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Activity-based proteomics profiling for identification and quantification of Trichoderma reesei cellulases Chen, Chi Fan 2011

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ACTIVITY-BASED PROTEOMICS PROFILING FOR IDENTIFICATION AND QUANTIFICATION OF TRICHODERMA REESEI CELLULASES by Chi Fan Chen B.Sc., The University of British Columbia, 2008  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Chemistry)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  November 2011  © Chi Fan Chen, 2011  ii Abstract  Cellulosic ethanol holds great promise as a renewable fuel to supplement gasoline. The complete conversion of cellulose involves multiple steps, one of which is the enzymatic degradation of cellulose to glucose. Trichoderma reesei (recently renamed as Hypocrea jecorina) secretes a large variety of cellulases that work synergistically in the hydrolysis of cellulose into glucose. However, enzymes tend to “die off” during biomass conversion, decreasing the efficiency of degradation, yet it is difficult to determine which enzyme in the mixture loses activity. Furthermore, significant diversity in the composition of plant cell walls requires optimization of enzyme mixture used for hydrolysis of each feedstock. In order to address the problems, tools to identify and quantitate the active enzyme species present in the enzyme mixtures are required, and would characterize the composition of the active enzymes in the hydrolysis mixtures. We proposed an activity-based protein profiling (ABPP) approach. It involves a set of chemical probes, each containing a cellulase-specific inactivating functionality to label the active enzymes in the hydrolysis mixture, and a reporter group to quantitate the modified enzymes using such reagents, the concentration of one specific enzyme could be determined based on the amount of labeled enzymes detected using the reporter group. A cellulase- specific inactivating functionality is either an affinity label or a mechanism-based inactivator incorporating a specificity-determining chemical group. To evaluate the potential inactivating functionalities, six cellobiose-based affinity labels based on reactive groups that have proved useful for other glycosidases, and two mechanism-based inactivators have been tested with the 4 principal T. reesei cellulases (provided by our collaborator) for their efficiencies. Seven  iii of them inactivated at least one of the cellulases; among these, C-epoxypentyl cellobioside had the best overall performance. It had relatively good binding affinity and efficiency toward three cellulases, and it will be included in a second generation of ABPP probe. An enlarged inactivator library will be required to target other cellulases. Ultimately, at least one ABPP for each of the T. reesei cellulase will be generated and hopefully will prove useful to the biofuel industries.  iv Table of Contents  Abstract .................................................................................................................................... ii Table of Contents ................................................................................................................... iv List of Tables ........................................................................................................................... x List of Figures ......................................................................................................................... xi List of Abbreviations ........................................................................................................... xvi Acknowledgements .............................................................................................................. xxi Chapter  1: General Introduction ......................................................................................... 1 1.1 Benefits and Applications of Biofuels ...................................................................... 1 1.2 Lignocellulosic Biomass Conversion Process .......................................................... 2 1.2.1 Pretreatment: Removal of the Lignin and Hemicellulose Protecting Layer ......... 5 1.2.2 Cellulolytic System Used for Cellulose Degradation ............................................ 8 1.2.2.1 Problems Associated with the T. reesei Cellulolytic System and a Potential Solution ........................................................................................................ 9 1.3 Glycoside Hydrolases (Glycosidases)..................................................................... 10 1.3.1 Classification of Glucoside Hydrolases .............................................................. 11 1.3.1.1 Classification by the Location of Cleavage: Exoglucanase and Endoglucanase ............................................................................................ 11 1.3.1.2 Carbohydrate Active enZyme (CAZy) Classification ................................ 13 1.3.2 Catalytic Mechanism of Glycosidases ................................................................ 15 1.3.2.1 Single Displacement (Inverting) ................................................................. 16 1.3.2.2 Double Displacement (Retaining) ............................................................... 17  v 1.3.3 Glycoside Hydrolase Subsite Nomenclature ....................................................... 18 1.4 Methods of Measuring Active Enzyme Concentrations in a Protein Mixture ........ 19 1.4.1 Active Site Titration ............................................................................................ 20 1.4.2 Activity Based Protein Profiling (ABPP) ............................................................ 21 1.4.2.1 Irreversible Inactivators for Glycosidases .................................................. 23 1.4.2.2 Linker Arms and Reporter Groups ............................................................. 26 1.4.3 A Method for Improving the Specificity of Individual T. reesei Cellulases ....... 27 1.5 Aims of This Study ................................................................................................. 28 Chapter  2: Results and Discussion ..................................................................................... 30 2.1 Introduction to T. reesei Cellulases: Cellobiohydrolase I, Cellobiohydrolase II, Endoglucanase I and Endoglucanase II .................................................................. 30 2.1.1 CBHI and CBHII Cellulases ............................................................................... 31 2.1.2 EGI and EGII Cellulases ..................................................................................... 33 2.2 Abg Glycosynthase in Carbohydrate Synthesis ...................................................... 35 2.3 Synthesis of Cellobioside Inactivators and 2,4-Dinitrophenyl (2,4-DNP) Substrates  ................................................................................................................................ 37 2.3.1 Synthesis of N-Bromoacetyl β-Cellobiosylamine (2.1) ...................................... 37 2.3.2 Synthesis of O-Epoxyalkyl β-Cellobiosides (2.2, 2.3, and 2.4) .......................... 39 2.3.3 Synthesis of C-Epoxyalkyl β-Cellobiosides (2.5 and 2.6) .................................. 43 2.3.4 Synthesis of 2,4-Dinitrophenyl 2-deoxy-2-fluoro β-cellobioside (2FDNPC, 2.7) and 2,4-Dinitrophenyl 2-deoxy-2-fluoro β-lactoside (2FDNPL, 2.8) ................. 47 2.3.5 Synthesis of 2,4-Dinitrophenyl Trisaccharides (2,4-DNP Trisaccharides) ......... 51  vi 2.4 Kinetic Evaluations of Cellobiose-based Inactivators and Substrates of Cellulases  ................................................................................................................................ 53 2.4.1 2,4-Dinitrophenyl β-cellobioside as a Standard Substrate .................................. 53 2.4.2 Kinetic Evaluation of Potential Active Site-Directed Inactivators and Mechanism-Based Inactivators for T. reesei Cellulases ..................................... 59 2.4.2.1 Kinetic Evaluation of N-Bromoacetyl β-Cellobiosylamine (2.1) as a Potential Inactivator for T. reesei Cellulases .............................................. 