Science, Faculty of
Chemistry, Department of
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UBCV
Reid, Stephen P.
Master of Science - MSc
University of British Columbia
Tay Sachs and Gaucher diseases are lysosomal storage disorders that are caused by deficiencies in the lysosomal enzymes β-hexosaminidase A and glucocerebrosidase, respectively. The glycolipid substrates of deficient β-hexosaminidase A and glucocerebrosidase accumulate in lysosomal neurons, resulting in neurodegenerative disorders. The enzyme deficiencies are caused by heritable amino acid point mutations that result in unstable and potentially misfolded enzymes. The mutant enzymes, although catalytically active in many cases, are destroyed by cellular quality control mechanisms in the endoplasmic reticulum (ER). A potential therapy for adult chronic Tay Sachs disease and Gaucher disease is the use of enzyme inhibitors as chemical chaperones. To act as a chemical chaperone the enzyme-specific inhibitor must stabilize the folded conformation of the enzyme and thus prevent enzyme destruction by cellular quality control mechanisms. The enzyme/inhibitor complex can then be transferred from the E R to the lysosome where the substrate concentration is significantly high that some enzyme activity is restored. In the chronic adult disease forms, only 10 % of normal enzyme activity is required for a patient to become asymptomatic. A competitive inhibitor for β-hexosaminidase A, NAG-thiazoline, was synthesized and shown to be a potent inhibitor o f β-hexosaminidase A and the related enzyme β-hexosaminidase B, with Ki values of 270 nM and 190 nM , respectively. Chemical denaturation studies showed that NAG-thiazoline stabilizes a bacterial hexosaminidase from Streptomyces plicatus (Sp. Hex.) and human P-hexosaminidase B against guanidine hydrochloride denaturation. Thermal denaturation studies showed that NAG-thiazoline stabilizes Sp. Hex., β-hexosaminidase B, and β- hexosaminidase A against thermal denaturation. These results provide a chemical explanation for recent results from collaborator Dr. Mike Tropak (The Hospital for Sick Children, Toronto), which showed that NAG-thiazoline increases Phexosaminidase A activity in Tay Sachs fibroblasts. A mechanism based inactivator for glucocerebrosidase, 2-deoxy-2-fluoro- β-Dglucopyranosyl fluoride, was synthesized and shown to stabilize glucocerebrosidase against guanidine hydrochloride denaturation. N-Octyl-l-epivalienamine was synthesized and shown to be a potent competitive inhibitor o f glucocerebrosidase with a Ki value of 75 nM and to stabilize glucocerebrosidase against thermal denaturation. These results show that 2-deoxy-2-fluoro- β-D-glucopyranosyl fluoride and N-octyl-l-epivalienamine stabilize the folded conformation of glucocerebrosidase and may act as chemical chaperones in therapies for Gaucher disease. An additional study was performed in which the synthetic capacity of a glycosynthase developed from a β-glucuronidase from Thermotoga maritima was explored. Using α-D-glucopyranuronosyl fluoride or α-D-galactopyranuronosyl fluoride as donors, and pNP-glucoside, pNP-xyloside, and pNP-cellobioside as acceptors, the E476A β-glucuronidase mutant catalyzed the formation of six novel oligosaccharides.
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T H E U S E O F I N H I B I T O R S T O S T A B I L I Z E F O L D E D C O N F O R M A T I O N S O F E N Z Y M E S By STEPHEN P. REID B . S c , The University of British Columbia, 2000 A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF T H E REQUIREMENTS OF THE D E G R E E OF M A S T E R OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Chemistry) We accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH C O L U M B I A March 2004 © Stephen P. Reid, 2004 Library Authorization In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Name of Author (please print) Date (dd/mm/yyyy) Title of Thesis: jy U>" •••/* k.'b; fu ,•-i W ^)f>bJi^ Degree: M , Sc, Year: 3 / t o u ^ Department of CWmo+,-<•-) The University of British Columbia Vancouver, B C Canada Abstract Tay Sachs and Gaucher diseases are lysosomal storage disorders that are caused by deficiencies in the lysosomal enzymes (3-hexosaminidase A and glucocerebrosidase, respectively. The glycol ipid substrates o f deficient P-hexosaminidase A and glucocerebrosidase accumulate in lysosomal neurons, resulting in neurodegenerative disorders. The enzyme deficiencies are caused by heritable amino acid point mutations that result in unstable and potentially misfolded enzymes. The mutant enzymes, although catalytically active in many cases, are destroyed by cellular quality control mechanisms in the endoplasmic reticulum (ER) . A potential therapy for adult chronic Tay Sachs disease and Gaucher disease is the use o f enzyme inhibitors as chemical chaperones. To act as a chemical chaperone the enzyme-specific inhibitor must stabilize the folded conformation o f the enzyme and thus prevent enzyme destruction by cellular quality control mechanisms. The enzyme/inhibitor complex can then be transferred from the E R to the lysosome where the substrate concentration is significantly high that some enzyme activity is restored. In the chronic adult disease forms, only 10 % of normal enzyme activity is required for a patient to become asymptomatic. A competitive inhibitor for P-hexosaminidase A , NAG- th iazo l ine , was synthesized and shown to be a potent inhibitor o f p-hexosaminidase A and the related enzyme P-hexosaminidase B , with K\ values o f 270 n M and 190 n M , respectively. Chemical denaturation studies showed that NAG-th iazo l ine stabilizes a bacterial hexosaminidase from Streptomyces plicatus (Sp. Hex.) and human P-hexosaminidase B against guanidine hydrochloride denaturation. Thermal denaturation studies showed that NAG-th iazo l ine stabilizes Sp. Hex., P-hexosaminidase B , and 0-hexosaminidase A against thermal denaturation. These results provide a chemical explanation for recent results from collaborator Dr. M i k e Tropak (The Hospital for Sick Children, Toronto), which showed that NAG- th iazo l ine increases P-hexosaminidase A activity in Tay Sachs fibroblasts. A mechanism based inactivator for glucocerebrosidase, 2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride, was synthesized and shown to stabilize glucocerebrosidase against guanidine hydrochloride denaturation. 7V-Octyl-l-epivalienamine was synthesized and shown to be a potent competitive inhibitor of glucocerebrosidase with n a K\ value o f 75 n M and to stabilize glucocerebrosidase against thermal denaturation. These results show that 2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride and /V-oc ty l - l -epivalienamine stabilize the folded conformation of glucocerebrosidase and may act as chemical chaperones in therapies for Gaucher disease. A n additional study was performed in which the synthetic capacity o f a glycosynthase developed from a P-glucuronidase from Thermotoga maritima was explored. Us ing cc-D-glucopyranuronosyl fluoride or ot-D-galactopyranuronosyl fluoride as donors, and pNP-glucoside, pNP-xyloside, and pNP-cellobioside as acceptors, the E 4 7 6 A P-glucuronidase mutant catalyzed the formation o f six novel oligosaccharides. in Table of Contents Abstract >> Table of Contents iv List of Figures v>> List of Tables i x List of Schemes x List of Abbreviations x ' Acknowledgments x ' " 1 General Introduction 1 1.1 Glycosidases I /. /. / The Catalytic Mechanism of Retaining ji-Clycosidases 2 1.1.2 The A nch imeric Assistance Mechanism ". 5 1.1.3 Clycosidase Inhibitors 8 1.1.4 Mechanism Based Inactivators 9 1.2 Lysosomal Storage Disorders 10 1.2.1 Tay Sachs Disease 13 1.2.1.1 The Human Hexosaminidases 13 1.2.1.2 The Cause of the Deficiency in Hex A 16 1.2.2 Gaucher Disease 17 1.2.2.1 Glucocerebrosidase 18 1.2.3 Therapies for Lysosomal Storage Disorders 19 1.2.3.1 The Chemical Chaperone Approach 19 1.3 Aims of this Thesis 21 2 The Stabilizing Effect of NAG-Thiazoline on Family 20 Hexosaminidases 22 2.1 NAG-Thiazoline: a Competitive Inhibitor of Family 20 Hexosaminidases 22 2.2 The Synthesis of NAG-Thiazoline 24 2.3 Inhibition Results 25 2.4 Denaturation Experiments 31 2.4.1 Sp. Hex. Thermal Denaturation Studies 32 2.4.2 Sp. Hex. Guanidine Hydrochloride Denaturation Studies 33 2.4.3 Hex B Thermal Denaturation Studies 37 2.4.4 Hex B Guanidine Hydrochloride Denaturation Studies 38 2.4.5 Hex A Denaturation Studies 41 2.5 Conclusion/Future Outlook 42 I V 3 The Stabilizing Effect of 2-Deoxy-2-Fluoro-|3-D-Glucopyranosyl Fluoride and /V-Octyl-l-Epivalienamine on Glucocerebrosidase 43 3.1 Stabilization of Glucocerebrosidase by a Mechanism-Based lnactivator 43 3.1.1 The Synthesis of 2-Deoxy-2-Fluoro-/3-D-Glucopyranosyl Fluoride 45 3.1.2 Inactivalion of Glucocerebrosidase with 2-Deoxy-2-Fluoro-/3-D-Glucosyl Fluoride 46 3.1.3 Denaturation Studies with Glucocerebrosidase in the Presence and Absence of 2-Deoxy-2-Fluoro-fJ-D-Glucosyl Fluoride 47 3.2 Stabilization of Glucocerebrosidase by a Competitive Inhibitor 51 3.2.1 N-Octyl-1-Epivalienamine: a Competitive Inhibitor of Glucocerebrosidase 51 3.2.2 The Inhibition of Glucocerebrosidase by N-Octyl-1-Epivalienamine 53 3.2.3 Denaturation Studies with Glucocerebrosidase in the Presence and Absence of N-Octyl-1-Epivalienamine 54 3.3 Conclusion 56 4 The Development of a Glucuronidase Glycosynthase 57 4.1 Introduction 57 4.1.1 Backgound on Glycosynthases 57 4.1.2 Thermotoga maritima 59 4.1.3 ^-Glucuronidases 59 4.2 Development of a Glucuronosynthase from the P-Glucuronidase from Thermotoga maritima 60 4.2.1 Synthesis of Donor Sugars: a-D-Glucopyranuronosyl Fluoride and a-D-Galactopyranuronosyl fluoride 61 4.2.2 Glucuronosynthase Reactions 62 4.2.3 Attempts to Synthesize Glycosaminoglycan Fragments 66 4.3 Conclusion 70 5 Materials and Methods 71 5.1 Enzymology 71 5.1.1 Sp. Hex. Kinetics 71 5.1.2 Human ^-Hexosaminidase A and B Kinetics 71 5.1.2.1 Determination ofkcJKm Values for pNP-GlcNAc and pNP-GalNAc 72 5.1.3 Glucocerebrosidase Kinetics 73 5.1.4 Competitive Inhibition 73 5.1.5 Inactivalion of Glucocerebrosidase 74 5.1.6 Thermal Denaturation Experiments 74 5.1.7 Guanidine Hydrochloride Denaturation Experiments 76 5.1.8 General Procedure for Glucuronidase Glycosynthase Reactions 78 5.2 Syntheses 79 v 5.2.1 General Synthesis 79 5.2.2 Generous Gifts 79 5.2.3 NAG-Thiazoline (2.2) :. 80 5.2.4 2-Deoxy-2-Fluoro-f3-D-Glucopyranosyl Fluoride (3.3) 82 5.2.5 N-Octyl-l-Epivalienamine (3.6) 84 5.2.6 a-D-GalactopyranuronosylFluoride (4.6) 86 5.2.7 a-D-Glucopyranuronosyl Fluoride (4.9) 89 5.2.8 General Procedure for Derivitization of Glycosynthase Products 90 5.2.9 Assignment of Protons in ' H NMR Spectra of Derivatized Glycosynthase Products 91 5.2.10 Characterization of Glycosynthase Products 91 Appendix 95 References 96 V I List of Figures Figure 1.1 Diagram showing the glycone and aglycone components of a D-glucoside, and the numbering scheme used for carbohydrates in this thesis 1 Figure 1.2 Stereochemical outcome of reactions catalyzed by inverting and retaining glycosidases 2 Figure 1.3 The ion pair intermediate proposed by Phillips 4 Figure 1.4 Examples of competitive inhibitors of glycosidases 9 Figure 1.5 Diagram of terminal A^-acetyl-(3-D-glucosamine (1.6) and A'-acetyl-P-D-galactosamine (1.7) residues 14 Figure 1.6 Diagram of 4-methylumbelliferyl (3-D-yV-acetylglucosamine-6-sulfate (1.8) ( M U G S ) 15 Figure 1.7 Diagram of yV-butyl deoxynojirimycin (1.9) 19 Figure 1.8 Diagram of compounds 1.10-1.12 20 Figure 2.1 Diagram showing the oxazolinium ion intermediate and NAG-thiazoline (2.2) 22 Figure 2.2 Diagram of 4-methylumbelliferyl 2-thioacetamido glucoside (2.3) 23 Figure 2.3 Diagram of GalNAc-isofagomineHCl (2.4) 24 Figure 2.4 Diagram of 4-methylumbelliferyl (3-D-A'-acetylglucosaminide ( M U G ) (2.5) 26 Figure 2.5 Dixon plots for a series of family 20 inhibitors 28 Figure 2.6 Diagram of pNP-GlcNAc (2.8) and pNP-GalNAc (2.9) 29 Figure 2.7 The folded and unfolded slates of a protein 31 Figure 2.8 The relative activity of Sp. Hex. in the presence or absence of NAG-thiazoline during thermal denaturation at 55 °C 33 Figure 2.9 The intrinsic fluorescence spectrum of Sp. Hex. in buffer and in 5 M guanidine hydrochloride 34 Figure 2.10 The intrinsic fluorescence spectrum of Sp. Hex. in the absence and presence of N A G -thiazoline 34 Figure 2.11 Chemical denaturation of Sp. Hex. in the absence and presence of NAG-thiazoline, as monitored by intrinsic fluorescence 35 Figure 2.12 The C D spectrum of Sp. Hex 36 Figure 2.13 Chemical denaturation of Sp. Hex. in the absence and presence of NAG-thiazoline, as monitored by C D spectrometry 37 Figure 2.14 The relative activity of Hex B in the presence or absence of NAG-thiazoline during thermal denaturation at 60 °C 38 Figure 2.15 The intrinsic fluorescence spectrum of folded and denatured Hex B 39 Figure 2.16 The intrinsic fluorescence spectrum of Hex B incubated with NAG-thiazoline, folded and denatured 39 Figure 2.17 Chemical denaturation of Hex B in the absence and presence of NAG-thiazoline, as monitored by intrinsic fluorescence (A.m l x) 40 Figure 2.18 The relative activity of Hex A in the presence or absence of thiazoline during thermal denaturation at 56 °C 41 Figure 3.1 Diagram of 2-deoxy-2-fluoro-(3-D-glucopyranosyl fluoride (3.3) 43 vii Figure 3.2 The time-dependent inactivation of glucocerebrosidase at room temperature by 2-deoxy-2-fluoro-fS-D-glucopyranosyl fluoride (3.3) 47 Figure 3.3 The intrinsic fluorescent spectra of folded and denatured glucocerebrosidase 48 Figure 3.4 Chemical denaturation of glucocerebrosidase in the absence and presence of 2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride (inactivator), as monitored by intrinsic fluorescence at a fixed wavelength of 330 nm 49 Figure 3.5 Chemical denaturation of glucocerebrosidase in the absence and presence of 2-deoxy-2-fluoro-(3-D-glucopyranosyl fluoride (inactivator), as monitored by intrinsic fluorescence at A . m a x 49 Figure 3.6 Diagram of yV-octyl-l-epivalienamine (3.6) and 1-epivalienamine (3.7) 51 Figure 3.7 Dixon plot for the inhibition of glucocerebrosidase by /V-octyl-1 -epivalienamine 54 Figure 3.8 Lineweaver-Burk plot for glucocerebrosidase inhibition by A'-octyl-l-epivalienamine 54 Figure 3.9 The relative activity of glucocerebrosidase in the presence or absence of A'-octyl-l-epivalienamine during thermal denaturation at 53 °C 55 Figure 4.1 The structures of glucuronic acid (4.1) and hyaluronic acid (4.2) 59 Figure 4.2 Diagram of UDP-glucuronic acid 60 Figure 4.3 Diagram of 2-deoxy-2-fluoro-P-D-glucopyranuronosyl fluoride 61 Figure 4.4 Diagram of cc-D-galactopyranuronosyl fluoride (4.6) and cc-D-glucopyranuronosyl fluoride (4.9) 61 Figure 4.5 The compounds that did not function as acceptors for the E476A glucuronosynthase 67 Figure 4.6 Molecular model of T. maritima P-glucuronidase with bound 2-deoxy-2-fluoro galactosyl moiety 69 Figure 4.7 Space-filled model of T. maritima P-glucuronidase 69 V l l l List of Tables Table 2.1 K\ values for a series of family 20 glycosidase inhibitors 27 Table 2.2 Family 20 hexosaminidase substrate specificities 30 Table 4.1 E476A Glucuronidase glycosynthase reaction yields 64 Table 4.1 E476A Glucuronidase glycosynthase products selected for characterization 65 Table 5.1 Concentrations and temperatures used during thermal denaturation experiments 75 Table 5.2 Concentrations used during guanidine hydrochloride denaturation experiments 77 ix List of Schemes Scheme 1.1 The mechanism of a retaining P-glucosidase 3 Scheme 1.2 The mechanism of family 20 P-hexosaminidases 7 Scheme 1.3 Inactivation of a retaining P-glucosidase with 2,4-dinitrophenyl 2-deoxy-2-fluoro-P-D-glucopyranoside (1.5) 10 Scheme 1.4 The degradation of gangliosides by lysosomal enzymes 12 Scheme 1.5 The reaction catalyzed by lysosomal human p-hexosaminidase A 15 Scheme 1.6 The reaction catalyzed by glucocerebrosidase 18 Scheme 2.1 The synthesis of NAG-thiazoline (2.2) 25 Scheme 3.1 Inactivation of a retaining P-glycosidase with 2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride (3.3) 44 Scheme 3.2 The synthesis of 2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride (3.3) 46 Scheme 3.3 The synthesis of A'-octyl-l-epivalienamine (3.6) 53 Scheme 4.1 a. A trans-glycosylation reaction 58 b. A glycosynthase reaction 58 Scheme 4.2 The synthesis of galactopyranuronosyl fluoride (4.6) 62 Scheme 4.3 A glycosynthase reaction catalyzed by the E476A mutant of P-glucuronidase 63 Scheme 4.4 The acetylation and esterification of compound 4.13 66 X List of Abbreviations Abg: Agrobacterium sp. (3-glucosidase Ar: Aryl a.u.: Absorbance units Bn: Benzyl C D : Circular dichroism C H O : Chinese hamster ovary C M C : Critical micelle concentration C O S Y : Correlation spectroscopy D N P : Dinitrophenyl E. coli Escherichia coli ER: Endoplasmic reticulum F: Folded GalNAc: ^-acetylgalactosamine GalNAc-isofagamine: (2i?,3/?,45',5i?)-2-acetamido-3,4-dihydroxy-5-hydroxymethyl-piperid G l c N A c : jV-acetylglucosamine GlcNAc-6-S0 4 " : jV-acetylglucosamine-6-sulfate G M P : Good manufacturing practices h: Hour(s) Hex A : P-Hexosaminidase A Hex B: P-Hexosaminidase B H e x S : P-Hexosaminidase S I: Inhibitor k Ik "•cat' ^non-Ratio of rate constants for catalyzed and non-catalyzed reactions /cH//cD: Ratio of rate constants for protio and deutero substrates K{. Inhibition constant Km- Michaelis constant of a substrate L C - M S / M S : Liquid chromatography-tandem mass spectrometry M : Moles per litre min: Minutes M U G : 4-methylumbelliferyl-p-D-AT-acetylglucosaminide M U G S : 4-Methylumbelliferyl-P-D-jV-acetylglucosamine-6-sulfate m/z: Mass to charge ratio NAGal-thiazoline: (3a«,55 >65,7/J,7a/?)-5-(acetoxymethyl-6,7-diacetoxy-2-methyl-5,6,7,7a-tetrahydro-3a//-pyrano[3,2-<i]thiazole) NAG-thiazoline: (3a7e,57?,6S,,7^,7a7?)-5-(acetoxymethyl-6,7-diacetoxy-2-methyl-5,6,7,7a-tetrahydro-3a//-pyrano[3,2-rf]thiazole) N M R : Nuclear magnetic resonance O A c : Acetate PET: Positron emission tomography pNP: para-nitrophenyl pNP-GalNAc: pora-nitrophenyl P-D-ALacetylgalactosaminide pNP-GlcNAc: para-nitrophenyl P-D-A'-acetylglucosaminide RT: Room temperature s: Seconds S: Substrate Sp. Hex.: P-Hexosaminidase from Streptomycesplicatus T: Temperature THF: Tetrahydrofuran T F A : Trifluoroacetic acid T L C : Thin layer chromatography U : Unfolded U D P : Uridine diphosphate U V : Ultraviolet light v: Reaction velocity V m M : Maximum reaction velocity Vis : Visible light XylNAc-isofagamine: (27?,3/?,47?)