1,2 = 1.7, J1,3 = 1.1 Hz, 1H, H-1), 5.44 (br. q, J2,3 = 1.7 Hz, 1H, H-2), 5.20 (br. dt, 1H, H-3), 3.79 (s, 3H, OMe), 2.06 (s, 6H, 2xOAc) ppm.  13 C-NMR  (75 MHz; CD3 OD): δ 171.5  (OAc), 170.6 (OAc), 163.4 (C-6), 148.8 (C-5), 144.0 (Ar), 132.1 (Ar), 131.1 (Ar), 130.2 (Ar), 107.9 (C-4), 98.2 (C-1), 68.3 (C-3), 64.5 (C-2), 53.1 (OMe), 20.74 (OAc), 20.58 (OAc) ppm MS : Calcd. for C17 H15 Cl3 NaO8 : 475.0/477.0; found: 475.2/477.2 Methyl (3-nitrophenyl 2,3-di-O-acetyl-4-deoxy-α-l-threo-hex-4enopyranosid)uronate (61) COOMe AcO  O O OAc  NO2  Globally protected 3-nitrophenyl β-d-glucuronide (60, 0.103 g, 0.232 mmol) was subjected to the general method for DBU-catalysed elimination (page 186). Puri-  213  7.2. Synthesis fication by flash column chromatography (3:1 hexanes/ethyl acetate) gave a white powder (0.064 g, 0.166 mmol, 72%). 1 H-NMR (300 MHz; CDCl3 ): δ 7.93-7.90 (m, 2H, H-2 ,4 ), 7.50-7.41 (m, 2H, H-5 ,6 ), 6.30 (dd, J3,4 = 4.0, J2,4 = 1.9 Hz, 1H, H-4), 5.88 (dd, J1,2 = 1.9, J1,3 = 1.1 Hz, 1H, H-1), 5.30-5.26 (m, 2H, H-2,3), 3.77 (s, 3H, OMe), 2.12 (s, 3H, OAc), 2.10 (s, 3H, OAc) ppm. Methyl (4-chlorophenyl 2,3-di-O-acetyl-4-deoxy-α-l-threo-hex-4enopyranosid)uronate (63) COOMe AcO  O O OAc  Cl  Globally protected 4-chlorophenyl β-d-glucuronide (62, 0.103 g, 0.232 mmol) was subjected to the general method for DBU-catalysed elimination (page 186). Purification by flash column chromatography (3:1 hexanes/ethyl acetate) gave a white powder (0.064 g, 0.166 mmol, 72%). 1 H-NMR (300 MHz; CDCl3 ): δ 7.26 (d, J2 ,3 = J5 ,6 = 9.0 Hz, 2H, H-3 ,5 ), 7.03 (d, 2H, H-2 ,6 ), 6.29 (dd, J3,4 = 4.5, J2,4 = 1.5 Hz, 1H, H-4), 5.77 (dd, J1,2 = 2.7, J1,3 = 1.0 Hz, 1H, H-1), 5.29-5.26 (m, 2H, H-2,3), 3.80 (s, 3H, OMe), 2.13 (s, 3H, OAc), 2.11 (s, 3H, OAc) ppm.  13 C-NMR  (101  MHz; CD3 OD): δ 170.4 (OAc), 169.7 (OAc), 162.1 (C-6), 154.9 (C-5), 142.1 (Ar), 129.4 (Ar), 128.1 (Ar), 118.5 (Ar), 107.7 (C-4), 95.1 (C-1), 68.2 (C-3), 64.9 (C-2), 51.9 (OMe), 19.47 (OAc), 19.32 (OAc) ppm.  214  7.2. Synthesis Methyl (4-tert-butylphenyl 2,3-di-O-acetyl-4-deoxy-α-l-threo-hex-4enopyranosid)uronate (65) COOMe AcO  O O OAc  Globally protected 4-tert -butylphenyl β-d-glucuronide (64, 0.158 g, 0.338 mmol) was subjected to the general method for DBU-catalysed elimination (page 186). Purification by flash column chromatography (4:1 hexanes/ethyl acetate) gave a white powder (0.061 g, 0.150 mmol, 44%). 1 H-NMR (300 MHz; CDCl3 ): δ 7.31 (d, J2 ,3 = J5 ,6 = 8.9 Hz, 2H, H-3 ,5 ), 7.02 (d, 2H, H-2 ,6 ), 6.28 (dd, J3,4 = 4.2, J2,4 = 1.6 Hz, 1H, H-4), 5.78 (dd, J1,2 = 2.6, J1,3 = 1.4 Hz, 1H, H-1), 5.31-5.26 (m, 2H, H-2,3), 3.81 (s, 3H, OMe), 2.13 (s, 3H, OAc), 2.11 (s, 3H, OAc), 1.29 (s, 9H, tert-butyl) ppm.  13 C-NMR  (101 MHz; CD3 OD): δ 171.7 (OAc), 171.0 (OAc), 163.4  (C-6), 155.4 (C-5), 147.4 (Ar), 143.6 (Ar), 127.5 (Ar), 117.8 (Ar), 108.7 (C-4), 96.8 (C-1), 69.7 (C-3), 66.4 (C-2), 53.1 (OMe), 35.1(tBu), 31.9 (tBu), 20.73 (OAc), 20.59 (OAc) ppm. Methyl (1-O-benzyl-2,3-di-O-acetyl-4-deoxy-α-l-threo-hex-4enopyranosid)uronate (67) COOMe AcO  O O OAc  Globally protected benzyl β-d-glucuronide (66, 32 mg, 0.075 mmol) was subjected to the general method for DBU-catalysed elimination (page 186). Purification by flash column chromatography (5:2 petroleum ether/ethyl acetate) gave a colourless syrup (0.020 g, 0.055 mmol, 73%). 1 H-NMR (300 MHz; CDCl3 ): δ 7.37-7.28 (m, 5H,  215  7.2. Synthesis H-2 ,3 ,4 ,5 ,6 ), 6.23 (dd, J3,4 = 4.5, J2,4 = 1.1 Hz, 1H, H-4), 5.30 (d, J1,2 = 2.5 Hz, 1H, H-1), 5.20 (dd, J2,3 = 1.9 Hz, 1H, H-3), 5.14 (br. q, 1H, H-2), 4.87 (d, J7 a ,7 b = 12.3 Hz, 1H, H-7 a ), 4.68 (d, 1H, H-7 b ), 3.82 (s, 3H, OMe), 2.08 (s, 3H, OAc), 2.06 (s, 3H, OAc) ppm.  13 C-NMR  (75 MHz; CDCl3 ): δ 170.2 (OAc), 169.5 (OAc),  162.3 (C-6), 142.4 (C-5), 136.8 (Ar), 128.5 (Ar), 128.0 (Ar), 127.5 (Ar), 107.5 (C-4), 95.7 (C-1), 70.6 (OCH2 Ar), 68.5 (C-3), 64.4 (C-2), 52.7 (OMe), 21.03 (OAc), 20.88 (OAc) ppm. MS : Calcd. for C18 H20 NaO8 : 387.1; found: 387.3 2,4-Dinitrophenyl 4-deoxy-α-l-threo-hex-4-enopyranosiduronic acid (47) COOH HO  O O OH  NO2  O2N  Globally protected 2,4-dinitrophenyl unsaturated β-d-glucuronide (55, 30 mg, 0.068 mmol) was dissolved in acetone (3.4 mL). Aqueous 1 M HCl (3.4 mL) was added and the reaction mixture stirred at ambient temperature for 20 days, with monitoring by TLC (3:3:1:1 toluene/ethyl acetate/methanol/acetic acid). The reaction was stopped by removing the organic solvent under vacuum and immediately purified by HPLC over a C-18 stationary phase, eluting with water (5 min) then a linear gradient to 100% acetonitrile over 1 hour. Product fractions were identified by UV-vis absorbance on-line and confirmed by TLC (3:3:1:1 toluene/ethyl acetate/methanol/acetic acid), then pooled and lyophilised to give an off-white powder (7.8 mg, 0.022 mmol, 32%). 1 H-NMR (600 MHz; CD3 OD): δ 8.78 (d, J3 ,5 = 2.8 Hz, 1H, H-5 ), 8.53 (dd, J2 ,3 = 9.3 Hz, 1H, H-3 ), 7.86 (d, 1H, H-2 ), 6.30 (dd, J3,4 = 4.2, J2,4 = 0.7 Hz, 1H, H-4), 6.07 (d, J1,2 = 4.1, J1,3 = 0.7 Hz, 1H, H-1), 4.15 (br. td, J2,3 = 3.8 Hz, 1H, H-3), 4.09-4.08 (br. td, 1H, H-2) ppm.  13 C-NMR  (151 MHz;  CD3 OD): δ 164.67 (C-6), 155.14 (C-5), 143.26 (Ar), 141.15 (Ar), 141.07 (Ar), 130.16 216  7.2. Synthesis (Ar), 122.67 (Ar), 119.74 (Ar), 114.11 (C-4), 99.78 (C-1), 70.56 (C-3), 66.89 (C-2) ppm. HRMS : Calcd. for C12 H9 N2 O10 : 341.0257; found: 341.0260 2,5-Dinitrophenyl 4-deoxy-α-l-threo-hex-4-enopyranosiduronic acid (48) COOH HO  O O OH  NO2  O2N  Globally protected 2,5-dinitrophenyl unsaturated β-d-glucuronide (57, 24 mg, 0.050 mmol) was dissolved in acetone (2.5 mL). Aqueous 1 M HCl (2.5 mL) was added and the reaction mixture stirred at ambient temperature for 16 days, with monitoring by TLC (3:3:1:1 toluene/ethyl acetate/methanol/acetic acid). The reaction was stopped by removing the organic solvent under vacuum and immediately purified by HPLC over a C-18 stationary phase, eluting with water (5 min) then a linear gradient to 100% acetonitrile over 1 hour. Product fractions were identified by UV-vis absorbance on-line and confirmed by TLC (3:3:1:1 toluene/ethyl acetate/methanol/acetic acid), then pooled and lyophilised to give an off-white powder (9.9 mg, 0.028 mmol, 56%). 1 H-NMR (600 MHz; CD3 OD): δ 8.43 (d, J4 ,6 = 1.0 Hz, 1H, H-6 ), 8.03 - 8.11 (m, 2 H, H-3 ,4 ), 6.27 (d, J3,4 = 4.1 Hz, 1 H, H-4), 5.97 (d, J1,2 = 4.1 Hz, 1 H, H-1), 4.13 (br. t, J2,3 = 3.8 Hz, 1 H, H-3), 4.05 (br. t, 1 H, H-2) ppm.  13 C-NMR  (151MHz; CD3 OD): δ 163.4 (C-6), 150.4 (C-5), 149.5 (Ar), 144.6  (Ar), 139.9 (Ar), 126.1 (Ar), 118.0 (Ar), 114.5 (Ar), 112.9 (C-4), 99.2 (C-1), 69.4 (C-3), 65.6 (C-2) ppm. HRMS : Calcd. for C12 H9 N2 O10 : 341.0257; found: 341.0251  217  7.2. Synthesis 2,4,6-Trichlorophenyl 4-deoxy-α-l-threo-hex-4enopyranosid)uronate (49) COOH HO  O O OH  Cl  Cl  Cl  Globally protected 2,4,6-trichlorophenyl unsaturated β-d-glucuronide (59, 21 mg, 0.044 mmol) was deprotected as per the general method for ester saponification (page 187). Purification was by HPLC over a C-18 stationary phase, eluting with water (5 min) then a linear gradient to 100% acetonitrile over 1 hour. Product fractions were identified by UV-vis absorbance on-line and confirmed by TLC (3:3:1:1 toluene/ethyl acetate/methanol/acetic acid) then pooled and lyophilised to give a white powder (9.9 mg, 0.028 mmol, 63%). 1 H-NMR (400 MHz; D2 O): δ 7.50 (s, 2H, H-3 ,5 ), 6.34 (dd,J3,4 = 4.9, J2,4 = 1.3 Hz, 1H, H-4), 6.74 (dd, J1,2 = 2.2, J1,3 = 0.9 Hz, 1H, H-1), 4.30 (br. q, J2,3 = 1.8 Hz, 1H, H-2), 4.88 (br. dt, 1H, H-3) ppm.  13 C-NMR  (101  MHz; D2 O): δ 149.5 (Ar), 131.8 (Ar), 131.4 (Ar), 130.1 (Ar), 112.0 (C-4), 102.3 (C1), 70.1 (C-3), 65.9 (C-2) ppm (carbon 5 and 6 signals too weak to detect, but seen in protected form). MS : Calcd. for C12 H8 Cl3 O6 : 376.9/378.9; found: 377.1/379.1 3-Nitrophenyl 4-deoxy-α-l-threo-hex-4-enopyranosiduronic acid (50) COOH HO  O O OH  NO2  Globally protected 3-nitrophenyl unsaturated β-d-glucuronide (61, 44 mg, 0.111 mmol) was subjected to the general method for Zemplén deprotection (page 187), but using a slight excess of sodium methoxide instead of a catalytic amount. Deacetylation was complete within 5 min, at which time water (200 µL) was added and the 218  7.2. Synthesis reaction mixture stirred at 0 °C for a further 60 min at 0 °C. The reaction was quenched with Sephadex ion exchange resin (H+ form) then filtered. Purification by flash column chromatography (7:2:1 ethyl acetate/methanol/water) then 5 g C-18 Sep-pak, washed with water, 40% acetonitrile in water and 100% acetonitrile, gave a slightly yellow powder following lyophilisation (28.1 mg, 0.095 mmol, 85%).  1 H-  NMR (600 MHz; D2 O): δ 7.88 - 7.96 (m, 2 H, H-2 ,4 ), 7.45 - 7.55 (m, 2 H, H-5 ,6 ), 6.24 (d, J3,4 = 4.1 Hz, 1 H, H-4), 5.84 (d, J1,2 = 4.6 Hz, 1 H, H-1), 4.27 (t, J2,3 = 4.1 Hz, 1 H, H-3), 4.10 (br. t, 1 H, H-2) ppm.  13 C-NMR  (151 MHz; D2 O): δ  164.7 (C-6), 155.6 (C-5), 148.1 (Ar), 139.9 (Ar), 130.1 (Ar), 123.7 (Ar), 118.0 (Ar), 111.9 (Ar), 111.8 (C-4), 97.4 (C-1), 68.6 (C-3), 64.9 (C-2) ppm. HRMS : Calcd. for C12 H10 NO8 : 296.0406; found: 296.0411 4-Chlorophenyl 4-deoxy-α-l-threo-hex-4-enopyranosiduronic acid (51) COOH HO  O O OH  Cl  Globally protected 4-chlorophenyl unsaturated β-d-glucuronide (63, 32 mg, 0.083 mmol) was deprotected by the general method for ester saponification (page 187). The product was purified by HPLC over a C-18 stationary phase, eluting with water (5 min) then a linear gradient to 100% acetonitrile over 1 hour. Product fractions were identified by UV-vis absorbance on-line and confirmed by TLC (3:3:1:1 toluene/ethyl acetate/methanol/acetic acid), then pooled and lyophilised to give an off-white powder (10.8 mg, 0.038 mmol, 46%).  1 H-NMR  (400 MHz; D2 O): δ 8.09  (d, J2 ,3 = J5 ,6 = 9.2 Hz, 2H, H-3 ,5 ), 7.17 (d, 2 H, H-2 ,6 ), 6.22 (d, J3,4 = 4.1 Hz, 1 H, H-4), 5.84 (d, J1,2 = 4.8 Hz, 1 H, H-1), 4.29 (br. t, J2,3 = 4.5 Hz, 1 H, H-3), 4.07 (br. t, 1 H, H-2) ppm.  13 C-NMR  (101 MHz; D2 O): δ 165.1 (C-6), 161.1  (Ar), 142.7 (Ar), 126.1 (Ar), 116.9 (Ar), 112.5 (C-4), 97.3 (C-1), 69.3 (C-3), 65.7 219  7.2. Synthesis (C-2) ppm (carbon 5 signal too weak to detect, but seen in protected form). HRMS : Calcd. for C12 H10 ClO6 : 285.0166; found: 285.0161 4-Tert-butylphenyl 4-deoxy-α-l-threo-hex-4-enopyranosiduronic acid (52) COOH HO  O O OH  Globally protected 4-tert -butylphenyl unsaturated β-d-glucuronide (65, 33 mg, 0.081 mmol) was deprotected by the general method for ester saponification (page 187). Purification was by 5 g C-18 Sep-pak, washed with 10% acetonitrile in water, 40% acetonitrile in water, 60% acetonitrile in water, 75% acetonitrile in water and 100% acetonitrile. All fractions determined by TLC (3:3:1:1 toluene/ethyl acetate/methanol/acetic acid) to contain pure product were pooled and lyophilised, while those containing impure product were pooled, lyophilised and purified again to give a white powder (13.8 mg, 0.044 mmol, 54%). 1 H-NMR (600 MHz; CD3 OD): δ 7.34 (d, J2 ,3 = J5 ,6 = 8.7 Hz, 2H, H-3 ,5 ), 7.10 (d, 2H, H-2 ,6 ), 6.13 (d, J3,4 = 3.6 Hz, 1 H, H-4), 5.52 (d, J1,2 = 5.6 Hz, 1 H, H-1), 4.20 (br. t, J2,3 = 4.9 Hz, 1 H, H-3), 3.90 (br. t, 1 H, H-2), 1.30 (s, 9 H, tert-butyl) ppm.  13 C-NMR  (151 MHz; CD3 OD):  δ 165.38 (C-6), 156.26 (C-5), 146.90 (Ar), 127.28 (Ar), 117.95 (Ar), 113.17 (C-4), 100.99 (C-1), 72.03 (C-3), 68.37 (C-2), 35.04 (s, tBu), 31.91 (s, tBu) ppm. HRMS : Calcd. for C16 H19 O6 : 307.1186; found: 307.1182  220  7.2. Synthesis 1-O-Benzyl-4-deoxy-α-l-threo-hex-4-enopyranosiduronic acid (53) COOH O O OH  HO  Globally protected benzyl unsaturated β-d-glucuronide(67, 20 mg, 0.055 mmol) was deprotected by the general method for ester saponification (page 187). Purification was by 5 g C-18 Sep-pak, washed with water, 40% acetonitrile in water twice and 100% acetonitrile. All fractions determined by TLC (3:3:1:1 toluene/ethyl acetate/methanol/acetic acid) to contain pure product were pooled and lyophilised to give a white powder (14.1 mg, 0.053 mmol, 96%). 1 H-NMR (400 MHz; D2 O): δ 7.35-7.28 (m, 5H, H-2 ,3 ,4 ,5 ,6 ), 6.06 (d, J3,4 = 4.0 Hz, 1H, H-4), 5.16 (d, J1,2 = 4.5 Hz, 1H, H-1), 4.77 (d, J7 a ,7 b = 10.5 Hz, 1H, H-7 a ), 4.71 (d, 1H, H-7 b ), 4.06 (br. t, J2,3 = 4.1 Hz, 1H, H-3), 3.80 (br. t, 1H, H-2) ppm.  13 C-NMR  (101 MHz; D2 O): δ  136.86 (Ar), 128.82 (Ar), 128.51 (Ar), 128.46 (Ar), 111.63 (C-4), 99.53 (C-1), 71.54 (s, OCH2 Ar), 69.57 (C-3), 65.76 (C-2) ppm (carbon 5 and 6 signals too weak to detect, but seen in protected form). HRMS : Calcd. for C13 H14 NaO6 : 289.0688; found: 289.0690  7.2.6  Substrates for with varied heteroatoms  Methyl (2,3,4-tri-O-acetyl-β-d-glucopyranosyl fluoride)uronate (68) MeOOC AcO AcO  O OAc  F  Globally protected α-d-glucuronyl bromide (3, 0.142 g, 0.358 mmol) was dissolved in acetonitrile (4 mL), then silver (I) fluoride (0.214 g, 1.687 mmol, 4.7 eq.) added and stirred at ambient temperature overnight in the dark under a nitrogen atmosphere. The reaction mixture was filtered through Celite, then purified by flash  221  7.2. Synthesis column chromatography (2:1 petroleum ether/ethyl acetate) to give a white powder (0.103 g, 0.306 mmol, 85%). 