60 2.4.2.2 Kinetic Evaluation of O-linked and C-linked Epoxyalkyl β-Cellobiosides 60 2.4.2.2.1 Discussion of Inactivation Results of the Four T. reesei Celluases ....... 63 2.4.2.2.2 Summary of the Studies of Epoxide-based Cellulase Inactivators ........ 65 2.4.2.2.3 Internal Cleavage of Cellobiosides by EGI ........................................... 66 2.4.2.2.4 Inactivation of CBHII  by EPO3C (2.2) and EPC4C (2.5) Did Not Occur According to a Single Exponential Decay ............................................. 69 2.4.2.3 Kinetic Evaluation of 2,4-Dinitrophenyl 2-deoxy-2-fluoro β-cellobioside (2FDNPC, 2.7) and 2,4-Dinitrophenyl 2-deoxy-2-fluoro β-lactoside (2FDNPL, 2.8) as Mechanism-based Inactivators for T. reesei Retaining Cellulases .................................................................................................... 71 2.4.3 Summary of Inactivation Studies with T. reesei Cellulases ................................ 73 2.4.4 Kinetic Evaluation of 2,4-Dinitrophenyl Trisaccharides with T. reesei Cellulases  ............................................................................................................................. 75 2.4.4.1 Comparison of Kinetic Parameters for 2,4-DNP Substrates on CBHII ...... 78 2.4.4.1.1 Comparison of kinetic results of 2,4-DNPC and 2,4-DNP trisaccharides with CBHII............................................................................................. 78  vii 2.4.4.1.2 Comparison of kinetic results of four 2,4-DNP trisaccharides (2.9, 2.10, 2.11, and 2.12) with CBHII ................................................................... 79 2.4.4.2 Comparison of Kinetic Parameters for 2,4-DNP Substrates on EGII: ........ 81 2.4.4.2.1 Comparison of kinetic results of 2,4-DNPC and 2,4-DNP trisaccharides with EGII ............................................................................................... 81 2.4.4.2.2 Comparison of kinetic results of four 2,4-DNP trisaccharides (2.9, 2.10, 2.11, and 2.12) with EGII ...................................................................... 81 2.4.4.3 Comparison of Kinetic Parameters for 2,4-DNP Trisaccharide Substrates on CBHI and EGI ....................................................................................... 83 2.5 Other Work Done to Probe Substrate Specificity ................................................... 85 2.6 Summary of the Kinetic Studies from the Synthetic Compounds with T. reesei Cellulases ................................................................................................................ 87 Chapter  3: Materials and Methods .................................................................................... 90 3.1 Generous Gifts ........................................................................................................ 90 3.2 General Synthesis.................................................................................................... 90 3.2.1 General Methods ................................................................................................. 91 3.2.1.1 Acetylation of Free Sugars.......................................................................... 91 3.2.1.2 General Deacetylation ................................................................................. 91 3.2.1.2.1 Deacetylation by Sodium Methoxide..................................................... 91 3.2.1.2.2 Deacetylation with Ammonia ................................................................ 92 3.2.1.2.3 Deacetylation in Acidic Condition......................................................... 92 3.2.1.3 De-benzylation of C-linked Glucosides ...................................................... 92 3.2.1.4 General Procedure for Synthesis of α-Glycosyl Fluorides ......................... 93  viii 3.2.1.5 General Procedure for Synthesis of α-Glycosyl Bromides ......................... 93 3.2.1.6 General Procedure for Alkenyl Alcohol Coupling with α-Cellobiosyl Trichloroacetimidates ................................................................................. 94 3.2.1.7 General Procedure for Epoxidation of Alkenyl Glycosides ....................... 94 3.2.1.8 General Procedure for Oligosaccharide Synthesis with Abg 2F6 Glycosynthase ............................................................................................ 95 3.2.2 Synthesis and Characterization ........................................................................... 96 3.3 Enzymology .......................................................................................................... 135 3.3.1 Production of Abg 2F6 Glycosynthase ............................................................. 135 3.3.2 Purification of T. reesei Cellulases by Iogen Corp. CBHI, CBHII, EGI, and EGII  ........................................................................................................................... 135 3.3.3 Enzyme Kinetics ................................................................................................ 136 3.3.3.1 General Methods ....................................................................................... 136 3.3.3.2 Steady-State Kinetic Measurements ......................................................... 137 3.3.3.3 Irreversible Inactivation Kinetics .............................................................. 138 3.3.3.4 Competitive Inhibition Studies To Determine Inhibition Constant Ki ..... 139 3.3.3.5 Substrate Depletion Assay for Rapid Determination of kcat/Km ................ 139 References……………………………………………………………………………..…141 Appendix A CONCENTRATIONS OF COMPOUNDS USED IN INACTIVATION ASSAYS (PARTIAL DATA) .............................................................................. 151 Appendix B GRAPHICAL PRESENTATION OF DATA .............................................. 152 B.1 Steady State Kinetics for the Hydrolysis of 3,4-DNP cellobioside and 2,4-DNP trisaccharides ..................................................................................................... 152  ix B.2 Time Dependent Inactivation Kinetics of T.reesei Cellulases with the Inactivators ........................................................................................................ 157 B.3 TLC and Gel Results ......................................................................................... 164 Appendix C BASIC ENZYME KINETICS ..................................................................... 165 C.1 Fundamentals of Enzyme Kinetics .................................................................... 165 C.2 Irreversible Inactivation and Mechanism-based Inhibition Kinetics ................ 167 C.3 Competitive Inhibition Kinetics ........................................................................ 168 C.4 Alternative Cleavage in This Thesis ................................................................. 169 C.4.1 Multiple Binding Modes Does Not Affect kcat/Km (or ki/Ki) .............................. 170    x List of Tables Table 1.1! Common reporter groups and linkers used in ABPP probes ............................... 27! Table 2.1! Properties of the four principal Trichoderma reesei cellulases. .......................... 30! Table 2.2! Physical properties of, p-nitrophenolate, 2,4-dinitrophenolate and 3,4- dinitrophenolate. .................................................................................................. 54! Table 2.3! Kinetic parameters for hydrolysis of three !-aryl cellobioside substrates. .......... 55! Table 2.4! Kinetic parameters for the inactivation of T. reesei cellulase by the O-epoxyalkyl !-cellobiosides and C-epoxyalkyl !-cellobiosides. .............................................. 62! Table 2.5! Kinetic parameters for the inactivation of EGI by 2FDNPC (2.7) and 2FDNPL (2.8). ..................................................................................................................... 71! Table 2.6!  Kinetic parameters for hydrolysis of 2,4-DNP glycoside substrates with T. reesei cellulases determined by absorbance change at 400 nm. ..................................... 