-2-acetamido-3,4-dihydroxy-piperidine A . m a x : Wavelength of maximum fluorescence A N : Difference in refractive index x i i Acknowledgements I would like to thank my supervisor, Professor Stephen Withers, for his great knowledge and enthusiasm towards science, and for creating a great environment for working and learning. I also thank my collaborators at the Hospital for Sick Children in Toronto: Dr. Don Mahuran, Dr. Mike Tropak, and Brigitte Rigat. I thank Professor R . V . Stick (The University of Western Australia) and Professor Spencer Knapp (Rutgers, New Jersey) for providing compounds. I extend my gratitude to the staff at the U B C N M R facility for technical assistance: Lianne Darge, Marietta Austria and Dr. N ick Burlinson. Thanks to Mcintosh group member Dr. Mark Okon for running a 600 M H z N M R . I thank the staff at the U B C mass spectrometry lab and Dr. Fred Rosell for assistance with C D spectroscopy. In the Withers group I'd like to thank Karen Rupitz for assistance with enzyme kinetics and Shouming He for assistance with mass spectrometry. I would also like to thank my summer student, Fathima Shaikh for allowing me to learn some supervision skills and doing a nice job on some syntheses. I am seriously indebted to many people who provided invaluable advice and guidance along the way, and became my good friends. I would like to thank all Withers group members that I have had the pleasure of working with, past and present. I must give extra thanks to Dr.'s Andrew Watts, Michael Jahn, Chris Tarling, Hongming Chen, David Vocadlo, Carl Rye, Tanja Wrodnigg, Ian Greig, Hannes Muellegger, James Macdonald, Mark Vaughn, and soon to be Dr.'s Dave Dietrich, Luke Lairson, Omid Hekmat, Seung Lee, David Poon, and Brian Rempel. Finally I thank my non-scientist friends in Vancouver, Calgary and elsewhere, and my family for continuing love and support in my adventures. x i i i 1 General Introduction 1.1 Glycosidases To the average person the word carbohydrate brings to mind spaghetti and maybe a 'carb free' diet. To a scientist, carbohydrates have countless important roles, such as involvement in cel l-cel l interactions, protein signaling, and as the monomeric units found in cellulose and starch. A l o n g with nucleotides and amino acids, carbohydrates are the molecules most important for carrying out the tasks responsible for life itself. Carbohydrates in l iv ing systems are synthesized, metabolized, transferred, and modified with the help of enzymes. Glycosidases catalyze the cleavage o f the glycosidic bond, that is the C - 0 bond between the glycone (carbohydrate) and the aglycone (may or may not be a carbohydrate) (Figure 1.1). i 11 i Glycone Aglycone Figure 1.1 Diagram showing the glycone and aglycone components of a D-glucoside, and the numbering scheme used for carbohydrates in this thesis. The reactions carried out by glycosidases can be classified according to three criteria: 1. The structure of the glycone. A particular glycosidase usually demonstrates maximum activity on a specific glycone. For example a galactosidase more rapidly cleaves a galactoside than a glucoside. 2. The anomeric configuration of the glycone. Each glycosidase specifically catalyzes the cleavage o f either a or P glycosidic linkages. 1 3. The stereochemical outcome of the reaction catalyzed. A glycosidase may catalyze the cleavage o f the glycosidic bond with either retention or inversion of configuration of the anomeric centre (Figure 1.2). In addition, Henrissat has developed a system classifying glycosidases into 92 different families based on amino acid sequence similarities (http://afrnb.cnrs-m r s . f r / C A Z Y / ) 1 . Members of each family have the same three-dimensional fold and catalyze reactions with the same stereochemical outcome. Figure 1.2 Stereochemical outcome of reactions catalyzed by inverting and retaining glycosidases. The ability o f glycosidases to enhance reaction rates is truly remarkable. The glycosidic bond is extremely stable; for example the glycosidic bond in cellulose has a half-life o f 5 mi l l ion years under physiological conditions. Enzymes are able to catalyze the cleavage o f glycosidic bonds with rate enhancements 2 (kcJknon) o f up to 10 1 7 , an amazing feat considering that an enzyme is simply a polymer o f amino acids. 1.1.1 The Catalytic Mechanism of Retaining /3-Glycosidases The mechanism of retaining p-glycosidases, first proposed in 1953 by Koshland, 3 requires the presence o f two carboxylic acid groups in the active site. One carboxylic acid group acts as a general acid/base catalyst, and the other acts as the catalytic nucleophile (Scheme 1.1). In the first step o f the mechanism, the glycosylation step, the catalytic nucleophile attacks the anomeric center. The 2 mechanism proceeds through an oxocarbenium ion transition state in which the sugar adopts a distorted conformation with partial positive charge on the anomeric carbon and the endocyclic oxygen. The general acid/base residue aids the departure o f the aglycone by protonating the glycosidic oxygen. The glycosidic bond is broken and a glycosyl enzyme intermediate, in which the sugar is covalently bound to the enzyme, is formed. In the second step, the deglycosylation step, an incoming nucleophile, water or another carbohydrate (in the case o f transglycosylation), attacks the anomeric centre with general base catalysis from the general acid/base residue. A second oxocarbenium ion transition state is formed, again forming partial positive charge on the anomeric carbon and the endocyclic oxygen. Final ly , the glycosyl enzyme intermediate is released and the product is formed with overall retention o f anomeric configuration. Scheme 1.1 The mechanism of a retaining P-glucosidase. The double displacement mechanism for retaining P-glycosidases is now widely accepted due to considerable experimental evidence, but historically this was not always the case. A n alternative mechanism, proposed by Phi l l ips , 4 involved an 3 sp 2 hybridized ion-pair intermediate rather than a glycosyl-enzyme intermediate (Figure 1.3). v / W W \ . Figure 1.3. The ion pair intermediate proposed by Phillips. 4 Early evidence against the ion pair mechanism came in the form of kinetic isotope effects. Sinnott and Souchard found that when an activated substrate for which independent studies had shown that the second step was rate-limiting was used, (&H/&D) > 1, indicating that the second (deglycosylation) step involved a decrease in hybridization from intermediate to transition state (sp to sp"-like). I f the mechanism did in fact proceed through an ion pair, an inverse kinetic isotope effect would be expected since the ion pair intermediate is sp 2 hybridized, and an increase in hybridization would be needed to reach the more sp 3 - l ike transition state. Further evidence for the double displacement mechanism has been provided by X- ray crystallography. The X-ray crystal structures o f numerous retaining P-glycosidases have been published which show the presence o f two carboxylic acid residues in the active site, positioned approximately 5 A apart, a space suitable for the proposed mechanism to take place. 6 In several cases the covalent intermediate has been trapped using a deactivated substrate, which has allowed identification o f the catalytic nucleophile through X- ray crystallography, 7 and through proteolytic digestion 8 9 followed by L C - M S / M S analysis o f the fragments. ' Mutagenesis studies in which the acid/base carboxyl group was replaced with a different amino acid have shown that the glycosylation step was slowed for substrates with poor leaving groups which need acid catalysis for departure (reviewed by Zechel and Withers) . 1 0 The deglycosylation step was also slowed considerably for all substrates, since base catalysis was not available to aid the incoming nucleophile. For substrates with reactive leaving groups, the glycosylation step was not affected. However the deglycosylation step was slowed, resulting in accumulation of the covalent 4 intermediate and resulting in unusually low substrate Km values and high initial reaction rates in the pre-steady state. The addition of nucleophilic anions such as azide, formate and acetate resulted in an increase in rate o f cleavage o f the glycosyl enzyme intermediate, thus o f steady state rates for activated substrates. N o such 'rescue' was seen with wild-type enzymes. In order for the acid/base residue to function as an acid catalyst in the glycosylation step and a base catalyst in the deglycosylation step, the p K a o f the carboxylic acid must change during the course o f the reaction. A 1 3 C N M R titration study on a xylanase from Bacillus circulans showed that the p K a o f the acid/base residue dropped from 6.7 to 4.2 from the glycosylation to step to the deglycosylation step, indicating that the acid/base carboxylate undergoes " p K a cycl ing" in order to act as both an acid and a base catalyst. 1 1 The results o f a study on a P-glycosidase from Agrobacterium sp. ( A b g ) 1 2 are also consistent with a double displacement mechanism. A Bronstead plot (log kcal vs. p K a ) for a series o f aryl P-glucosides showed a biphasic, concave downward Bronstead relationship, indicating that the deglycosylation step is rate l imit ing for active substrates, and the glycosylation step is rate l imit ing for less active substrates. The same study also demonstrated that there are normal (kn/ku > 1) a secondary deuterium kinetic isotope effects for both the glycosylation and deglycosylation steps, as was the case in the earlier study by Sinnott and Souchard. 1.1.2 The Anchimeric Assistance Mechanism The body o f evidence summarized above provides excellent evidence that retaining P-glycosidases proceed through a double displacement mechanism. However, there is a class of p-glycosidases that employ an intramolecular catalytic nucleophile rather than an enzymatic nucleophile. The family 20 hexosaminidases proceed through an anchimeric assistance mechanism in which the substrate 2-acetamido group acts as the catalytic nucleophile (Scheme 1.2).1 3 A s is the case in the standard double displacement mechanism, two carboxylic acid groups (labeled A and B) are present in the active site. In this case however, neither carboxylate acts as the catalytic nucleophile. Instead the oxygen atom on the 2-acetamido group attacks the anomeric centre in the glycosylation step. The mechanism proceeds through an 5 oxocarbenium ion transition state in which the sugar adopts a distorted conformation with partial positive charge on the anomeric carbon, endocyclic oxygen and the nitrogen o f the acetamido group. General acid catalysis from carboxylic acid B aids the departure o f the aglycone, the glycosidic bond is broken, and an oxazolinium ion intermediate is formed. Carboxylate A stabilizes the positive charge that forms on the nitrogen in the oxazolinium ion intermediate. In the deglycosylation step, general base catalysis from carboxylate B aids the incoming nucleophile, in this case water, to attack the anomeric centre and the mechanism proceeds through a second oxocarbenium ion intermediate to give the hydrolyzed product with retention o f anomeric configuration. > 6 Scheme 1.2 The mechanism of family 20 P-hexosaminidases. 7 1.1.3 Glycosidase Inhibitors Glycosidase inhibitors are useful tools for probing enzyme mechanism. Since competitive inhibitors interact with the active site, kinetic and structural studies can provide information about the interactions involved in catalysis. Enzymes essentially function by lowering the activation energy o f a chemical reaction by stabilizing the transition state. To achieve this, enzymes must bind the transition state even better than the ground state. 1 4 In this vein, competitive glycosidase inhibitors are often designed to mimic at least one feature o f the oxocarbenium ion transition state. In addition to providing insight into enzyme mechanism, glycosidase inhibitors have the potential to act as drugs in many diseases, such as H I V , 1 5 inf luenza, 1 6 and cancer. 1 7 Nitrogen-containing sugar mimics (Figure 1.4) are potent inhibitors o f many 18 19 20 glycosidases. ' Acarbose (1.1), a natural product, is a transition state mimic that 21 is a potent a-amylase inhibitor and is used as a treatment for diabetes mellitus. The key unit o f acarbose is the carba-sugar at the non-reducing end, termed 'va l ienamine ' . 2 2 Because o f the double bond, the molecule adopts a flattened conformation, while the nitrogen provides the ability to carry positive charge, thus mimick ing the proposed oxocarbenium ion-like transition state. N o j i r i m y c i n 2 3 (1.2), another natural product, and its related analogs, contain a nitrogen atom in place o f the endocyclic oxygen found in sugars. Sugar mimics like nojir imycin which contain an endocyclic nitrogen are termed 'aza-sugars'. When charged, the nitrogen binds tightly to the active site, which contains various amino acids positioned to stabilize the positive oxocarbenium ion-like transition state. Despite the tight binding qualities of nojirimycins, studies comparing the changes in free energy associated with binding o f substrates and related inhibitors have shown that the nojirimycins are not true transition state analogs. 2 4 Another class o f aza-sugars are the isofagomines, which contain a nitrogen atom at the pseudo anomeric centre that can accommodate the positive charge found at the anomeric centre in the oxocarbenium ion-like transition state. The discovery that the natural product Siastatin B 2 5 (1.3) was a potent sialidase inhibitor inspired the synthesis o f isofagomine 2 6 (1.4) and related derivat ives. 2 7 ' 2 8 Interestingly, the 8 nojirimycin class of inhibitors are generally potent inhibitors of ct-glycosidases, while 29 the isofagomine class of inhibitors are generally potent inhibitors of P-glycosidases. 1.2 Nojirimycin 1.3 Siastatin B 1.4 Isofagomine Figure 1.4 Examples of competitive glycosidase inhibitors. 1.1.4 Mechanism Based Inactivators Mechanism-based inactivators react with a key catalytic residue in the active site irreversibly or with an extremely slow turnover rate. In 1987 the Withers group introduced a novel class of mechanism-based inactivators, the 2-deoxy-2-fluoroglucosides. 3 0 The first such inactivator was 2,4-dinitrophenyl 2-deoxy-2-fluoro-P-D-glucopyranoside (1.5, Scheme 1.3). This compound has an electronegative fluorine atom at C-2 , which destabilizes the oxocarbenium ion-like transition states 9 for both the glycosylation and deglycosylation steps. The incorporation o f a highly reactive leaving group at C - l speeds up the glycosylation step sufficiently to overcome the destabilizing effect caused by the fluorine at C-2 , and the glycosyl-enzyme intermediate is formed. The deglycosylation step is slowed down sufficiently to allow accumulation o f the glycosyl enzyme intermediate, resulting in rapid, time-dependent inactivation. Slow hydrolysis Scheme 1.3 Inactivation of a retaining P-glucosidase with 2,4-dinitrophenyl 2-deoxy-2-fluoro-P-D-glucopyranoside (1.5). This technique has proven useful in identifying the catalytic nucleophiles o f several enzymes through X-ray crystallography, 7 and through proteolytic digestion followed by L C - M S / M S analysis o f the fragments. 8 ' 9 1.2 Lysosomal Storage Disorders Lysosomes are the organelles within the cell responsible for destruction and recycling o f cellular components. The degradation o f cellular components within the lysosome is carried out by various lysosomal enzymes. Lysosomal storage disorders are caused by deficiencies in lysosomal enzymes, which results in accumulation o f the deficient enzyme's substrate. One class o f molecules that are recycled in the lysosome are gangliosides, which are glycosphingolipids consisting of a ceramide unit attached to an oligosaccharide chain. Gangliosides are attached to animal cell membranes through their hydrophobic ceramide moiety. The hydrophilic oligosaccharide chains extend into the extracellular space, where they are involved in cel l differentiation and cel l -cell interactions. The highest ganglioside content has been found in the brain . 3 1 10 Gangliosides are degraded in an orderly fashion by specific enzymes within the lysosome (Scheme 1.4). A deficiency in any one of these enzymes results in disease due to accumulation of the particular ganglioside associated with that enzyme. 3 2 Scheme 1.4 shows the degradation o f gangliosides by enzymes in the lysosome, and the diseases associated with each enzyme deficiency. This thesis focuses on two o f the lysosomal enzymes, P-hexosaminidase A and glucocerebrosidase, and the diseases associated with these enzymes, Tay Sachs and Gaucher disease. 11 O H - O H O H - O H H O ~ S ^ » ^ - - 0 - \ ^ ^ ^ ? r"OH , _ - - O H O H O H G M 1 ganglioside O H O H G M 1 (3-galactosidase X . G a l GM1 gangliosidosis H O 9 r - O H O H A c H N Ic-J--0, ^ ^ V - V - O O H O H HO\H%/^j( ° M 2 ganglioside N H A c ^-Hexosaminidase A ^ G a l N A c Tay Sachs disease OH OH OH OH Globoside ?Hr0H cOH - E ^ i ' 0 ^ ^ ° H ° ^ w r 0 C e r H° ACHN ° ° r 0 H r°H "\H%X/C G M 3 ganglioside H o - S ^ ^ < £ - ^ ^ O C e r OH OH O H I Ganglioside neuraminidase IP Hexosaminidase A and B ( V N A N A J ^ G a l N A c Sandhoff disease OIH,OH ? H r " O H c O H a-Galactosidase A L \_ -o OH OH / Fabry disease 9 r-"OH ^ 0 H Lactosyl ceramide A H O - \ ^ ^ ° H O W ; ^ ' ' I p-Galactosidase O C e r O H O H I ^ Gal OH ? H ^ O H Sulfatide Glucocerebroside OH OH I Arylsulfatase A Gaucher disease Glucocerebrosidase Galactocerebrosidase leukodystrophy q j c J^-~ S 0 4 2 " Metachromatic X - C H 3 *7^~ f" O H C e r = ^ Gal JgL Ceramide ° H " C H 3 Krabbe disease OH Galactocerebroside Scheme 1.4 The degradation of gangliosides by lysosomal enzymes. Enzymes are shown in blue, and the diseases associated with each enzyme deficiency are shown in red. Adapted from Voet and Voet. 3 j 12 1.2.1 Tay Sachs Disease Dr. Warren Tay first described what would later be known as Tay Sachs disease in 1881. He observed a cherry-red spot on the retina o f a mentally and physically retarded one-year-old child. The cl inical symptoms first observed by Tay were observed again six years later by Bernhard Sachs and termed familial amaurotic i d iocy . 3 4 The condition was named Tay Sachs disease after the clinicians that first described it. Tay Sachs disease is caused by accumulation o f a compound in the brains o f patients, and in 1962 Lars Svennerholm identified that compound as G M 2 ganglioside 3 5 (Scheme 1.5). In 1969 Okada discovered that a deficiency in the enzyme P-hexosaminidase A was the cause of the accumulation of GM2 ganglioside. 3 6 The deficiency in P-hexosaminidase A is hereditary and most common among Ashkenazi Jews. Tay Sachs occurrs at an estimated carrier rate o f 1 in 300 in the general population and 1 in 35 among the Ashkenazi Jewish population. 3 1 There is currently no cure for Tay Sachs disease, though incidences o f the disease have dropped due to international carrier detection programs. 3 7 1.2.1.1 The Human Hexosaminidases Hexosaminidases catalyze the cleavage o f p-(l ,4) l inked A'-acetylglucosamine (1.6) ( G l c N A c ) or ^-acetylgalactosamine ( G a l N A c ) (1.7) residues from the non-reducing end of oligosaccharides and glycoconjugates (Figure 1.5). 13 HO' OH OR HO OR NH NH O 1.6 1.7 Figure 1.5 Diagram of terminal /V-acetyl-|3-D-glucosamine (1.6) and /V-acetyl-(3-D-galactosamine (1.7) residues. R = oligosaccharide or glycoconjuagate. Unl ike most glycosidases, which are specific for only one substrate, hexosaminidases catalyze the cleavage o f both jV-acetylglucosamine and 7V-acetylgalactosamine residues. The name 'hexosaminidase' encompasses both the glucosaminidase and galactosaminidase activity. Three P-hexosaminidase isozymes are found in human tissue. The isozymes are dimers composed o f combinations o f two subunits, a and p. P-Hexosaminidase A (Hex A ) is a heterodimer composed o f one a and one p subunit, and P-hexosaminidase B (Hex B) is a homodimer composed of two p subunits. The third isozyme is the rare P-hexosaminidase S (Hex S) composed o f two a subunits. Only Hex A and Hex S are capable o f efficiently hydrolyzing negatively charged substrates such as the artificial substrate 4 -methylumbelliferyl P-D-/V-acetylglucosamine-6-sulfate ( M U G S ) (1.8, Figure 1.6), while only Hex A is capable of cleaving the terminal G a l N A c from the negatively charged GM2 ganglioside in vivo (Scheme 1.5). In order to do so Hex A requires a cofactor, the G M 2 activator protein, which extracts G M 2 ganglioside from the 38 intralysosomal membrane and presents the terminal G a l N A c residue to Hex A . Both Hex A and Hex B are family 2 0 hexosaminidases which proceed through the anchimeric assistance mechanism (Scheme 1.2). 14 Scheme 1.5 The reaction catalyzed by lysosomal human P-hexosaminidase A . 1.2.1.2 The Cause of the Deficiency in Hex A The HEXA and HEXB genes encode the a and p subunits, respectively. Heritable naturally occurring point mutations in the HEXA gene result in mutant a subunits, which may result in an unstable, misfolded Hex A enzyme. L i k e many other proteins, Hex A is synthesized in the endoplasmic reticulum (ER) . Misfolded and incompletely assembled proteins are common side-products o f protein synthesis in the E R . Whi le correctly folded proteins are secreted and go on to perform their function, misfolded and incompletely assembled proteins are retained in the E R and degraded through quality control mechanisms. Thus, unstable, potentially misfolded Hex A is not transported through the golgi apparatus to the lysosome, but instead is retained in the E R and destroyed by quality control mechanisms in the c e l l . 