1 H-NMR (300 MHz; CDCl3 ): δ 5.41 (dd, J1,F = 51.0, J1,2 = 5.2 Hz, 1H, H-1), 5.39 (t, J3,4 = J4,5 = 8.2 Hz, 1H, H-4), 5.22 (t, J2,3 = 8.2 Hz, 1H, H-3), 5.08 (ddd, J2,F = 9.9 Hz, 1H, H-2), 4.27 (d, 1H, H-5), 3.77 (s, 3H, OMe), 2.09 (s, 3H, OAc), 2.03 (s, 6H, 2xOAc) ppm.  13 C-NMR  (75 MHz; CDCl3 ): δ 169.86  (OAc), 169.34 (OAc), 169.14 (OAc), 167.00 (C-6), 105.93 (d, JC1 ,F = 223.9 Hz, C-1), 72.23 (d, JC5 ,F = 3.6 Hz, C-5), 70.72 (d, JC2 ,F = 27.2 Hz, C-2), 70.48 (d, JC3 ,F = 3.0 Hz, C-3), 68.01 (C-4), 53.14 (OMe), 20.61 (2xOAc), 20.55 (OAc) ppm.  19 F-NMR  (282 MHz; CDCl3 ): δ -135.35 (dd, F-1) ppm. MS : Calcd. for C13 H17 FNaO9 : 359.1; found: 359.3 Methyl (2,3-di-O-acetyl-4-deoxy-α-l-threo-hex-4-enopyranosyl fluoride) uronate (69) COOMe AcO  O F OAc  Globally protected β-d-glucuronyl fluoride (68, 0.103 g, 0.306 mmol) was subjected to the general method for DBU-catalysed elimination (page 186). Purification by flash column chromatography (4:1 petroleum ether/ethyl acetate) gave a colourless syrup (0.028 g, 0.101 mmol, 33%).  1 H-NMR  (400 MHz; CDCl3 ): δ 6.35 (dd,  J3,4 = 4.8, J2,4 = 1.6 Hz, 1H, H-4), 5.93 (dt, J1,F = 49.7, J1,2 = J1,3 = 1.6 Hz, 1H, H-1), 5.17-5.16 (m, 2H, H-2,3), 3.85 (s, 3H, OMe), 2.09 (s, 3H, 2xOAc) ppm. 13 C-NMR  (101 MHz; CDCl3 ): δ 169.83 (OAc), 169.21 (OAc), 161.51 (C-6), 141.98  (C-5), 107.47 (C-4), 101.38 (d, JC1 ,F = 227.7 Hz, C-1), 66.18 (d, JC2 ,F =36.7 Hz, C2), 62.31 (C-3), 52.94 (OMe), 20.81 (OAc), 20.66 (OAc) ppm.  19 F-NMR  (282 MHz;  CDCl3 ): δ -140.59 (d, F-1) ppm. MS : Calcd. for C11 H13 FNaO7 : 299.1; found: 299.3  222  7.2. Synthesis (4-Deoxy-α-l-threo-hex-4-enopyranosyl fluoride)uronic acid (70) COOH HO  O F OH  Globally protected unsaturated β-d-glucuronyl fluoride (69, 28 mg, 0.101 mmol) was subjected to the general method for Zemplén deprotection (page 187) followed by that for hydrolysis by lithium hydroxide (page 187). The product was purified by 5 g C-18 Sep-pak, eluting with water, 10 % acetonitrile in water, 20 % acetonitrile in water, 40 % acetonitrile in water and 100% acetonitrile. All fractions determined by TLC to contain product were pooled and purified further by flash column chromatography (7:2:1 ethyl acetate/methanol/water) then lyophilised to an off-white powder (12.5 mg, 0.071 mmol, 70%). 1 H-NMR (300 MHz; D2 O): δ 6.18 (dd, J3,4 = 4.7, J2,4 = 1.2 Hz, 1H, H-4), 5.95 (dd, J1,F = 51.4, J1,2 = 2.3 Hz, 1H, H-1), 4.18 (dd, J2,3 = 1.2 Hz, 1H, H-3), 4.11 (dt, 1H, H-2) ppm.  13 C-NMR  (101 MHz; D2 O):  δ 167.37 (C-6), 108.70 (C-4), 105.10 (d, JC1 ,F = 222.7 Hz, C-1), 67.30 (d, JC2 ,F = 30.3 Hz, C-2), 63.58 (C-3) ppm.  19 F-NMR  (282 MHz; CDCl3 ): δ -142.35 (d, F-1)  ppm. MS : Calcd. for C6 H6 FO5 : 177.0199; found: 177.0202 Methyl (2,3,4-tri-O-acetyl-α-d-glucopyranosyl fluoride)uronate (71) MeOOC AcO AcO  O AcO F  Globally protected glucuronic acid (2, 0.560 g, 1.488 mmol) was dissolved in 70% HF in pyridine (10 mL) in a plastic container at 0 °C, then stirred overnight at 4 °C. The reaction was quenched with solid NaHCO3 on ice, then diluted with EtOAc. The organic phase was washed with water, sat. NaHCO3 and brine before drying over MgSO4 . Purification by flash column chromatography (5:2 petroleum 223  7.2. Synthesis ether/ethyl acetate) followed by crystallisation from toluene/n-heptane yielded sticky white prisms (0.102 g, 0.303 mmol, 20%). 1 H-NMR (300 MHz; CDCl3 ): δ 5.78 (dd, J1,F = 52.5, J1,2 = 2.5 Hz, 1H, H-1), 5.51 (t, J2,3 = J3,4 = 9.9 Hz, 1H, H-3), 5.19 (t, J4,5 = 9.9 Hz, 1H, H-4), 4.94 (ddd, J2,F = 24.1 Hz, 1H, H-2), 4.43 (d, 1H, H-5), 3.72 (s, 3H, OMe), 2.06 (s, 3H, OAc), 2.00 (s, 6H, 2xOAc) ppm.  13 C-NMR  (75  MHz; CDCl3 ): δ 169.89 (OAc), 169.79 (OAc), 169.48 (OAc), 166.99 (C-6), 103.61 (d, JC1 ,F = 231.2 Hz, C-1), 69.97 (d, JC3 ,F = 4.4 Hz, C-3), 69.94 (d, JC2 ,F = 24.1 Hz, C-2), 68.68 (C-4), 68.54 (C-5), 53.13 (OMe), 20.63 (OAc), 20.53 (OAc), 20.47 (OAc) ppm.  19 F-NMR  (282 MHz; CDCl3 ): δ -150.37 (dd, F-1) ppm. MS : Calcd.  for C13 H17 FNaO9 : 359.1; found: 359.3 Methyl (2,3-di-O-acetyl-4-deoxy-β-l-threo-hex-4-enopyranosyl fluoride) uronate (72) COOMe O  AcO AcO F  Globally protected α-d-glucuronyl fluoride (71, 0.080 g, 0.238 mmol) was subjected to the general method for DBU-catalysed elimination (page 186). Purification by flash column chromatography (5:2 petroleum ether/ethyl acetate) gave a colourless syrup (0.022 g, 0.080 mmol, 34%). 1 H-NMR (300 MHz; CDCl3 ): δ 6.15 (d, J3,4 = 2.3 Hz, 1H, H-4), 5.88 (dd, J1,F = 53.7, J1,2 = 1.9 Hz, 1H, H-1), 5.71 (dd, J2,3 = 8.9 Hz, 1H, H-3), 5.21 (ddd, J2,F = 24.1 Hz, 1H, H-2), 3.83 (s, 3H, OMe), 2.14 (s, 3H, OAc), 2.10 (s, 3H, OAc) ppm.  13 C-NMR  (75 MHz; CDCl3 ): δ 170.16 (OAc), 170.07  (OAc), 161.05 (C-6), 141.06 (C-5), 109.69 (C-4), 103.43 (d, JC1 ,F = 232.7 Hz, C-1), 68.39 (d, JC2 ,F = 21.4 Hz, C-2), 65.64 (d, JC3 ,F = 4.6 Hz, C-3), 52.99 (OMe), 20.96 (OAc), 20.77 (OAc) ppm.  19 F-NMR  (282 MHz; CDCl3 ): δ -146.30 (dd, F-1) ppm.  MS : Calcd. for C11 H13 FNaO7 : 299.1; found: 299.3 224  7.2. Synthesis (4-deoxy-β-l-threo-hex-4-enopyranosyl fluoride)uronic acid (73) COOH O  HO HO F  Globally protected unsaturated α-d-glucuronyl fluoride (72, 22 mg, 0.080 mmol) was subjected to the general method for Zemplén deprotection (page 187). The intermediate was purified by flash column chromatography (9:1 dichloromethane/methanol), then further deprotected by the general method for hydrolysis by lithium hydroxide (page 187). The final product was purified by flash column chromatography (7:2:1 ethyl acetate/methanol/water) and lyophilised to a white powder (11.3 mg, 0.063 mmol, 79%).  1 H-NMR  (300 MHz; D2 O): δ 5.92 (d, J3,4 = 2.2 Hz, 1H, H-4),  5.85 (dd, J1,F = 54.8, J1,2 = 1.5 Hz, 1H, H-1), 4.45 (dt, J2,3 = 8.7, J3,F = 2.8 Hz, 1H, H-3), 3.87 (ddd, J2,F = 26.4 Hz, 1H, H-2) ppm.  13 C-NMR  (75 MHz; D2 O): δ 169.41  (C-6), 109.84 (C-4), 105.39 (t, JC1 ,F = 191.3 Hz, C-1), 69.72 (d, JC2 ,F = 21.7 Hz, C-2), 65.30 (d, JC3 ,F = 5.2 Hz, C-3) ppm.  19 F-NMR  (282 MHz; CDCl3 ): δ -147.46  (br. dd, F-1) ppm. HRMS : Calcd. for C6 H6 FO5 : 177.0199; found: 177.0202 Methyl 3,4-di-O-acetyl-d-glucuronal (74) MeOOC AcO AcO  O  Globally protected α-d-glucuronyl bromide (3, 3.488 g, 8.78 mmol) was dissolved in acetone (17.5 mL) and saturated monobasic sodium phosphate (35 mL), then zinc dust (6.68 g) added and the reaction mixture stirred vigorously overnight at ambient temperature. The reaction slurry was then filtered through Celite, the solvent evaporated in vacuo, and the product dissolved in ethyl acetate before washing with water, saturated NaHCO3 and brine. Purification by flash column chromatography  225  7.2. Synthesis yielded an off-white solid (2.00 g, 7.75 mmol, 88%).  1 H-NMR  (300 MHz; CDCl3 ):  δ 6.57 (d, J1,2 = 5.9 Hz, 1H, H-1), 5.31 (br. q, J3,4 = J4,5 = 2.1, J2,4 = 1.9 Hz, 1H, H-4), 4.91 (td, J2,3 = 5.6 Hz, 1H, H-2), 4.87 (br. dd, 1H, H-3), 4.74 (dd, J3,5 = 1.2 Hz, 1H, H-5), 3.69 (s, 3H, OMe), 2.01 (s, 3H, OAc), 1.89 (s, 3H, OAc) ppm. 13 C-NMR  (75 MHz; CDCl3 ): δ 169.32 (OAc), 169.09 (OAc), 167.05 (C-6), 146.29  (C-1), 97.09 (C-2), 72.17 (C-5), 67.25 (C-3), 62.43 (C-4), 52.21 (OMe), 20.74 (OAc), 20.68 (OAc) ppm. MS : Calcd. for C11 H14 NaO7 : 281.1; found: 281.3 Methyl (2-deoxy-2-fluoro-1,3,4-tri-O-acetyl-α/β-dglucopyranosid)uronate (75) MeOOC AcO AcO  O F  OAc  Methyl 3,4-di-O-acetyl-d-glucuronal (74, 1.75 g, 6.14 mmol) was dissolved in 5:1 nitromethane/acetic acid (210 mL), then cooled to 0 °C. Selectfluor was added in portions, then the reaction mixture allowed to warm to ambient temperature while stirring over 4 days, then refluxed for 1.5hr. After cooling to ambient temperature, the solvents were evaporated in vacuo and the crude product dissolved in dichloromethane for washing with water, saturated NaHCO3 , water again, and brine. Purification by flash column chromatography (3:1, 2:1 then 3:2 petroleum ether/ethyl acetate), with impure fractions being purified a second time (3:1 petroleum ether/ethyl acetate), gave mixtures of alpha and beta gluco- (0.326 g, 0.969 mmol, 16 %, α/β 1:1.05) and manno- (0.481 g, crude, 23%, α/β 3:1) configured isomers as colourless syrups. 1 H-NMR (300 MHz; CDCl3 ): δ 6.37 (d, J1α,2α = 3.9 Hz, 1H, H-1α), 5.77 (dd, J1β,2β = 7.8, J1β,F = 3.5 Hz, 1H, H-1β), 5.49 (dt, J3α,F = 12.1, J2α,3α = J3α,4α = 9.8 Hz, 1H, H-3α), 5.38 (dt, J3β,F = 14.7, J2β,3β = J3β,4β = 9.8 Hz, 1H, H-3β), 5.06 (t, J4α,5α = J4β,5β = 9.8 Hz, 1H, H-4α, β), 4.61 (ddd, J2α,F = 48.3, 226  7.2. Synthesis 1H, H-2α), 4.38 (ddd, J2β,F = 51.8, 1H, H-2β), 4.28 (d, 1H, H-5α), 4.16 (d, 1H, H-5β), 3.63 (s, 6H, OMe α, β), 2.10 (s, 3H, OAc α), 1.99 (s, 6H, OAc α, β), 1.94 (s, 3H, OAc α), 1.93 (s, 3H, OAc β), 1.93 (s, 3H, OAc β) ppm.  19 F-NMR  (282 MHz; CDCl3 ): δ  -201.25 (ddd, F-2β), -203.10 (dd, F-2α) ppm. MS : Calcd. for C11 H14 NaO7 : 281.1; found: 359.3 Methyl (2,4-dinitrophenyl 2-deoxy-2-fluoro-3,4-di-O-acetyl-β-dglucopyranosid)uronate (76) MeOOC AcO AcO  O F  O O2N  NO2  Globally protected 2-deoxy-2-fluoro-glucuronic acid (75, 326 mg, 0.969 mmol) was dissolved in dichloromethane (5 mL), cooled to 0 °C, then acetic anhydride (2 mL) and HBr in acetic acid (33% w/v, 10 mL) added. The reaction was allowed to proceed at 4 °C overnight before being allowed to warm to ambient temperature and stirred a further 24 hours. The solvent was subsequently evaporated in vacuo, then the intermediate dissolved in dichloromethane and washed quickly with cold water, saturated NaHCO3 and brine before drying over MgSO4 and again removing the solvent in vacuo. Dinitrophenol (439 mg, 2.46 eq.) was dried thrice over toluene then dissolved in acetonitrile (15 mL) and added to the pale yellow brominated intermediate along with Ag2 O (1.00 g, 4.45 eq.) and the reaction mixture stirred vigorously at ambient temperature in the dark under an atmosphere of N2 . After three days the reaction mixture was filtered through Celite, the solvent evaporated in vacuo and the product dissolved in ethyl acetate and washed extensively with sat. NaHCO3 , water and brine before drying over MgSO4 . Purification by flash column chromatography (5:2 then 2:1 petroleum ether/ethyl acetate) followed by  227  7.2. Synthesis crystallisation from toluene/hexanes yielded white plates (164 mg, 0.356 mmol, 37% over two steps). 1 H-NMR (300 MHz; CDCl3 ): δ 8.68 (d, J3 ,5 = 2.7 Hz, 1H, H-5 ), 8.41 (dd, J2 ,3 = 9.3 Hz, 1H, H-3 ), 7.51 (d, 1H, H-2 ), 5.68 (dd, J1,F = 6.2, J1,2 = 5.8 Hz, 1H, H-1), 5.44-5.32 (m, 2H, H-3,4), 4.71 (dt, J2,F = 47.8, J2,3 = 5.8 Hz, 1H, H-2), 4.41 (d, J4,5 = 7.2 Hz, 1H, H-5), 3.63 (s, 3H, OMe), 2.09 (s, 3H, OAc), 2.04 (s, 3H, OAc) ppm.  13 C-NMR  (75 MHz; CDCl3 ): δ 169.58 (OAc), 169.44 (OAc),  166.65 (C-6), 153.46 (Ar), 141.96 (Ar), 139.71 (Ar), 128.84 (Ar), 121.56 (Ar), 117.71 (Ar), 97.06 (d, JC1 ,F = 29.5 Hz, C-1), 86.94 (d, JC2 ,F =188.7 Hz, C-2), 72.46 (C-5), 68.86 (d, JC3 ,F = 23.1 Hz, C-3), 67.35 (d, JC4 ,F =5.6 Hz, C-4), 53.05 (OMe), 20.49 (2xOAc) ppm.  19 F-NMR  (282 MHz; CDCl3 ): δ -197.40 (ddd, J3,F = 12.2 Hz, F-2)  ppm. MS : Calcd. for C17 H17 FN2 NaO12 : 483.1; found: 483.2 Methyl (2,4-dinitrophenyl 2,4-dideoxy-2-fluoro-3-O-acetyl-α-l-threo-hex4-enopyranosid)uronate (77) COOMe O O  AcO F  NO2 O2N  Globally protected 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-d-glucuronide (76, 26 mg, 0.057 mmol) was subjected to the general method for DBU-catalysed elimination (page 186), but reacted only 10 min at 0 °C. Purification by flash column chromatography (3:1 petroleum ether/ethyl acetate) gave a colourless syrup (17 mg, 0.042 mmol, 74%). 1 H-NMR (300 MHz; CDCl3 ): δ 8.71 (d, J3 ,5 = 2.6 Hz, 1H, H5 ), 8.49 (dd, J2 ,3 = 9.2 Hz, 1H, H-3 ), 7.76 (d, 1H, H-2 ), 6.37 (dd, J3,4 = 4.8, J2,4 = 1.0 Hz, 1H, H-4), 6.15 (d, J1,F = 4.0 Hz, 1H, H-1), 5.43 (dd, J3,F = 16.2 Hz, 1H, H-3), 5.06 (dd, J2,F = 43.2 Hz, 1H, H-2), 3.81 (s, 3H, OMe), 2.19 (s, 3H, OAc) ppm. 13 C-NMR  (75 MHz; CDCl3 ): δ 170.17 (OAc), 161.22 (C-6), 152.96 (Ar), 142.40 (C228  7.2. Synthesis 5), 141.32 (Ar), 140.22 (Ar), 129.15 (Ar), 121.57 (Ar), 118.00 (Ar), 107.87 (C-4), 93.92 (d, JC1 ,F = 34.8 Hz, C-1), 83.88 (d, JC2 ,F = 178.3 Hz, C-2), 61.89 (d, JC3 ,F = 33.4 Hz, C-3), 53.02 (OMe), 20.75 (OAc) ppm.  19 F-NMR  (282 MHz; CDCl3 ): δ  -196.59 (ddd, F-2) ppm. MS : Calcd. for C15 H13 FN2 NaO10 : 423.1; found: 423.3 2,4-Dinitrophenyl 2,4-dideoxy-2-fluoro-α-l-threo-hex-4enopyranosiduronic acid (78) COOH O O  HO F  NO2 O2N  Globally protected 2,4-dinitrophenyl unsaturated 2-deoxy-2-fluoro-β-d-glucuronide (77, 17 mg, 0.042 mmol) was deprotected using the general method for hydrolysis by lithium hydroxide (page 187). A significant proportion remained acetylated after quenching, so the crude partially deprotected product was subjected to the general method for Zemplén deprotection (page 187). Purification was by flash column chromatography (4:1 ethyl acetate/methanol) followed by 5 g C-18 Sep-pak, eluting with water then 50% methanol. The solvent was evaporated in vacuo, the product dissolved in water and lyophilised to a slightly off-white powder (8 mg, 0.023 mmol, 55%).  