77! Table 2.7! Michaelis-Menten parameters for the hydrolysis of DNPC and its derivatives by T. reesei. ............................................................................................................... 86!   xi List of Figures Figure 1.1! (a) Structure and composition of lignocelluloses: lignin, hemicellulose and cellulose. (b) Structure of a cellulose chain with an arrow indicating !-(1,4) glycosidic bonds, and the reducing/non-reducing terminus.. ............................... 3! Figure 1.2! Overall production process of cellulosic ethanol conducted at Iogen Corp. (http://www.iogen.ca/). ......................................................................................... 4! Figure 1.3! (a) Chemical structure of lignin. (b) Partial structure of hemicellulose. .............. 6! Figure 1.4! Schematic of the pretreatment process on lignocellulosic material. .................... 7! Figure 1.5! Diagram of a !-monosaccharide with glycosidic bond indicated with an arrow.   ............................................................................................................................ 10! Figure 1.6 ! Hydrolysis catalyzed by a !-glycosidase. ........................................................... 11! Figure 1.7! (a) The locations in cellulose where endoglucanase and exoglucanase act. (b) Van der Waals surface representations of the active-site tunnels (arrowed) in GH 6 CBH Cel 6A from Humicola  insolens (b.1), and endoglucanase E2 from Thermomonospora fusca (b.2). ........................................................................... 13! Figure 1.8! Products produced by retaining or inverting glycosidases. ................................ 16! Figure 1.9! Mechanism of enzymatic reaction of inverting glycosidases. ............................ 17! Figure 1.10! Mechanism of enzymatic reaction of !-retaining glycosidases. ......................... 18! Figure 1.11 ! Nomenclature of sugar binding subsites in a glycosidase active site. ................ 19! Figure 1.12! (a) Basic structure of an ABPP Probe and (b) potential ABPP probes for cellulases. ............................................................................................................ 22! Figure 1.13! A general strategy for activity-based protein profiling. In-gel fluorescence analysis is employed in this example. ................................................................ 23!  xii Figure 1.14! Examples of some group specific labels and irreversible inactivators. (a) Structures of EAC (b) Woodward’s reagent K (c) Cyclophellitol (d) 2-deoxy-2- fluoro glucosides. ............................................................................................... 24! Figure 1.15! Structures of potential inactivators of cellulases synthesized in this work. ....... 29! Figure 2.1 ! Schematic representation of the CBHI of T. reesei catalytic domain with cellulose bound.. ................................................................................................. 31! Figure 2.2! Catalytic domain of T. reesei CBHII with a bound cellohexaose molecule.. ..... 32! Figure 2.3! (a) Crystal structure of catalytic domain of T. reesei EGI. (b) The catalytic domain of CBHI. The green loops which enclose the glycosyl binding subsites are missing in EGI of T. reesei ........................................................................... 33! Figure 2.4! Catalytic domain of Cel 5A of T. aurantiacus. .................................................. 34! Figure 2.5! Synergistic action of endoglucanases, cellobiohydrolases, and !-glucosidase in the conversion of a celloheptaose into glucose. ................................................. 35! Figure 2.6! Proposed glycosynthase reaction catalyzed by a nucleophile mutant of a specific glucosidase.. ....................................................................................................... 36! Figure 2.7! Potential inactivation of cellulases with N-bromoacetyl !-cellobiosylamine. ... 37! Figure 2.8! Synthesis of N-bromoacetyl !-cellobiosylamine (2.1).. ..................................... 39! Figure 2.9! (a) Structures of aziridine-based and epoxy-based inactivators. (b)Potential inactivation reaction occurs in cellulases with an O-epoxyalkyl !-cellobioside.  ............................................................................................................................ 40! Figure 2.10! O-Epoxyalkyl !-cellobiosides 2.2, 2.3, and 2.4.. ............................................... 41! Figure 2.11! Synthesis of (R,S)-O-epoxypropyl !-cellobioside (2.2), (R,S)-O-epoxybutyl !- cellobioside (2.3), and (R,S)-O-epoxypentyl !-cellobioside (2.4).. .................... 42!  xiii Figure 2.12! C-Epoxyalkyl !-cellobiosides 2.5 and 2.6. ......................................................... 43! Figure 2.13! Attempted synthesis of (a) O-tetraacetyl-C-allyl !-glucoside and (b) O- tetraacetyl-C-allyl !-cellobioside. ...................................................................... 44! Figure 2.14! Synthesis of (R,S)-C-epoxybutyl !-cellobioside (2.5) and (R,S)-C-epoxypentyl !-cellobioside (2.6). ............................................................................................ 46! Figure 2.15! Synthesis of 2,4-dinitrophenyl 2-deoxy-2-fluoro !-cellobioside (2.7) and 2,4 dinitrophenyl 2-deoxy-2-fluoro !-lactoside (2.8).. ............................................. 49! Figure 2.16 ! Syntheses of 2,4-dinitrophenyl trisaccharides 2.9, 2.10, 2.11 and 2.12. ............ 52! Figure 2.17! Chemical structures of three substrate candidates: pNPC, 2,4-DNPC and 3,4- DNPC. ................................................................................................................ 53! Figure 2.18! TLC of hydrolytic reactions of 2,4-DNPC with T. reesei cellulases. (a) Samples were taken after 5 minutes of enzyme addition. (b) Hydrolytic reaction of 2,4- DNPC with EGI. ................................................................................................. 57! Figure 2.19! The possible hydrolysis of the reducing terminal glycosidic bond. ................... 61! Figure 2.20! A histogram of inactivator efficiencies of epoxyalkyl cellobiosides to T. reesei cellulases. ............................................................................................................ 66! Figure 2.21! (a) TLC results of inactivation reactions of CBHI, CBHII and EGI by EPC5C (2.6). (b) Mass spectrometric data of inactivation of EGI by EPC5C. ............... 68! Figure 2.22! Natural logarithm (Ln) values of relative residual CBHII activity vs. time for inactivation of CBHII by EPO3C (2.2) and EPC4C (2.5). ................................. 69! Figure 2.23! The “flattening-out” phenomenon in the reaction of 2FDNPC (2.7) and 2FDNPL (2.8) with EGI. .................................................................................... 72!  xiv Figure 2.24!  (a) Possible transglycosylation of the glycosyl-enzyme intermediate by 2FDNPC (2.7). (b) No transglycosylation of glycosyl-enzyme intermediate by C4" axial hydroxyl group of 2FDNPL (2.8). ...................................................... 73! Figure 2.25! Histograms displaying inactivation rate constants (ki/Ki) for each inactivator/cellulase combination grouped by (a) enzyme; (b) inactivator. ....... 74! Figure 2.26! Structures of 2,4-DNP trisaccharides (2.9, 2.10, 2.11 and 2.12). ....................... 76! Figure 2.27! TLC of reaction mixture of 2,4-DNP trisaccharides with T. reesei cellulases.   ............................................................................................................................ 78! Figure 2.28! (a) Reaction coordination diagram of classical mechanism of an inverting glycosidase. (b) Energy diagram of classical double displacement mechanism of a retaining glycosidase. ...................................................................................... 80! Figure 2.29! Protein interactions with cellobiose in the active site (-3 and -2 subsites) of Cel 5A of B. agaradherans. (a) A schematic representation of Cel 5A-cellobiose interaction. (b) Electron density figure for cellobiose bound in the active site.   ............................................................................................................................ 