3 9 Consequently, the mutant Hex A , though catalytically active, does not reach its target substrate and G M2 ganglioside accumulates. Tay Sachs disease is the most common of three similar diseases known as the GM2 gangliosidoses. Mutations in the P subunit, encoded by the HEXB gene, cause Sandhoff disease. The third and most rare disease is known as the AB-variant form of GM2 gangliosidosis and is caused by mutations in the GM2A gene, which codes for the G M 2 activator protein. There are several different levels o f severity and age o f onset o f Tay Sachs disease. The acute, infantile form is caused by mutations in the a subunit that prevent any Hex A from reaching the lysosome, and is characterized by rapidly progressing neurological deterioration beginning at 3-5 months o f age and resulting in death by the age o f 4 years. 3 7 The less severe adult onset form of Tay Sachs is characterized by slowly progressing neurodegenerative disorders. This milder phenotype is seen in mutations that al low only 1-5 % of wild-type Hex A activity. The majority of Ashkenazi Jewish patients with adult Tay Sachs disease have an otG269S mutation which causes the protein to be slightly unstable. Interestingly, mutations that al low for only 5-10 % of wild-type activity have been seen in patients who are asymptomatic 4 0 . Thus it is believed that a "crit ical threshold" o f 5-10 % of wild-type Hex A activity is needed to prevent accumulation o f G M 2 ganglioside. 4 0 The a and P subunits of Hex A are inactive as monomers. The recently published crystal structure for Hex B and modeling data for Hex A 4 1 provided insight 16 into why this is the case. Although each subunit contains an active site, the crystal structure o f Hex B showed that the two active sites are found at the interface o f the two monomers, and that several residues from one subunit stabilize and structurally complete the active site residues o f the other subunit. The a and p subunits share a primary structure that is 60 % identical, and most o f the conservation between the subunits is found within their catalytic domains. Model ing studies showed that like Hex B , the active site of Hex A is found at the dimer interface. The modeling studies also showed a large groove at the a,p dimer interface where the G M 2 activator protein l ikely docks, and that the active sites o f the a and P subunits are essentially identical except for three amino acid changes: PAsp426 -> aGlu394 , pAsp452 ctAsn423, and pLeu453 -> ctArg424. The presence o f an arginine residue (Arg424) in the a active site instead o f the leucine residue (Leu453) appears to help create the electrostatic environment needed to accommodate negatively charged substrates such as GM2 ganglioside. This result is supported by previous kinetic studies that showed that an Arg424Gln mutation affected the enzyme's ability to remove G l c N A c - 6 - S C V from an artificial substrate. 4 2 It was later shown that the same site in P-hexosaminidase A is responsible for binding both the 6-sulfate group on artificial substrates and the sialic acid moiety of G M 2 ganglioside. 4 3 1.2.2 Gaucher Disease The second lysosomal storage disorder discussed in this thesis is Gaucher disease. Gaucher disease was named after French dermatologist Phi l l ipe Gaucher, who described the first case o f the disease in 1882 in his medical thesis. 4 4 It was found that cells o f the spleen were peculiar due to a storage material, which was later characterized as glucosylceramide by another French scientist, A g h i o n . 4 4 Gaucher disease is the most common of the lysosomal storage disorders, with a prevalence o f 1/50 000 in the Caucasian population. 4 5 L i k e Tay Sachs disease, Gaucher disease is heritable and most common among Ashkenazi Jews. L i k e Tay Sachs disease, Gaucher disease is caused by point mutations resulting in unstable, misfolded enzymes that are destroyed by quality control mechanisms in the cell before they can reach their target substrates in the lysosome. Interestingly, the phenotypes for the disease are highly variable, though they are al l caused by 17 accumulation o f glucosylceramide. Gaucher disease is classified into 3 types based on central nervous system involvement and age of onset. 4 4 Type 1 is characterized by the absence o f central nervous system involvement, while type 2 is characterized by onset in infancy and rapidly progressing neurodegenerative disorders resulting in death before the age of 2 years. Type 3 is characterized by early childhood or adolescent onset and slowly progressing neurodegenerative disorders. A l l three types result in degrees o f enlargement of the liver and spleen. The only currently available treatment for Gaucher disease is enzyme replacement therapy, 4 6 which is expensive with a cost o f between $100 000 and $750 000 per patient per year, and is only effective towards type 1 Gaucher disease, since injected enzyme cannot cross the blood brain barrier. 4 7 1.2.2.1 Glucocerebrosidase Glucocerebrosidase, a retaining P-glycosidase from family 30, catalyzes the cleavage o f the glycosidic bond in glucosylceramide with retention o f anomeric configuration (Scheme 1.6).4 8 The proposed mechanism for glucocerebrosidase is that shown for general retaining P-glycosidases in Scheme 1.1. o Scheme 1.6 The reaction catalyzed by glucocerebrosidase. Glucocerebrosidase is a membrane-associated enzyme, which attaches to the negatively charged phospholipids on the lysosomal membrane once it is delivered from the E R to the lysosome. A n activator protein, saposin C , induces a further conformational change rendering the enzyme active. Substrate docking with the recently published X-ray crystal structure of glucocerebrosidase shows that the ceramide chain does not appear to fit in the active site, suggesting that the ceramide chain either remains embedded in the l ip id bilayer during catalysis or interacts with the saposin C cofactor. 4 9 Extensive kinetic characterization has shown that the 18 enzyme requires detergents such as Triton X-100 and sodium taurocholate for activity 50 in vitro. 1.2.3 Therapies for Lysosomal Storage Disorders Enzyme replacement therapy is proving to be an effective, albeit expensive, therapy for type 1 Gaucher disease. 4 7 The search for a more universal therapy has led to the study o f 'substrate deprivation therapy': inhibition o f the synthesis of glycosphingolipids. B lock ing the ganglioside production pathway should theoretically prevent ganglioside accumulation. Indeed, 7V-(n-butyl)deoxynojirimycin (1.9, Figure 1.7), an inhibitor o f the ceramide-specific glucosyl transferase involved in ganglioside synthesis, has shown promise in cl inical trials as a therapy for Gaucher disease. 5 1 However, side-effects such as osmotic diarrhea were experienced due to inhibition o f intestinal a-glucosidases and other glycosidases, and more importantly 52 toxic effects were observed on the liver and spleen in treated humans. Figure 1.7 Diagram of A^n-butyOdeoxynojirimycin (1.9). 1.2.3.1 The Chemical Chaperone Approach The chemical chaperone approach brings to mind rescuing someone from themselves. The body's own internal watchdogs are responsible for preventing a slightly unstable, yet still catalytically active enzyme from fulfi l l ing its all-important role. Studies o f the quality control mechanisms in the E R have shown that there are E R chaperones and folding enzymes that assist the folding process and serve as retention anchors for immature proteins, preventing these immature proteins from being degraded. 3 9 This knowledge spawned the idea that an artificial or chemical chaperone could be introduced that could theoretically stabilize an unstable, misfolded protein and allow that protein to escape degradation by the quality control OH OH 19 mechanisms. This concept has been applied to several systems, including the most common protein mutation causing cystic fibrosis. The mutant cystic fibrosis transmembrane conductance regulator protein was stabilized by glycerol, al lowing it to escape the quality control mechanisms in the E R , and restoring protein act ivi ty . 5 4 The chemical chaperone approach has also been applied to lysosomal storage disorders. In the first case, 1-deoxy-galactonojirimycin (1.10, Figure 1.8) was found to enhance oc-galactosidase A activity in Fabry lymphoblasts. 5 5 Later it was shown that Af-(n-nonyl)deoxynojirimycin (1.11) increases glucocerebrosidase activity in Gaucher fibroblasts. 4 7 Most recently the galacto-like 4 epimer o f 7V-octyl-l-epivalienamine (1.12) was shown to increase activity in a mouse model o f G M I gangliosidosis. 5 6 1.10 1.11 l l L Figure 1.8 Diagram of compounds 1.10-1.12. In each o f these cases an enzyme inhibitor was used to specifically target and stabilize the mutant enzyme in question. The binding o f the inhibitor in the active site provides enough stabilization through non-covalent hydrogen bonding interactions to al low the mutant enzyme to pass the quality control mechanisms in the E R and reach the lysosome. The use o f an enzyme inhibitor to increase enzyme activity seems counterintuitive. In this case, however, the inhibitor is tightly and specifically binding to the enzyme in the E R , then chaperoning the enzyme to the lysosome, where the concentration o f the substrate is much higher than the concentration o f the inhibitor (especially in cases where the substrate has accumulated). Once in the lysosome, some enzyme activity is restored when the substrate replaces the inhibitor in the active site. A modest increase of enzyme activity to only 5-10% of normal would be necessary for a Tay Sachs patient to be asymptomatic. 4 0 Side-effects should be minimal since the inhibitor is necessary at lower concentrations than the 'substrate 20 deprivation therapy' approach where a larger dose is necessary to inhibit the ganglioside synthesizing enzymes. 1.3 Aims of this Thesis The recent work published in this field has shown the potential for the use o f enzyme inhibitors as chemical chaperones in lysosomal storage disorders. The goal o f this thesis is ultimately to take the first steps towards chemical chaperone therapies for Tay Sachs and Gaucher diseases. The initial goal is to synthesize and test the inhibitory power o f small molecule inhibitors for these enzymes. The second aim is to show that these inhibitors have the ability to stabilize the folded conformation of the enzyme and provide a biochemical explanation for the stabilizing effect provided by chemical chaperones. The third goal is to provide the compounds and chemical expertise to the group o f collaborator Dr. Don Mahuran (Hospital for Sick Children, Toronto) in their ongoing research towards therapies for Tay Sachs and Gaucher disease. 21 2 The Stabilizing Effect of NAG-Thiazol ine on Family 20 Hexosaminidases 2.1 NAG-Thiazoline: a Competitive Inhibitor of Family 20 Hexosaminidases Until 1996 there was little evidence that family 20 hexosaminidases proceed through an anchimeric assistance mechanism instead of the standard double displacement mechanism. However, the work of Vocadlo, Withers et. al.n provided undeniable evidence that family 20 hexosaminidases do proceed through an anchimeric assistance mechanism. A stable analog of the proposed oxazolinium ion intermediate, NAG-thiazoline (systematic name (3a/?,5i?,6S,7i?,7ai?)-5-(acetoxymethyl-6,7-diacetoxy-2-methyl-5,6,7,7a-tetrahydro-3ar7-pyrano[3,2-djthiazole)) (2.2) was synthesized. As shown in Figure 2.1, NAG-thiazoline contains a sulfur atom in the place of the oxygen atom seen in the proposed oxazolinium ion intermediate. Inhibition studies showed that NAG-thiazoline is a powerful inhibitor of jack bean hexosaminidase (K\ = 280 nM) , 1 3 and a hexosaminidase from 57 Streptomycesplicatus (Sp. Hex.) (K\ = 20 uM). Figure 2.1 Diagram showing the oxazolinium ion intermediate and NAG-thiazoline (2.2). Further support for the anchimeric assistance mechanism was provided when incubation of jack bean hexosaminidase with 4-methylumbelliferyl-2-thioacetamido-P-D-glucoside (2.3, Figure 2.2) resulted in release of 4-methylumbelliferone and time-dependent loss of enzyme activity. The loss of enzyme activity was shown to be due to the reversible build-up of NAG-thiazoline. These results suggested that the enzyme catalyzes the conversion of 2.3 to NAG-thiazoline, through nucleophilic attack of the thioacetamido group on the anomeric centre. 22 2.3 Figure 2.2 Diagram of 4-methylumbelliferyl-2-thioacetamido-(3-D-glucoside (2.3). Structural data from X-ray crystal structures of Sp. Hex.5S and Hex B 4 1 with bound NAG-thiazoline have since provided insight into which residues are involved in the positioning of the 2-acetamido group for nucleophilic attack at the anomeric centre and the stabilization of the positive charge formed on the oxazolinium ring upon cyclization. X-ray crystal structures of Sp. Hex.59 and Hex B 4 1 with bound GalNAc -isofagomine (2.4, Figure 2.3), have also been published. Azasugars of the isofagomine class are generally strong inhibitors of P-retaining glycosidases, in part because of a strong electrostatic interaction that forms between the catalytic nucleophile and positively charged nitrogen at the pseudo anomeric centre.59 Since family 20 hexosaminidases lack a catalytic nucleophile, isofagomine type azasugars were not expected to be powerful inhibitors of these enzymes. Surprisingly, GalNAc -isofagomine was found to be a powerful inhibitor of Sp. Hex. (K\ = 2.1 uM). In this case, however, the X-ray crystal structures with bound GalNAc-isofagomine have shown a strong electrostatic hydrogen-bonding interaction between the protonated nitrogen atom of the ring and the general acid-base residue. 23 OH nu > e -H CI S H AcHN Figure 2.3 Diagram of GalNAc-isofagomine-HCl (2.4) (systematic name (2R,3R,4S,5R)-2-acetamido-3,4-dihydroxy-5-hydroxymethyl-piperidinium hydrochloride) 2.2 The Synthesis of NAG-Thiazoline Although there are several other known family 20 hexosamidase inhibitors, NAG-thiazoline was selected as the best candidate for further studies, as its relatively easy synthesis allows for rapid and economical scale-up. NAG-thiazoline was synthesized similarly to the published method (Scheme 2.1). 1 3 2-Acetamido-2-deoxy-P-D-glucopyranose 1,3,4,6-tetra-acetate was refluxed with Lawesson's reagent in toluene to provide the cyclized per-O-acetylated thiazoline (2.1) in 70 % yield. The reaction with Lawesson's reagent creates the thio-acetamide, and heating promotes cyclization through an SN2-like displacement of the P-OAc group by the thio-acetamide group. The per-O-acetylated thiazoline was then deprotected with sodium methoxide in methanol. Careful neutralization with glacial acetic acid provided the desired NAG-thiazoline product (2.2) in 75 % yield. Neutralization with the harsher Amberlite® H + I R 120 resin led to opening of the ring and formation of the hydrolysis product. 24 O A c OAc Lawesson's reagent* toluene 120 °C A c O A c O OAc S NH NaOMe MeOH H 3 C O , O C H 3 HO S 2.2 N Scheme 2.1 The synthesis of NAG-thiazoline (2.2). 2.3 Inhibition Results Hex A and Hex B enzyme activity can be measured by monitoring the release of methylumbelliferone from the artificial substrate 4-methylumbelliferyl P-D-A^-acetylglucosaminide (MUG) (2.5) by fluorescence (Figure 2.4). Alternatively, para-nitrophenyl P-D-TV-acetylglucosaminide (pNP-GlcNAc) can be used as an artificial substrate for the human P-hexosaminidases using a spectrophotometric assay with a U V - V i s spectrometer. However the concentration of pNP-GlcNAc needed is in the milli-molar range, resulting in difficulties with substrate solubility and high background absorbances. The use of a fluorescent substrate overcomes these complications and allows the use of less enzyme, but requires a stopped assay. At the pH used for the assay (pH 4.5), methylumbelliferone is protonated and hence does not fluoresce. The enzyme/substrate mixture can be diluted into a solution of high pH and the fluorescence of this solution can then be measured. Although this assaying technique is labour intensive, it is effective, and hence was used for this study. 25 C H ; Figure 2.4 Diagram of 4-methylumbelliferyl P-D-/V-acetylglucosaminide ( M U G ) (2.5) NAG-thiazoline was tested as an inhibitor of Hex A and Hex B by measuring the rates of hydrolysis of M U G at a series of NAG-thiazoline concentrations. For the purpose of comparison, two other potential inhibitors were also tested in the same fashion: NAGal-thiazoline (2.6) (systematic name (3ar?,56,,6.S,,7i?,7a/?)-5-(acetoxymethyl-6,7-diacetoxy-2-methyl-5,6,7,7a-tetrahydro-3a//-pyrano[3,2-ofjthiazole)), and XylNAc-isofagomineHCl (2.7) (systematic name (2R,3R,4R)-2-acetamido-3,4-dihydroxy-piperidinium hydrochloride). The structures of these compounds are shown in Table 2.1. NAGal-thiazoline is the galacto- version of NAG-thiazoline, and has a reported K\ value of 100 u M for Sp. Hex.51 K\ values were obtained for the three inhibitors by fitting the data to the equation for competitive inhibition (Equation 2.1) using GraFit version 4.0.19.6 0 1 + m K (Equation 2.1) The data can be graphically represented with Dixon plots (the inverse of the reaction rate vs. inhibitor concentration). The line for 1/V m a x intersects the lines representing different substrate concentrations at K\ = -[I]. The results of the inhibition studies are shown in Table 2.1, and the corresponding Dixon plots are shown in Figure 2.5. 26 Table 2.1 K\ values for a series of family 20 glycosidase inhibitors. Name Structure Sp. Hex. Hex B Hex A NAG-thiazoline (2.2) HO-^V N H ^0 S Y 20 u M 5 6 190 n M 270 n M NAGal-thiazoline (2.6) OH OH \ s / 100 u M 5 6 860 n M 820 n M XylNAc-isofagomineHCl (2.7) H O H O ^ ^ ^ © H a AcHN H 38 u M Not Done 90 u M 27 0.18 0.16 0.14 _ 0.12 c 'E. 0.1 <D 2 0.08 " 0.06 0.04 0.02 0 Inhibition of Hex B with NAG-thiazoline 1 I 1 I 1 I 1 I M 1 I 1 I 1 I 1 ! 1 Inhibition of Hex A with NAG-thiazoline [MUG]= 0.519 mM [MUG]= 1.20 mM 1A/max 0.14 0.12 0.1 1 0.08 2 0.06 0.04 0.02 0 1 I I I 1 i | 1 | 1 | 1 | 1 | 1 | 1 | 1 y | i [MUG] = - 0.519 mM / '_ - [MUG] = 1.20 mM - oy 1/Vmax -i h l i l i h l i h l i l i l o o o X X X ^ *? "f . - • > O o o O O O O ^ ,_ * * , x * *~ og -5 co CM ^ (D ID . . . [NAG-thiazoline] (M) [NAG-thiazoline] (M) Inhibition of Hex B with NAGal-thiazoline Inhibition of Hex A with NAGal-thiazoline [MUG] = 0.987 mM _ # _ [MUG] = 2.37 mM - o - [MUG] = 0.474 mM — 1/Vmax - O - [MUG] = 0.395 mM _ D _ [MUG] = 0.987 mM _ # _ [MUG] = 2.37 mM 1/Vmax [NAGal-thiazoline] (M) [NAGal-thiazoline] (M) Figure 2.5 Dixon plots for a series of family 20 inhibitors. 