1 H-NMR  (300 MHz; D2 O): δ 8.91 (d, J3 ,5 = 2.8 Hz, 1H, H-5 ), 8.60 (dd,  J2 ,3 = 9.4 Hz, 1H, H-3 ), 7.84 (d, 1H, H-2 ), 6.40 (br. t, J3,4 = 3.8, J2,4 = 3.4 Hz, 1H, H-4), 6.15 (d, J1,F = 4.4 Hz, 1H, H-1), 5.12 (d br. t, J2,F = 45.1, J2,3 =3.1 Hz, 1H, H-2), 4.51 (d br. t, J3,F = 17.9 Hz, 1H, H-3) ppm.  13 C-NMR  (101 MHz; D2 O): δ  153.82 (C-6), 142.20 (Ar), 139.76 (Ar), 139.52 (Ar), 130.14 (Ar), 122.40 (Ar), 119.32 (Ar), 107.48 (C-4), 95.18 (d, JC1 ,F = 33.4 Hz, C-1), 86.93 (d, JC2 ,F = 176.2 Hz, C-2), 62.21 (d, JC3 ,F = 29.2 Hz, C-3) ppm.  19 F-NMR  (282 MHz; CDCl3 ): δ -196.3 (ddd,  F-2) ppm. HRMS : Calcd. for C12 H8 FN2 O9 : 343.0214; found: 343.0213 229  7.2. Synthesis 1,2,3,6-Tetra-O-benzoyl-4-deoxy-4-fluoro-α-d-glucopyranose (80) OBz F BzO  O BzO  OBz  (Dimethylamino)sulfur trifluoride (690 µL, 8 eq.) was added dropwise to a solution of 1,2,3,6-Tetra-O-benzoyl-α-d-galactopyranose 216,217 (79, 0.516 g, 0.865 mmol) in dichloromethane (10 mL) at -30 °C, the reaction mixture was stirred for 5 hours, then the reaction was quenched with methanol. The reaction mixture was diluted with dichloromethane then washed with ice cold water, saturated NaHCO3 and brine before drying over MgSO4 . Purification by flash column chromatography (4:1 petroleum ether/ethyl acetate) gave the product as a white foam (0.496 g, 0.829 mmol, 96%).  1 H-NMR  (300 MHz; CDCl3 ): δ 8.22-7.83 (m, 8H, Ar), 7.71-7.27 (m, 12H,  Ar), 6.81 (d, J1,2 = 3.5 Hz, 1H, H-1), 6.31 (dt, J3,F = 13.9, J2,3 = J3,4 = 9.9 Hz, 1H, H-3), 5.61 (dd, 1H, H-2), 4.98 (dt, J4,F = 50.4, J4,5 = 9.9 Hz, 1H, H-4), 4.77-4.63 (m, 2H, H-5,6a ), 4.55 (dd, J6a ,6b = 9.6, J5,6b = 3.3 Hz, 1H, H-6b ) ppm.  13 C-NMR  (75 MHz; CDCl3 ): δ 166.16 (RCOOR), 165.80 (RCOOR), 165.49 (RCOOR), 164.38 (RCOOR), 134.15 (Ar), 133.73 (Ar), 133.68 (Ar), 133.46 (Ar), 130.11 (Ar), 129.98 (Ar), 129.92 (Ar), 129.63 (Ar), 128.95 (Ar), 128.62 (Ar), 128.58 (Ar), 128.49 (Ar), 89.83 (C-1), 86.85 (d, JC4 ,F = 188.0 Hz, C-4), 70.57 (d, JC3 ,F = 19.8 Hz, C-3), 70.09 (d, JC5 ,F = 10.6 Hz, C-5), 69.88 (d, JC2 ,F = 4.9 Hz, C-2), 62.13 (C-6) ppm.  19 F-NMR  (282 MHz; CDCl3 ): δ -198.00 (dd, F-4) ppm. MS : Calcd. for C34 H27 FNaO9 : 621.2; found: 621.4  230  7.2. Synthesis 4-Nitrophenyl 2,3,6-tetra-O-benzoyl-4-fluoro-β-d-glucopyranoside (81) OBz F BzO  O OBz  O NO2  Globally benzoylated 4-deoxy-4-fluoro-glucose (80, 496 mg, 0.829 mmol) was dissolved in dichloromethane (2 mL), cooled to 0 °C, then acetic anhydride (100 µL) and HBr in acetic acid (33% w/v, 10 mL) added. The reaction was allowed to proceed at 4 °C overnight. The reaction was quenched on ice/water, extracted with four portions of dichloromethane and the pooled organic phase washed with cold water, sat. NaHCO3 , and brine before drying over MgSO4 and removing the solvent in vacuo. The intermediate bromide and 4-nitrophenol (126 mg, 1.1 eq.) were dried twice over toluene then dissolved in acetonitrile (20 mL), Ag2 O (620 mg, 3 eq.) added, and the reaction mixture stirred vigorously overnight at ambient temperature in the dark under an atmosphere of argon. The reaction mixture was subsequently filtered through Celite, the solvent evaporated in vacuo and the product purified by flash column chromatography (6:1 petroleum ether/ethyl acetate) to afford white powder (322 mg, 0.523 mmol, 63% over two steps).  1 H-NMR  (300 MHz; CDCl3 ):  δ 8.16-7.96 (m, 6H, OBz), 7.68-7.37 (m, 9H, OBz), 7.06 (d, J2 ,3 = J5 ,6 = 9.2 Hz, 2H, H-3 ,5 ), 6.94 (d, 2H, H-2 ,6 ), 5.99 (dt, J3,F = 14.4, J2,3 = J3,4 = 9.1 Hz, 1H, H-3), 5.77 (dd, J1,2 = 7.6 Hz, 1H, H-2), 5.55 (d, 1H, H-1), 4.94 (dt, J4,F = 50.6, J4,5 = 9.1 Hz, 1H, H-4), 4.85 (br, t, J5,6a = 12.1 Hz, 1H, H-5), 4.65 (dd, J6a ,6b = 6.4 Hz, 1H, H-6a ), 4.37 (td, J5,6b = 2.9 Hz, 1H, H-6b ) ppm.  13 C-NMR  (75 MHz; CDCl3 ):  δ 166.19 (RCOOR), 165.76 (RCOOR), 165.25 (RCOOR), 162.25 (Ar), 161.00 (Ar), 143.28 (Ar), 133.85 (Ar), 130.01 (Ar), 129.94 (Ar), 129.77 (Ar), 128.71 (Ar), 128.65 (Ar), 128.59 (Ar), 126.30 (Ar), 125.78 (Ar), 116.86 (Ar), 115.74 (Ar), 98.21 (C-1), 87.25 (d, JC4 ,F = 189.7 Hz, C-4), 72.78 (d, JC5 ,F = 20.1 Hz, C-5), 72.28 (d, JC3 ,F 231  7.2. Synthesis = 23.8 Hz, C-3), 71.16 (d, JC2 ,F = 7.8 Hz, C-2), 62.52 (C-6) ppm.  19 F-NMR  (282  MHz; CDCl3 ): δ -199.03 (dd, F-4) ppm. MS : Calcd. for C33 H26 FNNaO10 : 638.1; found: 638.5 4-Nitrophenyl 4-fluoro-β-d-glucopyranoside (82) OH F HO  O OH  O NO2  Globally benzoylated 4-nitrophenyl 4-deoxy-4-fluoro-β-d-glucoside (81, 288 mg, 0.468 mmol) was subjected to the general method for Zemplén deprotection (page 187). The reaction was quenched with Sephadex ion exchange resin (H+ form) then filtered. Purification by flash column chromatography (7% methanol in dichloromethane) gave a white powder (63 mg, 0.208 mmol, 44%).  1 H-NMR(400  MHz;  MeOD): δ 8.21 (d, J2 ,3 = J5 ,6 = 9.2 Hz, 2H, H-3 ,5 ), 7.23 (d, 2H, H-2 ,6 ), 5.14 (d, J1,2 = 7.8 Hz, 1H, H-1), 4.36 (dt, J4,F = 50.8, J3,4 = J4,5 = 9.2 Hz, 1H, H-4), 3.89-3.72 (m, 4H, H-3,5,6a ,6b ), 3.56 (dd, J2,3 = 9.1 Hz, 1H, H-2) ppm.  13 C-NMR  (101 MHz; MeOD): δ 163.61 (Ar), 143.93 (Ar), 126.59 (Ar), 117.68 (Ar), 101.31 (C-1), 90.05 (d, JC4 ,F = 181.0 Hz, C-4), 75.65 (C-2), 75.43 (d, JC3 ,F = 7.7 Hz, C-3), 74.36 (d, JC5 ,F = 8.4 Hz, C-5), 61.39 (C-6) ppm.  19 F-NMR  (282 MHz; MeOD): δ  -201.66 (dd, J3,F = 16.1 Hz, F-4) ppm. MS : Calcd. for C12 H14 FNNaO7 : 326.1; found: 326.1  232  7.2. Synthesis 4-Nitrophenyl 4-fluoro-β-d-glucopyranosiduronic acid (83) HOOC F HO  O OH  O NO2  4-nitrophenyl 4-deoxy-4-fluoro-β-d-glucoside (82, 63 mg, 0.208 mmol), NaBr (15 mg, 0.7 eq.), and TEMPO (3 mg, 0.1 eq.) were dissolved in THF/water (1:2, 15 mL), cooled to 0 °C, then a solution of sodium hypochlorite (5%, 800 µL) added slowly. Basicity of the reaction was maintained with 0.2 M NaOH. After 10 min, the reaction was quenched with ethanol, acidified with Sephadex ion exchange resin (H+ form), then filtered and the solvent evaporated in vacuo. Purification by 5 g C-18 Sep-pak (eluting with 0%, 30%, 50%, and 100% methanol in water) followed by lyophilisation gave the product as a white powder (63 mg, 0.199 mmol, 95%). 1 H-NMR(400  MHz; MeOD): δ 8.19 (d, J2 ,3 = J5 ,6 = 9.2 Hz, 2H, H-3 ,5 ), 7.22 (d,  2H, H-2 ,6 ), 5.24 (d, J1,2 = 7.8 Hz, 1H, H-1), 4.48 (dt, J4,F = 49.9, J3,4 = J4,5 = 9.2 Hz, 1H, H-4), 4.37 (dd, J5,F = 4.1 Hz, 1H, H-5), 3.85 (dt, J3,F = 16.0, J2,3 = 9.2 Hz, 1H, H-3), 3.61 (dd, 1H, H-2) ppm.  13 C-NMR  (101 MHz; MeOD): δ 170.67  (C-6), 163.23 (Ar), 144.07 (Ar), 126.61 (Ar), 117.65 (Ar), 101.11 (C-1), 91.50 (d, JC4 ,F = 185.5 Hz, C-4), 75.21 (C-2), 75.03 (C-5), 73.95 (d, JC2 ,F = 8.7 Hz, C-3) ppm.  19 F-NMR  (282 MHz; MeOD): δ -200.57 (ddd, F-4) ppm. MS : Calcd. for  C12 H11 FNO8 : 316.1; found: 316.3  233  7.2. Synthesis Methyl (4-nitrophenyl 2,3-di-O-acetyl-4-fluoro-β-dglucopyranosid)uronate (84) MeOOC F AcO  O OAc  O NO2  4-nitrophenyl 4-deoxy-4-fluoro-β-d-glucopyranosiduronic acid (83, 57 mg, 0.180 mmol) was dissolved in methanol (5 mL) and acetyl chloride (50 µL) added. The reaction mixture was stirred for two hours then the solvent evaporated in vacuo and replaced with acetic anhydride (5 mL) acidified with Sephadex ion exchange resin (H+ form). After 2.5 hours the reaction mixture was filtered and the solvent again evaporated in vacuo. Purification by flash column chromatography (3:1 petroleum ether/ethyl acetate) yielded a white powder (63 mg, 0.152 mmol, 73%). 1 H-NMR(400  MHz; CDCl3 ): δ 8.20 (d, J2 ,3 = J5 ,6 = 9.2 Hz, 2H, H-3 ,5 ), 7.08 (d,  2H, H-2 ,6 ), 5.44 (dt, J3,F = 14.3, J2,3 = J3,4 = 8.6 Hz, 1H, H-3), 5.36 (d, J1,2 = 6.9 Hz, 1H, H-1), 5.23 (dd, 1H, H-2), 4.93 (dt, J4,F = 49.3, J4,5 = 8.6 Hz, 1H, H-4), 4.36 (dd, J5,F = 6.6 Hz, 1H, H-5), 3.75 (s, 3H, Me), 2.11 (s, 3H, OAc), 2.06 (s, 3H, OAc) ppm.  19 F-NMR  (282 MHz; CDCl3 ): δ -198.11 (ddd, F-4) ppm. MS : Calcd.  for C17 H18 FNNaO10 : 438.1; found: 438.3 Methyl (4-nitrophenyl 2,3-di-O-acetyl-4-fluoro-5-bromo-β-dglucopyranosid)uronate (85) MeOOC F AcO  O Br OAc  O NO2  Globally protected methyl (4-nitrophenyl 4-deoxy-4-fluoro-β-d-glucopyranosid)uronate (84, 63 mg, 0.152 mmol), CaCO3 (72 mg), and bromine (100 µL, 15 eq.) were dissolved in carbon tetrachloride (10 mL) and irradiated with a 300 W light 234  7.2. Synthesis bulb. After one hour, the reaction mixture was diluted with dichloromethane and washed with water, 1 M Na2 S2 O3 , saturated NaHCO3 , and brine then dried over MgSO4 . Purification by flash column chromatography (4:1 petroleum ether/ethyl acetate) yielded a white foam (64 mg, 0.129 mmol, 85%).  1 H-NMR  (400 MHz;  CDCl3 ): δ 8.25 (d, J2 ,3 = J5 ,6 = 9.2 Hz, 2H, H-3 ,5 ), 7.15 (d, 2H, H-2 ,6 ), 5.80 (d, J1,2 = 8.4 Hz, 1H, H-1), 5.71 (dt, J3,F = 11.8, J2,3 = J3,4 = 9.2 Hz, 1H, H-3), 5.37 (dd, 1H, H-2), 4.84 (dd, J4,F = 48.2 Hz, 1H, H-4), 3.92 (s, 3H, OMe), 2.13 (s, 3H, OAc), 2.09 (s, 3H, OAc) ppm.  19 F-NMR  (282 MHz; CDCl3 ): δ -185.92 (dd, F-4)  ppm. MS : Calcd. for C17 H17 BrFNNaO10 : 516.0/518.0; found: 516.2/518.2  7.2.7  Substrates for kinetic isotope effects  Methyl (1,5-anhydro-2,3,4-tri-O-acetyl-d-glucaropyran)uronate (90) MeOOC AcO AcO  O AcO  O  Globally protected glucuronyl hemiacetal (7, 0.748 g, 2.24 mmol) was dissolved in dichloromethane (25 mL), then Dess-Martin periodinane (1.432 g, 1.5 eq.) added and the reaction allowed to proceed overnight at ambient temperature. Following dilution with dichloromethane the product was washed with water, sat. NaHCO3 and brine, then dried over MgSO4 . Purification by flash column chromatography (2:1 petroleum ether/ethyl acetate) yielded a colourless syrup (0.620 g, 1.87 mmol, 83%). 1 H-NMR (400 MHz; CDCl3 ): δ 5.48 (d, J4,5 = 7.0 Hz, 1H, H-5), 5.31 (t, J2,3 = J3,4 = 3.1 Hz, 1H, H-3), 5.12 (ddd, J2,4 = 1.3 Hz, 1H, H-4), 5.00 (dd,1H, H-2), 3.80 (s, 3H, OMe), 2.08 (s, 3H, OAc), 2.05 (s, 3H, OAc), 1.98 (s, 3H, OAc) ppm. MS : Calcd. for C13 H16 NaO10 : 355.1; found: 355.3  235  7.2. Synthesis Methyl (2,3,4-tri-O-acetyl-1-{2 H}-α-d-glucopyran)uronate (91) MeOOC AcO AcO  O AcO  D OH  Globally protected glucuronic acid 1,5-lactone (90, 0.620 g, 1.87 mmol) in THF at 0 °C was slowly added an ice-cold solution of NaBD4 (40 mg, 0.956 mmol) in D2 O (150µL), and the reaction then allowed to proceed with stirring for a further 5 min before quenching with 300 µL acetic acid. The reaction mixture was then filtered and the solvent evaporated in vacuo, followed by co-evaporation with methanol twice to remove boric acid. Purification by flash column chromatography (1:1 petroleum ether/ethyl acetate) yielded a white foam (0.364 g, 1.09 mmol, 58%). 1 H-NMR (300 MHz; CDCl3 ): δ 5.59 (t, J2,3 = J3,4 = 9.7 Hz, 1H, H-3), 5.18 (t, J4,5 = 9.7 Hz, 1H, H-4), 4.90 (d, 1H, H-2), 4.60 (d, 1H, H-5), 3.75 (s, 3H, OMe), 2.09 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.04 (s, 3H, OAc) ppm. MS : Calcd. for C13 H17 DNaO10 : 358.1; found: 358.2 Methyl (1,2,3,4-tetra-O-acetyl-1-{2 H}-α-d-glucopyranosid)uronate (92) MeOOC AcO AcO  O AcO  D OAc  Globally protected 1-{2 H}-glucuronyl hemiacetal (91, 0.364 g, 1.09 mmol) was dissolved in dichloromethane (10 mL) then acetic anhydride (2.25 mL) followed by tifluoroacetic acid (270 µL) added, and stirred at ambient temperature until TLC indicated complete reaction (1:1 petroleum ether/ethyl acetate). The reaction was then quenched with ice/water, diluted with dichloromethane and washed with sat. NaHCO3 , and brine then dried over MgSO4 . Purification by flash column chromatography (2:1 to 1:1 petroleum ether/ethyl acetate) yielded a white foam (0.374 g, 236  7.2. Synthesis 0.991 mmol, 91%). MS : Calcd. for C15 H19 DNaO11 : 400.1; found: 400.3 Methyl (bromo 2,3,4-tri-O-acetyl-1-{2 H}-α-d-glucopyranosid)uronate (93) MeOOC AcO AcO  O D  AcO Br  Globally protected 1-{2 H}-glucuronic acid (92, 0.374 g, 0.991 mmol) was dissolved in dichloromethane (2.5 mL) then acetic anhydride (0.5 mL) and 33% HBr in acetic acid (10 mL) were added at 0 °C. This was stirred at 4 °C until the reaction was complete as judged by TLC (2:1 petroleum ether/ethyl acetate). The reaction was quenched in ice/water, the aqueous layer extracted thrice with dichloromethane and this pooled organic phase then extracted quickly with cold water, cold sat. NaHCO3 twice and brine, followed by drying over MgSO4 . Purification by flash column chromatography (2:1 petroleum ether/ethyl acetate) yielded a colourless syrup that was used immediately for the subsequent step (0.339 g, 0.852 mmol, 86%) Methyl (2,4,6-trichlorophenyl 2,3,4-tri-O-acetyl-1-{2 H}-α-dglucopyranosid)uronate (94) MeOOC AcO AcO  Cl  O O  AcO D  Cl  Cl  2,4,6-Trichlorophenol (0.109 g, 0.564 mmol, 1.3 eq.) was reacted with globally protected 1-{2 H}-glucuronyl bromide (93, 0.173 g, 0.434 mmol) by the general method for Koenigs-Knorr glycosylation (page 186). Purification by flash column chromatography (2:1 petroleum ether/ethyl acetate) followed by recrystallisation from ethanol yielded small white needles (0.112 g, 0.217 mmol, 50%). 1 H-NMR (300 237  7.2. Synthesis MHz; CDCl3 ): δ 7.32 (s, 2H, H-3 ,5 ), 5.38-5.30 (m, 3H, H-2,3,4), 3.97-3.94 (m, 1H, H-5), 3.72 (s, 3H, OMe), 2.09 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.02 (s, 3H, OAc) ppm. MS : Calcd. for C19 H18 DCl3 NaO10 : 536.0/538.0; found: 536.1/539.1 Methyl (2,4,6-trichlorophenyl 2,3-di-O-acetyl-4-deoxy-1-{2H}-α-l-threohex-4-enopyranosid)uronate (95)  AcO  COOMe Cl O O AcO D Cl  Cl  Globally protected 2,4,6-trichlorophenyl 1-{2 H}-glucuronide (94, 43 mg, 0.0835 mmol) was subjected to the general method for DBU-catalysed elimination (page 186). Purification by flash column chromatography (3:1 petroleum ether/ethyl acetate) gave a white powder (32 mg, 0.070 mmol, 84%). 