83! Figure B.3.1! TLC of (a) inactivation mixture of CBHI, CBHII and EGI with EPC4C. SM: EPC4C; (b) inactivation mixture of CBHI, CBHII and EGI with EPO3C. SM: EPO3C; (c) reaction mixture of DNPC and EGI with cyclophellitol. SM: DNPC, C: control (EGI and buffer only); Rxn: reaction (EGI, cyclophellitol, and buffer); Ref1: cellobiose; Ref2: glucose and DNPG. (d) Electrophoresis gel of the four T. reesei cellulases. C1: CBHI; C2: CBHII; E1: EGI; E2: EGII. . 164 Figure C.1.1 Plot of initial rate (v) versus substrate concentration [S] for a typical Michaelis- Menten kinetics……………………………………………………………...166  xv Figure C.4.1 Alternative cleavage enzymatic reaction with an (a) inactivator or (b) aryl substrate……………………………………………………………….…….170 Figure C.4.2 Productive and non-productive binding. The red arrow indicates the glycosidic bond that is hydrolyzed.    ……………………………………………….…171   xvi List of Abbreviations Abg - Agrobacterium sp. β-D-glucosidase (glycosynthase) ABPP - activity Based Protein Profiling Ac - acetyl group β-arabF - β-L-arabinosyl fluoride B. agaradherans - Bacillus agaradherans BSA - bovine serum albumin Bn - benzyl CAZy - carbohydrate active enzyme calcd. - calculated cat. - catalytic amount CBH I - cellobiohydrolase I CBH II - cellobiohydrolase II δ - chemical shift d - doublet DABCO - 1,4-diazobicyclo-[2.2.2]octane DBU - 1,8-Diazabicycloundec-7-ene DCM - dichloromethane dd - doublet of doublets ddd - doublet of doublet of doublets DMF - N, N-dimethylformamide DMSO - dimethyl sulfoxide DNFB - 1-fluoro-2,4-dinitrobenzene  xvii 2,4-DNP - 2,4-dinitrophenyl 2,4-DNPC - 2,4-dinitrophenyl β-cellobioside 2,4-DNPG - 2,4-dnitrophenyl β-glucoside L-arabinosylDNPC - 2,4-dnitrophenyl [(α-L-arabinopyranosyl)-(1→4)-O- β-D-glucopyranosyl]-(1→4)-O-β-D-glucopyranoside  D-fucosylDNPC - 2,4-dnitrophenyl [(β-D-fucopyranosyl)-(1→4)-O-β- D-glucopyranosyl]-(1→4)-O-β-D-glucopyranoside  D-galactosylDNPC - 2,4-dnitrophenyl [(β-D-galactopyranosyl)-(1→4)-O- β-D-glucopyranosyl]-(1→4)-O-β-D-glucopyranoside  D-xylosylDNPC - 2,4-dnitrophenyl [(β-D-xylopyranosyl)-(1→4)-O-β- D-glucopyranosyl]-(1→4)-O-β-D-glucopyranoside  dt - double of triplets E. coli - Escherichia coli EGI - endoglucanase I EGII - endoglucanase II EI - Michaelis complex with inactivator/inhibitor EPO3C - (R,S)-1,2-O-epoxypropyl β-cellobioside EPO4C - (R,S)-1,2-O-epoxybutyl β-cellobioside EPO5C - (R S)-1,2-O-epoxypentyl β-cellobioside EPC4C - (R,S)-1,2-C-epoxybutyl β-cellobioside EPC5C - (R,S)-1,2-C-epoxypentyl β-cellobioside eqv. - equivalent ES - Michaelis complex ESI - electrospray ionization ESI MS - electrospray ionization mass spectrometry  xviii EtOH - ethanol 2FDNPC - 2,4-dinitrophenyl 2-deoxy-2-fluoro β-cellobioside 2FDNPL - 2,4-dinitrophenyl 2-deoxy-2-fluoro β-lactoside α-fucF - α-D-fucosyl fluoride ∆G - Gibbs free energy α-galF - α-D-galactosyl fluoride α-D-glcF - α-D-glucosyl fluoride HPLC - high performance liquid chromatography HRMS - high resolution mass spectrometry Iogen Corp. - Iogen Corporation IUBMB - International Union of Biochemistry and Molecular Biology  J - coupling constant Ka - acid dissociation constant kcat - catalytic rate constant Ki - Michaelis constant of an enzymatic inhibitor/inactivator Km - Michaelis constant of an enzymatic substrate kobs - apparent inactivation rate in an irreversible inactivation  LB - Luria-Bertani media LC - liquid chromatography m/z - mass-to-charge ratio M - molarity (mol/L) m - multiplet  xix mCPBA - meta-chloroperoxybenzoic acid MeOD - deuterated methanol MeOH - methanol MS - mass spectrometry MW - molecular weight NMR - nuclear magnetic resonance pNPC - para-nitrophenyl β-cellobioside Pd/C - palladium on charcoal ppm - parts per million Rf - retention factor RT - room temperature s - singlet SN2 - bimolecular nucleophilic substitution SDS-PAGE - sodium dodeccyl sulfate polyacrylamide gel electrophoresis SelectFluorTM -  1-chloromethyl-4-fluoro-1,4- diazoniabicyclo[2,2,2]octane bis(tetrafluoroborate) SM - starting material t - triplet td - triplet of doublet TCA - trichloroacetimidate TIM - triose phosphate isomerase TLC - thin layer chromatography THF - tetrahydrofuran T. reesei - Trichoderma reesei  xx T. aurantiacus - Thermoascus aurantiacus UV-Vis - ultraviolet-visible light v/v - volume to volume Vmax - maximum velocity of an enzyme-catalyzed reaction w/w - weight to weight α-xylF - α-D-xylosyl fluoride Note: the three letter codes for the amino acids follow the recommendations of IUPAC. J. Biol. Chem. 1968, 243, 3557-3559 and J. Biol. Chem. 1972, 247, 977-983.                       xxi Acknowledgements  First and foremost, I would like to thank my supervisor, Professor Stephen G. Withers, for his inspiration, patience, enthusiasm and guidance over the past few years, and provided me a great opportunity to conduct research in his lab. Further, I would like to extend my gratitude to all the past and present Withers lab members, for all the unselfish support and wonderful memories, which had made my MS.c life unforgettable. I’d especially like to thank Dr. Hongming Chen, Dr. Jamie Rich and Dr. Ethan Goddard-Borger for their constant advice in organic carbohydrate synthesis, as well as Dr. Jacqueline Wicki, Dr. Emily Kwan and Dr. David Kwan for their generous help with Abg glycosynthase expression.  Without their guidance, this work would have been much harder. Besides, I would like to thanks our collaborator, Iogen Corporation for the supply of T. reesei cellulases. My lab coffee buddies, Aidha Shaikh, Joy Ratananikom and Seino Jongkees, we can’t start a day without a cup of Blue chip coffee. Moreover, I would like to give my appreciation to Ms. Miranda Joyce for organizing any big and little things in the lab. I would like to also show my gratitude to the staff in Chemistry Department especially NMR lab and MS lab staff. Lastly, much appreciate to all my friends in this Department especially 3rd floor and 4th floor A-wing chemists. I would never forget all the fun time spending with all you, sharing our life stories in the lab and in the hallway. Lastly but certainly not the least, I would like to thanks all my family members who gave me a considerable support when I was struggling with research or thesis writing. I couldn’t have accomplished this work without being cheered up by my family. Thanks!  1 Chapter  1: General Introduction  1.1 Benefits and Applications of Biofuels In the past two centuries global energy production has relied heavily on fossil fuels1-3, leading to growing concerns over environmental impacts and the continued depletion of non- renewable fossil fuel. Furthermore, the burning of fossil fuels has been associated with extensive detrimental effects such as increasing atmospheric carbon dioxide and global warming. In the short term, burning of fossil fuels leads to air pollution, elevated temperatures, and acid rain while the long term impacts could result in a dire imbalance of the global biome. For this reason, significant efforts have been made to develop alternative energy supplies to get away from the consumption of fossil fuels. Development of clean alternative energy supplies is highly desired to relieve the dependency on fossil fuels and to reduce adverse environmental impacts. One of the solutions to alleviating the consumption of fossil fuels is through the use of bioethanol. Bioethanol is ethanol produced by the fermentation of any carbohydrate-containing biomaterial. The ethanol produced is often used as a blending additive with gasoline to boost octane and as an alternative energy source. The addition of ethanol reduces green house gas emissions.4-6 Commonly used biomaterials for bioethanol production include agricultural crops (corn, potatoes, and sugarcane, which primarily consist of simple sugars or starch polymers), or lignocelluloses (leaves and wood, which are rich in cellulose).7,8 In recent years, 80% of ethanol is produced from the fermentation of simple sugar-based feedstock in Brazil and from corn (starch) based feedstock in the USA.9,10  2 The process of bioethanol generation generally involves the release (depolymerization) of sugar components from the biomass via chemical or