28 A s expected, NAG-th iazo l ine was found to be a potent competitive inhibitor o f both Hex A and Hex B , with Kt values of 270 n M and 190 n M , respectively, K\ values for NAGal - th iazo l ine (820 n M for Hex A and 860 n M for Hex B ) are approximately three fold higher. The previous work o f V o c a d l o 5 7 had shown that Sp. Hex. also binds NAG-th iazo l ine (K\ = 20 u M ) tighter than NAGal - th iazo l ine (K\ = 100 u M ) . The specificity constants (kcaJKm) for each enzyme for the artificial substrates para-nitrophenyl P-D-/V-acetylglucosaminide ( p N P - G l c N A c ) (2.8, Figure 2.6) and /para-nitrophenyl P-D-A^-acetylgalactosaminide ( p N P - G a l N A c ) (2.9, Figure 2.6) were examined to see whether the lower K, for the thiazoline o f gluco-configuration is paralleled by increased substrate specifity towards substrates of gluco- configuration. Figure 2.6 Diagram of pNP-GlcNAc (2.8) and pNP-GalNAc (2.9). The specificity constants provide a means of comparison o f the catalytic efficiency o f an enzyme towards a substrate. A higher kcJKm indicates a higher catalytic efficiency. For each enzyme, kcJKm for p N P - G l c N A c is slightly higher than that for p N P - G a l N A c , indicating that the enzymes show higher substrate specificity towards p N P - G l c N A c . Whi le this difference is small, it is supported by results from two previously published studies that showed that both Hex A and Hex B are more active towards artificial substrates of the gluco- configurat ion. 3 6 ' 6 1 Thus the increased selectivity towards NAG-th iazo l ine over NAGal- th iazo l ine parallels the increased substrate specificity for p N P - G l c N A c over p N P - G a l N A c demonstrated by a l l three hexosaminidases. N H A c 2.8 2.9 29 Table 2.2 Fami ly 20 hexosaminidase substrate specificities. Enzyme p N P - G l c N A c hJKm (x 10 5 M-'s" 1 ) p N P - G a l N A c kcJKm (x 10 5 M - ' s - 1 ) Sp. Hex.51 37.0 6.70 Hex B 20.4 14.5 Hex A 5.25 4.10 XylNAc- i sofagomine (2.7) was found to inhibit Hex A and Sp. Hex. wi th Kx values o f 90 u M and 38 u M respectively. Comparison o f the K\ values o f X y l N A c -isofagomine and NAG-th iazo l ine for Hex A (90 u M and 270 n M , respectively) reveals that NAG-th iazo l ine binds Hex A approximately 330 times tighter. Whi l e XylNAc- i so fagomine is not a powerful inhibitor o f Hex A , its related analog G a l N A c -isofagomine (2.4, Figure 2.3) may be better. Previously published results for inhibition o f Sp. Hex. by GalNAc-isofagomine {K\ = 2.1 u M ) 5 9 indicate that G a l N A c -isofagomine binds to Sp. Hex. tighter than the three inhibitors discussed here. However, the data in Table 2.1 shows that XylNAc- i so fagomine binds to Hex A less tightly than to Sp. Hex. I f the same trend holds, GalNAc-isofagomine w i l l not be a tighter binder to Hex A than N A G - and NAGal- th iazol ine . Regardless o f the potential inhibitory power o f GalNAc-isofagomine towards Hex A , the fact remains that the isofagomines are not trivial to synthesize (starting from an advanced precursor: 12 steps, 17 % overall yield for GalNAc- isofagomine) , 5 9 which makes the thiazolines more attractive therapeutic candidates. To summarize, NAG-th iazo l ine binds to Hex A approximately 3 times tighter than NAGal- th iazo l ine and 330 times tighter than XylNAc- i so fagomine . Because o f its ease of synthesis and tight-binding properties to Hex A , NAG- th iazo l ine was chosen for further studies. 30 2.4 Denaturation Experiments Globular proteins such as Sp. Hex., Hex A , and Hex B exist in equilibrium between the folded and unfolded states (Figure 2.7). The equilibrium can be shifted to the right, thereby denaturing the protein, by environmental changes such as rise in temperature, variation o f p H , or the addition of chemical denaturants such as guanidine hydrochloride. A m i n o acid mutations, of course, can also have this effect. Protein denaturation may or may not be reversible. For example, proteins denatured with guanidine hydrochloride can often be refolded by removing the guanidine hydrochloride through dialysis, but proteins denatured with heat often aggregate and precipitate, becoming irreversibly unfolded. In the simplest model, unfolding is a cooperative two state process. In this model a protein that is 50 % unfolded has 50 % of the molecules folded and 50 % unfolded; unfolding intermediates are less stable than either the folded or unfolded states and therefore do not exist for a significant length o f time. Folded (F) , Unfolded (U) *- Irreversibly Unfolded Figure 2.7 The folded and unfolded states of a protein. The idea o f using an enzyme inhibitor as a chemical chaperone is centred on the hypothesis that the interactions that form between enzyme and inhibitor provide extra enzyme stability. Should this be the case, one might expect an enzyme with bound inhibitor to be slightly more stable and hence slightly more resistant to conditions that tend to denature proteins such as heat and chaotropic agents. To test this hypothesis, we set out to see whether NAG-th iazo l ine stabilizes family 20 p-hexosaminidases against thermal and chemical denaturation. O f greatest interest was to see whether NAG-th iazo l ine stabilizes Hex A towards denaturation. Project collaborators from the group o f Dr. Don Mahuran (Hospital for Sick Children, Toronto) have successfully purified human Hex A that has been expressed in Chinese hamster ovary ( C H O ) cells. However, the purification proved difficult and only small quantities o f Hex A were made available to us for this study. Ideally, experiments would be performed with Hex A containing a mutation such as the Gly269Ser mutation common in adult Tay Sachs disease. Unfortunately, mutant Hex A has 31 proven sufficiently unstable that it has yet to be successfully purified. Hex B is more stable than Hex A 3 1 and has been purified in larger quantities. Thus init ial studies were performed on the easily obtainable bacterial Sp. Hex., and additional studies were performed with Hex B and where possible Hex A . Given that the three hexosaminidases are from the same family, it should be reasonable to extrapolate results from one to another. 2.4.1 Sp. Hex. Thermal Denaturation Studies Thermal denaturation studies were performed by first determining a temperature at which the enzyme loses substantial activity within an hour. The enzyme was then incubated at this temperature in the presence or absence o f inhibitor, and aliquots were removed at fixed time intervals and the enzyme activity was measured using the artificial substrate p N P - G l c N A c by U V / V i s spectrometry. The concentration of NAG-th iazo l ine selected for al l o f the denaturation experiments was at least ten times its K\ value to ensure that the inhibitor occupied the vast majority o f enzyme active sites. It was also ensured that the absolute concentration o f NAG-th iazo l ine was always greater than the enzyme concentration. In al l cases, when an aliquot o f the enzyme/NAG-thiazoline mixture was diluted into a buffered solution o f substrate for the kinetic assay, the dilution factor was large enough for the concentration o f NAG-th iazo l ine to become significantly less than K\ so that a reaction rate could be measured. Sp. Hex. thermal denaturation studies were performed by incubating the enzyme at a temperature o f 55 °C. Aliquots were removed and the residual activity o f Sp. Hex. was measured using the artificial substrate p N P - G l c N A c . A parallel experiment was performed in which Sp. Hex. was incubated in the presence o f 240 u M NAG- th iazo l ine (K\ = 20 u M ) . Figure 2.8 shows a plot o f percent enzyme activity remaining against incubation time at 55 °C. The figure shows that Sp. Hex. retains more activity when incubated with NAG-th iazo l ine , indicating that the NAG-th iazo l ine stabilizes Sp. Hex. to thermal denaturation. 32 40 240 uM thiazoline no thiazoline 0 20 40 60 time (min) Figure 2.8 The relative activity of Sp. Hex. in the presence or absence of NAG-thiazoline during thermal denaturation at 55 °C. 2.4.2 Sp. Hex. Guanidine Hydrochloride Denaturation Studies The resistance of Sp. Hex. to chemical denaturation was examined using guanidine hydrochloride as denaturant. The mechanism of action of chaotropic agents such as guanidine hydrochloride is not fully understood, although they are believed to disrupt hydrophobic interactions within the protein.6 2 In order to measure the degree of folding or unfolding, the intrinsic fluorescence of the enzyme was examined. The aromatic amino acids phenylalanine, tyrosine and tryptophan fluoresce when excited with radiation at the appropriate wavelength. Tyrosine and tryptophan absorb at wavelengths near 280 nm, which coincidentally is the wavelength where phenylalanine emits 6 2. Additionally, tryptophan emission is much stronger than tyrosine emission. Consequently when a protein is excited near 280 nm, it is mainly the tryptophan emission that is observed. Protein unfolding often leads to a change in emission intensity and a shift in the wavelength of maximum emission to a higher wavelength. When in a hydrophobic environment such as the buried interior of a protein, tryptophan has an emission maximum near 320 nm. The fluorescence maximum of tryptophan in the unfolded state often approaches that of tryptophan in aqueous solution, with a maximum near 350 nm. The fluorescence emission spectrum of Sp. Hex. shows an emission maximum of 333 nm when folded and 356 nm when incubated in 5 M guanidine hydrochloride (Figure 2.9). Of note is that NAG-thiazoline seems to quench some of 33 the fluorescence emission intensity when bound in the active site o f the enzyme, though the curve maintains the same shape (Figure 2.10). The unfolded spectra in the absence or presence o f NAG-thiazol ine are identical. At a certain concentration o f guanidine hydrochloride (2.5 M for Sp. Hex.), the fluorescence emission spectrum no longer changes with increasing guanidine hydrochloride concentrations, and thus the enzyme wil l be considered to be denatured. Figure 2.9 The intrinsic fluorescence spectrum of Sp. Hex. in buffer (red) and in 5 M guanidine hydrochloride (blue). 700 - i 600 A —1 —I 1 r -320 340 360 380 Wavelength (nm) Figure 2.10 The intrinsic fluorescence spectrum of Sp. Hex. in the absence (blue) and presence (red) of NAG-thiazoline. 34 In order to create a denaturation curve, Sp. Hex. was incubated at a series o f guanidine hydrochloride concentrations and the intrinsic enzyme fluorescence was measured at a fixed emission wavelength of 325 nm. A t this wavelength the difference between the emission intensity o f the spectra o f the folded and unfolded enzyme is greatest. A denaturation curve was created by plotting the percent fluorescence signal at 325 nm relative to the signal o f the folded enzyme, against the concentration o f guanidine hydrochloride (Figure 2.11). Comparison of the curves in the presence or absence o f NAG-th iazo l ine shows that incubating Sp. Hex. wi th NAG- th iazo l ine caused the denaturation curve to shift to the right. In the absence o f inhibitor Sp. Hex. began to unfold at a guanidine hydrochloride concentration o f approximately 0.9 M and unfolded completely at a concentration o f approximately 2 M . When pre-incubated with NAG-th iazo l ine , Sp. Hex. began to unfold at approximately 1.5 M guanidine hydrochloride and unfolded completely at approximately 2.5 M . Thus a higher concentration of guanidine hydrochloride is necessary to denature the enzyme when the inhibitor is bound. Figure 2.11 Chemical denaturation of Sp. Hex. in the absence and presence of N A G -thiazoline, as monitored by intrinsic fluorescence. Another technique that can be used to monitor protein unfolding is circular dichroism (CD) . The C D spectrum of folded Sp. Hex. is shown in Figure 2.12. 35 20 CD[mdeg] 197 250 300 Wavelength[nm] Figure 2.12 The C D spectrum of Sp. Hex. Folded proteins generally have a strong C D signal at 222 nm, the characteristic wavelength o f alpha helical content. The C D signal at this wavelength drops significantly in denatured proteins, which adopt a random c o i l . 6 2 A denaturation curve was created by incubating Sp. Hex. at a series o f guanidine hydrochloride concentrations and measuring the C D signal at 222 nm. The percent C D signal at 222 nm relative to the folded enzyme was plotted against the concentration o f guanidine hydrochloride (Figure 2.13). A s expected, the denaturation curve obtained is virtually identical to that obtained by fluorescence. When incubated with NAG-th iazo l ine , Sp. Hex. maintained a stronger C D signal at 222 nm in the 1-3 M guanidine range, again suggesting that NAG- th iazo l ine stabilizes Sp. Hex. against guanidine hydrochloride denaturation. 36 Figure 2.13 Chemical denaturation of Sp. Hex. in the absence and presence of N A G -thiazoline, as monitored by C D spectrometry. 2.4.3 Hex B Thermal Denaturation Studies For the thermal denaturation study with Hex B, the enzyme was incubated at 60 °C and the activity was measured using the artificial substrate 4-methylumbelliferyl P-D-A^-acetylglucosaminide (2.5, Figure 2.4). Figure 2.14 shows that over 30 minutes Hex B retained more activity when pre-incubated with N A G -thiazoline, demonstrating the ability of NAG-thiazoline to provide a stabilizing effect. 37 no NAG-thiazoline 2.4 nM NAG-thiazoline 40 0 20 40 time (min) Figure 2.14 The relative activity of Hex B in the presence or absence of NAG-thiazoline during thermal denaturation at 60 °C. 2.4.4 Hex B Guanidine Hydrochloride Denaturation Studies In addition to the thermal denaturation study, a guanidine denaturation study was undertaken with Hex B . The degree o f folding/unfolding was measured by intrinsic fluorescence. A s was the case with Sp, Hex., the fluorescence emission spectrum shifted to the right with increasing guanidine concentrations (Figure 2.15). In this case it was not possible to follow the enzyme unfolding at a fixed wavelength because the spectra o f the partially unfolded states maintained a high fluorescence emission signal at wavelengths near 325 nm (the wavelength used for the Sp. Hex. study). This was particularly troublesome when the enzyme was incubated with NAG- th iazo l ine since the inhibitor was again found to partially quench the fluorescence, in this case causing the fluorescence emission at 325 nm to be very similar for the folded and partially unfolded enzymes (Figure 2.16). 38 Figure 2.15 The intrinsic fluorescence spectrum of folded (red) and denatured (green) Hex B. 500-1 — i 1 1 1— 320 340 360 380 Wavelength (nm) Figure 2.16 The intrinsic fluorescence spectrum of Hex B incubated with NAG-thiazoline, folded (red) and denatured (purple). A n alternative to examining the fluorescence at a fixed wavelength is to monitor the change in wavelength o f maximum fluorescence (Xmax). A s the enzyme unfolds, the wavelength o f maximum fluorescence shifts to the right. The fact that NAG-thiazol ine quenches fluorescence does not significantly affect this parameter. 39 Figures 2.15 and 2.16 show that the Xmax for folded Hex B is 328 nm in the absence or presence o f NAG-th iazo l ine . A denaturation curve was obtained by plotting the wavelength o f maximum fluorescence against the concentration o f guanidine hydrochloride (Figure 2.17). no NAG-thiazoline 2.5 uM NAG-thiazoline 0 1 2 3 4 [Guanidine hydrochloride] (M) Figure 2.17 Chemical denaturation of Hex B in the absence and presence of NAG-thiazoline, as monitored by intrinsic fluorescence (A, m a x). The denaturation curve o f enzyme that was pre-incubated with N A G -thiazoline is shifted to the right relative to the denaturation curve o f the free enzyme. The stabilizing effect o f the NAG-th iazo l ine is particularly noticeable at guanidine hydrochloride concentrations below 2 M . Be low 2 M guanidine hydrochloride, the enzyme pre-incubated with NAG-th iazo l ine maintains a Xm3K below 330 nm, indicating that the enzyme remains completely folded. In the absence o f N A G -thiazoline, the enzyme begins to denature significantly at guanidine hydrochloride concentrations higher than 1 M . Above 3 M guanidine hydrochloride, both the free enzyme and the enzyme pre-incubated with NAG- th iazo l ine have the same A , m a x , indicating that the denaturing conditions are powerful enough to overpower the stabilizing effect o f NAG-th iazo l ine . 40 2.4.5 Hex A Denaturation Studies Unfortunately there was insufficient Hex A to perform a chemical denaturation study using guanidine hydrochloride. Had sufficient enzyme been available, it may have been possible to gather enough data points to noticeably see the denaturation o f each sub-unit separately in the dissociation curve. The a subunit is slightly less stable than the P subunit 3 7 and thus should denature before the P subunit. Such a study was not possible, but a thermal denaturation study, which required much less enzyme, was possible. The study was performed at 56 °C, a temperature slightly lower than the 60 °C used for the Hex B study. The lower temperature was necessary because the less stable Hex A denatured very quickly at 60 °C. Figure 2.18 shows that in the absence o f NAG-th iazo l ine , Hex A retains less than 30 % o f enzymatic activity when incubated at 56 °C for 30 minutes. However, in the presence o f N A G -thiazoline, Hex A retains approximately 60 % of enzymatic activity when incubated at 56 °C for 30 minutes. The data indicates that Hex A retains more activity over a 30 minute period when pre-incubated with NAG-th iazo l ine , suggesting that N A G -thiazoline stabilizes Hex A against thermal denaturation. > 100 i i 1 ' 1 1 i 80 60 - o \ • — • • 40 "—" n 20 -0 i I i i i I 0 10 20 30 time (min) Figure 2.1 3 The relative activity of Hex A no NAG-thiazoline 4.1 uM NAG-thiazoline during thermal denaturation at 56 °C. 41 2.5 Conclusion/Future Outlook The results o f the studies with Sp. Hex., Hex B and Hex A presented here show that NAG-th iazo l ine stabilizes these enzymes against denaturation. These results provide some insight into why a competitive inhibitor may act as a chemical chaperone in a therapy for a lysosomal storage disorder. Further studies by collaborator Dr. M i k e Tropak (Hospital for Sick Children, Toronto) with adult Tay Sachs and Sandhoff fibroblasts have shown that NAG- th iazo l ine has a stabilizing effect wi th the mutant enzymes in cell l ines . 6 3 Tropak showed that NAG- th iazo l ine increases Hex A activity above the critical threshold o f 10 % of w i l d type activity in adult Tay Sachs and Sandhoff fibroblasts. Tropak also showed that the increased Hex A activity is in fact found in the lysosome, indicating that NAG- th iazo l ine is chaperoning the mutant Hex A from the E R to the lysosome. These extremely promising results have been further boosted by studies with mice in which large doses of NAG- th iazo l ine have been administered without any resulting toxicity effects. Mahuran and Tropak are planning on contracting out a quality assured ( G M P ) large-scale synthesis o f NAG-th iazo l ine for continued studies with mice, and potentially monkeys. I f no complications or major side effects are detected, NAG- th iazo l ine could soon be tested on humans as a therapy for Tay Sachs disease. 4 2 3 The Stabilizing Effect of 2-Deoxy-2-Fluoro-P-D-Glucopyranosyl Fluoride and /V-Octyl-l-Epivalienamine on Glucocerebrosidase 3.1 Stabilization of Glucocerebrosidase by a Mechanism-Based Inactivator The deficient enzyme in Gaucher disease is glucocerebrosidase, a family 30 retaining P-glycosidase that operates through the standard double displacement mechanism (see Scheme 1.1). Glucocerebrosidase is a 497 amino acid membrane-associated glycoprotein with a mass o f 59-67 kDa , depending upon the state o f glycosylat ion. 4 4 A s is the case with Tay Sachs disease and Hex A , we believe that an enzyme inhibitor could provide enough stabilization to al low an unstable mutant glucocerebrosidase to escape the quality control mechanisms in the E R and reach its substrate, glucosylceramide, in the lysosome. Unfortunately, glucocerebrosidase containing a Gaucher-causing mutation was not available for this study. However, a relatively large amount of wild-type enzyme was available, thanks to the donation of used vials from a patient currently undergoing enzyme replacement therapy. Each vial contains approximately 100 u L o f 1.4 mg/mL leftover enzyme. Due to the abundance o f enzyme, there was no need to do initial studies with a bacterial enzyme. A previous study had shown that glucocerebrosidase was covalently inactivated by 2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride (2FpF) (3.3, Figure Figure 3.1 Diagram of 2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride (3.3) 2-Deoxy-2-fluoro-D-glycosyl f luorides 6 5 function on the same principle as the original 2-fluoro mechanism based inactivators 3 0 (mechanism shown in Scheme 1.3), but the 2,4-dinitrophenyl leaving group has been replaced by a fluoride, which is also a good leaving group (Scheme 3.1). The catalytic nucleophile displaces the P-fluoride and forms a covalent bond with the 2-deoxy-2-fluoro-D-glucosyl moiety. Once 43 labelled by the inactivator, the enzyme was proteolytically digested and through L C -M S / M S analysis o f the fragments the catalytic nucleophile was identified as glutamate 340 . 