1 H-NMR (300 MHz; CDCl3 ): δ 7.32 (s, 2H, H-3 ,5 ), 6.40 (dd, J3,4 = 5.0, J2,4 = 1.2 Hz, 1H, H-4), 5.47 (br. s, 1H, H-2), 5.23 (dd, J2,3 = 1.2 Hz, 1H, H-3), 3.83 (s, 3H, OMe), 2.11 (s, 3H, OAc), 2.10 (s, 3H, OAc) ppm.  13 C-NMR  (75 MHz; CDCl3 ): δ 170.2 (OAc), 169.3 (OAc),  162.1 (C-6), 147.6 (C-5), 142.9 (Ar), 131.1 (Ar), 130.2 (Ar), 129.1 (Ar), 106.9 (C-4), 67.2 (C-3), 63.1 (C-2), 52.8 (OMe), 21.01 (OAc), 20.84 (OAc) ppm. MS : Calcd. for C17 H14 DCl3 NaO8 : 477.0/479.0; found: 476.2/478.2  238  7.2. Synthesis 2,4,6-Trichlorophenyl 4-deoxy-1-{2 H}-α-l-threo-hex-4enopyranosiduronic acid (96) COOH  Cl  O O  HO  Cl  HO D Cl  Globally protected 2,4,6-trichlorophenyl unsaturated 1-{2 H}-glucuronide (95, 32 mg, 0.070 mmol) was deprotected by the general method for ester saponification (page 187). Purification was by 5 g C-18 Sep-pak, washed with water, 40% acetonitrile in water and 100% acetonitrile. All fractions containing product as determined by TLC (3:3:1:1 toluene/ethyl acetate/methanol/acetic acid) were pooled and lyophilised to give a white powder (16.7 mg, 0.047 mmol, 67%). Final deuterium incorporation level measured to be 94.2% as determined by 1 H-NMR integrals. 1 HNMR (400 MHz; CD3 OD): δ 7.50 (s, 2H, H-3 ,5 ), 6.36 (dd, J3,4 = 4.9, J2,4 = 1.4 Hz, 1H, H-4), 4.30 (t, J2,3 = 1.7 Hz, 1H, H-2), 4.09 (dd, 1H, H-3) ppm. HRMS : Calcd. for C12 H8 DCl3 NaO6 : 377.9425; found: 377.9423 Methyl (2,4,6-trichlorophenyl 2,3-di-O-acetyl-4-deoxy-4,5-{2H}-β-dglucopyranosid)uronate (97) MeOOC D AcO  Cl  O D  OAc  O Cl  Cl  Globally protected 2,4,6-trichlorophenyl unsaturated β-d-glucuronide (59, 97 mg, 0.214 mmol) was dissolved in ethyl acetate and a suspension of palladium on carbon (10% w/w) was added. The reaction vessel was purged with a vacuum aspirator, flushed with D2 gas (three times each), then left sealed to react overnight. The catalyst was removed by filtration through Celite and the product purified by flash 239  7.2. Synthesis column chromatography (3:1 petroleum ether/ethyl acetate) to give a white powder (62 mg, 0.135 mmol, 63% C6 equatorial + 36 mg, 0.079 mmol, 37% C6 axial; overall quantitative). 1 H-NMR (300 MHz; CDCl3 ): δ 7.31 (s, 2H, H-3 ,5 ), 5.29 (dd, J2,3 = 9.4, J1,2 = 7.6 Hz, 1H, H-2), 5.11 (dd, J3,4 = 11.6 Hz, 1H, H-3), 5.11 (d, 1H, H-1), 3.74 (s, 3H, OMe), 2.11 (s, 3H, OAc), 2.07 (s, 3H, OAc), 1.93 (d, 1H, H-4) ppm. 13 C-NMR  (75 MHz; CDCl3 ): δ 170.4 (OAc), 169.7 (OAc), 168.6 (C-6), 148.29 (Ar),  130.9 (Ar), 130.5 (Ar), 129.12 (Ar), 101.5 (C-1), 72.01 (C-3), 70.1 (C-2), 60.53 (C-5), 52.7 (OMe), 21.0 (2xOAc), 14.35 (C-4) ppm. MS : Calcd. for C17 H15 D2 Cl3 NaO: 479.0/481.0; found: 479.2/481.2 Methyl (2,4,6-trichlorophenyl 2,3-di-O-acetyl-4-deoxy-5-bromo-4-{2H}β-d-glucopyranosid)uronate (98) MeOOC D AcO  Cl  O Br  OAc  O Cl  Cl  Methyl (2,4,6-trichlorophenyl 2,3-di-O-acetyl-4-deoxy-4,5-{2 H}-β-d-glucopyranosid)uronate (97, 62 mg, 0.135 mmol) was dissolved in carbon tetrachloride, N bromosuccinimide (29 mg, 1.2 eq.) and benzoyl peroxide (0.8 mg, 2.4 mol %) added, and the reaction mixture heated under reflux for 5 hours before cooling and concentrating. Purification by flash column chromatography (gradient 5:1 to 4:1 petroleum ether/ethyl acetate) gave a white powder (47 mg, 0.088 mmol, 65%). 1 H-NMR (300 MHz; CDCl3 ): δ 7.33 (s, 2H, H-3 ,5 ), 5.59 (d, J1,2 = 7.9 Hz, 1H, H-1), 5.56 (dd, J3,4 = 10.9, J2,3 = 9.7 Hz, 1H, H-3), 5.38 (dd, 1H, H-2), 3.86 (s, 3H, OMe), 2.32 (d, 1H, H-4), 2.13 (s, 3H, OAc), 2.06 (s, 3H, OAc) ppm.  13 C-NMR  (75 MHz; CDCl3 ):  δ 170.0 (OAc), 169.7 (OAc), 165.6 (C-6), 146.6 (Ar), 131.3 (Ar), 130.2 (Ar), 129.3 (Ar), 101.3 (C-1), 87.3 (C-5), 71.4 (C-3), 68.4 (C-2), 53.9 (OMe), 20.94 (C-4), 20.91  240  7.2. Synthesis (2xOAc) ppm. MS : Calcd. for C17 H15 DBrCl3 NaO8 : 555.9/557.9/559.9; found: 556.1/558.1/560.2 Methyl (2,4,6-trichlorophenyl 2,3-di-O-acetyl-4-deoxy-4-{2H}-α-l-threohex-4-enopyranosid)uronate (99) D AcO  COOMe Cl O O OAc  Cl  Cl  Methyl (2,4,6-trichlorophenyl 2,3-di-O-acetyl-4-deoxy-4-{2 H}-5-bromo-β-d-glucopyranosid)uronate (98, 29 mg, 54 µmol) was subjected to the general method for DBU-catalysed elimination (page 186). Purification by flash column chromatography (4:1 petroleum ether/ethyl acetate) gave a white powder (15 mg, 33 µmol, 72%). 1 HNMR (300 MHz; CDCl3 ): δ 7.32 (s, 2H, H-3 ,5 ), 5.83 (dd, J1,2 = 1.9, J1,3 = 1.1 Hz, 1H, H-1), 5.49 (br. t, J2,3 = 1.7 Hz, 1H, H-2), 5.23 (br. t, 1H, H-3), 3.83 (s, 3H, OMe), 2.11 (s, 3H, OAc), 2.11 (s, 3H, OAc) ppm.  13 C-NMR  (75 MHz; CDCl3 ):  δ 170.3 (OAc), 169.3 (OAc), 166.1 (C-6), 147.6 (C-5), 142.9 (Ar), 131.1 (Ar), 130.2 (Ar), 129.2 (Ar), 96.8 (C-1), 67.2 (C-3), 63.0 (C-2), 52.8 (OMe), 21.04 (OAc), 20.87 (OAc) ppm (carbon 4 signal too weak to detect, but seen in non-deuterated form). MS : Calcd. for C17 H14 DCl3 NaO8 : 476.0/478.0; found: 476.1/478.2  241  7.2. Synthesis 2,4,6-Trichlorophenyl 4-deoxy-4-{2 H}-α-l-threo-hex-4enopyranosiduronic acid (100) COOH  D  Cl  O O OH  HO  Cl  Cl  Globally protected 2,4,6-trichlorophenyl unsaturated 4-{2 H}-β-d-glucuronide (99, 15 mg, 33 µmol) was deprotected by the general method for ester saponification (page 187). Purification was by 5 g C-18 Sep-pak, washed with water, 10% acetonitrile in water, 40% acetonitrile in water and 100% acetonitrile. All fractions containing product as determined by TLC (3:3:1:1 toluene/ethyl acetate/methanol/acetic acid) were pooled and lyophilised to give a white powder (11 mg, 31 µmol, 54%). Final deuterium incorporation level measured to be 90.5% as determined by 1 H-NMR integrals.  1 H-NMR  (400 MHz; CD3 OD): δ 7.49 (s, 2H, H-3 ,5 ), 5.74 (dd, J1,2 =  2.3, J1,3 = 0.9 Hz, 1H, H-1), 4.30 (t, J2,3 = 2.3 Hz, 1H, H-2), 4.08 (dd, 1H, H3) ppm.  13 C-NMR  (101 MHz; CD3 OD): δ 172.19 (C-6), 149.57 (Ar), 131.72 (Ar),  131.44 (Ar), 130.07 (Ar), 102.25 (C-1), 69.98 (C-3), 65.90 (C-2) ppm (carbon 4 and 5 signals too weak to detect, but seen in non-deuterated form). HRMS : Calcd. for C12 H8 DCl3 NaO6 :377.9425; found: 377.9427  7.2.8  Potential inhibitors of UGL  2-Deoxy-2,3-didehydro-neuraminic acid (108) 177 HO  OH  TFA-.+H3N HO HO  COOH O  Protected 2-deoxy-2,3-didehydro-N -acetyl-neuraminic acid (104, 36 mg, 0.076  242  7.2. Synthesis mmol) was deprotected by the method of Gervay et al. 176,177 Final purification by 5 g C8 Sep-pak (eluted with water in 1 mL fractions) and subsequent lyophilisation yielded a white powder (21.9 mg, 60 µmol, 79% over 4 steps). NMR characterisation matched literature values. 218  7.2.9  Potential trapping reagents for UGL  2-Keto-3-deoxy-3-fluoro-d-glycero-d-galactonononic acid (109) 180 OH HO HO  HO HO  OH O  COOH F  β-Fluoropyruvic acid (0.504 g, 4.75 mmol) and D-Mannose (1.703 g, 2 eq.) were dissolved in water (45 mL), Neuraminic acid aldolase (36 mg) was added, and the reaction allowed to proceed at ambient temperature until  19 F-NMR  indicated con-  sumption of the fluoropyruvic acid (12 days, with a second 44 mg portion of aldolase added after 4 days). The product was then filtered through a cotton plug and purified by Dowex 1XB ion exchange resin (pre-equilibrated with 6 M formic acid then water) eluting with water until no more mannose elutes, then 1 M formic acid to elute the product. Fractions containing product were pooled and the solvent evaporated in vacuo until the volume was sufficiently low for lyophilisation, which yielded a white foam (1.07 g, 3.74 mmol, 79 %, 4.35:1 ax/eq F).  19 F-NMR  (282 MHz; H2 O+D2 O):  δ -199.85 (dd, JF,3 = 50.0, JF,4 = 13.3 Hz, F-3eq ), -207.09 (dd, JF,3 = 48.5, JF,4 = 30.1 Hz, F-3ax ) ppm. MS : Calcd. for C9 H14 FO9 : 285.1; found: 285.3  243  7.2. Synthesis Methyl 2,4,5,7,8,9-hexa-O-acetyl-3-deoxy-3-fluoro-d-glycero-d-galacto-2nonulopyranosonate (110) 180 OAc AcOAcO AcOAcO  OAc O  COOMe F  A solution of 2-keto-3-deoxy-3-fluoro-d-glycero-d-galactonononic acid (109, 1.07 g, 3.74 mmol) in methanol (13.4 mL) and trifluoroacetic acid (670 µL) was stirred at ambient temperature for four hours. The solvents were then evaporated in vacuo, the intermediate methyl ester dissolved in pyridine (13 mL) and acetic anhydride (4 mL), and allowed to react overnight at ambient temperature. Solvents were again evaporated in vacuo, the product dissolved in ethyl acetate, and washed with water, saturated NaHCO3 , 1 N HCl and brine. Purification by flash column chromatography (1:1 petroleum ether/ethyl acetate) yielded a white foam (1.19 g, 2.15 mmol, 58%).  1 H-NMR  (300 MHz; CDCl3 ): δ 5.39-5.27 (m, 3H, H-4,5,7), 5.16 (td, J7,8 =  J8,9b = 5.8, J8,9a = 2.1 Hz, 1H, H-8), 4.96 (dd, J3,F = 48.7, J3,4 = 2.0 Hz, 1H, H-3), 4.55 (dd, J9a ,9b = 12.5 Hz, 1H, H-9a ), 4.17 (dd, 1H, H-9b ), 4.12-4.08 (m, 1H, H-6), 3.84 (s, 3H, OMe), 2.18 (s, 3H, OAc), 2.12 (s, 3H, OAc), 2.09 (s, 3H, OAc), 2.04 (s, 3H, OAc), 2.03 (s, 3H, OAc), 2.02 (s, 3H, OAc) ppm.  19 F-NMR  (282 MHz; CDCl3 ):  δ -208.19 (dd, JF,4 = 29.4 Hz, F-3) ppm. MS : Calcd. for C22 H29 FNaO15 : 575.1; found: 575.3  244  7.2. Synthesis Methyl 4,5,7,8,9-penta-O-acetyl-3-deoxy-3-fluoro-d-glycero-d-galacto-2nonulopyranosonate (111) 180 OAc AcOAcO AcOAcO  OH O  COOMe F  Methyl 2,4,5,7,8,9-hexa-O-acetyl-3-deoxy-3-fluoro-d-glycero-d-galacto-2-nonulopyranosonate (110, 0.960 mg, 1.74 mmol) was dissolved in methanol (15 mL), hydrazine acetate (0.331 g, 2 eq.) added at 0 °C and the reaction mixture stirred at ambient temperature for 1 hour. The solvent was evaporated in vacuo then the product dissolved in ethyl acetate and washed with water, 1 N HCl, and brine before drying over MgSO4 . Purification by flash column chromatography (3:2 petroleum ether/ethyl acetate) yielded a white foam (0.620 g, 1.21 mmol, 70 %), while 85 mg of unreacted starting material was recovered (9%). 1 H-NMR (300 MHz; CDCl3 ): δ 5.45-5.15 (m, 4H, H-4,5,7,8), 4.91 (dd, J3,F = 49.5, J3,4 = 1.6 Hz, 1H, H-3), 4.57 (dd, J9a ,9b = 11.9, J8,9a = 1.3 Hz, 1H, H-9a ), 4.27 (d, J5,6 = 9.6 Hz, 1H, H-6), 4.07 (dd, J8,9b = 6.4 Hz, 1H, H-9b ), 3.79 (s, 3H, OMe), 2.06 (s, 3H, OAc), 2.02 (s, 3H, OAc), 2.01 (s, 3H, OAc), 1.98 (s, 3H, OAc), 1.96 (s, 3H, OAc) ppm.  13 C-NMR  (75  MHz; CDCl3 ): δ 171.15 (OAc), 170.75 (OAc), 170.27 (br. s, 2xOAc), 169.60 (OAc), 167.48 (C-1), 94.43 (d, JC2 ,F = 25.6 Hz, C-2), 87.73 (d, JC3 ,F = 184.9 Hz, C-3), 70.62 (C-6), 70.43 (d, JC4 ,F = 16.9 Hz, C-4), 69.65 (C-7), 67.20 (C-8), 64.29 (C-5), 62.70 (C-9), 53.55 (OMe), 21.03 (OAc), 20.84 (OAc), 20.76 (OAc), 20.70 (br. s, 2xOAc) ppm.  19 F-NMR  (282 MHz; CDCl3 ): δ -205.08 (dd, J4,F = 26.8 Hz, F-3) ppm. MS :  Calcd. for C20 H27 FNaO14 : 533.1; found: 533.2  245  7.2. Synthesis Methyl (fluoro 4,5,7,8,9-penta-O-acetyl-3-deoxy-3-difluoro-d-glycero-dgalacto-2-nonulopyrano)sonate (112) 180 OAc AcOAcO AcOAcO  COOMe O  F F  (Dimethylamino)sulfur trifluoride (120 µL) was added dropwise to a solution of methyl 4,5,7,8,9-penta-O-acetyl-3-deoxy-3-fluoro-d-glycero-d-galacto-2-nonulopyranosonate (111, 0.375 g, 0.735 mmol) in dichloromethane (10 mL) at 0 °C, the reaction mixture was stirred for 35 min, then the reaction was quenched with methanol. The reaction mixture was diluted with dichloromethane then washed with ice cold water, saturated NaHCO3 and brine before drying over MgSO4 . Purification by flash column chromatography (2:1 petroleum ether/ethyl acetate) gave the product as a colourless syrup (0.164 g, 0.320 mmol, 44%), with the axial anomer also recovered (0.129 g, 0.252 mmol, 34%).  1 H-NMR  (300 MHz; CDCl3 ): δ 5.31-4.98 (m, 5H, H-  2,3,4,7,8), 4.28-4.07 (m, 3H, H-6,9a,9b), 3.86 (s, 3H, OMe), 2.06 (br. s, 6H, OAc x 2), 2.04 (s, 3H, OAc), 1.99 (s, 3H, OAc), 1.98 (s, 3H, OAc).  13 C-NMR  (75 MHz;  CDCl3 ): δ 170.55 (OAc), 169.90 (OAc), 169.78 (OAc), 169.60 (OAc), 169.16 (OAc), 164.22 (dd, J = 30.1, 4.1 Hz, C-1), 104.64 (dd, J = 227.7, 17.3 Hz, C-2), 85.80 (dd, J = 194.5, 19.6 Hz, C-3), 72.04 (d, J = 4.3 Hz, C-6), 69.99 (dd, J = 16.6, 5.5 Hz, C-4), 68.58 (C-7), 66.09 (C-8), 63.59 (d, J = 3.3 Hz, C-5), 61.73 (C-9), 53.98 (OMe), 20.98 (OAc), 20.77 (OAc), 20.64 (br. s, 2xOAc), 20.59 (OAc) ppm.  19 F-NMR  (282  MHz; CDCl3 ): δ -124.95 (d, JF2 ,F3 = 10.5 Hz, F-2), -216.13 (ddd, J3,F3 = 50.1, J4,F3 = 24.2 Hz, F-3) ppm. MS : Calcd. for C20 H26 F2 NaO13 : 535.1; found: 535.3  246  7.2. Synthesis Methyl (fluoro 3-deoxy-3-difluoro-d-glycero-d-galacto-2nonulopyrano)sonate (113) 180 OH  COOMe O  HO HO  HO HO  F F  Methyl (fluoro 4,5,7,8,9-penta-O-acetyl-3-deoxy-3-difluoro-d-glycero-d-galacto-2nonulopyrano)sonate (112, 55 mg, 0.107 mmol) was subjected to the general method for Zemplén deprotection (page 187). Purification by flash column chromatography (5:1 ethyl acetate/methanol) gave a colourless syrup (21 mg, 0.069 mmol, 65%). 