biochemical treatment (e.g. enzyme treatments) prior to fermentation. The overall process of bioethanol generation from simple sugar and starch based crops, particularly the depolymerization process, is more cost-effective and less complicated than it is for lignocellulose (fibrous plant) sources. This is attributed to the relatively complex nature of fibrous plant sources comprised of lignin and hemicelluloses, which complicate cellulose isolation, despite lignocelluloses being far more abundant in nature than food crop sources.11,12 However, given the increasing cost of the food supply and the time required for land recovery and crop re-planting, the demand for bioethanol production from lignocellulosic biomass has gradually been increasing. With the continued improvements of available biochemical tools and techniques, bioethanol generation can be greatly accelerated while further lowering the cost of conversion of lignocellulosic biomass. Given the current advances in cellulosic ethanol generation, lignocellulosic bioethanol is showing a great promise as an alternative energy source.13  1.2 Lignocellulosic Biomass Conversion Process Lignocellulosic biomass comes from a variety of places such as forests, agriculture, and yard waste. Lignocellulose mainly consists of a heterogeneous complex of cellulose, hemicellulose and lignin (structures of each component are given in Section 1.2.1). The relative percentage of the three components depends highly on the source of lignocellulosic material (Figure 1.1(a), and Section 1.2.1).3  3  Figure 1.1 (a) Structure and composition of lignocelluloses: lignin, hemicellulose and cellulose.14 Figure adapted from reference 14. (b) Structure of a cellulose chain with an arrow indicating !-(1,4) glycosidic bonds, and the reducing/non-reducing terminus. (The reducing end of an oligosaccharide chain refers to the terminuses where the hemiacetal or hemiketal group present.  4 Cellulose is a linear carbohydrate polymer consisting of 1,4-!-D-glucosyl units linked (Figure 1.1(b)). It must first be isolated from the surrounding lignin and hemicellulose structures via a chemical or biochemical pre-treatment. The isolated cellulose is then degraded enzymatically. Finally, fermentation of the degraded cellulose products generates ethanol. Our collaborator, Iogen Corporation, has developed a lignocellulosic biomass conversion system with the following four procedures (Figure 1.2)(www.iogen.ca)15,16:  Figure 1.2 Overall production process of cellulosic ethanol conducted at Iogen Corp. (http://www.iogen.ca/).16 Figure adapeted from Iogen Corp. website: http://www.iogen.ca; reproduced with permission.  5 1. Pretreatment of biomass. In order to optimize the subsequent enzymatic degradation step, liberation of cellulose from complex hemicellulose and lignin is required. This process is usually conducted via chemical, biochemical, or physical treatments.17,18 2. Cellulase degradation. One of the key steps in the biomass conversion, during which cellulose is broken down into the monosaccharide glucose (also referred to as saccharification) via a cocktail of cellulase enzymes secreted from the filamentous fungus Trichoderma reesei (recently re-named as Hypocrea jecorina). The overall goal of this biomass project, within our group, is to design a tool to aid in solving the problems encountered in this step. 3. Fermentation. The major step of biomass conversion. The fermentation of glucose generates ethanol. It is usually carried out by yeasts or other microorganisms. 4. Distillation. This is the final stage of the biomass conversion process where ethanol is distilled and isolated for fuel grade application.  The following Sections will highlight in further detail the pretreatment (Step 1) and cellulose degradation processes (Step 2) as they are the most pertinent to the subject matter of this thesis.  1.2.1 Pretreatment: Removal of the Lignin and Hemicellulose Protecting Layer Lignin and hemicellulose interact with cellulose creating a protective shield that protects plants against microbial degradation and various pathogens. This extensive linking network provides mechanical strength and structural rigidity, while the structural complexity of the lignocelluloses makes enzymatic degradation challenging. Since cellulose is embedded inside the  6 protective shield formed by lignin and hemicelluloses (Figure 1.1), it has to be liberated from this protective material before cellulases can act efficiently. Hemicelluloses are shorter polysaccharide chains composed of different types of monomers and sugar derivatives, such as D-xylose, L-arabinose, and 4-O-methyl-D-glucuronic acid. They bind strongly with and cross-link the cellulose fibrils. Lignin is a non-carbohydrate- containing polyphenolic biopolymer (Figure 1.3) that is extensively cross-linked with itself and with other cell wall components such as hemicelluloses. It assists in forming the protective layer shielding the cellulosic material from biodegradation.19,20  Figure 1.3 (a) Chemical structure of lignin. Adapted from “Detailed Chemical Structure of a Portion of a Lignin Molecule” by Taiz, L. and Zeiger, E. 2010. 21(b) Partial structure of hemicellulose.   7 Pretreatment (Figure 1.4) is a process of breaking down the protective shield (i.e. removal of lignin and hemicellulose from cellulose) and reducing the crystallinity of the cellulose component.11,19 The pretreatment can be carried out in different ways, such as chemically or mechanically.17,18 It has been shown that the hydrolysis of crude lignocellulosic biomass without any form of pretreatment results in an extremely low yield of degraded cellulosic products; however, the high cost associated with removing the protecting layer is one of the main challenges for producing bioethanol in an economically sensible fashion. Although this is not the focus of our current project, improving the pretreatment technique is one of the focal points in cellulosic ethanol development.   Figure 1.4 Schematic of the pretreatment process on lignocellulosic material (adapted from reference 22).22     8 1.2.2 Cellulolytic System Used for Cellulose Degradation Since cellulose is a homogenous polymer consisting of a single type of glycosidic linkage, depolymerization should be fairly simple. However, the large diversity of lignocellulosic feedstock complicates the enzymatic degradation process. Moreover, cellulose can exist in different morphologies, which can further complicate depolymerization. Hence, different types of degradation systems are utilized to accommodate a variety of lignocellulosic biomass to achieve an efficient degradation of cellulose. Saccharification of cellulose in nature is primarily done by microorganisms such as fungi and bacteria.23 Some microorganisms are able to secrete large quantities of highly diverse extracellular glycosidases, creating a cellulolytic system to efficiently degrade the cellulose polymer. The filamentous fungus Trichoderma reesei has attracted much attention for its cellulolytic system and is utilized extensively in lignocellulosic biomass conversion.24,25 The T. reesei cellulolytic system is capable of secreting a large quantity of glycosidases and cellulases required for cellulose degradation, and has been shown to be exceptionally efficient in cellulosic glucose production. Additionally, there are no co-secreted fungal toxins or antibiotics associated with T. reesei enzyme production; therefore, it is popular among the food and pharmaceutical industries.26 More details of the T. reesei cellulolytic system used in the enzymatic breakdown of cellulose, and information on the four principal T. reesei cellulases, are given below and in Chapter 2.    