6 4 Slow hydrolysis Scheme 3.1 Inactivation of a retaining (3-glycosidase with 2-deoxy-2-fluoro-|3-D-glucopyranosyl fluoride (3.3). 2-Deoxy-2-fluoro-P-D-glucopyranosyl fluoride (3.3) was considered as a good candidate for a chemical chaperone since the inactivator forms a covalent bond to the enzyme within the active site, possibly stabilizing the enzyme significantly. A previous study in the Withers lab had shown that P-glucosidases from Agrobacterium faecalis and Caldocellum saccharolyticum were stabilized against urea denaturation by a 2-deoxy-2-fluoro-D-glucosyl moiety bound in the active si te. 6 6 Another important consideration is that 2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride has been shown to reach organs such as the brain, spleen, liver and kidney when injected into rats and to inhibit the enzymes therein. 6 7 A l s o important is that this inhibited P-glycosidase activity was restored within 48 hours, showing that the trapped glycosyl enzyme intermediate was slowly turned over via hydrolysis or transglycosylation. Thus in theory i f the compound was administered to a patient, it would reach the target organs, and the free glucocerebrosidase enzyme would be released from the glycosyl enzyme intermediate within a reasonable time scale. Another advantage o f 18 the use o f a fluorine-containing inactivator is the option o f incorporation o f F , which would allow monitoring of the location of the labeled enzyme through positron emission tomography (PET). In a previous study in the Withers laboratory, John McCarter successfully labelled a P-glucosidase from Agrobacterium sp. (Abg) with 2-deoxy-2-[ l 8 F]fluoro-P-D-mannopyranosyl [ 1 8 F]fluoride and performed preliminary in vivo P E T imaging studies with rats. 6 8 In a later study, Alexander W o n g attempted to 44 radiolabel glucocerebrosidase with 2,6-dideoxy-2-fluoro-6-[ 1 8 F]-fluoro-P-D-glucopyranosyl f luor ide . 6 9 ' 7 0 Although the cold version o f compound was found to 1 8 inactivate the enzyme, the radiolabelling of glucocerebrosidase with the F compound was not successful. Despite the fact that the initial attempt to radiolabel glucocerebrosidase did not succeed, the successful radiolabeling o f A b g by McCarter has shown the potential for this technique, and the possibility o f radiolabeling 18 glucocerebrosidase with an F compound remains. 3.1.1 The Synthesis of 2-Deoxy-2-Fluoro-B-D-Glucopyranosyl Fluoride 2-Deoxy-2-fluoro-P-D-glucopyrariosyl fluoride (3.3) was synthesized according to literature methods (Scheme 3 .2) . 7 1 ' 7 2 Tri-O-acetyl-D-glucal was reacted with acetic anhydride/acetic acid and Selectfluor® at 60 °C to provide a mixture o f three compounds; the a - and P- anomers o f the gluco isomer, and the oc-manno isomer. The gluco and manno compounds were separated by column chromatography with some difficulty providing a mixture of the a and P gluco isomers in a poor yield o f 10 %. This material was then converted to the ct-bromide (3.1) with 80 % yield using HBr/acetic acid. The bromide was displaced with silver fluoride to provide the P-fluoride (3.2) in 70 % yield. Deprotection was carried out with sodium methoxide in methanol to provide 2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride (3.3) in 85 % yield. 45 H B r / C H 3 C O O H C H 2 C I 2 I F Scheme 3.2 The synthesis of 2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride (3.3) 3.1.2 Inactivation of Glucocerebrosidase with 2-Deoxy-2-Fluoro-f3-D-Glucopyranosyl Fluoride Glucocerebrosidase was incubated with an excess o f 2-deoxy-2-fluoro-p-D-glucopyranosyl fluoride at room temperature. Aliquots o f the enzyme/inactivator mixture were removed after 45 minutes, 3 hours, and 16 hours and enzyme activity was monitored using the artificial substrate 2,4-dinitrophenyl P-D-glucopyranoside (2,4-DNP-glucopyranoside). A continuous spectrophotmetric assay was used to monitor the release o f 2,4-dinitrophenol at 400 nm. Figure 3.2 shows that glucocerebrosidase was inactivated in a time-dependent fashion, and that after 16 hours the enzyme had lost 90 % of its activity. 46 0.4 1 i I i I i I 1 I 1 I 1 I r 'E o o 3? 5 < 0 0 2 4 6 8 10 12 14 16 time (h) Figure 3.2 The time-dependent inactivation of glucocerebrosidase at room temperature by 2-deoxy-2-fluoro-|3-D-glucopyranosyl fluoride (3.3). [Glucocerebrosidase] = 0.44 |j.M. [2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride] = 2.5 m M 3.1.3 Denaturation Studies with Glucocerebrosidase in the Presence and Absence of 2-Deoxy-2-Fluoro-B-D-Glucopyranosyl Fluoride Ideally, the stabilizing effect of 2-deoxy-2-fluoro-p-D-glucopyranosyl fluoride on glucocerebrosidase could be measured with both thermal and chemical denaturation studies like those performed previously with the hexosaminidases. However, performing a thermal denaturation study by monitoring enzyme activity is complicated by the fact that 2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride is a covalent inactivator and not a competitive inhibitor l ike NAG- th iazo l ine . In the case of a competitive inhibitor, the inhibitor is held in the active site through non-covalent interactions. B y diluting the enzyme/inhibitor mixture so that [I] « K,, a large proportion o f active sites become vacant, al lowing the substrate to enter the active site, and making an assay for enzyme activity using an artificial substrate possible. However, in the case o f a covalent inactivator like 2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride, the inactivator is covalently linked in the active site, and remains covalently linked even after dilution. Thus the substrate cannot replace the inactivator in the active site and an assay for enzyme activity is not possible. Consequently thermal enzyme denaturation cannot be measured by monitoring enzyme activity. However, a chemical denaturation study analogous to those performed with the hexosaminidases is possible. Intrinsic fluorescence was chosen over C D spectroscopy as the method o f choice to monitor enzyme unfolding since it requires 47 the use of less enzyme. Figure 3.3 shows the intrinsic fluorescence spectra o f folded and denatured glucocerebrosidase. 400H - 200-1 c CD =! 300H iooH 320 340 360 Wavelength (nm) 380 Figure 3.3 The intrinsic fluorescent spectra of folded (red) and denatured (blue) glucocerebrosidase. A s was the case for the hexosaminidases, the intrinsic fluorescence spectrum o f denatured glucocerebrosidase is decreased in fluorescence intensity and is shifted to the right, with a higher X , i m x . When the flurorescence spectra o f glucocerebrosidase incubated in various concentrations o f guanidine hydrochloride are examined, a trend is apparent. A s the concentration o f guanidine hydrochloride increases, A , m a x shifts to the right and the fluorescence signal decreases in intensity as the tryptophan residues move from the hydrophobic buried interior o f the folded protein to the solvent exposed denatured state. A denaturation curve can be created by either plotting the fluorescence intensity at a fixed wavelength or A , m a x against guanidine hydrochloride concentration. In this case, both the fluorescence emission at a fixed wavelength o f 330 nm (Figure 3.4) and the wavelength o f maximum fluorescence (A . m a x ) (Figure 3.5) were examined. The fixed wavelength o f 330 nm was chosen because it is at this wavelength that the difference in fluorescence intensity between the folded and unfolded spectra is greatest. 48 Figure 3.4 Chemical denaturation of glucocerebrosidase in the absence and presence of 2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride (inactivator), as monitored by intrinsic fluorescence at a fixed wavelength of 330 nm. Figure 3.5 Chemical denaturation of glucocerebrosidase in the absence and presence of 2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride (inactivator), as monitored by intrinsic fluorescence at A . m a x . Examining Figure 3.4 first, in the absence o f the 2-deoxy-2-fluoro-D-glucosyl moiety (which forms after incubating glucocerebrosidase with 2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride) the relative fluorescence at 330 nm drops rapidly above 1 M guanidine hydrochloride, indicating that the enzyme begins to unfold at these low guanidine hydrochloride concentrations. However, in the presence o f the 2-deoxy-2-fluoro-D-glucosyl moiety the relative fluorescence at 330 nm stays near 100 % up to 3 49 M guanidine hydrochloride, indicating that the enzyme remains mainly folded in this range o f guanidine hydrochloride concentrations. Figure 3.5 shows that in the absence o f the 2-deoxy-2-fluoro-D-glucosyl moiety, the Ximii increases rapidly above 1 M guanidine hydrochloride. This increase in A , m a x is the result o f more tryptophan residues becoming exposed to solvent, indicating that the protein is beginning to unfold. In the presence o f the 2-deoxy-2-fluoro-D-glucosyl moiety, Xnwx remains near 336 nm up to 3 M guanidine hydrochloride, indicating that the tryptophan residues remain sheltered from solvent as in the completely folded enzyme. Interestingly, in both Figure 3.4 and 3.5, the data for denaturation after pre-incubation with 2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride fit the equation for a sigmoidal curve, as expected for a cooperative two state unfolding process. However, in the absence o f the 2-deoxy-2-fluoro-D-glucosyl moiety, the data do not fit a sigmoidal curve. Instead the relationship seems more linear. This could be because glucocerebrosidase, a membrane-associated protein, may be resistant to complete denaturaton as is the case for some other membrane-associated proteins 7 3 . Guanidine hydrochloride, which is believed to act by disrupting hydrophobic interactions within the folded protein, 7 4 may not be able to penetrate the long sequences o f hydrophobic amino acid residues present in some membrane-associated proteins. Another consideration is that the buffer used for kinetics and the unfolding studies with glucocerebrosidase contains detergents such as thesit and sodium taurocholate, which are hydrophobic in nature. For full enzyme activity, these detergents must be present at or above their critical micelle concentration ( C M C ) . 5 0 It has been shown that certain detergents present at concentrations higher than their critical micelle concentration stabilize partially folded protein intermediates 7 5. The fact that the data obtained after pre-incubation with 2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride does fit a sigmoidal curve suggests that over the guanidine hydrochloride range o f 0 to 3 M , the 2-deoxy-2-fluoro-D-glucosyl moiety stabilizes the enzyme sufficiently to keep the enzyme completely folded. A t concentrations higher than 3 M , the conditions are powerful enough to unfold the enzyme completely, essentially skipping the unfolded intermediates seen in the absence of the 2-deoxy-2-fluoro-D-glucosyl moiety, and approximating a cooperative two-state unfolding process. 50 Regardless o f the explanation for the shapes o f the curves, both Figures 3.4 and 3.5 clearly show that the presence o f the 2-deoxy-2-fluoro-D-glucosyl moiety in the active site stabilizes glucocerebrosidase against denaturation in the 0 to 4 M guanidine hydrochloride concentration range. 3.2 Stabilization of Glucocerebrosidase by a Competitive Inhibitor 3.2.1 N-Octyl-l-Epivalienamine: a Competitive Inhibitor of Glucocerebrosidase Having tested the stabilizing effect o f a covalent enzyme inactivator, it was o f interest to see how a competitive inhibitor would compare. In a study by the group o f Ogawa 7 6 , a series o f TV-alkyl-P-valienamines were synthesized and tested as inhibitors o f mouse liver glucocerebrosidase. The study showed that A^-octyl-l-epivalienamine (3.6) was the most potent mouse liver glucocerebrosidase inhibitor o f those inhibitors tested. The 1-epivalienamine unit (3.7) is an isomer o f the valienamine unit present in the naturally occurring compound acarbose (Figure 1.4). The attachment o f an octyl chain to the amine provides the l ipophilici ty present in glucosylceramide, the natural substrate for glucocerebrosidase. A collaboration with the group o f Professor R. V . Stick (the Universi ty o f Western Australia), provided tetra-O-benzyl-1-epivalienamine (3.4, Scheme 3.4) (systematic name (li?,47?,55,65)-4,5,6-tribenzyloxy-3-(benzyloxymethyl)-cyclohex-2-enylamine), an advanced precursor to Af-octy l - l -epivalienamine. The synthesis o f tetra-O-benzyl-1-epivalienamine is lengthy, requiring 12 steps. 7 7 OH OH HO HO' (CH 2 ) 7 C H 3 HO N H 2 HO 3.6 3.7 Figure 3.6 Diagram of TV-octyl-l-epivalienamine (3.6) and 1-epivalienamine (3.7). 5 1 Initially the synthesis of the AZ-octyl product was attempted following the scheme published by Ogawa 7 6 in which the free amine was reacted with octanoyl chloride, creating an amide bond. The amide bond was then reduced using lithium aluminum hydride. Unfortunately, the desired product was not achieved, perhaps due to our use of benzyl ether protecting groups rather than the isopropylidene group used by Ogawa. There is precedent in the literature for lithium aluminum hydride causing 78 hydrogenolysis of a primary benzyl ether group, resulting in the alcohol. A n alternate strategy towards the synthesis of 7V-octyl-l-epivalienamine had to be employed. Tetra-O-benzyl-l-epivalienamine (3.4) was treated with octyl aldehyde in the presence of a reducing agent in an attempted reductive amination (Scheme 3.3). Water was distilled off with a Dean Stark apparatus, driving the equilibrium from the hemi-aminal to the imine. Addition of sodium borohydride in the same pot reduced the imine to the amine to provide the iV-octyl product (3.5) with an overall yield of 65 %. Deprotection was carried out with sodium and liquid ammonia in THF (a Birch reduction), to afford the deprotected A/-octyl-l-epivalienamine (3.6). The Birch reduction reaction was quenched with ammonium chloride, causing the final product to be contaminated with sodium chloride and excess ammonium chloride. Unfortunately, the TV-octyl-1 -epivalienamine proved to be very difficult to purify from these salts. A combination of Sep-Pak® C-18 resin and ion exchange columns was necessary to eliminate the salts. 52 •OBn •OBn B n O octyl aldehyde benzene B n O BnO-B n O N ^ r c , - ( C H 2 ) 6 O B n H O B n 3.4 N a B H 4 Na O B n B n O ' B n O • ( C H 2 ) 7 O B n O H 3.5 3.6 Scheme 3.3 The synthesis of 7V-octyl-l-epivalienamine (3.6). 3.2.2 The Inhibition of Glucocerebrosidase by N-Octyl-l-Epivalienamine The inhibition o f glucocerebrosidase by Af-octyl-l-epivalienamine was measured by monitoring enzyme activity using the artificial substrate 2 ,4 -DNP-glucopyranoside at a series o f iV-octyl-1 -epivalienamine concentrations. The data were fit to the equation for competitive inhibition (Equation 2.1, p. 26), revealing that iV-octyl-1 -epivalienamine is a potent inhibitor o f human glucocerebrosidase with a K\ value o f 75 n M . A graphical representation o f the data is shown in Figure 3.7 (Dixon plot). A Lineweaver-Burk plot (the inverse o f reaction rate vs. the inverse o f substrate concentration for different inhibitor concentrations) (Figure 3.8) can be used to verify that the inhibition is competitive. The Michael is-Menten equation rearranges to: Thus a plot of l / v 0 versus 1/[S] w i l l have a slope o f K m / V m a x , and an intercept o f 1/Vmax- For competitive inhibitors, V m a x remains constant, and the lines on the Lineweaver-Burk plot intersect at the same spot on the Y axis. Figure 3.8 shows that this is the case for glucocerebrosidase inhibition by //-octyl-1-epivalienamine, indicating that the inhibition is indeed competitive. (Equation 3.1) 53 Figure 3.7 Dixon plot for the inhibition of glucocerebrosidase by yV-octyl-l-epivalienamine. Figure 3.8 Lineweaver-Burk plot for glucocerebrosidase inhibition by A'-octyl-l-epivalienamine. 3.2.3 Denaturation Studies with Glucocerebrosidase in the Presence and Absence of N-Octyl-l-Epivalienamine A chemical denaturation study using guanidine hydrochloride and monitoring unfolding by fluorescence was performed. Interestingly, the enzyme pre-incubated with N-octyl- l-epivalienamine did not show increased resistance to guanidine hydrochloride denaturation. A n explanation for this unexpected result is that the guanidine hydrochloride may be interacting with the TV-octyl-l-epivalienamine and preventing it from binding as tightly. This could be due to the fact that guanidine 54 hydrochloride is hydrophobic in nature, as is the octyl chain in yV-octyl-1-epivalienamine. Although //-octyl-1-epivalienamine does not protect glucocerebrosidase against guanidine hydrochloride-induced denaturation, the fol lowing study showed that it does provide a stabilizing effect against thermal denaturation and this is probably more relevant to the physiological situation. Glucocerebrosidase was incubated at 53 °C in the presence and absence o f TV-octyl-l-epivalienamine, and the residual activity at a series o f time points was measured using the artificial substrate 2,4-DNP-glucopyranoside. Figure 3.9 shows that in the absence o f A/ -oc ty l - l -epivalienamine, enzyme activity dropped rapidly; with almost zero detectable activity after 30 minutes. The data was fit to a first order decay equation, which provided a calculated half-life o f 5.6 minutes. In the presence o f 7V-octyl-l-epivalienamine, loss o f activity was gradual, and the calculated half-life is 32 minutes. The data demonstrates that /V-octyl-1 -epivalienamine has a substantial stabilizing effect when glucocerebrosidase is incubated at an elevated temperature over time. This result shows that although no stabilizing effect against guanidine hydrochloride-induced denaturation could be detected, /V-octyl-l-epivalienamine does have an ability to protect glucocerebrosidase against thermal denaturation. 0 10 20 30 time (minutes) Figure 3.9 The relative activity of glucocerebrosidase in the presence or absence of A'-octyl-l-epivalienamine during thermal denaturation at 53 °C. 55 3.3 Conclusion The results presented here indicate that both a mechanism based inactivator (2-deoxy-2-fluoro-[3-D-glucopyranosyl fluoride) and a competitive inhibitor (,/V-octyl-1-epivalienamine) stabilize glucocerebrosidase against denaturation. Both compounds could potentially function as chemical chaperones in therapies for Gaucher disease. Further studies with Gaucher fibroblasts are necessary to evaluate the ability o f these inhibitors to enhance mutant glucocerebrosidase activity in the lysosome. The presence o f the octyl chain would increase the specificity o f TV-octyl-l-epivalienamine towards glucocerebrosidase over other glycosidases, reducing the potential for side effects, and could also help the compound cross cellular membranes. However the difficulty o f synthesis o f TV-octyl-l-epivalienamine would result in a costly therapy, although l ikely less costly than the presently used enzyme replacement therapy. In contrast, 2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride is relatively easy to synthesize, but would l ikely have less specificity for glucocerebrosidase. This may or may not be a problem since the inhibitor is being used at sub-inhibitory concentrations needed to provide the modest increase in enzyme activity to remove disease symptoms. I f need be, the fluoride at C - l could be replaced with an activated l ipophil ic chain, in an effort to increase the specificity o f the compound towards glucocerebrosidase. 