19 F-NMR  (282 MHz; D2 O): δ -122.61 (d, JF2 ,F3 = 11.1 Hz, F-2), -218.33 (ddd, J3,F3  = 50.3, J4,F3 = 29.3 Hz, F-3) ppm. MS : Calcd. for C10 H16 F2 NaO8 : 325.1; found: 325.3 Fluoro 3-deoxy-3-difluoro-d-glycero-d-galacto-2-nonulopyranosonic acid (114) 180 OH HO HO  HO HO  COOH O  F F  To a solution of methyl (fluoro 3-deoxy-3-difluoro-d-glycero-d-galacto-2-nonulopyrano)sonate (113, 21 mg, 0.069 mmol) in tetrahydrofuran (1.5 mL) and water (5 mL) was added 1 N NaOH (150 µL), and the reaction allowed to proceed at ambient temperature for 10 min then quenched with Sephadex ion exchange resin (H+ form). Following filtration and removal of the organic solvent in vacuo, the product was purified by 5 g C-18 Sep-pak eluted with water. Lyophilisation yielded a white foam (15.4 mg, 0.054, 78%). 1 H-NMR (300 MHz; D2 O): δ 5.19 (dt, J3,F3 = 50.9, J3,F2 = 3.0 Hz, 1H, H-3), 4.10-3.64 (m, 7H, H-4,5,6,7,8,9a,9b) ppm.  19 F-NMR  247  7.2. Synthesis (282 MHz; D2 O): δ -122.27 (dd, JF2 ,F3 = 11.1 Hz, F-2), -217.57 (ddd, J4,F3 = 28.0 Hz, F-3) ppm. HRMS : Calcd. for C9 H13 F2 O8 : 287.0578; found: 287.0580 Phenacyl (2,3-di-O-acetyl-4-deoxy-α-l-threo-hex-4-enopyranosyl fluoride)uronate (116) O O AcO  O  Ph  O F OAc  Phenacyl (fluoro 2,3,4-tri-O-acetyl-β-d-glucopyranosyl)uronate 181 (115, 0.507 g, 1.151 mmol) was subjected to the general method for DBU-catalysed elimination (page 186). Purification by flash column chromatography (3:1 petroleum ether/ethyl acetate) gave a colourless syrup (0.366 g, 0.962 mmol, 84%).  1 H-NMR  (400 MHz;  CDCl3 ): δ 7.88 (d, J2 ,3 = J5 ,6 = 7.4 Hz, 2H, H-2 ,6 ), 7.58 (t, J3 ,4 = J4 ,5 = 7.4 Hz, 1H, H-4 ), 7.45 (t, 2H, H-3 ,5 ), 6.46 (d, J3,4 = 4.9 Hz, 1H, H-4), 5.93 (d, J1,F = 49.6 Hz, 1H, H-1), 5.52-5.43 (m, 2H, CH2 C(O)Ar), 5.19-5.17 (m, 2H, H-2,3), 2.08 (s, 3H, Ar), 2.06 (s, 3H, Ar) ppm.  13 C-NMR  (101 MHz; CDCl3 ): δ 190.92 (C=O),  169.73 (OAc), 169.22 (OAc), 160.41 (C-6), 141.49 (d, JC5 ,F = 1.4 Hz, C-5), 134.17 (Ar), 133.87 (Ar), 128.99 (Ar), 127.80 (Ar), 108.43 (C-4), 101.31 (d, JC1 ,F = 228.3 Hz, C-1), 66.99 (CH2 C(O)Ar), 66.14 (d, JC2 ,F = 36.6 Hz, C-2), 62.22 (C-3), 20.71 (OAc), 20.59 (OAc) ppm.  19 F-NMR  (282 MHz; CDCl3 ): δ -140.41 (d, F-1) ppm.  MS : Calcd. for C18 H17 FNaO8 : 403.1; found: 403.3  248  7.2. Synthesis Phenacyl (2,3-di-O-acetyl-4-deoxy-β-d-glucopyranosyl fluoride) uronate (117) O Ph O O AcO  O OAc  F  Phenacyl (fluoro 2,3-di-O-acetyl-4-deoxy-α-l-threo-hex-4-enopyranosid)uronate (116, 0.323 g, 0.849 mmol) was dissolved in ethyl acetate (10 mL) and a spatula tip of palladium on carbon (10% catalyst) was added. The vessel was degassed under vacuum then flushed with hydrogen, and allowed to react at ambient temperature overnight. The crude intermediate was then filtered through Celite, the solvent evaporated in vacuo, and dissolved in fresh ethyl acetate (5 mL). 2-Bromoacetophenone (208 mg, 1.2 eq.) and triethylamine (160 µL, 1.4 eq.) were added and the reaction mixture stirred at ambient temperature for 3 hours. The reaction mixture was subsequently diluted and washed with water, 1 N HCl, saturated NaHCO3 , and brine, then dried over MgSO4 and purified by flash column chromatography (2:1 petroleum ether/ethyl acetate) and crystallisation to yield a white powder (0.157 g, 0.411, 48% over two steps). 1 H-NMR (300 MHz; acetone-d6): δ 8.02 (d, J2 ,3 = J5 ,6 = 7.4 Hz, 2H, H-2 ,6 ), 7.70 (t, J3 ,4 = J4 ,5 = 7.4 Hz, 1H, H-4 ), 7.57 (t, 2H, H-3 ,5 ), 5.67 (d, J7 a ,7 b = 16.1 Hz, 1H, H-7 a ), 5.57 (d, 1H, H-7 b ), 5.55 (dd, J1,F = 52.2, J1,2 = 6.2 Hz, 1H, H-1), 5.21 (ddd, J3,4ax = 11.1, J2,3 = 8.8, J3,4eq = 5.3 Hz, 1H, H-3), 4.98 (ddd, J2,F = 11.7 Hz, 1H, H-2), 4.79 (dd, J4ax ,5 = 11.7, J4eq ,5 = 2.8 Hz, 1H, H-5), 2.60 (ddd, J4eq ,4ax = 13.2 Hz, 1H, H-4eq ), 2.08 (s, 3H, OAc), 2.04 (ddd, 1H, H-4ax ), 2.03 (s, 3H, OAc) ppm.  19 F-NMR  (282 MHz; acetone-d6): δ -139.95 (dd, F-1) ppm.  MS : Calcd. for C18 H19 FNaO8 : 405.1; found: 405.3  249  7.2. Synthesis Phenacyl (2,3-di-O-acetyl-4-deoxy-5-fluoro-α-l-idopyranosyl fluoride)uronate (118) F  O  AcO O  O  F OAc O Ph  N -Bromosuccinimide (0.274 g, 4 eq.) and phenacyl (fluoro 2,3-di-O-acetyl-4deoxy-β-d-glucopyranosyl)uronate (117, 0.147 g, 0.384 mmol) were dissolved in carbon tetrachloride (5 mL) and irradiated with a 300 W light bulb. After two hours, the reaction mixture was diluted with dichloromethane and washed with water, saturated NaHCO3 , and brine then dried over MgSO4 . Purification was attempted by column chromatography (4:1 to 3:1 petroleum ether/ethyl acetate) but the product was found to have partially decomposed under these conditions and thus was not completely pure; the partially purified intermediate was used immediately for the next step. This intermediate (87 mg, 0.188 mmol) was dissolved in acetonitrile (2 mL) along with silver (I) fluoride (27 mg, 1.1 eq.), and stirred at ambient temperature overnight in the dark. On completion, the reaction mixture was filtered through a plug of silica (eluting with ethyl acetate) and then purified by flash column chromatography (3:1 to 2:1 petroleum ether/ethyl acetate) to yield a colourless syrup (60 mg, 0.150 mmol, 39% over 2 steps). This product was seen by  19 F-NMR  to  have contaminants remaining (approximately 10% by integrals) so crystallisation was attempted from toluene/petroleum ether and from ethanol/water, with no success. Further chromatography (5:2 petroleum ether/ethyl acetate, 1% methanol in dichloromethane, 0.8% acetone in dichloromethane) also proved unsuccessful in removing these contaminants. 1 H-NMR (400 MHz; CDCl3 ): δ 7.90 (d, J2 ,3 = J5 ,6 = 7.4 Hz, 2H, H-2 ,6 ), 7.64 (t, J3 ,4 = J4 ,5 = 7.4 Hz, 1H, H-4 ), 7.51 (t, 2H, H-3 ,5 ), 5.72 (dd, J1,F1 = 48.3, J1,2 = 0.9 Hz, 1H, H-1), 5.55-5.47 (m, 2H, H-7 a ,7 b ), 5.24-5.13 250  7.2. Synthesis (m, 2H, H-2,3), 2.78 (ddd, J4ax ,F5 = 26.6, J4ax 4eq = 15.2, J3,4ax = 4.2 Hz, 1H, H-4ax ), 2.58 (ddd, J4eq ,F5 = 9.9, J3,4eq = 5.6 Hz, 1H, H-4eq ), 2.14 (s, 3H, OAc), 2.12 (s, 3H, OAc) ppm.  13 C-NMR  (101 MHz; CDCl3 ): δ 190.33 (C=O), 170.00 (OAc), 169.32  (OAc), 164.72 (d, J = 33.1 Hz, C-6), 134.44 (Ar), 133.75 (Ar), 129.16 (Ar), 127.88 (Ar), 105.42 (d, JC5 ,F5 = 239.3 Hz, C-5), 104.65 (d, JC1 ,F1 = 232.3 Hz, C-1), 67.53 (s, CH2 C(O)Ar), 66.55 (d, JC2 ,F1 = 35.3 Hz, C-2), 64.31 (C-3), 29.90 (d, JC4 ,F5 = 25.9 Hz, C-4), 21.00 (OAc), 20.82 (OAc) ppm.  19 F-NMR  (282 MHz; CDCl3 ): δ  -102.64 (ddd, JF1 ,F5 = 17.4 Hz, F-5), -124.57 (ddd, JF1 ,2 = 5.6 Hz, F-1), -140.41 (d, J = 49.5 Hz, unknown contaminant) ppm. MS : Calcd. for C18 H18 F2 NaO8 : 423.1; found: 423.3 (2,3-Di-O-acetyl-5-fluoro-4-deoxy-α-l-idopyranosyl fluoride)uronic acid (119) F AcO HOOC  O OAc  F  Phenacyl (fluoro 5-fluoro-2,3-di-O-acetyl-4-deoxy-α-l-idopyranosyl)uronate (118, 41 mg, 0.102 mmol) was dissolved in 8:1 methanol/water (4.5 mL) and a spatula tip of palladium on carbon (10% catalyst) was added. The vessel was degassed under vacuum then flushed with hydrogen, and allowed to react at ambient temperature for 3 hours. The crude intermediate was then filtered through Celite, the solvent evaporated in vacuo, and the product purified by flash column chromatography (5 % methanol in dichloromethane) to give a colourless syrup (20 mg, 0.071 mmol, 69%). The contaminant visible in 1 H-NMR  19 F-NMR  from the previous reaction remained.  (300 MHz; CD3 OD): δ 5.68 (d, J1,F1 = 50.2 Hz, 1H, H-1), 5.15-5.07 (m,  2H, H-3,4), 2.63 (ddd, J4ax ,F5 = 21.7, J4ax ,4eq = 14.9, J3,4ax = 5.9 Hz, 1H, H-4ax ), 2.26 (ddd, J4eq ,F5 = 11.7, J3,4eq = 6.1 Hz, 1H, H-4eq ), 2.10 (s, 3H, OAc), 2.06 (s, 3H,  251  7.2. Synthesis OAc) ppm.  19 F-NMR  (282 MHz; CD3 OD): δ -100.68 (ddd, JF1 ,F5 = 7.2 Hz, F-5),  -124.89 (ddd, JF1 ,2 = 14.9 Hz, F-1), -141.55 (d, J = 50.3 Hz, contaminant) ppm. 4-Deoxy-5-fluoro-α-l-idopyranosyl fluoride)uronic acid (120) F HO HOOC  O OH  F  Fluoro 2,3-di-O-acetyl-5-fluoro-4-deoxy-α-l-idopyranosyluronic acid (119, 20 mg, 0.071 mmol) was dissolved in dry methanol (5 mL) and ammonia gas bubbled through until the solution was saturated. The reaction vessel was sealed and the reaction mixture stirred at 4 °C over three days. The solvent and ammonia were then evaporated in vacuo, and the product purified by 5 g C-18 Sep-pak eluted with water, passed through a plug of Sephadex ion exchange resin (H+ form), then by flash column chromatography (17:2:1 then 7:2:1 ethyl acetate/methanol/water). The product was observed to decompose on silica gel, but a single pure fraction was isolated and lyophilised to give a white powder (1.7 mg, 8.5 µmol, 12%) with a further 4.5 mg of impure product recovered. 1 H-NMR (400 MHz; D2 O): δ 5.71 (dt, J1,F1 = 52.2, J1,2 = 2.4 Hz, 1H, H-1), 4.03 (q, J2,3 = J3,4ax = J3,4eq = 5.3 Hz, 1H, H-3), 3.98 (ddd, J2,F1 = 8.7 Hz, 1H, H-2), 2.53 (ddd, J4ax ,F5 = 31.0, J4ax ,4eq = 15.3 Hz, 1H, H-4ax ), 2.18 (ddd, J4eq ,F5 = 13.2 Hz, 1H, H-4eq ) ppm.  19 F-NMR  (282 MHz; D2 O):  δ -97.61 (ddd, JF1 ,F5 = 16.3 Hz, F-5), -124.72 (ddd, F-1) ppm. HRMS : Calcd. for C6 H7 F2 O5 : 197.0262; found: 197.0258  252  7.2. Synthesis Methyl (2,3,4-tri-O-acetyl-1-fluoro-d-glucopyranosyl fluoride) uronate (121) MeOOC AcO AcO  O F  AcO F  and Methyl (2,3,4-tri-O-acetyl-d-gluc-1-enopyranosyl fluoride)uronate (122) MeOOC AcO AcO  O F AcO  Globally protected glucuronic acid 1,5-lactone (150, 0.502 g, 1.511 mmol) was dissolved in dichloromethane (15 mL), flushed with argon and cooled to -30 °C, then (dimethylamino)sulfur trifluoride (0.29 mL, 2 eq.) added and the reaction mixture allowed to stir at ambient temperature for 4 days. A second equal portion of (dimethylamino)sulfur trifluoride was added, and the reaction again allowed to proceed at ambient temperature for 3 days before quenching with methanol at 0 °C. The product was washed with saturated NaHCO3 , water and brine before drying over MgSO4 . Partial purification by flash column chromatography (5:2 petroleum ether/ethyl acetate) yielded the product 121 as a colourless syrup (0.088 g, 0.248 mmol, 16%) with the vinyl fluoride 151 as a minor contaminant (0.052 g, 0.156 mmol, 10%, by methyl peak integrals), along with 0.130 g (26%) starting material recovered. For 121, 1 H-NMR (300 MHz; CDCl3 ): δ 5.40-5.24 (m, 3H, H-2,3,4), 4.39 (d, J4,5 = 9.8 Hz, 1H, H-5), 3.72 (s, 3H, OMe), 2.06 (s, 3H, OAc), 1.98 (s, 3H, OAc), 1.97 (s, 3H, OAc) ppm.  13 C-NMR  (75 MHz; CDCl3 ): δ 169.50 (OAc), 169.18 (OAc),  168.84 (OAc), 165.45 (C-6), 120.12 (dd, JC1 ,Fax = 273.5, JC1 ,Feq = 257.6 Hz, C-1),  253  7.2. Synthesis 71.97 (dd, JC5 ,Fax = 4.5, JC5 ,Feq = 2.3 Hz, C-5), 69.98 (t, JC3 ,Fax = JC3 ,Feq = 9.0 Hz, C-3), 68.49 (t, JC2 ,Fax = JC2 ,Feq = 30.0 Hz, C-2), 68.19 (C-4), 53.28 (OMe), 20.36 (OAc), 20.27 (OAc), 20.22 (OAc) ppm.  19 F-NMR  (282 MHz; CDCl3 ): δ -83.46 (d,  JFax ,Feq = 147.8 Hz, F-1eq ), -86.02 (dd, JFax ,2 = 15.8 Hz, F-1ax ) ppm. MS : Calcd. for C13 H16 F2 NaO9 : 377.1; found: 377.2 Methyl (2,3-di-O-acetyl-4-deoxy-1-fluoro-l-threo-hex-4-enopyranosyl fluoride)uronate (123) COOMe O F  AcO AcO F  Globally protected glucuronyl 1,1-difluoride (121, 88 mg, 0.248 mmol, contaminated with globally protected 1-vinyl glucuronyl fluoride (122, 52 mg, 0.156 mmol) was subjected to the general method for DBU-catalysed elimination (page 186). Purification by flash column chromatography (4:1 petroleum ether/ethyl acetate) gave a white powder (53 mg, 0.180 mmol, 73%).  1 H-NMR  (300 MHz; CDCl3 ): δ  6.27 (d, J3,4 = 3.6 Hz, 1H, H-4), 5.53-5.43 (m, 2H, H-2,3), 3.85 (s, 3H, OMe), 2.14 (s, 3H, OAc), 2.09 (s, 3H, OAc) ppm.  13 C-NMR  (101 MHz; CDCl3 ): δ 169.73 (OAc),  168.79 (OAc), 160.16 (C-6), 141.96 (C-5), 119.93 (t, JC1 ,Fax = JC1 ,Feq = 264.2 Hz, C-1), 109.02 (C-4), 66.49 (t, JC2 ,Fax = JC2 ,Feq = 31.4 Hz, C-2), 66.48 (d, JC3 ,Feq = 8.9 Hz, C-3), 53.21 (OMe), 20.75 (OAc), 20.53 (OAc) ppm.  19 F-NMR  (282 MHz;  CDCl3 ): δ -83.81 (d, J = 158.6 Hz, F-1eq ), -84.87 (ddd, JFeq ,Fax = 158.6, J2,Fax = 9.0, J3,Fax = 4.5 Hz, F-1ax ) ppm. MS : Calcd. for C11 H12 F2 NaO7 : 317.0; found: 317.3  254  7.2. Synthesis (4-Deoxy-1-fluoro-l-threo-hex-4-enopyranosyl fluoride)uronic acid (124) COOH O F  HO HO F  Globally protected unsaturated glucuronyl 1,1-difluoride (123, 17 mg, 0.058 mmol) was deprotected by the general method for acidic trans-esterification (page 187). The intermediate was purified by flash column chromatography (5 % methanol in dichloromethane) before further deprotection by the general method for hydrolysis by lithium hydroxide (page 187). The final product was purified by flash column chromatography (7:2:1 ethyl acetate/methanol/water) then 5 g C-18 Sep-pak, eluting with water, and lyophilised to give a white powder (11.2 mg, 0.057 mmol, 98%). 1 H-NMR  (400 MHz; D2 O): δ 6.00 (d, J3,4 = 3.2 Hz, 1H, H-4), 4.45 (dt, J2,3 = 6.7,  J3,Fax = 3.2 Hz, 1H, H-3), 4.11 (ddd, J2,Fax = 11.6, J2,Feq = 5.0 Hz, 1H, H-2) ppm. 13 C-NMR  (101 MHz; D2 O): δ 166.88 (C-6), 122.53 (dd, JC1 ,Fax = 264.1, JC1 ,Feq =  260.0 Hz, C-1), 109.60 (C-4), 69.60 (t, JC2 ,Feq = JC2 ,Fax = 27.4 Hz, C-2), 67.09 (d, JC3 ,Feq =3.2 Hz, C-3) ppm (C-5 signal too weak).  19 F-NMR  (282 MHz; D2 O): δ  -86.07-85.94 (m, 2F, Fax ,Feq ) ppm. MS : Calcd. for C6 H5 F2 O5 : 195.0; found: 195.3  255  7.2. Synthesis  7.2.10  Glucuronides for extension to heparanase substrates.  Methyl (trifluoromethylumbelliferyl 2,3,4-tri-O-acetyl-β-dglucopyranosid)uronate (140) MeOOC AcO AcO  O OAc  O  CF3 O O  Methyl (bromo 2,3,4-tri-O-acetyl-α-d-glucopyranosid)uronate (3, 0.821 g, 2.067 mmol) was reacted with trifluoromethylumbelliferone (0.522 g, 1.1 eq.) by the general method for Koenigs-Knorr glycosylation (page 186). Purification by flash column chromatography (2:1 petroleum ether/ethyl acetate) then crystallisation from ethanol yielded a white powder (0.483 g, 0.884 mmol, 43%).  