9 1.2.2.1 Problems Associated with the T. reesei Cellulolytic System and a Potential Solution One of the major problems often encountered for bioethanol production incorporating enzymatic saccharification is the “die off” of cellulases over time. The “die off” refers to a marked decrease in efficiency of the degradation process over time, likely as the result of one of the enzyme components becoming rate limiting. However, the exact reason for the observed “die off” effect remains unclear.27 Identifying and quantitating the active/inactive cellulase components in the current system may be the key to optimizing the cellulolytic system, by identifying the deficient enzymes which lead to activity loss. Unfortunately, there currently remains a lack of tools to determine which enzyme(s) are “dying off”, since all of the enzymes carry out the same chemical reaction. We have proposed an “activity-based profiling” approach to identify the active enzyme species in the mixture based on the previous success of this approach within our group.28 In this approach, a set of ABPP probes (Section 1.4.2) will be introduced for quantitation and identification of the labelled enzymes, ideally one of which is specific for each individual enzyme. Presumably only active cellulases with an appropriate active site topology can accommodate our probes and will be labelled. Hence the active enzyme concentration could be determined by quantitation of the corresponding appended tag. More details about the ABPP probe design will be given below in Section 1.4.2.      10 1.3 Glycoside Hydrolases (Glycosidases) Carbohydrates and their conjugates, are a group of essential biological molecules that are ubiquitous throughout nature. Nature utilizes carbohydrates for energy storage, structural support (e.g. cell walls), cellular communication, etc. Furthermore, carbohydrate conjugates are involved in numerous biochemical processes.29 The glycosidic bond, which forms the backbone of polysaccharides, is a covalent bond formed at the anomeric carbon (the carbon where the hemiacetal or hemiketal is located) of one glycoside and a hydroxyl group of another glycoside counterpart (Figure 1.5). Glycosidic bonds are one of the most kinetically stable linkages between monomer units in nature. For instance, the glycosidic bonds within cellulose have a half life for spontaneous hydrolysis at room temperature in water estimated at around five million years.30,31  Figure 1.5 Diagram of a !-monosaccharide with glycosidic bond indicated with an arrow. The blue coded site is the anomeric center of a glycoside. An aglycone may or may not be a carbohydrate group.  Nature utilizes glycoside hydrolases to break down sugar macromolecules into simpler saccharides. Glycoside hydrolases are enzymes that catalyze the hydrolysis of glycosidic bonds. They can accelerate the reaction rate upwards of 1017 fold.32 Glycosidases catalyze the hydrolysis of a glycosidic bond with the consumption of a water molecule, resulting in the formation of an additional hydroxyl group and a hemiacetal group (Figure 1.6).  11  Figure 1.6  Hydrolysis catalyzed by a !-glycosidase.  1.3.1 Classification of Glucoside Hydrolases Given the high degree of complexity and diversity of glycans found in nature, the considerable number of carbohydrate active enzymes also found in nature should not be surprising. It was therefore necessary to establish some means of classification in regard to their functionality, sequence, structure, etc. To understand the cellulases involved in this current project, three classifications are introduced here: (1) according to the stereochemical outcome of the product (Section 1.3.2)33, (2) by the location where the enzyme acts on the substrate, either endo (in the middle) or exo (at the end) of the glycan chain (Figure 1.7(a)), or (3) by the primary sequence similarity of the enzymes.34 Each of these classification methods will be discussed individually below.  1.3.1.1 Classification by the Location of Cleavage: Exoglucanase and Endoglucanase  Endo and exo refers to the site where the glycoside hydrolases act. Endoglucanases hydrolyze the internal glycosidic bonds of polysaccharides, whereas exoglucanases remove one or more sugar units from the terminus of the polysaccharide chain (Figure 1.7(a)). This difference mainly results from the architecture of the substrate binding site at the catalytic center. Examples of exo and endo-glycosidases are cellobiohydrolase II (CBHII) and endoglucanase I (EGI), respectively. The exo-cellulase, CBHII, of T. reesei contains extended  12 loops that enclose the catalytic site, forming a tunnel 20 Å in depth for cellulose binding. This results in the hydrolysis occurring from the terminus of the polysaccharide chain.  On the other hand the endo-cellulase, EGI, lacks an enclosed loop active site and therefore has an open cleft- like active site structure (Figure 1.7(b)), which allows for a certain amount of flexibility for substrate binding. This added flexibility results in different sites of cleavage within the internal glycan chain, which provides an explanation for the random lengths of oligosaccharides released from EGI.35-37 Some studies have` also shown that a truncated version of the exo-cellulase, cellobiohydrolase I (CBHI) from T. reesei, which has a similar tunnel-like active site structure to that of CBHII, behaves partially like an endoglucanase when the extended loops enclosing the catalytic site are removed.38 The crystal structures of three of the T. reesei cellulases CBHI, CBHII and EGI have been determined and are referred to in Chapter 2, Section 2.1.   13  Figure 1.7 (a) The locations in cellulose where endoglucanase and exoglucanase act. (b) Van der Waals surface representations of the active-site tunnels (arrowed) in GH 6 CBH Cel 6A from Humicola  insolens (b.1), and endoglucanase E2 from Thermomonospora fusca (b.2).39 This research was originally published in Biochemical Journal. Varrot, A., Hastrup, S., Schulein, M. and Davies, G. Crystal structure of the catalytic core domain of the family 6 cellobiohydrolase II, Cel6A, from Humicola insolens, at 1.92 Å resolution. Biochemical Journal. 1999; 337: 297-304 © copyright holder.  1.3.1.2 Carbohydrate Active enZyme (CAZy) Classification Glycoside hydrolases are conventionally classified by the International Union of Biochemistry and Molecular Biology (IUBMB) system, which is based on substrate specificity, and sometimes the catalytic mechanism of the enzyme. However, this nomenclature does not  14 reveal information about the primary sequence. Furthermore, it does not provide any information about the structural properties of the enzyme.40 In 1991, Bernard Henrrissat41 devised a more sophisticated classification system, based on the amino acid sequence of the identified Carbohydrate Active enzymes (CAZy). The up-to- date CAZy system (http://www.cazy.org) contains approximately 300 CAZy families, including enzymes involved in carbohydrate degradation, synthesis and modification. The glycosidases have been classified into some 125 different families through the efforts of carbohydrate scientists from around the world.42 It has been observed that the products generated by glycosidases within the same family share the same stereochemistry, suggesting that these glycosidases proceed through the same catalytic mechanism32, and that they are also structurally very similar. This assumption is supported by crystal structures of glycosidases grouped within the same family, which display common folding patterns and conserved active site architecture. Therefore, the generality of the CAZy classification allows for a good prediction of enzyme function based on the primary sequence similarity. Under the CAZy classification the T. reesei cellulase families tested in this thesis belong to GH 5, 6, and 7. Each of these families will be discussed briefly below. GH 543: a large family containing around twenty five hundred members to date. Enzymes in this family are classified as retaining glycosidases and follow Koshland’s double displacement mechanism (Section 1.3.2.2). The two catalytic residues were both found to be glutamates. The 3D structure of the family members is a classical ("/!)8 TIM barrel fold. GH 644: a relatively small family with approximately 700 glycosidases, cleaves !-(1,4) glycosidic bonds in an inverting manner. The catalytic acid residues in this family are aspartates.  