56 4 The Development of a Glucuronidase Glycosynthase 4.1 Introduction 4.1.1 Background on Glycosynthases Oligosaccharides are o f considerable therapeutic interest, 7 9 with disease targets including metabolic and cardiovascular diseases, and cancer. Unfortunately, the chemical synthesis o f oligosaccharides is hampered by the tedious use o f protecting groups and the challenge of controlling the stereochemistry o f the anomeric l inkage. 8 0 This difficulty has hindered the development of oligosaccharides for use as therapeutics. The problems faced with chemical synthesis can be overcome by the use o f enzymes, which have built-in regio- and stereo-selectivity. Glycosy l transferases, which catalyze the transfer of a glycoside from an activated donor sugar to a saccharide acceptor, are the obvious best candidates. However, the donor is usually a nucleotide sugar, and the cost o f nucleotide donor sugars is a limitation for industrial use. 8 1 Another possibility is the use o f glycosidases 'run in reverse' in transglycosylation reactions. The glycosyl enzyme intermediate in the standard double displacement mechanism can be intercepted with an acceptor sugar instead of water, creating a transglycosylation product instead of a hydrolysis product (Scheme 4.1a). This is achieved by using donor sugars with good leaving groups to increase the concentration o f glycosyl enzyme, and adding a high concentration o f acceptor sugar. The effective concentration of water can also be reduced with the use o f co-solvents. However, this technique suffers from poor yields since the oligosaccharide product o f the transglycosylation reaction is a substrate for the enzyme. The Withers group found a solution to this problem with the introduction of glycosynthases in 1998 (Scheme 4.1b). 8 2 The catalytic glutamate nucleophile in a retaining glycosidase from Agrobacterium sp. (Abg) was replaced with an alanine residue, resulting in a correctly folded but hydrolytically inactive enzyme. However, the mutant enzyme was able to catalyze the formation o f p-( l ,4) linkages between oc-glucopyranosyl fluoride and a variety of acceptors. The cc-glucosyl fluoride donor, which is o f the opposite anomeric configuration to that o f the regular substrate, mimics the glycosyl-enzyme intermediate found in the reaction pathway o f retaining 57 P-glycosidases. The acceptor binds in such a way that the 4-hydroxyl is positioned for nucleophilic attack on the anomeric center. General base catalysis is provided by the catalytic acid/base residue, increasing the nucleophilicity o f the acceptor hydroxyl group. Fluoride, a good leaving group, departs and the glycosidic bond is formed, creating an oligosaccharide product. a. Scheme 4.1 a. A trans-glycosylation reaction, b. A glycosynthase reaction. The obvious advantage o f this technique is that the oligosaccharide product is not a substrate for the hydrolytically inactive glycosynthase enzyme, and as a consequence yields are very good (70-90 %). Another advantage is that the glycosyl fluoride donors are relatively simple and inexpensive to synthesize. The glycosynthase technique has since been applied to other glycosidases by the Withers group and by others to create a variety o f products and linkages, which have been 83 catalogued in a review by Wi l l i ams and Withers. 58 At present, however, there are no reported glycosynthases that can incorporate glucuronic acid residues into oligosaccharides. The work presented here describes the first such glycosynthase, developed from a glucuronidase from Thermotoga maritima. 4.1.2 Thermotoga maritima T. maritima is a thermophilic bacterium isolated from geothermally heated marine sediment with an optimum growth temperature of 80 °C. 8 4 The complete O f genomic sequence of T. maritima has been reported. Numerous enzymes have been purified from T. maritima, including several glycosidases that are resistant to high temperatures, denaturing agents, and alcohols.8 6 This resistance to harsher conditions has created interest in thermophilic enzymes due to their potential application in biotechnology and in industrial processes that require stability at high temperatures. 4.1.3 B-Glucuronidases Family 2 P-glucuronidases are retaining P-glycosidases that catalyze the removal of glucuronic acid (4.1, Figure 4.1) units from the non-reducing end of oligosaccharides. Glucuronic acid has several important biological roles. It is a component of glycosaminoglycans, which are found in the ground substance that makes up the extracellular spaces in cartilage, tendon, skin, and blood vessel walls. One such glycosaminoglycan is hyaluronic acid (4.2, Figure 4.1), which consists of p-(1-4) linked repeating units of glucuronic acid (3-(l-3) linked to 7V-acetyl-D-87 glucosamine, and is used for eye surgeries and other medicinal applications. 4.2 Figure 4.1. The structures of glucuronic acid (4.1) and hyaluronic acid (4.2). 59 Another glucuronic acid-containing glycosaminoglycan is heparin, which inhibits enzymes involved in blood coagulation and is widely used as an anti-coagulant in surgery. 8 8 Glucuronic acid also plays a key role in drug metabolism. Drugs are often metabolized through the transfer o f glucuronic acid from U D P -glucuronic (Figure 4.2) acid to the drug. This transfer o f glucuronic acid is a detoxification reaction that terminates the activity o f the drug and results in excret ion. 8 9 o OH OH Figure 4.2 Diagram of UDP-glucuronic acid The creation o f a glycosynthase from a glucuronidase (a glucuronosynthase) would provide the ability to incorporate glucuronic acid monosaccharides into oligosaccharides thus creating the possibility o f synthesizing glycosaminoglycans, glucuronoconjugates, or potential drugs. The chemical synthesis o f such compounds involves the use o f cumbersome protecting group manipulations 9 0 and complications due to the formation o f side-products. 9 1 A glucuronidase from T. maritima was selected for conversion into a glucuronosynthase in the hope that the thermophilic properties o f T. maritima would lead to a robust glycosynthase useful for a range of applications. 4.2 Development of a Glucuronosynthase from the p-Glucuronidase from Thermotoga maritima The development of a glucuronosynthase was a collaborative effort with undergraduate student Jiyoung Hwang and post-doctoral fellow Dr. Hamzah M o h d . Salleh. The P-glucuronidase from T. maritima was cloned, overexpressed in Escherichia coli, and purified by Jiyoung Hwang. Dr . Hamzah M o h d . Salleh labeled the enzyme with the mechanism based inactivator 2-deoxy-2-fluoro-p-D-60 glucopyranuronosyl fluoride (Figure 4.3). 2-Deoxy-2-fluoro-P-D-glucopyranuronosyl fluoride inactivates the glucuronidase by reacting with the catalytic nucleophile, as described previously (Scheme 1.3). The catalytic nucleophile was identified as glutamate 476 fol lowing enzyme digestion and L C - M S / M S analysis o f the fragments by Shouming He (not published). Dr. Hamzah M o h d . Salleh then carried out site-directed mutagenesis to generate three mutant enzymes in which glutamate 476 was replaced with glycine, alanine, and serine. F Figure 4.3 Diagram of 2-deoxy-2-fluoro-P-D-glucopyranuronosyl fluoride. 4.2.1 Synthesis of Donor Sugars: a-D-Galactopyranuronosyl Fluoride and a-D-Glucopyranuronosyl Fluoride The donor sugars a-D-galactopyranuronosyl fluoride (4.6) and oc-D-glucopyranuronosyl fluoride (4.9) were synthesized for use in the glycosynthase reaction (Figure 4.4). Both fluoro-sugars were synthesized using the same synthetic route, beginning with the corresponding acid. C02H 4.9 Figure 4.4 Diagram of a-D-galactopyranuronosyl fluoride (4.6) and a-D-glucopyranuronosyl fluoride (4.9). Shown in Scheme 4.2 is the synthesis o f a-D-galactopyranuronosyl fluoride (4.6). D-Galacturonic acid was dissolved in dry methanol and (TMS)diazomethane was added drop wise to yield the methyl ester 4.3. The methyl ester 4.3 was 61 subsequently acetylated to provide the acetylated methyl ester 4.4 in 60 % yield over the two steps. Reaction with HF/pyridine yielded the protected a-fluoride 4.5 in 32 % yield after purification by recrystallization. The anomeric stereochemistry was assigned on the basis of 'Ft N M R coupling constants. Deprotection proceeded with the addition of hydrogen peroxide and lithium hydroxide in 7:3 THF:H20 to first saponify the ester. Once the methyl ester was converted to the acid salt, the remaining acetyl groups were deprotected with sodium hydroxide in methanol to yield a-D-galactopyranuronosyl fluoride (4.6) in 49 % yield. These specific conditions are important because the methyl ester causes H-5 to be relatively acidic. Addition of a strongly basic deprotecting agent would risk elimination of the 4-OAc group, particularly in the case of the galacto-isomer, where the 4-OAc group is trans-diaxial to H-5. O H O A c Scheme 4.2 The synthesis of galactopyranuronosyl fluoride (4.6). 4.2.2 Glucuronosynthase Reactions Test reactions performed by Dr. Hamzah Mohd. Salleh indicated that the E476A and E476G mutants catalyze a glycosynthase reaction between a-D-glucopyranuronosyl fluoride and pNP-glucoside. The E476S mutant did not function 92 as a glycosynthase. Interestingly, with the original Abg glycosynthase and a mannosynthase from Cellulomonas fimi,93 the serine mutant is a better glycosynthase 62 than the alanine mutant. It is not clear why the serine mutant is a better glycosynthase, yet this is not the case with the glucuronosynthase. The alanine mutant was selected for reaction scale-up and further study. A glycosynthase reaction using the E476A mutant is shown in Scheme 4.3. Scheme 4.3 A glycosynthase reaction catalyzed by the E476A mutant of P-glucuronidase. Although the enzyme is a glucuronidase, the donor could be of either gluco- or galacto- stereochemistry; both oc-D-glucopyranuronosyl fluoride (a-GluF, 4.9) and a-D-galactopyranuronosyl fluoride (cc-GalF, 4.6) functioned as donors. PNP P-D-glucoside (4.10), pNP P-D-xyloside (4.11), and pNP P-D-cellobioside (4.12) were good acceptors. Glycosynthase reactions with the two donors and three acceptors were initally carried out by Dr. Hamzah Mohd. Salleh and later repeated by the author to produce more compound for product characterization. Table 4.1 shows glucuronosynthase reaction yields obtained by Dr. Hamzah Mohd. Salleh. The product of a glycosynthase reaction can act as an acceptor in additional glycosynthase reactions (provided excess donor is present), resulting in longer oligosaccharides over time. Thus tri and tetrasaccharides were isolated where two to three donor units were added to the oligosaccharide chain (Table 4.1). 63 Table 4.1 E476A Glucuronidase Glycosynthase Reaction Yields Donor Acceptor disaccharide trisaccharide tetrasaccharide ot-GalF (4.6) pNP p-D-glucoside (4.10) 83 % 7 % n.d.' pNP p-D-xyloside (4.11) 32% 6 % n.d. pNP p-D-cellobioside (4.12) - 60% n.d. ct-GluF (4.9) pNP p-D-glucoside (4.10) 55 % 43 % 1 % pNP p-D-xyloside (4.11) 12% 48 % 4 % pNP p-D-cellobioside (4.12) — 72 % 10% n.d. = not done The products resulting from the addition of one glucuronic acid or galacturonic acid unit to an acceptor (disaccharides for pNP P-D-glucoside and pNP p-D-xyloside, and trisaccharides for pNP P-D-cellobioside, 4.13-4.18) were selected for characterization (Table 4.2). 64 Table 4.2 E 4 7 6 A Glucuronidase glycosynthase products selected for characterization Donor Acceptor Product selected for characterization O H 1 C O , H "" \ Ti H O | F 4.6 C 0 H «===\ \ 0 H ^"^ 4.10 H°COH ,^ -OH OH OH 4.13 \H ^ 4.11 HO C Q H O - ^ i ^ ^ O ^ ^ ^ ^ OH OH 4.14 H O 4.12 H O C Q H OH Q H V ^ ° \ HO-TV -^A „ ~ ^ X ^ ^ O /^:5XV-NO2 H°-'--^-' , , , , ,'T*^-^0~--^ 0"V I OH OH H \ ^ ^--^ HO 4.15 C0 2 H H O " - ^ ^ - ' 0 \ H O 1 F 4.9 C02H C 0 H H O ^ V H O ^ r VN°2 OH OH 4.16 \H ^ 4.11 C02H « s s = \ H 0 - ^ U H 0 ^ ^ ° \ i fN°2 OH OH 4.17 H O 4.12 OH OH \ N ^ - " HO 4.18 65 The products 4.13-4.18 were derivatized to their acetylated methyl esters 4.19-4.24 for characterization. The reaction scheme for the derivatization o f compound 4.13 is shown in Scheme 4.4. Salts and enzyme were removed from the lyophil ized mixture by passing the material through a C-18 Sep-Pak® cartridge. The material was acetylated with acetic acid and catalytic trifluoroacetic acid, and then esterified with (TMS)diazomethane in methanol. Through ' F I - ' H C O S Y N M R the linkages formed between donor and acceptor were identified as P-( l ,3) in all cases. Scheme 4.4 The acetylation and esterification of compound 4.13. 4.2.3 Attempts to Synthesize Glycosaminoglycan Fragments In order to synthesize glycosaminoglycan fragments, an acceptor containing a 2-amino group would be necesserary. Unfortunately, Af-acetyl-D-glucosamine ( G l c N A c ) (4.25), p N P /V-acetyl-p-D-glucosaminide ( p N P - G l c N A c ) (4.26), p N P p -D-glucosamine (4.27) and NAG-th iazo l ine (2.2) do not function as acceptors (Figure 4.5). 66 Figure 4.5 The compounds that did not function as acceptors for the E476A glucuronosynthase. A n additional test showed that glucose does not function as an acceptor, suggesting that the p N P group is necessary for acceptor binding. Because glucose did not function as an acceptor, it is not surprising that G l c N A c (4.25) and N A G -thiazoline (2.2), which lack the apparently needed p N P group, also did not act as acceptors. The fact that p N P - G l c N A c and pNP-glucosamine do not function as acceptors suggests that the active site o f the glucuronosynthase does not tolerate the added size o f an amino or /V-acetyl group relative to a hydroxyl group at the 2 position. The problems presented by the fact that the E 4 7 6 A P-glucuronidase does not use 2-amino sugars as acceptors could be overcome with further enzyme mutations. The amino acid residues involved in binding the acceptor could be mutated to create space for the needed amino or //-acetyl group. Examining the active site in an X- ray crystal structure could provide candidate amino acids for mutation. Unfortunately, the structure o f the T. maritima P-glucuronidase is not available. However, the structures o f the related family 2 human P-glucuronidase 9 4 and E. coli P-galactosidase 9 5 ' 9 6 are available, making a molecular modell ing study possible. A molecular model o f the wild-type P-glucuronidase from T. maritima was created using DeepView/Swiss -PdbViewer 9 7 based on the structure o f the human p-glucuronidase 9 4 , which is the most closely related enzyme with a structure available (34 % identity and 50 % similarity o f amino acid sequence) (Figure 4.6). To provide 67 further insight into binding, a bound carbohydrate was added to the model. There are no structures available for the human P-glucuronidase with bound inhibitor, however a structure o f an enzyme/inhibitor complex o f the homologous E. coli P-galactosidase 9 6 is available. The structure contains a 2-deoxy-2-fluoro galactosyl moiety covalently l inked to the catalytic nucleophile formed by pre-incubating E. coli P-galactosidase with the inactivator 2,4-dinitrophenyl 2-deoxy-2-fluoro-P-D-galactopyranoside. The model o f the T. maritima P-glucuronidase was overlaid onto the structure o f the E. coli P-galactosidase with covalently bound 2-deoxy-2-fluoro galactosyl moiety. The overlaid structure of the P-galactosidase was then removed from the model, leaving a picture o f the 2-deoxy-2-fluoro galactosyl moiety attached to the catalytic nucleophile E476 o f the T. maritima p-glucuronidase (Figure 4.6). The geometry o f the covalently bound 2-deoxy-2-fluoro galactosyl moiety approximates the geometry o f an a fluoride donor sugar bound in the active site of the E 4 7 6 A nucleophile mutant used in the glycosynthase reactions. A space fil led version of the model (Figure 4.7) shows the pocket where the acceptor sugar binds, and the residues involved in binding the acceptor sugar. The acceptor sugar could sit 'right side up' wi th the 2-hydroxyl (or desired 2-amino) group pointed downward in the picture, or the acceptor sugar could sit 'up side down', with the 2-group pointed upwards in the picture. The residues in the model that create the pocket where the acceptor sugar binds are Phe l40 , Tyr444, Tyr440, Thr475, Asn438, and Met426 (Figure 4.7). These residues are candidates for further site-directed mutagenesis studies on the E 4 7 6 A glucuronosynthase to allow the use o f acceptors with 2-amino groups. 68 M4 E476 T475 Figure 4.6 Molecular model of T. maritima P-glucuronidase with bound 2-deoxy-2-fluoro galactosyl moiety (green). Shown in yellow is the catalytic nucleophile (E476) and shown in blue is the catalytic acid/base residue (E383). M 4 2 6 T 4 7 5 Figure 4.7 Space-fdled model of T. maritima P-glucuronidase. 69 Another approach to creating mutants that can accommodate 2-amino containing acceptors is directed evolution. Members o f the Withers group are currently working on directed evolution o f glycosynthases to improve reaction rates, using a screen with a coupled assay that recognizes the product once it is formed. This approach could lead to mutations far removed from the active site that cause the small changes in active site geometry needed to accommodate acceptors with 2-amino groups and allow the synthesis o f glycosaminoglycan fragments. 4.3 Conclusion A glycosynthase was developed from the P-glucuronidase from T. maritima. Although the synthesis o f a glycosaminoglycan fragment was not achieved, six novel oligosaccharides were synthesized and characterized, thus adding to the repertoire o f compounds that can be synthesized by glycosynthases. 70 5 Materials and Methods 5.1 Enzymology 5.1.1 Sp. Hex. Kinetics 58 Sp. Hex. was cloned and overexpressed in E. coli as described previously by Jiyoung Hwang, an undergraduate student in the Withers laboratory. Continuous spectrophotometric assays for Sp. Hex. were performed on a Var ian Cary 4000 or Var ian Cary 300 U V - V i s spectrophotometer, using para-nitrophenyl p-D-7V-acetylglucosaminide ( p N P - G l c N A c ) as substrate. The buffer used for kinetic studies was 25/50 m M sodium citrate/sodium phosphate, 75 m M N a C l , p H 5.0. Kinet ic assays were carried out at 37 °C. The concentration o f enzyme used was 1.18 n M . The release o f /wra-nitrophenol was measured at 360 nm. The extiction coefficient used forpara-ni t rophenol 5 7 was 2.05 m N f ' c m ' ' . Michael is-Menten parameters were determined using GraFit version 4.0.19. 6 0 The graphs and resulting Michael i s -Menten parameters are in the Appendix. 5.1.2 Human B-Hexosaminidase A and B Kinetics Human P-hexosaminidases A and B were provided by the group o f Dr. Don Mahuran (Hospital for Sick Children, Toronto). The buffer used was 20/40 m M sodium citrate/sodium phosphate, 0.1 % H S A , p H 4.50. 4-Methylumbelliferyl-/V-acetyl-P-D-glucosaminide ( M U G ) was used as substrate. Stopped assays for Hex A and Hex B were performed as follows. Substrate/buffer solutions were pre-incubated at 37 °C in eppendorf tubes. Enzyme was added to a concentration o f 0.23 n M for Hex A and 0.084 n M for Hex B . A t fixed time intervals 20 u L aliquots o f reaction mixture were removed and diluted into a cuvette containing 1 m L of 0.1 M 2-amino-2-methyl-l-propanol (pH 10.0). The fluorescence resulting from release o f methylumbelliferone was measured on a Var ian Cary Eclipse fluorimeter. 71 The instrument parameters were set as follows: Ex. Wavelength (nm) Em. Wavelength (nm) Ex. S l i t (nm) Em. S l i t (nm) Ave Time (sec) E x c i t a t i o n f i l t e r E m i s s i o n f i l t e r PMT V o l t a g e (V) 365.00 450.00 5 5 0.1000 Auto Open Medium A n equation (equation 5.1) to convert data from units of fluorescence to units of concentration was created by measuring the fluorescence of solutions of known concentrations of methylumbelliferone, plotting the data, and fitting the data to a linear fit using GraFit version 4.0.19.6 0 Michaelis-Menten parameters were calculated using GraFit version 4.0.19.DU The graphs and resulting Michaelis-Menten parameters are in the Appendix. 