1 H-NMR  (300 MHz;  CDCl3 ): δ 7.64 (d, J5 ,6 = 8.7 Hz, 1H, H-5 ), 7.00-6.97 (m, 2H, H-6 ,7 ), 6.67 (s, 1H, H-3 ), 5.45-5.29 (m, 4H, H-1,2,3,4), 4.27 (d, J4,5 = 7.2 Hz, 1H, H-5), 3.72 (s, 3H, OMe), 2.04 (s, 9H, 3xOAc) ppm. ppm.  13 C-NMR  19 F-NMR  (282 MHz; CDCl3 ): δ -65.17 (s, CF3 )  (75 MHz; CDCl3 ): δ 170.07 (OAc), 169.41 (OAc), 169.21 (OAc),  166.64 (C-6), 159.93 (Ar), 158.95 (Ar), 155.86 (Ar), 126.73 (Ar), 114.84 (Ar), 113.94 (Ar), 113.90 (dAr), 113.78 (Ar), 109.13 (Ar), 104.75 (Ar), 98.11 (C-1), 72.74 (C-3), 71.58 (C-2), 70.85 (C-4), 68.88 (C-5), 53.21 (OMe), 20.68 (2xOAc), 20.59 (OAc) ppm. MS : Calcd. for C23 H21 F3 NaO12 : 569.1; found: 569.2  256  7.2. Synthesis Trifluoromethylumbelliferyl β-d-glucopyranosiduronic acid (138) HOOC HO HO  O OH  O  CF3 O O  Globally protected trifluoromethylumbelliferyl unsaturated β-d-glucuronide(140, 30 mg, 54.9 µmol) was deprotected using the general methods for acidic transesterification followed by hydrolysis by lithium hydroxide (page 187). Purification by flash column chromatography (15:1 to 9:1 dichloromethane/methanol after transesterification and 7:2:1 ethyl acetate/methanol/water after methyl ester hydrolysis) yielded a white powder (5.3 mg, 13.0 µmol, 24% over two steps, with significant losses to lactone hydrolysis on the second). 1 H-NMR (400 MHz; CDCl3 ): δ 7.80 (d, J5 ,6 = 8.3 Hz, 1H, H-5 ), 7.20-7.18 (m, 2H, H-6 ,7 ), 6.84 (s, 1H, H-3 ), 5.28 (d, J1,2 = 7.1 Hz, 1H, H-1), 4.02 (d, J4,5 = 8.6 Hz, 1H, H-5), 3.72-3.64 (m, 3H, H-2,3,4) ppm. 19 F-NMR  (282 MHz; CDCl3 ): δ -65.21 (s, CF3 ) ppm. MS : Calcd. for C16 H12 F3 O9 :  405.0; found: 405.3 2,4-Dinitrophenyl 2-deoxy-2-fluoro-β-d-glucopyranosiduronic acid (139) HOOC HO HO  O2N  O F  O NO2  Methyl (2,4-dinitrophenyl 2-deoxy-2-fluoro-3,4-di-O-acetyl-β-d-glucopyranosid)uronate (76, 18 mg, 39 µmol) was deprotected using the general methods for acidic trans-esterification followed by hydrolysis by lithium hydroxide (page 187). Purification was by 5 g C-18 Sep-pak, washed with water, 40% acetonitrile in water and 100% acetonitrile. All fractions determined by TLC (3:3:1:1 toluene/ethyl acet257  7.3. Biochemistry. ate/methanol/acetic acid) to contain pure product were pooled and lyophilised to give a white powder (10.6 mg, 29.3 µmol, 75% over 2 steps).  1 H-NMR  (400 MHz;  D2 O): δ 8.84 (d, J3 ,5 = 2.7 Hz, 1H, H-5 ), 8.48 (dd, J2 ,3 = 9.3 Hz, 1H, H-3 ), 7.58 (d, 1H, H-2 ), 5.77 (dd, J1,2 = 7.4, J1,F = 3.4 Hz, H-1), 4.62 (ddd, J2,F = 50.8, J2,3 = 9.3 Hz, H-2), 4.30 (d, J4,5 = 9.3 Hz, H-5), 3.99 (dt, J3,F = 15.6, J3,4 = 9.3 Hz, H-3), 3.79 (t, H-4) ppm.  13 C-NMR  (101 MHz; D2 O): δ 171.58 (C-6), 153.73 (Ar), 141.88  (Ar), 139.00 (Ar), 129.92 (Ar), 122.24 (Ar), 117.92 (Ar), 97.65 (d, JC1 ,F = 25.5 Hz, C-1), 90.87 (d, JC2 ,F = 186.4 Hz, C-2), 74.90 (C-5), 73.38 (d, JC3 ,F = 18.4 Hz, C-3), 70.44 (d, JC4 ,F = 9.1 Hz, C-4) ppm. MS : Calcd. for C12 H10 FN2 O10 : 361.0; found: 361.3  7.3 7.3.1  Biochemistry. Cloning of UGL from Clostridium perfringens.  The gene for UGL from Clostridium perfringens strain ATCC13124 genomic DNA (ATCC, Virginia) was amplified by PCR using Pwo ‘superyield’ polymerase in Pwo buffer (Roche, Switzerland) with 5 % DMSO and 3 mM MgCl2 . Primer sequences (Integrated DNA technologies, Illinois) were CACACAGCTAGCATGATTAAGGAAATAAGAGTTGAAGAGATTGC (forward) and CACACACTCGAGTTACCAATAAAGGTTCCAATCTTTATAAAATCTTATTAAGG (reverse), showing restriction sites (XhoI and NheI) in bold and start codon underlined. The thermocycler was run for 30 cycles of 30 s 95 °C melting, 30 s of 48 °C annealing and 1 min of 72 °C extension, after which the product was gel purified and used as a template for another round of PCR under the same conditions, except annealing at 51 °C and with a final extension step of 7 min. The amplified gene was gel purified, digested with XhoI and NheI (Fermentas, Maryland) in NEB buffer 1 (New England Biolabs, Massachu-  258  7.3. Biochemistry. setts) and ligated in rapid ligation buffer using T4 ligase (Fermentas, Maryland) into pET28a(+), which had been digested with the same restriction enzymes, and the ligation product was again gel purified. This plasmid, termed pET28a::UGL, was transformed by electroporation in a Gene Pulser II (<5 ms, 25 µF, 200 Ω, 2.5 kV, 0.2 cm cuvette; Bio-Rad, California) into Escherichia coli BL21(DE3) which was plated on TYP kanamycin (50 μg.ml-1 ) and a single colony selected for overnight culture to store as a stock in 10% DMSO at -80 °C. Sequence was confirmed by commercial sequencing with T7 and T7terminal primers (NAPS unit, University of British Columbia, Canada).  7.3.2  Testing of expression conditions  Expression was tested with variation in growth medium (TYP or LB), expression temperature (30 or 37 °C), concentration of IPTG (1 or 0.1 mM), and cell density at induction (exponential or stationary growth phase, low or high O.D. respectively). A set of 8 overnight 3 mL cultures, half in TYP and half in LB media with 50 µg.mL-1 kanamycin, were subcultured 1 in 100 into a further set of 8 tubes with the same volume of fresh growth media and grown at 37 °C to an optical density of 0.5 (averaged across all tubes). Cells were induced with an appropriate amount of IPTG then expression allowed to continue at the appropriate temperature for either 4 hours for high O.D. induction or overnight for low O.D. induction. On completion of expression, 1.8 mL of each culture was pelleted at 13 200 rpm for 2 min, the supernatant discarded, and the pellet frozen until all samples were ready. Cells were then resuspended in 300 µL of Bugbuster protein extraction reagent (Novagen, Massachusetts) and incubated at ambient temperature for 20 min before centrifuging at 13 200 rpm for 20 min. The supernatant was separated from the pellet, to which 20 µL of LDS loading buffer was added and then boiled for 10 min. Supernatant  259  7.3. Biochemistry. and partially-dissolved pellet were then analysed by SDS-PAGE.  7.3.3  Heterologous expression of UGL in Escherichia coli.  BL21(DE3) cells, transformed with pET28a::UGL, grown in an overnight culture (1 mL TYP, 50 µg.mL-1 kanamycin) were sub-cultured 1 in 1000 into 500 mL TYP media with 50 µg.mL-1 kanamycin, and shaken (225 rpm) at 37 °C until middle to late log phase. The culture was then induced by adding IPTG (Invitrogen, California) to a final concentration of 100 µM and shaken overnight (225 rpm) at 37 °C. Cells were harvested by centrifugation at 3800 rcf, 4 °C, 25 min then lysed by three passes of a 40 K manual fill French pressure cell at 1000 psi (SLM instruments, Illinois) in 15 mL lysis buffer (buffer A, below, with benzonase; Novagen, Germany, and EDTA free protease inhibitor; Roche, Switzerland). The crude lysate was clarified by centrifugation at 1700 rcf, 4 °C, 30 min and filtered at 5.0 µm with a millex SV sterile filter (Millipore, Massachusetts). Clarified supernatant was then purified by immobilised-metal affinity chromatography with a Qiagen NiNTA superflow 1 mL column on an Äkta 900 purifier as follows: The clarified supernatant was loaded using an auxiliary pump at 1 mL.min-1 , and rinsed with 5 mL buffer A. This was then eluted stepwise: 20 mL buffer A, 15 mL 2.5% buffer B in A, 15 mL 40% buffer B in A and 5 mL buffer B, protein elution was monitored at 280 nm (buffer A: 20 mM Tris.HCl pH 8, 20 mM imidazole, 25 mM NaCl, 1 mM DTT, buffer B: 20 mM Tris.HCl pH 8, 400 mM imidazole, 25 mM NaCl, 1 mM DTT). Fractions confirmed to contain UGL by SDS-PAGE (4-20% in Tris/glycine buffer, Invitrogen, California) were pooled and concentrated then exchanged into Tris.HCl 20 mM pH 8, 1 mM DTT in a 30 kDa cut off centrifugal filter (final dilution estimated at 1 in 50 000; Millipore, Massachusetts), then stored at 4 °C. Final protein purity was assessed by SDS-PAGE and concentration determined by UV-vis absorbance using an ε280 of  260  7.3. Biochemistry. 106 230 M.-1 .cm.-1 calculated using the ProtParam tool from the ExPASy website (http://web.expasy.org/protparam/).  7.3.4  Michaelis-Menten kinetics  Kinetic parameters were determined by measurement of initial rates in MES.NaOH buffer 50 mM pH 6.6 with 1 mg.mL-1 BSA and 1 µM UGL. Extinction coefficients were determined by allowing a 1 mM reaction to run to completion overnight, and wavelength was selected based on the largest difference in absorbance within the linear range of the spectrophotometer (total Abs≤3.5). Initial rates were measured at a range of at least five different substrate concentrations, ranging from at least Km /5 to 5 × Km (Km /7 to 7 × Km where possible), and the MichaelisMenten equation (equation 7.1) fit by non-linear regression. Where reactions were linear over a sufficiently long time, several rates were measured simultaneously using an automated cell changer. To measure first order rates by substrate depletion, reactions at low substrate concentrations ([S] ≤ Km /5) were allowed to proceed for at least 5 half lives and fit to a first order rate expression using the spectrophotometer’s proprietary software.  V0 =  kcat [E][S] Km + [S]  (7.1)  With the heparin- and chondroitin-derived natural substrates (11, 12, 13, 14, 15, and 16), the axial phenyl substrate (43), Kdn2en (40), and the fluorinated substrate (70) saturation kinetics were not able to be attained. In the case of the axial phenyl substrate (43) this was a result of apparent inhibition at higher concentration giving a strong deviation from the curve, while with the non-chromogenic substrates (11, 12, 13, 14, 15, 16, 40, and 70) rates for substrate concentrations over 5 mM could not be reliably measured due to high initial absorbance, since the 261  7.3. Biochemistry. reaction was monitored via decrease in absorbance from a high initial peak. The particularly poorly binding substrates heparan 6-sulfate disaccharide (14), Kdn2en (40), and equatorial ΔGlcA fluoride (70) showed very little deviation from linear dependence of rate on substrate concentration in this range, so was fit with a linear model in GraFit 5.0 to determine kcat /Km , with Km approximated through measurement of Ki for the latter two (see section 7.3.11). Plots of rate against substrate concentration are provided in Appendix D.  7.3.5  1  H-NMR monitoring of UGL-catalysed reaction.  Hydrolysis of thiophenyl ΔGlcA (46, 3.9 mg) by UGL (14 µM) was monitored using 1 H-NMR  in phosphate buffer (45 mM, pD 7.1 by direct pH meter measurement)  with β-mercaptoethanol (0.9 mM) and BSA (0.1 % w/v). Before addition of the enzyme to the substrate all locking, tuning, and shimming of the spectrometer was performed on the enzyme/buffer/BSA mix, then the enzyme was added to the substrate followed by fine-tuning of the shimming and recording of spectra after 0, 5, 10, 15, 45, 110, 170, and 266 minutes. The sample was then left at ambient temperature over approximately 72 hours before a final spectrum was recorded, to allow time for any slow equilibration. Each spectrum was recorded for 32 scans. A separate spectrum of the substrate in the same buffer conditions without enzyme was also recorded.  7.3.6  Profile of UGL activity at varied pH.  Buffers at varied pH (3.0–9.0, with increments of 0.5) were made using citric acid (0.1 M) and dibasic sodium phosphate (0.2 M), based on the table available from SigmaAldrich  (http://www.sigmaaldrich.com/life-science/core-bioreagents/biological-  buffers/learning-center/buffer-reference-center.html) with the exact pH determined  262  7.3. Biochemistry. by pH meter. The pH-activity profile was determined by measuring kcat /Km at each pH by substrate depletion, as detailed earlier (section 7.3.4, page 261), using pNP ΔGlcA (6, 50 µM) in 50% buffer (above, final concentration 50–100 mM) with BSA (0.1 % w/v), and UGL at 1 µM. The stability of UGL in each buffer was also tested by pre-incubation of the enzyme in the appropriate buffer for 20 min before 20× dilution into a solution of pNP ΔGlcA (6, 250 µM) in MES.NaOH (40 mM, pH 6.6) with BSA (0.1 % w/v) and the initial rate measured to determine the proportion of enzyme activity remaining.  7.3.7  Titration of benzyl ΔGlcA.  Benzyl ΔGlcA (53) was dissolved in the same buffers used for the enzymatic pH profile to a final concentration of 250 µM for a pH range from 2.7 to 7.2, then a scan taken of absorbance from 400–200 nm using the same buffer as a reference cell. The main spectral changes were in the lowest end of this range, where substantial noise was also seen. To control for this, scans were normalised for baseline using the averaged values from 340–340 nm then the ratio of the averaged absorbance for the 220–200 nm range against the 235 nm peak was plotted against pH and fit to obtain pKa .  7.3.8  Effect of temperature on UGL.  