15 However, the identity of the catalytic general base, which deprotonates water, remains controversial (Figure 1.9). Only endoglucanases and cellobiohydrolases have been reported in this family. The tertiary structure of GH 6 glycosidases reveals modified "/! barrel folds. GH 745: This family contains approximately 1700 members. The enzymes within this family follow the classical double displacement mechanism. Unlike GH 6, the cellobiohydrolases in this family cleave glycosidic bonds from the reducing end of sugar polymers. The two catalytic residues were both found to be glutamates, and the enzymes in this family share a !-jellyroll fold.  1.3.2 Catalytic Mechanism of Glycosidases Koshland46 (1953) first proposed the principal mechanisms for the two major classes of glycosidases, inverting and retaining (Figure 1.8), based on the stereochemistry of the products. The inverting glycosidases follow a direct displacement mechanism and generate products with inverted stereochemistry at the anomeric center. On the other hand, retaining glycosidases generate products with conserved (retained) stereochemistry at the anomeric center via a double displacement mechanism. Both classes of enzymes in their archetypal forms utilize two catalytic carboxylic acid residues and proceed through oxocarbenium ion-like transition states. Although the majority of glycosidases are categorized into these two classic inverting/retaining mechanisms, many structural and mechanistic studies have also shown different catalytic mechanisms occurring in a few other glycosidases. Nonetheless, all cellulases discussed within this thesis adopt either the standard inverting or retaining mechanism. Enormous numbers of mechanistic and structural studies have led to the acceptance of the two postulated mechanisms  16 as being the primary mechanisms associated with glycosidases. The details of these inverting/retaining glycosidase mechanisms will be discussed below.   Figure 1.8 Products produced by retaining or inverting glycosidases.  1.3.2.1 Single Displacement (Inverting) The inverting glycosidase goes through a direct displacement mechanism (Figure 1.9). The catalytic residues are typically carboxylic acid functionalities and are generally separated by 6-12 Å.47-49 A water molecule bound in the active site is activated by a catalytic base, a carboxylic acid residue (general base catalysis), for nucleophilic attack at the anomeric center of the sugar subunit at the -1 site (See Section 1.3.3 for a discussion of -1/+1 subsite nomenclature). This nucleophilic attack directly displaces the leaving group, which is protonated by the other carboxylic acid residue (general acid catalysis). The hydrolysis goes through an oxocarbenium ion-like transition state where the anomeric center coordinates with both the nucleophilic water molecule and the protonated leaving group. Sufficient room is necessary to accommodate the catalytic water molecule and therefore results in a larger distance between the two catalytic residues when compared to retaining glycosidases (5 Å) (Section 1.3.2.2).  17  Figure 1.9 Mechanism of enzymatic reaction of inverting glycosidases.  1.3.2.2 Double Displacement (Retaining) In contrast with inverting glycosidases, retaining glycosidases undergo a double displacement mechanism (Figure 1.10) involving a covalent glycosyl-enzyme intermediate. Similar to the inverting mechanism, two carboxylic acid residues take part in the retaining hydrolysis mechanism. One of the catalytic carboxylic acid residues activates the leaving group by protonation (general acid catalysis), while the other catalytic carboxylate residue performs a nucleophilic attack at the anomeric center of the glycosyl unit in the -1 subsite. This is known as the glycosylation step and generates a covalently linked glycosyl-enzyme intermediate. The second displacement reaction occurs when a bound water molecule, activated by the catalytic base residue, performs nucleophilic attack at the anomeric center to release the sugar from the enzyme (known as the deglycosylation step). Both glycosylation and deglycosylation steps proceed through oxocarbenium ion-like transition states. The two catalytic residues are typically 5 Å apart, shorter than is seen for inverting enzymes.  18  Figure 1.10 Mechanism of enzymatic reaction of !-retaining glycosidases.  1.3.3 Glycoside Hydrolase Subsite Nomenclature Substrate binding plays an important role in enzymatic catalysis. For many types of enzymes, substrate binding modes can strongly affect the catalytic activity. Therefore, appropriate nomenclature for substrate binding sites is needed for comparisons between enzymes with catalytic similarity or mutants of the same enzyme. This thesis adopts the nomenclature system of sugar binding subsites (a binding site which accommodates one glycosyl unit in the catalytic domain of the glycosidase) in which50 sugar binding subsites are labeled –n or +n, where n is an integer, and +/- indicates the sites as  19 towards the reducing end/non reducing end (Figure 1.11) of substrates respectively. Cleavage occurs between the -1 and +1 subsites. An example given below is from one of our cellulases of interest, cellobiohydrolase I (CBHI) from T. reesei, which is known to release cellobiose as a product from the reducing end (Figure 1.11).  Figure 1.11  Nomenclature of sugar binding subsites in a glycosidase active site. The example given here is T. reesei cellobiohydrolase I. The reducing end is shown on the right and is designated by +’s, while the non-reducing end is on the left and denoted by –’s.  1.4 Methods of Measuring Active Enzyme Concentrations in a Protein Mixture   Protein identification and quantification have long been essential and important techniques in biological research, especially in the field of proteomics, which refers to the identification, understanding and characterization of the proteome (the expressed set of proteins that are encoded by a genome) in various organisms and/or in specific biological conditions.51 The routine method to identify and quantitate the species usually involves purification of that protein of interest from the mixture, followed by characterization of enzyme activity, which could be a tedious process and hence not a suitable solution in the problem addressed in Section 1.2.2.1.  We are aiming to design a tool to quantify and identify the species of active cellulases in the enzyme mixture. The methodologies which allow for this application include active site titration and, our main focus, activity based protein profiling (ABPP).  20 1.4.1 Active Site Titration Active site titration is a method for measuring the absolute concentration of active enzymes.52 Active site titration usually involves the formation and accumulation of covalently linked enzyme species with a reagent designed to target the active site, with release of a chromogenic group upon covalently binding with the enzyme. For accurate active enzyme concentration measurements, turnover of the covalently bound species should not occur during the time of measurement. If one equivalent of substrate reacts with one equivalent of enzyme, the total concentration of the active site can be determined by measuring the amount of chromogenic product released.53 Examples of active site titration agents are the aryl 2-deoxy-2-fluoro glycosides, a general class of mechanism-based inactivators for retaining glycosidases (more information on this class of compounds is in Chapter 2 Section 2.3.4) used to determine the concentration of retaining glycosidases.53 The rate of the deglycosylation step is essentially slowed in the hydrolysis of 2-fluoro substrate when catalyzed by retaining glycosidases so that the glycosyl- enzyme intermediate accumulates. The formation of the glycosyl-enzyme intermediate paired with the release of aryl moieties, such as 2,4-dinitrophenol allows for accurate quantification spectrophotometrically. The amount of aryl group released from the enzymatic reaction is equal to the amount of active glycosidases, assuming only one active site is present. Recently, fluorescent leaving groups with higher sensitivity have been incorporated into the design of active site titration agents.54 Fluorescent groups with low detection limits allow for the accurate measurement of active enzymes with low concentrations.  