5.1.2.1 Determination of kcat/Km Values for pNP-GlcNAc and pNP-GalNAc hJKm values for /?ara-nitrophenyl P-D-Af-acetylglucosaminide (pNP-GlcNAc) and /rara-nitrophenyl p-D-TV-acetylgalactosaminide (pNP-GalNAc) were determined using the substrate depletion method as follows. A continuous spectrophotometric assay was performed at 37 °C on a Varian Cary 4000 U V - V i s spectrophotometer. The substrate concentration used was 13.0 u M (Km ~ 500 uM). Enzyme concentrations were 0.77 n M for Hex B and 1.4 n M for Hex A . The change in absorbance was measured at 360 nm over ~ 40 minutes. The data were fitted to a first-order rate equation using the Cary WinUV kinetics application version 3.00, giving values for the pseudo-first-order rate constant. Equation 5.2 shows a modified Michaelis-Menten equation for the conditions of low substrate concentration ([S] « K m ) . The / : 0 bs values correspond to \E\kcJKm, and thus the kczt/Km values can be extracted by dividing the observed rate constants by the enzyme concentration. Fluorescence = (5.83 x 10 ) x [methylumbelliferone] (Equation 5.1) v = kca,[E][S] (Equation 5.2) Km 72 5.1.3 Glucocerebrosidase Kinetics Glucocerebrosidase was donated in the form of used vials o f Cerezyme (produced by Genzyme) from a patient currently undergoing enzyme replacement therapy. The buffer used for kinetics was 50 m M acetate buffer, p H 5.5, containing 0.125 % Thesit® or Triton X-100®, 0.30 % sodium taurocholate, and 1 m M E D T A . 2,4-Dinitrophenyl P-D-glucopyranoside was used as substrate for continuous spectrophotometric assays. Kinet ic assays were carried out at 37 °C. The concentration o f enzyme used was 29.0 n M . The release o f the 2,4-dinitrophenol was measured at 400 nm. The extiction coefficient used for 2,4-dinitrophenol 7 0 was 10.7 m M ' c m " 1 . Michael is-Menten parameters were determined using GraFit version 4.0.19. 6 0 The graphs and resulting Michael is-Menten parameters are presented in the Appendix. 5.1.4 Competitive Inhibition A l l inhibition kinetics were performed by measuring the reaction rate at a fixed concentration o f artificial substrate while varying the concentration o f inhibitor. K\ values were determined by fitting the data to the equation for competitive inhibitrion (Equation 5.3) using GraFit version 4.0.19. 6 0 K.. (Equation 5.3) + [S] K\ values were graphically represented by creating a plot o f the inverse o f the reaction rate against the concentration of inhibitor (Dixon plot). The line for 1 / V m a x intersects the lines for different substrate concentrations at K\ = -[I]. The theory for this method o f representing K\, taken from Segel , 9 7 is presented below. 73 For competitive inhibition, Vmax[S] V = [S] + Km(l + [l]/K) The reciprocal o f this equation is 1 K»,[I] 1 V Vmax[S]A:,- V„ When - = 1 v V max 1 K,„[l] 1 Vmax Vtnax[S]./Y/ Vmax and ATmril , Km 1 = —+1 + [S]K [S] so, .TYm[I] Km ~[SJK> ~ [S] and M = -*< Km 1 + [S], 5.7.5 Inactivation of Glucocerebrosidase Glucocerebrosidase (0.441 u M ) was incubated with an excess o f 2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride (2.47 m M ) at room temperature. Aliquots o f the enzyme/inactivator mixture were removed after 45 minutes, 3 hours, and 16 hours and enzyme activity was monitored as described above (5.1.3). 5.1.6 Thermal Denaturation Experiments Thermal denaturation studies were performed by first determining a temperature at which the enzyme loses substantial activity within an hour. This was achieved by incubating the enzyme at 55 °C, and removing aliquots at fixed time intervals and measuring the enzyme activity with an artificial substrate. Different incubation temperatures were used until a suitable temperature was found. The enzyme was then incubated at this temperature in the presence or absence o f inhibitor, 74 and aliquots were removed at fixed time intervals and the enzyme activity with an artificial substrate was measured. The artificial substrates and assay conditions employed were identical to those used for previously described Michael is -Menten and inhibition kinetics for each enzyme. The concentration o f inhibitor selected for al l denaturation experiments was at least ten times its K\ value to ensure that the inhibitor occupied the vast majority o f enzyme active sites. It was also ensured that the absolute concentration o f inhibitor was always greater than the enzyme concentration. In a l l cases, when an aliquot o f the enzyme/inhibitor mixture was diluted into a buffered solution o f substrate for the kinetic assay, the dilution factor was large enough for the concentration o f inhibitor to become sub-inhibitory. The table below summarizes the temperatures and concentrations used. Table 5.1 Concentrations and temperatures used during thermal denaturation experiments. pre-incubation pre-incubation assay assay Enzyme Inhibitor [Enzyme] (nM) [Inhibitor] (uM) [Enzyme] (nM) [Inhibitor] (uM) Ki (uM) T (°C) [substrate] (mM) Sp. Hex. N A G -thiazoline 38.2 240 1.18 7.41 20 55 0.666 Hex A N A G -thiazoline 10.5 4.00 0.379 0.144 0.40 56 1.86 Hex B N A G -thiazoline 10.6 2.40 0.341 0.0774 0.24 60 2.90 Glucocerebrosidase TV-octyl-l-epivalienamine 250 1.25 3.09 0.0154 0.10 53 2.05 75 5.1.7 Guanidine Hydrochloride Denaturation Experiments Guanidine hydrochloride (99+%) was purchased from Sigma. The guanidine hydrochloride did not contain fluorescent impurities, and thus was not further purified. For each enzyme, the buffer used for kinetics was also used for denaturation experiments, except in the following cases where the buffers were found to interfere with fluorescence or CD. Phosphate buffers interfered with C D spectra, so the citrate/phosphate buffer used for Sp. Hex. was replaced with a 100 m M acetate buffer, pH 5.0, 75 m M NaCl , which did not affect enzyme activity. The Triton X-100® used in glucocerebrosidase buffer fluoresced significantly, and was replaced with Thesit , which did not affect enzyme activity. Guanidine hydrochloride concentrations were calculated by measuring the refractive index with a Spectronic Instruments model 334610 refractometer and using the following equation (from Creighton),6 2 where A N is the difference in refractive index between the guanidine hydrochloride/buffer solution and the buffer alone at the sodium D line. [Gdm-HCl] = 57.147(AN) + 38.68(AN)2 - 91.60(AN)3 (Equation 5.4) After adding enzyme or enzyme/inhibitor mixtures to the solutions of guanidine hydrochloride, solutions were allowed to equilibrate for at least 3 hours before fluorescence or CD readings were taken. Incubations for longer time periods showed no significant changes in fluorescence or C D measurements. For enzyme/inhibitor mixtures, the enzyme was pre-incubated with inhibitor for 10 minutes before addition to solutions of guanidine hydrochloride. In each case the inhibitor concentration was at least 10 x K\ to ensure that the vast majority of active sites were occupied. For glucocerebrosidase inactivation with 2-deoxy-2-fluoro-P-glucopyranosyl-fluoride, the enzyme/inactivator mixture was incubated at room temperature for 16 hours, at which time the enzyme was 90 % inactivated, prior to addition to solutions of guanidine hydrochloride. The table below summarizes the concentrations used. A l l data refer to fluorescence experiments except where noted. 76 Table 5.2 Concentrations used during guanidine hydrochloride denaturation experiments. Enzyme Inhibitor [Enzyme] (uM) [Inhibitor] ( u M ) Ki (uM) Sp. Hex. NAG-th iazo l ine 0.66 230 20 Sp. Hex* NAG-th iazo l ine 3.0 230 20 Hex A NAG-th iazo l ine 0.090 4.1 0.40 Hex B NAG-th iazo l ine 0.098 2.5 0.24 Glucocerebrosidase /V-octyl-1-epivalienamine 0.44 28 0.10 Glucocerebrosidase 2-deoxy-2-fluoro-(3-D-glucopyranosyl-fluoride 0.44 2500 N A a concentrations used for C D measurements. Circular dichroism measurements were taken on a J A S C O J-810 spectropolarimeter. Fluorescence measurements were taken on a Varian Cary Eclipse fluorimeter. A fluorescence excitation scan revealed a maximum at 280 nm, thus 280 nm was selected as the excitation wavelength. Fluorescence emission scans were corrected 98 with a Savitzky Golay equation using a filter size o f 19 and an interval o f 1.00. The instrument parameters were set as follows. Scan mode X M o d e Start (nm) Stop (nm) Ex. Wavelength (nm) Ex. Slit (nm) Em. Slit (nm) Scan rate (nm/min) Data interval (nm) Averaging Time (s) Emission Wavelength (nm) 300.00 400.00 280.00 10 10 600.00 1.0000 0.1000 77 Excitation filter Auto Emission filter Open P M T voltage (V) Medium Chemical denaturation curves were fit to the equation for a sigmoidal curve (Equation 5.5) using GraFit version 4.0.19. 6 0 5.1.8 General Procedure for Glucuronidase Glycosynthase Reactions In a typical preparative scale reaction 50 u L o f 300 m M glucuronyl or galacturonyl fluoride and 200 u L o f 50 m M acceptor were added to 750 u L o f 3.5 mg /mL E 4 7 6 A p-glucuronidase in 50 m M p H 7 sodium phosphate buffer. Reaction concentrations were 15 m M donor, 10 m M acceptor, and 2.8 mg /mL E 4 7 6 A p-glucuronidase. The reactions were incubated for 18 hours at 37 °C and lyophilized before work-up. Test scale reactions were performed with a total volume o f 100 u L , and preparative scale reactions were performed with a total volume o f 1 m L . Reaction yields were calculated by separating a small portion o f the material by H P L C (acetonitrile/water 9:1 to 1:1) and integrating the peaks o f the H P L C profile. Compounds were detected by U V absorption o f the para-nitrophenyl containing acceptors and products at 254 nm. The percent yield was calculated as the area o f the peak due to the glycosynthase product divided by the sum of the areas o f the glycosynthase product and acceptor peaks. Molecular weights o f the oligosaccharide products were confirmed by E S I - M S . a (Equation 5.5) 78 5.2 Syntheses 5.2.1 General Synthesis A l l reagents were purchased from Sigma/Aldr ich unless otherwise stated. Solvents used were either reagent, certified, or spectral grade. Anhydrous solvents were prepared as follows: toluene, benzene, and acetonitrile were distilled over calcium hydride. Methanol was distilled from magnesium turnings in the presence o f iodine. Tetrahydrofuran was distilled from sodium in the presence o f benzophenone. Deionized water, purified with a Mi l l ipore Milli-Q® system, was used for all aqueous solutions. Synthetic and enzymatic reactions were monitored by T L C using M e r c k Kieselgel F254 alumina based sheets (thickness 0.2 mm). Compounds were detected by ultraviolet light and/or by charring with 10 % ammonium molybdate in 2 M H 2 S O 4 . Co lumn chromatography was performed with M e r c k Kieselgel (230-400 mesh) si l ica gel. N M R spectra were recorded on a Bruker Avance 300 M H z , Bruker Avance 400 M H z , or a Varian Inova 600 M H z spectrometer. Chemical shifts were reported on the 8 scale in parts per mi l l ion from tetramethylsilane ( T M S ) . The abbreviations used in describing multiplicity are: s-singlet, d-doublet, t-triplet, m-multiplet, br-broad. I 9 F N M R spectra were referenced to trifluoroacetic acid at 0 ppm. Low-resolution mass spectra were measured on an ESI P E Sciex A P I 300 spectrometer. High-resolution mass spectra were measured on an ESI Micromass L C T spectrometer by the mass spectrometry laboratory at the Universi ty o f Bri t ish Columbia . In each case analytical data for previously reported compounds was identical to the data reported in the reference provided with the compound title. 5.2.2 Generous Gifts The group o f Professor R. V . Stick (The Universi ty o f Western Australia) provided the tetra-O-benzyl-l-epivalienamine for the synthesis o f /V-octyl-1-epivalienamine. The group o f Professor Spencer Knapp (Rutgers, N e w Jersey) provided the Xy lNAc- i so fagamine -HCl . NAGal- th iazol ine and a portion o f the N A G -thiazoline were synthesized by Dr. Hongming Chen in the Withers laboratory. A 79 precursor to oc-D-glucopyranuronosyl fluoride, methyl (1,2,3,4-tetra-O-acetyl-D-glucopyranosyl)uronate, was synthesized by Dr. Michae l Jahn in the Withers laboratory. 2,4-Dinitrophenyl |3-D-glucopyranoside was synthesized by Dr . Michae l Jahn and Fathima Shaikh in the Withers laboratory. 5.2.3 NAG-Thiazoline13 (2.2) Tri-0-acetyl-l,2-dideoxy-2'-methyl-a-D-glucopyranoso-[2,l-rf]-A2 '-thiazoline (tri-0-acetyl NAG-thiazoline) (2.1) 2-Acetamido-2-deoxy-P-D-glucopyranose 1,3,4,6-tetra-acetate (2.92 g, 7.50 mmol) was dissolved in dry toluene (35 mL) . Lawesson's reagent (2.5 g, 6.18 mmol) was added and the mixture was heated to reflux and stirred at 110 °C under argon for 3.5 hours at which point the reaction was judged complete by T L C . The solvent was evaporated in vacuo and the material was purified by column chromatography (petroleum ether/ethyl acetate 2:1 to 1:2) to yield tri-O-acetyl NAG- th iazo l ine (2.1) as an o i l (1.87 g, 5.41 mmol , 72 %). ' H N M R (300 M H z , C D C 1 3 ) 5: 6.23 (d, 1 H , J H 2 = 7.1 H z , H - l ) , 5.55 (m, 1 H , H-3), 4.94 (b rd , 1 H , J45 = 9.5 H z , H-4) , 4.48-4.43 (m, 1 H , H-2), 4.11 (d, 2 H , J 6 a , 5 = J 6 A > 5 = 4.4 H z , H - 6 a , H-6 b ) , 3.54 (dt, 1 H , H-5), 2.31 (d, 3 H , JMC,I = 2.1 H z , M e ) , 2.12, 2.07, 2.06 (3 s, 9 H , 3 O A c ) . 80 l,2-Dideoxy-2'-methyl-a-D-glucopyranoso-[2,l-</]-A2'-thiazoline ( N A G -thiazoline) (2.2) The per-O-acetylated thiazoline 2.1 (1.87 g, 5.41 mmol) was dissolved in dry methanol (50 m L ) and sodium methoxide (catalytic amount) was added. The mixture was stirred under argon for 2 hours at which time the reaction was judged complete by T L C . Acet ic acid was added dropwise until the mixture was acidic and the solvent was evaporated in vacuo. The material was dry loaded onto sil ica and purified by column chromatography (ethyl acetate/methanol/water 10:1:0 to 7:3:1) to yie ld N A G -thiazoline (2.2) as a gummy solid (0.902 g, 4.11 mmol , 76 %). ] H N M R (400 M H z , D 2 0 ) 8: 6.20 (d, 1 H , Jit2 = 7.0 H z , H - l ) , 4.35-4.32 (m, 1 H , H-2) , 4.14 (dd, 1 H , JX2 = 4.5, J 3 > 4 = 3.2 H z , H-3), 3.65 (dd, J6afib = 12.4, J6a>5 = 2.4 H z , H-6 a ) , 3.52 (dd, 1 H , J 4 , 5 = 9.1 H z , H-4), 3.51 (dd, 1 H , J 6 b ; 5 = 7.2 H z , H-6 b ) , 3.24 (ddd, 1 H , H-5), 2.16 (d, 3 H , JMC,2 = 2.1 H z , M e ) . 1 3 C N M R (100 M H z , D 2 0 , referenced to M e O H ) 8: 171.54 (SC=N), 87.83, 77.85, 74.11, 71.52, 69.41, 61.63 (6 ring C ' s ) , 19.47 ( C H 3 ) . H R M S (ESI) m/z 242.0462 [ M + N a ] + . Calculated for C 8 H i 3 N N a 0 4 S 242.0463. 81 5.2.4 2-Deoxy-2-Fluoro-fi-D-Glucopyranosyl Fluoride (3.3) 3,4,6-Tri-0-acetyl-2-deoxy-2-fluoro-a-D-glucopyranosyl bromide (3.1) Tri-O-acetyl-D-glucal (15.0 g, 55.1 mmol) was dissolved in acetic acid (100 m L ) and acetic anhydride (20 m L ) was added. The mixture was heated to 60 °C and Selectfluor® (20.0 g, 56.4 mmol) was added. After stirring for 1 hour at 60 °C D M F (10 m L ) was added to facilitate dissolving. Acet ic acid (30 m L ) and acetic anhydride (15 m L ) were added and the mixture was stirred for 6 hrs at 60 °C. The reaction was judged incomplete by T L C , so another portion o f Selectfluor® (5.0 g, 14 mmol) was added and the reaction mixture was stirred for 14 hours at 60 °C. The solvent was evaporated in vacuo, and the mixture was diluted with water (200 m L ) . The material was extracted with D C M (2 x 150 m L ) , and washed with water and saturated sodium bicarbonate solution. The organic layer was dried over M g S O ^ and concentrated to yield 14.5 g (41.4 mmol) o f a mixture o f the a-gluco, P-gluco and p-manno isomers o f l,3,4,6-tetra-0-acetyl-2-deoxy-2-fluoro-D-pyranose. The residue was purified by column chromatography (petroleum ether/ethyl acetate 3:1 to 1:1), yielding a mixture o f the a and P anomers o f l,3,4,6-tetra-0-acetyl-2-deoxy-2-fluoro-D-glucopyranose (2.0 g, 5.7 mmol , 10 %). The l,2,4,6-tetra-0-acetyl-2-deoxy-2-fluoro-D-glucopyranose (2.0 g, 5.7 mmol) was dissolved in dichloromethane (8 m L ) and cooled in an ice bath under N 2 . H B r in acetic acid (30 wt. %, 5.0 m L , 25 mmol) was added and the reaction mixture was stirred for 16 h at R T under N 2 . The material was diluted into ice water (100 mL) and ethyl acetate (40 mL) was added. The organic layer was washed with saturated sodium bicarbonate solution and brine, then dried over MgSCM and concentrated in vacuo to yield 3,4,6-tri-0-acetyl-2-deoxy-2-fluoro-cc-D-glucopyranosyl bromide (3.1) (1.7 g, 4.6 mmol , 80 %). ! H N M R (400 M H z , C D C 1 3 ) 5: 6.51 (d, 1 H , J , , 2 = 4.2 H z , H - l ) , 5.61 (ddd, 1 H , J 3 ,F = 11.2, J 3 , 4 = 9.9, J 3 , 2 = 9.4 H z , H-3), 5.10 (dd, 1 H , J 4 > 5 = 82 9.8 H z , H - 4 ) , 4 . 5 2 (ddd, 1 H , J 2 , F = 49 .4 H z , H - 2 ) , 4 . 34 -4 .28 (m, 2 H , H - 6 a , H-6 b ) , 4 .13 -4 .08 (m, 1 H , H - 5 ) , 2 .03 , 2 .05 , 2 .07 (3 s, 9 H , 3 O - A c ) . 3,4,6-Tri-0-acetyl-2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride (3.2) OAc AcO F The a-bromide 3.1 (1.7 g, 4.6 mmol) was dissolved in dry acetonitrile (50 mL) and silver fluoride (1.70 g, 13.4 mmol) was added. After stirring for 16 h the reaction was judged incomplete by T L C , so another portion o f silver fluoride (0.50 g, 3.9 mmol) was added. The mixture was stirred for 5 h at which point the reaction was judged complete by T L C . The material was fdtered through Celite and the solvent was evaporated in vacuo. The residue was dissolved in ethyl acetate (50 m L ) and washed with water and brine. The organic layer was dried over M g S 0 4 , the solvent was evaporated in vacuo, and the residue crystallized from ethyl acetate/heptane to yield 3,4,6-tri-0-acetyl-2-deoxy-2-fluoro-p-D-glucopyranosyl fluoride (3.2) (1.0 g, 3.2 mmol , 70 %). ' H N M R (300 M H z , C D C 1 3 ) 5: 5.42 (ddd, 1 H , y 1 > M = 51.9, Ji>2 = 6.2, y 1 > F . 2 = 3.8 H z , H - l ) , 5.31 (m, 1 H , H-3), 5.11 (dd, 1 H , J 4 , 5 = 9.7, J 4 > 3 = 9.3 H z , H-4), 4.49 (dddd, 1 H , y2,F-2 = 56.1, J 2 j F - i = 11.0, J2,i = 7.9 H z , H-2), 4.26 (dd, 1 H , J 6 a , 6 b = 12.5, J 6 a > 5 = 4.8 Hz , H-6 8 ) , 4.18 (dd, y 6 b > 5 = 2.7 H z , H-6 b ) , 3.88 (ddd, 1 H , H-5), 2.08 (s, 6 H , 2 O - A c ) , 2.03 (s, 3 H , O - A c ) . 2-Deoxy-2-fluoro-P-D-glucopyranosyl fluoride (3.3) A portion o f the 3,4,6-tri-0-acetyl-2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride (3.2) (0.422 g, 1.36 mmol) was dissolved in dry methanol (40 m L ) . Sodium OH F 83 methoxide (2 spatula tips) was added and the mixture was stirred for 1 hour at which point the reaction was judged complete by T L C . Amberli te® H + IR 120 resin was added, and the mixture was filtered and then the solvent was evaporated in vacuo. The residue was purified by column chromatography (ethyl acetate/methanol/water 27:2:1) to yield 2-deoxy-2-fluoro-P-D-glucopyranosyl fluoride as a solid (3.3) (0.250 g, 1.16 mmol , 85 %) . ' H N M R (400 M H z , D 2 0 ) 5: 5.38 (ddd, 1 H , J\t?.\ = 53.1, = 7.0, JI,F-2 = 3.3 H z , H - l ) , 4.21 (dddd, 1 H , J2,F.2 = 51.5, 72,F-I = 13.5, / 2 , 3 = 9.1 H z , H -2), 3.79 (ddd, 1 H , y 6 a , 6 b = 12.5, J6*,s = 2.2, y 6 a , F . , = 0.9 H z , H-6 a ) , 3.71 (dddd, 1 H , JXF. 2 = 15.4, y 3 > 4 = 9.0, / 3 > F- I = 0.8 H z , H-3), 3.64 (ddd, 1 H , J 6 b , 5 = 5.3, J 6 b , F - i = 1.0 H z , H -6 b), 3.50 (dddd, 1 H , J 5 , 4 = 9.9, Js, F-, = 0.8 H z , H-5), 3.41 (dd, 1 H , H-4). 1 9 F N M R (282 M H z , D 2 0 , referenced to T F A ) 5: -125.97 (dt, J F-2,F-I = 16.9 H z , F-2), -67.37 (dt, F - l ) . ES I M S m/z 207.1 [ M + N a ] + . Calculated for C 6 H 1 0 F 2 N a O 4 207.0. 