Temperature effects on the UGL-catalysed reaction were determined using pNP ΔGlcA (6) by measuring initial rates at saturating substrate conditions (1.875 µM) for the effect on kcat , and substrate depletion at low substrate concentration (50 µM) for kcat /Km , as detailed earlier (section 7.3.4, page 261). Rates were measured at 25–50 °C at 5 °C intervals in MES.NaOH buffer (40 mM, pH 6.6) containing BSA (0.1% w/v) using 0.625 µM UGL. Stability of the enzyme at 50 °C was tested by  263  7.3. Biochemistry. incubating the enzyme at this temperature for 20 min, with a control kept on ice, before adding to 250 µM pNP ΔGlcA and measuring the initial rate.  7.3.9  Reaction of unsaturated glucuronides with UGL in D2 O.  4-Nitrophenyl ΔGlcA (6, 3.37 mg, 11.3 µmol) was dissolved in D2 O (500 µL final), sodium phosphate buffer (100 mM final, pD 6.5), BSA (1 mg.mL-1 final). UGL stock was lyophilised thrice into D2 O then added to a final concentration of 0.16 mg.mL-1 /3.32 µM, and the reaction allowed to proceed at ambient temperature overnight. The reaction mixture was analysed directly by 1 H-NMR with a water suppression pulse sequence. The sample was then lyophilised into H2 O for 2 H-NMR.  7.3.10  Reaction of unsaturated glucuronides with UGL in 10% methanol.  4-Nitrophenyl or phenyl ΔGlcA (6, 10) (8.6 mg, 30 µmol) was dissolved in water, methanol (10 % v/v final), sodium phosphate buffer (50 mM final, pH 6.27), BSA (1 mg.mL-1 final), UGL (0.051 mg.mL-1 /1.05 µM final) to a total volume of 4 mL. The reaction was allowed to proceed at ambient temperature for 3 hours, at which point TLC indicated the reaction was largely complete (3:3:1:1 toluene/ethyl acetate/methanol/acetic acid, substrate Rf = 0.4). Enzyme and BSA were removed by centrifugal filtration (30 kDa cutoff, 5 000 rcf, 4 °C, 10 min, reaction vessel and filter washed once with 2 mL H2 O) then the flowthrough lyophilised and stored at -20 °C until ready for analysis. Reaction products were reconstituted in 700 µL D2 O and 1 H-NMR,  COSY and NOESY spectra recorded.  264  7.3. Biochemistry.  7.3.11  Testing of competitive inhibitors.  For those compounds which had been shown in other assays to act as a substrate it was assumed that inhibition was competitive. With these compounds, inhibition was approximated by measurement of at least 5 rates with varied inhibitor and a fixed substrate concentration at or slightly above Km . Plotting of the inverse of these rates against the inhibitor concentration allowed determination of an intercept with the inverse of Vmax , defined as −1/Ki . All other compounds were assayed with a matrix of at least 4 different substrate and inhibitor concentrations (more where possible, but at times limited by compound amounts available), bracketing Km and a previously determined approximate Ki (using the method outlined in the previous paragraph), respectively. These rates were then fit to modified Michaelis-Menten equations describing reaction in the presence of competitive (equation 7.2), non-competitive (equation 7.3) and mixed type inhibition (equation 7.4) by non-linear regression. The equation giving the lowest errors was deemed to be the most appropriate, which was confirmed by plotting 1/rate against inhibitor concentration (a Dixon plot) and observing the intersection of all plots at X =  −1 Ki  and Y =  1 Vmax ,  V0 =  V0 =  V0 =  and only that value reported. kcat [E][S]  Km (1 +  [I] Ki ) +  (7.2)  [S]  1 kcat [E][S] 1+[I]/K i  (7.3)  Km + [S]  kcat [E][S] Km (1 +  [I] Ki ) +  [S](1 +  [I] ) Ki  (7.4)  265  7.3. Biochemistry.  7.3.12  Testing of compounds as mechanism-based inactivators.  Compounds designed as potential mechanism-based inactivators of UGL were tested for their ability to induce time-dependent loss of enzyme activity. Samples at varied inactivator concentration were incubated at 30 °C with enzyme (4 µM), using the buffer described for standard Michaelis-Menten kinetics, and aliquots were removed at timepoints to test for residual enzyme activity. For each such timepoint, 20 µL of inactivator mix was added to 180 µL of a pre-prepared substrate/buffer mix at a substrate concentration in a large excess over Km (pNP ΔGlcA, 6, 1.875 mM), and absorbance change was monitored for 1–2 minutes at 37 °C. Data for each inactivator concentration were fit to an equation describing first order decay with offset. As no clear inactivation at varied concentrations was observed, these first order rates were not further processed. For all reactions where inactivation was observed, the pH was tested using a pH-fix strip (Macherey-Nagel, Germany) before and after incubation. For inactivator concentrations at or above the buffer concentration, the inactivator was adjusted to pH 6.5 by mixing equimolar solutions of the inactivator as a free acid and as its sodium salt.  7.3.13  Kinetic isotope effects.  Solvent kinetic isotope effect. UGL was exchanged into D2 O using repeated spin filtration through a 30 kDa cutoff spin filter at 4 °C, 4 000 rpm into 50 mM phosphate pD 7.1 with 1 mM β-mercaptoethanol. The final volume was adjusted to match the initial volume, and protein concentration was then determined by A280 to match the initial stock in H2 O. A stock of pNP ΔGlcA (152) was also prepared in D2 O. Reactions in H2 O were measured at pH 6.61 while those in D2 O were measured using buffer at pD 7.05, using phosphate buffers (direct pH meter readings). 266  7.3. Biochemistry. For isotope effects on kcat , initial rates were measured and fit to a linear rate, with 8 replicates, using 2 mM substrate. Averaging of these linear rates for each substrate allowed calculation of a ratio and its standard error. For isotope effects on kcat /Km , the first order rate constant was measured by substrate depletion as detailed earlier (section 7.3.4, page 261) using 50 µM substrate. Reactions were monitored using a cell changer containing four reactions, with a total of 8 replicates for each substrate. These first order rate constants were also averaged for each substrate, allowing calculation of a ratio and its standard error. Direct measurement of single isotope substitution effects. Using the 1- and 4-deuterated substrates 96 and 100, as well as the non-deuterated form 49, rates for isotopically substituted substrates were measured at high and low substrate concentrations relative to Km to determine isotope effects on kcat and kcat /Km . Measurements were alternated between substituted and unsubstituted substrates using a single stock of each substrate and enzyme for all reactions to avoid bias. Stock solutions were pre-incubated at 37 °C between assays. In order to minimise pipetting errors, each stock was made to a concentration that allowed for mixing of large volumes relative to the total, typically 100 µL each of substrate and enzyme stock for a 200 µL reaction. Ratios of kH to kD were determined as detailed for the solvent kinetic isotope effects (with 5 mM substrate used for the effects on kcat ). Confirmation of effect from single isotope substitution at carbon 4 by competition in NMR. An approximately 1:1 ratio of 4-1 H and 4-2 H substrates (96 and 100) were dissolved in phosphate buffer 40 mM, pD 7.1 (direct pH meter reading) and BSA 1 mg.mL-1  267  7.3. Biochemistry. all in D2 O to a total substrate concentration of 7.5 mM. The reaction was started by adding UGL stock to 2.4 μM (in H2 O), and monitored by 1 H-NMR. The fraction of reaction for the light isotope substrate in each spectrum was calculated from the integral of the proton signal at carbon 4 at 6.14 ppm over the sum of integrals of the aryl peaks at 7.53 ppm and 7.28 ppm, for starting material and product aryl groups respectively, all compared to that in the initial spectrum at time = 0. The corresponding ratio of substrate isotopomers was determined by the integral ratio of signals from protons at carbon 1 at 5.76 ppm and carbon 4 at 6.14 ppm for a given data point. The stated error represents the standard error from curve fitting by non-linear regression using equation 7.5, where F1 is the fraction of light isotope substrate reacted,  R R0  is the isotope ratio at a given fraction of light isotope substrate  reacted over that at t=0, and KIE is the kinetic isotope effect. 1 R = 10(log(1−F1 )( KIE −1)) R0  7.3.14  (7.5)  Attempted Rescue of D113G mutant with nucleophiles  UGLD113G (57 µM), purified as detailed earlier (section 7.3.3, page 260), was incubated at ambient temperature in phosphate buffer (50 mM, ph 5.0) with BSA (0.1% w/v), pNP ΔGlcA (6, 10 mM), and a range of potential alternate nucleophiles. Nucleophiles tested were sodium formate (pH 5.0, 1 M), sodium acetate (pH 5.0, 0.5 M), methanol (4 % v/v), β-mercaptoethanol (1 M), sodium azide (1 M), sodium cyanate (0.25 M), sodium thiocyanate (1 M), and potassium cyanide (1 M). Reactions were monitored over a week using TLC (3:2:2 1-butanol/Acetic acid/water).  268  7.3. Biochemistry. Testing in a spectrophotometer was carried out by monitoring of UGL-catalysed hydrolysis of 4-nitrophenyl ΔGlcA (6) through release of 4-nitrophenolate in the presence of sodium azide and sodium formate, pH 6.6, at a concentration of 100 mM. The enzyme was pre-incubated in the presence of azide/formate for 5 minutes before starting the reaction by addition of substrate to allow for the enzyme to equilibrate.  7.3.15  Heparanase kinetics  Arixtra As a positive control for heparanase activity, the assay of Hammond et al. was used. 209 Briefly, Arixtra (a kind gift from Prof. Jian Liu, University of North Carolina, U.S.A.) at 100 µM (142) was hydrolysed overnight (16–20 hours) in 40 mM sodium acetate buffer at pH 5.0 by 2 µg.mL-1 heparanase in a 100 µL reaction mixture. The reaction was quenched with an equal volume of WST-1 (141, Toronto Research Chemicals, Canada) at 1.69 mM in 0.1 M NaOH then developed for one hour at 60 °C in a sealed 384 well plate followed by reading of A584 . A standard curve of galactose on the same plate allowed quantification of this signal. This assay was also used on a shorter timeframe to monitor inhibition and inactivation of other compounds. Linearity on a shorter timeframe was confirmed by taking aliquots from a reaction over 20 minutes and plotting the concentration of reducing sugar equivalents against time. Hydrolysis of trisaccharides with β-glucuronidase Heparanase substrate trisaccharides (100–200 µM) were incubated with β-glucuronidase from bovine liver (10 kU.mL-1 ) in citrate buffer (50 mM, pH 4.5) at 37 °C. Reactions were monitored by HPLC using an analytic scale Zorbax SAX column 269  7.3. Biochemistry. eluting with a gradient of 0–100% 1 M potassium phosphate at pH 6.6 over 60 min at 1 mL.min-1 . On completion, typically overnight, the reaction was boiled for 5 minutes to precipitate the enzyme, centrifuged, then immediately used to start heparanase digestion. Fluorogenic substrate Hydrolysis of fluorogenic substrates was assayed by reaction of 100 µM substrate overnight in 100 µL 40 mM sodium acetate pH 5.0 with 2 µg.mL-1 heparanase. Reactions with a trifluoromethylumbelliferyl leaving group were quenched by addition of 100 µL Tris.NaOH pH 8.0 at 0.2 M then fluorescence read in a 384 well with excitation at 385 nm and emission at 502 nm. Reactions with a methylumbelliferyl leaving group were quenched by addition of 100 µL glycine.NaOH pH 10.0 at 0.2 M then fluorescence read in a 384 well with excitation at 355 nm and emission at 460 nm. A standard curve for each leaving group was prepared in the same buffers. For comparison, certain substrates, as discussed in Subsection 5.3.2, were also measured under conditions duplicating those of Pearson et al., 210 with pH 5.0 acetate buffer at 60 mM, BSA at 0.1 mg.mL-1 , substrate at 5 mM, and enzyme at 1.2 µg.mL-1 in a 50 µL reaction, quenching with 200 µL of pH 10.0 glycine at 0.2 M. Inactivation Inactivation assaying of heparanase was carried out in a similar manner to UGL (refer 7.3.12 on page 266), but at 37 °C. Inactivation was carried out in a 50 µL reaction volume in sodium acetate buffer (40 mM, pH 5.0) with the DNP 2F 6SNAc trisaccharide inactivator (149) at 1 mM and enzyme at 20 µg.mL-1 (10× the concentration used for substrate testing), along with a control reaction with no inactivator. At each timepoint 5 µL of the inactivation reaction mixture was added to 45 µL of  270  7.3. Biochemistry. Arixtra reaction mixture, in the same buffer and with the Arixtra substrate (142) at 250 µM, and reacted at 37 °C for 30 minutes before quenching with 30 µL of 0.2 M NaOH and freezing. After the final timepoint was taken, two sample Arixtra reactions with no enzyme and with and without inactivator were prepared as controls for a non-enzymatic background control in each case, and a standard curve of galactose prepared under the same conditions as the Arixtra reactions, then 30 µL of WST-1 at 3.38 mM was added to all timepoints and standard curve solutions and then incubated at 60 °C for one hour before reading A584 of 100 µL in a reducedvolume 384 well plate. The amount of Arixtra cleaved was quantified by subtracting the amount of reducing sugar equivalents in the relevant no enzyme background control from that in each timepoint reaction, and these values plotted against time for the inactivation and no inactivator control, with non-linear regression fitting to first order decay with offset.  