5.2.5 N-Octyl-1-Epivalienamine (3.6) (l/?,4/?,55,65)-4,5,6-tribenzyloxy-3-(benzyloxymethyl)-l-(octylamino)-2-cyclohexene (tetra-0-benzyWV-octyl-l-epivalienamine) (3.5) OBn Tetra-O-benzyl-1-epivalienamine" [(17^,4/^,5S,6S)-4,5,6-tribenzyloxy-3-(benzyloxymethyl)-l-amino-2-cyclohexene] (3.4) (0.408 g, 0.761 mmol) was dissolved in dry benzene (100 mL) . Octy l aldehyde (130 u L , 0.832 mmol) and p-toluenesulfonic acid monohydrate (catalytic amount) were added and the mixture was heated at reflux with a Dean-Stark apparatus. After approximately 50 % of the solvent had distilled off, another portion o f octyl aldehyde (100 u L , 0.640 mmol) was added and the mixture was stirred under reflux until a further 50 % of the solvent had distilled off. Dry methanol (20 m L ) and sodium borohydride (catalytic amount) were added. The mixture was stirred for 5 minutes and acetic acid was added dropwise until no fizzing was visible in the reaction mixture. The solvent was evaporated in 84 vacuo and the material was dissolved in dichloromethane (100 m L ) and washed with saturated sodium bicarbonate and brine. The organic layer was dried over M g S 0 4 , filtered, and the solvent was evaporated in vacuo. The residue was purified by column chromatography (toluene/ethyl acetate/triethylamine 95:5:1 to 50:50:1) to yield tetra-O-benzyl-N-octyl-l-epivalienamine (3.5) (0.319 g, 0.492 mmol , 65 %). ' H N M R (400 M H z , C D C 1 3 ) 5: 7.33-7.24 (m, 20 H , CH 2 -P / z ) , 5.70 (s, 1 H , H-2), 4.96 (t, 2 H , J= 11.7 H z , C / / 2 - P h ) , 4.84 (d, 1 H , 7 = 10.9 H z , C7/ 2 -Ph) , 4.81 (d, 1 H , J = 10.9 H z , C / / 2 - P h ) , 4.70 (t, 2 H , J = 10.9 H z , C / / 2 -Ph ) , 4.50 (d, 1 H , J= 11.9 H z , CH2-?h), 4.43 (d, 1 H , J = 11.9 H z , CH2-?h), 4.32 (br d, 1 H , J 4 ; 5 = 7.6 H z , H-4), 4.25 (br d, 1 H , y 7 a ,7b= 12.0 H z , H-7 a ) , 3.90-3.86 (m, 2 H , H - 7 b , H-5), 3.59 (dd, 1 H , J 6 , , = 9.8, J6,5 = 8.6 H z , H-6), 3.36-3.34 (br d, 1 H , H - l ) , 2.52-2.39 (m, 2 H , N H - C i f c ) , 1.35-1.23 (m, 12 H , N H 2 - C H 2 - ( C 7 / 2 ) 6 ) , 0.87 (t, 3 H , J = 6.6 H z , (CH2)7-CH3). 1 3 C N M R (75 M H z , CDCI3) 8: 138.70, 138.66 (4 quaternary Ph C) , 138.43 (C-2/3), 135.55 (C-2/3), 128.71, 128.59, 128.51, 128.31, 128.02, 127.99, 127.78, 127.69 (15 C , Ph), 85.30 (C-4/5/6), 81.07 (C-4/5/6), 80.45 (C-4/5/6), 75.32, 75.19, 74.54, 72.27, 70.51 (4 C , Ph-C H 2 , C-7), 59.71 ( C - l ) , 46.46 ( N H - C H 2 ) , 31.98, 30.55, 29.67, 29.41, 27.48, 22.81 (6 C , C H 2 ) , 14.24 ( C H 3 ) . H R M S (ESI) m/z 648.4061 [ M + H ] + . Calculated for C 4 3 H 5 4 N 0 4 648.4053. (li?,4i?,55',65)-4,5,6-hydroxy-3-(hydroxymethyl)-l-(octylamino)-2-cyclohexene (/V-Octyl-l-epivalienamine) 7 6 (3.6) The protected /V-octyl-l-epivalienamine 3.5 (0.319 g, 0.492 mmol) was dissolved in dry T H F (25 m L ) and stirred under nitrogen. A m m o n i a gas was condensed onto the mixture at -78 °C until a precipitate began to form. Sodium (4 small chunks) was added and the mixture was stirred for 1 hour after a deep blue colour had formed in the reaction mixture. A m m o n i u m chloride was added until the solution became colourless. The mixture was allowed to warm to room temperature OH OH 85 while stirring under nitrogen with a vent open to the atmosphere for the ammonia gas to vent. The remaining solvent was evaporated in vacuo. The residue was dissolved in methanol, filtered through Celite, and the solvent was again evaporated in vacuo. A portion o f the product/salt mixture was further purified. The material was first purified by column chromatography (dichloromethane/methanol 6:1, 0.5 % triethylamine). The solvent was evaporated in vacuo and the residue was dissolved in H 2 0 and loaded onto a Sep-Pak® C-18 column as per the Sep-Pak® instruction manual. The Sep-Pak® C-18 column was flushed with H 2 0 and then methanol was used to elute the compound. The solvent was evaporated in vacuo and the material was redissolved in methanol and purified by anion exchange chromatography with a B i o - R a d AG® 1-X8 resin 200-400 mesh anion exchange column. The solvent was evaporated in vacuo to yield pure iV-octyl-1 -epivalienamine (3.6) as a solid. H N M R (400 M H z D 2 0 ) 5: 5.57 (s, 1 H , H-2), 4.13-4.03 (m, 3 H , H - l , H - 7 a , H-7 b ) , 3.82 (dt, 1 H , J4,5 = 9.2, J 4 ;7a = A ? b = 2 - 5 H z , H-4), 3.63 (dd, 1 H , J5>6 = 10.2 H z , H-5), 3.48 (dd, 1 H , y 6 , i = 8.3 H z , H-6), 3.06-2.93 (m, 2 H , N H - C / 7 2 ) , 1.55-1.65 (m, 2 H , N H - C H 2 -CH2), 1.27-1.12 (m, 10 H , 5 C H 2 ) , 0.72 (t, 3 H , J = 7.0 H z , C H 3 ) . , 3 C N M R (100 M H z , D 2 0 , referenced to M e O H ) 5: 145.00 (C-4), 114.80 (C-5), 75.76 ( C - l ) , 71.51 (C-6/7), 69.97 (C-2), 61.03 (C6/7), 59.78 (C-3), 44.77 ( N H - C H 2 ) , 31.23, 28.40, 25.95, 22.21 (6 C H 2 ) , 13.63 ( C H 3 ) . H R M S (ESI) m/z 288.2173 [ M + H ] + . Calculated for C i 5 H 3 0 N O 4 288.2175. 5.2.6 a-D-Galactopyranuronosyl Fluoride (4.6) Methyl (2,3,4-tri-O-acetyl-a-D-galactopyranosyl fluoride)uronate (4.5) D-Galacturonic acid (2.10 g, 10.7 mmol) was dissolved in dry methanol and (TMS)diazomethane (10 m L , 2.0 M in hexanes, 20 mmol) was added dropwise until the solution retained a yel low colour. 10% Acet ic acid in methanol was added OAc F 86 dropwise until the solution became colourless. The solvent was evaporated, and then co-evaporated once with toluene yielding the methyl ester (4.3) as a thick brown oi l (2.71 g). The methyl ester was dissolved in pyridine (30 m L ) and cooled to 0 °C in an ice bath. Acet ic anhydride (15 m L , 160 mmol) was added and the reaction mixture was stirred for 4.5 hours at which time the reaction was judged complete by T L C . Methanol (15 m L ) was added slowly to quench the acetic anhydride. The solvent was evaporated to near dryness and the material was dissolved in dichloromethane (50 m L ) and washed with 1 M HC1, saturated sodium bicarbonate, and brine. The organic layer was dried over MgSCM and the solvent was evaporated. The material was purified by column chromatography (petroleum ether/ethyl acetate 4:1 to 1:3) yielding methyl (l,2,3,4-tetra-0-acetyl-D-galactopyranosyl)uronate (4.4) as a foam (2.42 g, 6.43 mmol , 60 %). The per-O-acetylated methyl ester 4.4 (2.42 g, 6.43 mmol) was dissolved in dichloromethane and transferred to a 50 m L Teflon tube. The solvent was evaporated and 70 % HF-pyridine (15 m L ) was added dropwise. The reaction mixture was stirred for 70 h, and then the reaction mixture was added dropwise to 400 m L of saturated sodium bicarbonate solution. The product was extracted with ethyl acetate (200 m L ) and washed with saturated sodium bicarbonate, 1 M HC1, and brine. The organic layer was dried over M g S C U and the solvent was evaporated. The material was purified by column chromatography (petroleum ether/ethyl acetate, 5:1 to 1:1), and crystallized from ethyl acetate/heptane to yield methyl (2,3,4-tri-O-acetyl-a-D-galactopyranosyl fluoride)uronate (4.5) (0.698 g, 2.08 mmol , 32 %) . *H N M R (400 M H z , CDCI3) 8: 5.90 (dd, 1 H , J U F = 52.5, J K 2 = 2.7 H z , H - l ) , 5.82 (m, 1 H , H-4), 5.39 (dd, 1 H , J 3 , 2 = 10.9, J 3 , 4 = 3.3 H z , H-3), 5.22 (ddd, 1 H , J 2 , F = 23.9 H z , H-2), 4.76 (d, 1 H , y 5 j 4 = 1.2 Hz , H-5), 3.76 (s, 3 H , 0-CH3), 2.10, 2.09, 2.00 (3 s, 9 H , 3 O-A c ) . 1 3 C N M R (100 M H z , C D C 1 3 ) 8: 169.95, 169.67, 169.45 (3 s, 3 C O ) , 166.09 (s, C-6), 104.24 (d, J i , F = 230.0 H z , C - l ) , 70.32, 68.32 (2 s, C-3/4/5), 66.83 (d, J2,F = 23.5 H z , C-2), 66.51 (s, C-3/4/5), 52.82 (s, O-CH3), 20.49 (s, 2 x C O - C H 3 ) , 20.36 (s, C O - C H 3 ) . 1 9 F N M R (282 M H z , C D C 1 3 ) 8: -75.62 (dd). ESI M S m/z 359.4 [ M + N a ] + . Calculated for C i 3 H i 7 F N a 0 9 359.3. 87 a-D-Galactopyranuronosyl Fluoride (4.6) OH The protected galactopyranuronosyl fluoride 4.5 (0.698 g, 2.08 mmol) was dissolved in 7:3 T H F : H 2 0 (23 m L ) and cooled in an ice bath. H2O2 (8.8 m L , 30 % in H 2 0 , 114 mmol) and 1.25 M L i O H (4.0 m L , 5.0 mmol) were added and the mixture was stirred for 3 hours at 0 °C and 2 hours at room temperature. Methanol (9.4 m L ) and 4.0 M N a O H (4.5 m L , 18 mmol) were added and the mixture was stirred at R T until the reaction was judged complete by T L C (3 hours). Amberli te® H + IR 120 resin was added, the mixture was filtered, and the solvent was evaporated in vacuo. The material was dry loaded on to si l ica with methanol and purified by column chromatography (ethyl acetate/petroleum ether/water + 0.2 % acetic acid, 5:1:0 to 6:3:1) to yield a-D-galactopyranuronosyl fluoride (4.6) as a solid (200 mg, 1.02 mmol , 49 %) . ' H N M R (400 M H z , D 2 0 ) 5: 5.68 (dd, 1 H , J , > F = 53.2 H z , Jh2 = 2.8 H z , H - l ) , 4.63 (d, 1 H , J 5 , 4 = 1.5 H z , H-5), 4.31 (dd, 1 H , J 4,3 = 3.3 H z , H-4) , 3.90 (dd, 1 H , J 3 ,2 = 10.0 H z , H-3), 3.78 (ddd, 1 H , J 2 ; F = 25.6 H z , H-2). 1 3 C N M R (100 M H z , D 2 0 , referenced to M e O H ) 8: 171.82 (s, C O ) , 107.43 (d, JhV = 224.9 H z , C - l ) , 72.69 (s, C-3), 69.77, 68.53 (s, C-4/5), 67.52 (d, J 2 , F = 24.2 H z , C-2). 1 9 F N M R (282 M H z , referenced to T F A ) 5: -77.17 (dd). ESI M S m/z 219.3 [ M + N a f . Calculated for C 6 H 9 F N a 0 6 219.1. 88 5.2.7 a-D-Glucopyranuronosyl Fluoride (4.9) Methyl (2,3,4-tri-O-acetyl-a-D-glucopyranosyl fluoride)uronate (4.8) C 0 2 M e A c O -AcO Methy l (l,2,3,4-tetra-0-acetyl-D-glucopyranosyl)uronate (4.7) (5.1 g, 13.5 mmol) was placed in a 50 m L Teflon tube. 70 % HF-pyridine (10 m L ) was added dropwise. The mixture was stirred at room temperature for 2 hours. The reaction was incomplete by T L C , so more 70 % HF-pyridine (7 m L ) was added and the mixture was stirred for 24 hours at which time the reaction was judged complete by T L C . The reaction mixture was added dropwise to 600 m L o f saturated sodium bicarbonate solution. The product was extracted with ethyl acetate (300 m L ) and washed with saturated sodium bicarbonate, 1 M HC1, and brine. The organic layer was dried over MgSCM and the solvent was evaporated in vacuo. The material was purified by column chromatography (petroleum ether/ethyl acetate, 5:1 to 1:1), and crystallized from ethyl acetate/heptane to yield methyl (2,3,4-tri-O-acetyl-a-D-glucopyranosyl fluoride)uronate (4.8) (2.62 g, 7.79 mmol , 58 %). ! H N M R (400 M H z , C D C 1 3 ) 5: 5.79 (dd, 1 H , y , ) F = 53.0, Jia = 2.7 H z , H - l ) , 5.53 (dd, 1 H , J 3 ; 2 = 10.2, J 3 ) 4 = 9.9 H z , H-3), 5.21 (dd, 1 H , 7 4 , 5 = 10.3 H z , H-4), 4.95 (ddd, 1 H , J2,F = 24.2 H z , H-2), 4.47 (d, 1 H , H-5), 3.74 (s, 3 H , 0 - C H 3 ) , 2.08 (s, 3 H , O - A c ) , 2.02 (s, 6 H , 2 O - A c ) . 1 9 F N M R (282 M H z , C D C 1 3 ) 5: -73.36 (dd). ESI M S m/z 359.0 [ M + N a ] + . Calculated for C i 3 H i 7 F N a 0 9 359.3. 89 a-D-Glucopyranuronosyl fluoride (4.9) The a-fluoride 4.8 (2.62 g, 7.79 mmol) was dissolved in 7:3 T H F : H 2 0 (80 m L ) and cooled in an ice bath. H 2 0 2 (30.5 m L , 30 % in H 2 0 , 394 mmol) and 1.25 M L i O H (14.0 m L , 17.5 mmol) were added and the mixture was removed from the ice bath and stirred for 2 hours at room temperature. Methanol (32.5 m L ) and 4.0 M N a O H (15.5 m L , 62.0 mmol) were added and the mixture was stirred at 4 °C for 2 hours, allowed to warm to room temperature, then stirred for a further 2 hours at room temperature. Amberli te® H + IR 120 resin was added, the mixture was filtered, and the solvent was evaporated in vacuo. The material was dry loaded on to s i l ica with methanol and purified by column chromatography (ethyl acetate/petroleum ether/water + 0.2 % acetic acid, 5:1:0 to 6:3:1) to yield a-D-glucopyranuronosyl fluoride (4.9) as a solid (460 mg, 2.35 mmol , 30 %). ' H N M R (400 M H z , D 2 0 ) 5: 5.59 (dd, 1 H , y i ; F = 53 .1 ,7 i , 2 = 2 . 7 H z , H - l ) , 4.05 (d, 1 H , J 5 A = 10.1 H z , H-5), 3.64 (dd, 1 H , J X 1 = 9.8, y 3 , 4 = 9.5 H z , H-3), 3.54 (ddd, 1 H , J2,F = 25.9 H z , H-2), 3.48 (dd, 1 H , H-4). 1 9 F N M R (282 M H z , D 2 0 ) 5: -73.81 (dd). ESI M S m/z 219.4 [ M + N a ] + . Calculated for C 6 H 9 F N a 0 6 219.1. 5.2.8 General Procedure for Derivatization of Glycosynthase Products Salts and enzyme were removed from the lyophil ized mixture by passing the material through a C-18 Sep-Pak® cartridge according to the instruction booklet. The C-18 cartridge was flushed with methanol (6 m L ) , followed by water (6 m L ) . The reaction mixture was loaded with water and the cartridge was flushed with water (6 m L ) . The compound was then eluted with methanol and the solvent was evaporated in vacuo. The residue was dissolved in a minimum amount o f acetic anhydride, and 3 drops of trifluoroacetic acid were added. The reaction mixture was allowed to stir at R T overnight, and then the solvent was evaporated in vacuo. A few drops o f 90 acetonitrile and 10 % HC1 were added to cleave the mixed anhydride that may have formed, then the material was dissolved in chloroform and washed with 10 % HC1 and brine. The organic layer was dried over MgSC>4, evaporated, and the residue dissolved in H P L C grade M e O H and (TMS)diazomethane added dropwise until a yel low colour persisted. The mixture was stirred for 5 minutes, and acetic acid (one drop) was added, causing the solution to become colourless. The solvent was evaporated in vacuo and the material was purified by column chromatography (toluene/ethyl acetate 3:1 to 3:2). 5.2.9 Assignment of Protons in !H NMR Specra of Derivatized Glycosynthase Products The protons o f the derivatized glycosynthase products were assigned using ] H N M R C O S Y spectra. Assignments were made by examining the couplings within the ring system. The location o f the glycosidic bond was determined on the basis o f the chemical shift o f the ring protons. The acetate groups are more electron withdrawing than a glycosidic likage; hence the proton attached to a carbon involved in the glycosidic bond is found more upfield. The configuration of the anomeric linkage ( a or P) was assigned based on the 71,2 coupling constants. 5.2.10 Characterization of Glycosynthase Products Para-nitrophenyl 3-0-(methyl 2,3,4-tri-0-acetyl-P-D-galacturonate)-2,4,6-tri-0-acetyl-P-D-glucoside (4.19) AcO OAc ' H N M R (400 M H z , C D C 1 3 ) : 8 8.18, 7.02 (2 d, 4 H , J = 9.2 H z , A r ) , 5.63 (d, 1 H , Jv>y = 3.5 H z , H-4') , 5.33-5.28 (m, 1 H , H-2), 5.14-5.08 (m, 2 H , H-2 ' , H-4), 5.03-4.99 (m, 2 H , H - l , H-3 ' ) , 4.59 (d, 1 H , JV>T = 7.8 H z , H - l ' ) , 4.26-4.20 (m, 3 H , H-5 ' , H - 6 a , H -6 b), 4.06 (t, 1 H , J 3 ) 4 = 73,2 = 9.5 H z , H-3), 3.96-3.88 (m, 1 H , H-5), 3.74, (s, 3 H , O-91 C H 3 ) 2.22, 2.11, 2.09, 2.06, 2.03, 1.97 (6 s, 18 H , 6 O - A c ) . H R M S (ESI) m/z 766.1802 [ M + N a ] + . Calculated for C 3 i H 3 7 N N a 0 2 o 766.1807. jPam-nitrophenyl 2,4-di-0-acetyl-3-0-(methyl 2,3,4-tri-0-acetyl-(3-D-galacturonate)-P-D-xyloside (4.20) AcO AcO AcO ' H N M R (J couplings from 400 M H z , assigned with 600 M H z , C D C 1 3 ) : 8 8.19, 7.12 (2 d, 4 H , .7=9.3 H z , A r ) , 5.69 (dd, 1 H , J4<>3< = 3.5, Jvty = 1.4 H z , H-4 ') , 5.36 (d, 1 H , J , , 2 = 3.4 H z , H - l ) , 5.26 (dd, 1 H , J2;y = 10.4, J2;y = 8.1 H z , H-2 ') , 5.11-5.02 (m, 3 H , H-4, H - 3 ' , H-2), 4.31 (d, 1 H , H-5') , 4.67 (d, 1 H , H - l ' ) , 4.20 (dd, 1 H , J 5a,5b = 12.6, J5aA = 3.3 H z , H-5 a ) , 4.03 (t, 1 H , J 3 ; 4 = J 3 , 2 = 5.3 Hz , H-3), 3.72 (s, 3 H , 0 - C H 3 ) , 3.64 (dd, 1 H , J5bA = 4.5 H z , H-5 b ) , 2.15, 2.13, 2.11, 2.04, 1.99 (5 s, 15 H , 5 O - A c ) . H R M S (ESI) m/z 694.1612 [ M + N a ] + . Calculated for C 2 8 H 3 3 N N a O i 8 694.1595. Arra-nitrophenyl 4-0-(3-0-[methyl 2,3,4-tri-0-acetyl-(3-D-galacturonate]-2,4,6-tri-0-acetyl-P-D-glucoside)-2,3,4-tri-0-acetyl-P-D-glucoside (4.21) OAc ' H N M R (400 M H z , C D C 1 3 ) : 8 8.18, 7.03 (2 d, 4 H , J = 9.2 H z , A r ) , 5.62 (dd, 1 H , J 4 " ; 3 " = 3.5, J 4 - , 5 " = 1.1 H z , H-4") , 5.26 (t, 1 H , J3A = J3,2 = 8.8 H z , H-3), 5.19 (dd, 1 H , J2J = 7.4 H z , H-2), 5.15 (d, 1 H , H - l ) , 5.05 (dd, 1 H , J 2 » ; 3 » = 10.4, J 2 " , i " = 7.8 H z , H-2") , 5.05-4.95 (m, 3 H , H-4 ' , 2', 3"), 4.52 (d, 1 H , H - l " ) , 4.46-4.43 (m, 1 H , H-6 a ) , 4.34-4.29 (m, 1 H , H-6 ' a ) , 4.32 (d, 1 H , Jv? = 8.1 H z , H - l ' ) , 4.23 (d, 1 H , H-5") , 4.12-4.05 (m, 2 H , H - 6 b , H-6 ' b ) , 3.90 (t, 1 H , J 3 - > 4 . = Jy>2> = 9.5 H z , H-3 ') , 3.85-3.78 (m, 2 H , H-4, H-5), 3.71 (s, 3 H , 0 - C H 3 ) , 3.67-3.63 (m, 1 H , H-5 ' ) , 2.18, 2.10, 2.09, 92 2.08, 2.07, 2.03, 2.02, 2.01, 1.96 (9 s, 27 H , 9 O - A c ) . H R M S (ESI) m/z 1054.2632 [ M + N a ] + . Calculated for C 4 3 H 5 3 N N a 0 2 8 1054.2652. Pf l ra-ni t rophenyl 3-0-(methyl 2,3,4-tr i -0-acetyl-R-D-glucuronate)-2,4,6-tr i -0-acetyl-p-D-glucoside (4.22) OAc OAc ' H N M R (400 M H z , C D C 1 3 ) : 5 8.18, 7.01 (2 d, 4 H , J = 9.2 H z , A r ) , 5.29 (dd, 1 H , J 2 ) 3 = 9.4, y 2 , i = 7.9 H z , H-2), 5.20 (dd, 1 H , JyA> = 9.6, Jy>2. = 9.2 H z , H-3 ') , 5.12 (dd, 1 H , J 4 ' , 5 ' = 9.7 H z , H-4') , 5.06 (dd, 1 H , J 4 , 5 = 9.6, J 4 , 3 = 9.1 H z , H-4), 5.02 (d, 1 H , 7 i > 2 = 7.9, H - l ) , 4.89 (dd, 1 H , J2;y = 8.1 H z , H-2 ') , 4.65 (d, 1 H , H - l ' ) , 4.20-4.18 (m, 2 H , H - 6 a , H-6 b ) , 3.99 (d, 1 H , H-5 ') , 3.98 (dd, 1 H , J= 9.4 H z , H-3) , 3.87-3.83 (m, 1 H , H-5), 3.72 (s, 3 H , O-CH3), 2.13, 2.12, 2.04, 2.02, 1.99, 1.98 (6 s, 18 H , 6 O - A c ) . H R M S (ESI) m/z 766.1813 [ M + N a f . Calculated for C 3 i H 3 7 N N a 0 2 o 766.1807. Pa ra -n i t ropheny l 2,4-di-0-acetyl-3-0-(methyl 2 ,3 ,4- t r i -O-acetyl-P-D-glucuronate)-p-D-xyloside (4.23) OAc OAc ' H N M R (400 M H z , C D C 1 3 ) : 8 8.18, 7.08 (2 d , 4 H , / = 9 . 2 H z , A r ) , 5.31 (d, 1 H , y , ; 2 = 4.4 H z , H - l ) , 5.24 (dd, 1 H , Jy# = 9.5, Jy>2- = 9.1 H z , H-3 ') , 5.19 (dd, 1 H , Jv>s. = 9.6 H z , H-4 ') , 5.06 (dd 1 H , J 2 , 3 = 6.5 H z , H-2), 5.04 (dd, 1 H , J2;v = 7.8 H z , H-2 ') , 5.01 (m, 1 H , H-4), 4.73 (d, 1 H , H - l ' ) , 4.16 (dd, 1 H , J 5a,5b = 12.7, J 5 a > 4 = 9.0 H z , H -5 a), 4.04 (d, 1 H , H-5 ') , 3.98 (dd, 1 H , / 3 > 4 = 5.9 H z , H-3), 3.73 (s, 3 H , O-CH3), 3.58 (dd, 1 H , y 5 b , 4 = 5.4 H z , H-5 b ) , 2.13, 2.11, 2.03 (3 s, 9 H , 3 O - A c ) , 2.01 (s, 6 H , 2 O-A c ) . H R M S (ESI) m/z 694.1614 [ M + N a ] + . Calculated for C 2 8 H 3 3 N N a O , 8 694.1595. 93 jPara-nitrophenyl 4-0-(3-0-[methyl 2,3,4-tri-0-acetyl-P-D-glucuronate]-2,4,6-tri-0-acetyI-P-D-glucoside)-2,3,4-tri-0-acetyl-P-D-glucoside (4.24) OAc ' H N M R (400 M H z , CDCI3): 8 8.18, 7.03 (2 d, 4 H , J= 9.2 H z , A r ) , 5.25 (t, 1 H , 7 3 ,2 = J 3 , 4 = 8.2 H z , H-3), 5.23-5.12 (m, 3 H , H-2 , H - l , H-3") , 5.10, (dd, 1 H , J 4 " , 5 " = 9.7 H z , y 4 " ,3" , H-4") , 4.88-4.83 (m, 2 H , H-2 ' , H-4') , 4.85 (dd, 1 H , J2-x = 9.3, J2",r = 8.1 H z , H-2") , 4.57 (d, 1 H , H - l " ) , 4.45 (m, 1 H , H-6 a ) , 4.32 (d, 1 H , JV>T = 8.0 H z , H - l ' ) , 4.31-4.26 (m, 1 H , H-6 ' a ) , 4.13-4.07 (m 1 H , H-6 b ) , 4.05 (dd, 1 H , J6%&a = 12.3, J 6 'b,5' = 2.1 H z , H-6'b), 3.98 (d, 1 H , H-5") , 3.85, 3.80 (m, 3 H , H-5 , H - 3 ' , H-4), 3.71 (s, 1 H , 0 - C H 3 ) , 3.62-3.58 (m, l H , H-5') , 2.12-1.98 (m, 27 H , 9 O - A c ) . H R M S (ESI) m/z 1054.2656 [ M + N a ] + . Calculated for C 4 3 H 5 3 N N a 0 2 8 1054.2652. 94 Appendix Michael is-Menten parameters and plots of reaction rate against substrate concentration Table A l . Michae l i s -Men ten Parmeters E n z y m e Substrate Km ( m M ) ^cat Sp. Hex. p N P - G l c N A c 0.072 3.2 x 10 2 s"1 Hex A M U G 0.85 4.5 x 1 0 2 s"1 H e x B M U G 0.92 1.5 x 10" s"1 Glucocerebrosidase 2 , 4 - D N P - g l u c o s i d e 1.6 4.3 x 1 0 ' m i n " 1 HexB 0 0.5 1 1.5 2 2.5 3 3.5 4 [MUG] (mM) Glucocerebrosidase 1.2 -i [2,4-DNP-glucoside] (mM) 95 References (1) Henrissat, B . ; Bairoch, A . Biochem. J. 1996, 316, 695-696. (2) Wolfenden, R.; L u , X . D . ; Young , G . J. Am. Chem. Soc. 1998, 120, 6814-6815. (3) Koshland, D . E . Biol. Rev. 1953, 28, 416-436. (4) Phil l ips, D . C. Proc. Natl. Acad. Sci. U. S. A. 1967, 15, 483-495. (5) Sinnot, M . L . ; Souchard, I. J. L . Biochem. J. 1973, 133, 89-98. (6) McCarter , J. D . ; Withers, S. G . Curr. Opin. Struct. Biol. 1994, 4, 885-892. 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The use of inhibitors to stabilize folded conformations of enzymes
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