271  References [1] Mathews, C. K.; van Holde, K. E.; Ahern, K. G. Biochemistry; BenjaminCummings, 2000. [2] Robinson, J. M. Geology 1990, 18, 607–610. [3] Robinson, J. 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Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  M............................I MNKSRTLIFSLLALMGTASSADAQIAKTPA M..........................MKI M.............................  2 30 4 1  Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  KEIRVEEIAKKDEFLKTKLLTRAEVKNAID KPLSDQMAATVMEIWPERAKKWSYDHGVVQ KPVKVESIENPKRFLDSRLLTKIEVEEAIE ..............W..........QQAIG  32 60 34 7  Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  LVIKQIDA...NMEYFK............. DGMDALWKRSGNASYFKYIQNDMDGFISAD KALKQLYI...NIDYFG............. DALGITAR...NLKKFG.............  46 90 48 21  291  Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  ...EKFPSSA..................TK GIIDTYSQEHVNIDNVKNGTVLLDLYKITG ...EEYPTPA..................TF ...DRFPHVS..................DG  55 120 57 30  Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  NNQYGIIENIEWTD..........GFWTGL QQKYFKAATTLWEQLKIQPRTKQGSFWHKK NNTYKVMDNTEWTN..........GFWTGC SNKYVLNDNTDWTD..........GFWSGI  75 150 77 50  Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  L.....WLAYEYTGDEKYRELA..DKNVAS IYPNQVWLDGLYMGQPFYAEYAALIGNKEA L.....WLAYEYNQDKKLKNIA..HKNVLS L.....WLCYEYTGDEQYREGA..VRTVAS  98 180 100 73  Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  catalytic residue ↓ FKNRVEKDIELDHHD....LGFLY...SLA FDDIANQFIWVEQNTRDARTGLLYHGWDES FLNRINNRIALDHHD....LGFLY...TPS FRERLDRFENLDHHD....IGFLY...SLS  121 210 123 96  Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  TVSGYKLTGSEDARE...........ASIK KTERWADPKTGLSPHIWARAMGWYAMALVE CTAEYRINGDAKALE...........ATIK AKAQWIVEKDESARK...........LALD  140 240 142 115  Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  AANKLISRYQEKGEFIQAWGELG....... TLDNFPKTHPKRQEMINILNRLAAAVKNTQ AADKLMERYQEKGGFIQAWGELG....... AADVLMRRWRADAGIIQAWGPKG.......  163 270 165 138  Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  catalytic residue ↓ .SKDH.YRFIID.......CLLNIPLLYWA NNKTGVWYDILDQPNRKGNYFESSASSMFV .YKEH.YRLIID.......CLLNIQLLFFA .DPENGGRIIID.......CLLNLPLLLWA  184 300 186 160  Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  SDETGDAKYRNIANKHFVTSCN........ YAIAKGVRLGYLPASYFVVASKGYKGIQQE YEQTGDEKYRQVAVNHFYASAN........ GEQTGDPEYRRVAEAHALKSRR........  206 330 208 182  292  Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  NVIRDDASAFH...TFYMDNETGKPLRGVT FIEQRAEGKINLKGTVSVSGLGGKPYRDGS NVVRDDSSAFH...TFYFDPETGEPLKGVT FLVRGDDSSYH...TFYFDPENGNAIRGGT  233 360 235 209  4-sulfate binding ↓ Clostridium_perfringens RQGYSDDSAWARGQAWGVYGIPLNYRYTRN Flavobacterium_heparinum YEYYMSEKVVSNDP.KGVGAFLMAANEMEI Streptococcus_agalactiae RQGYSDESSWARGQAWGIYGIPLSYRKMKD HQGNTDGSTWTRGQAWGIYGFALNSRYLGN Bacillus_sp_GL_1  263 389 265 239  Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  ESCFNLYEG....MTNYFLNRLPKD...NV AALPKPGLGKTVLLDSYFNNESRKDQSGNL YQQIILFKG....MTNYFLNRLPED...KV ADLLETAKR....MARHFLARVPED...GV  286 419 288 262  Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  CYWDLIFND..............GDDHSKD VSWHYKWDELANGGFSMWADQFNNAGFKTA SYWDLIFTD..............GSGQPRD VYWDFEVPQ..............EPSSYRD  302 449 304 278  Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  SSAAAIAVCGMHEMNKYL...PEVDENKEV TLKAAPTAANLKNASVYIIVDPDTEKETEK TSATATAVCGIHEMLKHL...PEVDPDKET SSASAITACGLLEIASQL...DESDPERQR  329 479 331 305  Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  YKYAMHNILRSLME................ PNFVAQNDIKAIAEWVKGGGILVLMANDTG YKYAMHTMLRSLIE................ FIDAAKTTVTALRD................  343 509 345 319  Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  ........................NYMNPE NVELDHFNQLAKTFGIEFNKDSKGRVVKSQ ........................QYSNSE ........................GYAERD  349 539 351 325  6-sulfate binding domain Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  IEPGKPVLLHGVYSWHSGKGV......... FEMGKVMVPAGNEIFKTAKQLYVKEYSSLK FIAGRPLLLHGVYSWHSGKGV......... DGEAEGFIRRGSYHVRGGISP.........  370 569 372 346  293  Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  .......................DEGNIW. LTTAAKAVLKDKDGDNVMAIAKYGKGAVFA .......................DEGNIW. .......................DDYTIW.  376 599 378 352  Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  .GDYFFLEALI...RFYKDWNLY....... IGDPWLYNEYVDGRKLPADYQNFEAGQDLV .GDYYYLEALI...RFYKDWELY....... .GDYYYLEALL...RLERGVTGY.......  395 629 397 371  Clostridium_perfringens Flavobacterium_heparinum Streptococcus_agalactiae Bacillus_sp_GL_1  .W........ NWIGKQLLKK .W........ .WYERGR...  396 639 398 377  294  Appendix B  2D-NMR spectra of UGL products and standards  Figure B.1: Expanded TOCSY spectrum of the UGL catalysed reaction of phenyl ΔGlcA (10) in 10% methanol.  295  Figure B.2: COSY45 spectrum of the UGL-catalysed reaction of phenyl ΔGlcA (10) in 10% methanol, to form 26.  296  Figure B.3: NOESY spectrum of the UGL catalysed reaction of phenyl ΔGlcA (10) in 10% methanol (26).  297  Figure B.4: COSY45 spectrum of 28, the carbon 5 epimeric synthetic standard for the UGL catalysed reaction of phenyl ΔGlcA (10) in 10% methanol.  298  Figure B.5: NOESY spectrum of 28, the carbon 5 epimeric synthetic standard for the UGL catalysed reaction of phenyl ΔGlcA (10) in 10% methanol.  299  Appendix C  Kinetic isotope effects Kinetic isotope effects are an important tool that can be used to probe the transition state of a reaction, without changing the reaction path. These effects arise from the difference in energy of the vibrational states of the two isotopomers. Substitution of an isotope for a heavier or lighter analogue will have an effect on the reduced mass of a diatomic system, influencing the energy of its vibrations, and thereby the ease with which its bond order can change. This influence on reduced mass and vibrational energy can be approximated by the equation for the energy of a harmonic oscillator, equation C.1, and the associated equations for vibrational frequency, equation C.2, and reduced mass, equation C.3. From these it can be seen that changes in the mass of one atom in the pair can have a small influence on the vibrational energy states. Example isotopologue energy levels for a quantum number of 0 are represented in the Morse potential curve in Figure C.1.  E = (n + 1/2)hv  v=  µ=  1 2π  k µ  mA mB mA + mB  (C.1)  (C.2)  (C.3)  (where E is energy, h is planck’s constant, n is quantum number, v is vibrational 300  Energy  frequency, k is vibrational force constant, µ is reduced mass, and m is mass)  BDED  BDEH  E0H E0D re  internuclear distance, r  Figure C.1: Hypothetical Morse potential curve, showing the zero point energy for a hydrogen and deuterium deuterated compound. The optimal internuclear distance is denoted re , and bond dissociation energies are denoted BDE. Any isotopic substitution can, in theory, lead to such an effect. However, the most common is a deuterium for hydrogen substitution, as the effect on reduced mass is substantial, synthesis is often reasonably simple, and no radio-isotopes are involved. Other common isotopes used for such experiments include 15 N,  and  17 O  or  18 O.  13 C  or  14 C,  It is important to note that, for an enzymatic reaction, if non-  chemical steps such as domain movements, substrate binding, or product release are rate limiting then no isotope effect will be observed. Kinetic isotope effects arising 301  from bond making or bond breaking to the isotope in question are termed primary effects, while those arising from indirect effects are termed secondary. Primary isotope effects can most easily be conceptualised from the difference in energy between the ground state, where a deuterated compound is lower in energy than a protonated compound, and the hypothetical limit of a bond breaking transition state, where the bond is completely broken and so both deuterated and protonated compounds must necessarily be of the same energy. This is represented in Figure C.2. Since this energy gap is smaller in a protonated than in a deuterated compound, the protonated compound reacts more quickly. In cases where there is higher bond order in the transition state the effect will be smaller. Primary kinetic isotope effects usually manifest as rate differences of between 2 and 7 fold faster reaction for the protonated compound over the deuterated. Secondary isotope effects can arise in a number of ways, but generally still come about from different effects on the energy at the ground state and at the transition state. Secondary effects are said to be α if the isotope is attached to the centre at which the reaction is taking place, and β if it is adjacent to this centre. Secondary effects have a less dramatic rate difference between isotopologues, typically ranging from 0.7 to 1.5 fold faster reaction of the hydrogen-substituted compound over that of the deuterium-substituted compound. Those cases where the effect is less than one are said to be inverse effects, with the deuterated compound reacting faster, while those above one are said to be normal effects. The most common way in which an α-secondary effect can arise is through changes in hybridisation. Because the energy of vibrational modes is dependent on hybridisation, a change in hybridisation at the transition state relative to the ground state can lead to a difference in activation energy for the two isotopomers, and thus a difference in rate. Changing hybridisation from sp3 to sp2 or sp2 to sp results in  302  ΔEH1<ΔED1 small primary KIE  Potential energy  XX  ΔEH2<<ΔED2 large primary KIE  H+ δ-  H  X-  D+ Transition states  δ+ δ-  X ΔEH1  D  δ+  ΔEH2 ΔED1  ΔED2  X H X D  Ground states  Figure C.2: Example transition states leading to primary kinetic isotope effects. In the case represented by ΔEH 1 and ΔED 1, the transition states have a relatively high bond order and the differences in energy between the deuterated and protonated cases remain at the transition state, and so a small primary kinetic isotope effect would be observed. In the case represented by ΔEH 2 and ΔED 2, the transition states are shown at the extreme limit, with no bond remaining, and so a large primary kinetic isotope effect would be expected.  303  a normal kinetic isotope effect, while the opposite change in hybridisation has the opposite kinetic isotope effect. These effects are illustrated in Figure C.3.  sp transition state  sp3 transition state EH  Energy  EH ED sp2 ground state  ΔED  ED  ΔEH  ΔED  ΔEH  EH ED ΔED>ΔEH Normal KIE  ΔED<ΔEH Inverse KIE  Figure C.3: Example energy differences between a hypothetic sp2 ground state and sp and sp3 transition states for a hydrogen and a deuterium substituted compound, which would give rise to a normal and an inverse KIE, respectively. A common means by which a normal β-secondary kinetic isotope effect can arise is by hyperconjugation. This is the donation of electron density from a carbon- or heteroatom-hydrogen bond to an adjacent vacant orbital to stabilise it. Because carbon-deuterium bonds are slightly stronger than a carbon-hydrogen bond, its electrons are less available for hyperconjugation. This means that if there is an increase in positive charge at the transition state at a carbon centre adjacent to a carbonhydrogen bond then substitution of this hydrogen for deuterium will give a slower reaction, as the charged centre, and thus the transition state in general, will be less stabilised. This electron donation is, however, very sensitive to the orientation of the  304  vacant orbital and the adjacent carbon- or heteroatom-hydrogen bond, as the bonding orbital must be able to overlap with the vacant atomic orbital for this stabilising electron donation to take place. This overlap is illustrated in Figure C.4. +  H(D) H(D)  X  Y  H(D)  H(D)  + H(D)  Figure C.4: Hypothetical kinetic isotope effect arising from hyperconjugation. The positive charge at the transition state is better stabilised by an adjacent hydrogen, arising from its weaker C−H bond. Other sources of kinetic isotope effects exist, but are less common. These arise from the same general principle, of a difference in the activation energy arising from isotopic substitution. In the previous case, with a hydrogen adacent to a developing charge, if the heteroatom-hydrogen bonding orbital is not able to overlap with the vacant orbital, an effect from induction can sometimes be seen. This gives a small inverse effect, as the deuterium atom is slightly less electron withdrawing compared to hydrogen. In a final example, steric effects can occasionally give rise to kinetic isotope effects, but only in highly restricted systems. For example in the bond rotation shown in Figure C.5, the larger deuterium atoms slow down interconversion of the two forms.  CD3 D3C  D3C CD3  Figure C.5: Steric interactions leading to a kinetic isotope effect.  305  Appendix D  Plots for Michaelis-Menten kinetics Plots of rate against substrate concentration, organised by compound number. Fits shown are to the Michaelis-Menten equation (equation 7.1 on page 261), with double recpirocal plots as insets (not used for analysis). 6  11  10  12  306  13  14  15  40  43  46  47  48  307  49  50  51  52  53  70  50 with D113G mutant  308