However, one of the problems with using active site titration methods for the determination of active cellulase concentration in biomass conversion is that the enzymatic  21 hydrolysis is performed in a complex dark colored mixture of pretreated biomass and T. reesei culture. The dark background color in the mixture limits the utility of fluorescent probes for accurate measurement of chromophore release with spectrophotometers.  1.4.2 Activity Based Protein Profiling (ABPP) Activity based protein profiling has been a useful tool to identify the specific classes of proteins in complex protein mixtures, especially in proteomics applications.55-59 The ABPP approach involves the use of chemical probes to label the active enzymes in a protein mixture. The class of ABPP chemical probes that targets specific classes of enzymes is referred to as directed ABPP. Directed ABPP probes contain three general features, (1) an irreversible inactivating group: an active site-directed inactivator (affinity label) or a mechanism-based inactivator, which forms a stable conjugate with a nucleophilic residue in the active site (Section 1.4.2.1) (2) a reporter group such as a fluorescent moiety for fast detection and quantification, and (3) a linker to prevent the reporter group (mentioned next) from sterically hindering binding of the inactivator (Figure 1.12).57,60,61 Since the irreversible inactivating group is either active site-directed or mechanism-based for the targeted enzymes, some mechanistic or structural information is required for the appropriate design of ABPP probes.  22  Figure 1.12 (a) Basic structure of an ABPP Probe and (b) potential ABPP probes for cellulases.  When an ABPP is directly applied to the enzyme mixture, the functional states of targeted enzymes can be detected relatively quickly. A general mechanism of directed probe binding involves first the binding of the substrate mimicking group to the active site of the enzyme and then the reaction of the reactive group with an adjacent nucleophilic residue. Once the covalent linkage is established, the covalently linked species can be analyzed via techniques such as gel electrophoresis, depending on the types of reporter used (Figure 1.13).  23   Figure 1.13 A general strategy for activity-based protein profiling. In-gel fluorescence analysis is employed in this example.  Activity-based protein profiling has been developed as a methodology to identify many types of glycosidases including exo and endoglucanases.62-70 In addition, many affinity labels/mechanism-based inactivators have already been developed for glycosidases over the years.71 These served as useful starting points for the design of the active site-directed inactivating component of ABPP probes for T. reesei cellulases.  For simplicity, these potential inactivators were initially synthesized on the smallest repeating unit, a cellobiose scaffold, but this can be extended to tri or tetrasaccharide versions where needed.  1.4.2.1 Irreversible Inactivators for Glycosidases Two key features are installed into the inactivating groups. The first is the natural substrate mimicking moiety (i.e. sugar moiety), which allows for efficient docking into the enzyme active site. The other feature is the chemically reactive group (Figure 1.14), which is essential for forming a covalent linkage with the enzyme active site. Chemically reactive groups which are able to form a stable conjugate with nucleophilic residues of glycosidases have served  24 as useful tools for catalytic residue identification.72 They are generally grouped into two categories: group-specific labels, and irreversible inactivators. Examples of group-specific labels for glycosidases are carbodiimide derivatives, such as 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide (EAC) and Woodward’s reagent K (Figure 1.14(a) and (b)), which have been used to identify some critical carboxylic groups at the active sites of xylanases and cellulases.72 However, group-specific labels do not necessarily discriminate between reactive residue located at the active site versus elsewhere in the protein, and therefore they are not applicable for ABPP design for the current project.  Figure 1.14 Examples of some group specific labels and irreversible inactivators. (a) Structures of EAC (b) Woodward’s reagent K (c) Cyclophellitol (d) 2-deoxy- 2-fluoro glucosides.  Irreversible inactivators are divided into active site-directed and mechanism-based. Active site-directed inactivators contain a reactive functional group such as an "-halocarbonyl, isothiocyanate or epoxide reactive group, which is able to form a stable conjugate with a nucleophilic residue in the active site. Glycosides carrying these functionalities tend to label active site histidines, carboxylic acids, cysteines and lysines. In most cases, the modified residue for retaining glycosidases turned out to be the catalytic nucleophile.71,72 In keeping with the aim  25 of our project (mainly for trapping the cellulases), it is not essential that the inactivators link with the catalytic residue; however, the inhibition of the enzymes has to be irreversible. On the other hand, a mechanism-based inactivator is a chemically stable molecule until activated by the catalytic reaction of the enzyme. A stable covalently linked species is formed upon activation by the enzyme’s catalytic activity. Examples of mechanism-based inactivators for glycosidases are cyclophellitol (Figure 1.14(c)) and activated fluorinated glycosides (Figure 1.14(d)).73-75 Both of these mechanism-based inactivators have been utilized extensively in mass spectrometric and crystallographic studies of many glycosidases. Cyclophellitol is an endocyclic epoxide structure mimicking the sugar ring and is therefore believed to bind the active site in a similar way to other substrates. The crystal structure of !-glucosidase from Thermotoga maritima determined with the cyclophellitol showed that the inactivator was bound to the catalytic nucleophile of the enzyme.76 Activated fluorinated glycosides such as 2-deoxy-2-fluoro and 5-fluoro derivatives, are a specific class of mechanism-based inactivators for retaining glycosidases. The inactivation is the result of the accumulation of stable glycosyl-enzyme intermediates followed by slow deglycosylation. Deglycosylation is often slow enough that the inactivation is apparently irreversible. More details for this class of inactivators will be given in Chapter 2 Section 2.3.4. Active site-directed and mechanism-based inactivators are only able to bind to active sites with a specific tertiary structure that allows for favorable docking. We are assuming that inactivator binding will only occur to active enzymes. Given this assumption, these inactivators would be valuable for the design of ABPP probes specific for cellulases. For the enzymatic  26 studies within this thesis, both active site-directed and mechanism-based inactivators were used to test the retaining cellulases.  1.4.2.2 Linker Arms and Reporter Groups A linker arm is required to minimize interference of the inactivation reaction by the tag. The linker is usually an alkyl chain incorporating some heteroatomic groups such as an amide or ether linkage to increase solubility. Some other features could also be employed in the linker for desired functionality. For example, a cleavable linker allows for the effective isolation of peptides.77 In addition, linkers containing either heavy or light isotopes can be utilized for MS analysis, especially if quantitative comparison of two enzyme samples is desired.78 The choice of reporter groups depends on the need. For instance, if purification of the targeted protein or peptide is the main objective, a biotin tag is usually incorporated for ease of purification by affinity chromatography.79 Alternatively, fluorophore tags are often utilized since they can be visualized on electrophoresis gel, providing fast estimations of target enzyme concentration by measuring the intensity of fluorescence. A list of commonly used linkers and tags is shown in Table 1.1.  27 Table 1.1 Common reporter groups and linkers used in ABPP probes79-81   1.4.3 A Method for Improving the Specificity of Individual T. reesei Cellulases Given the similar tertiary structures and catalytic activities of glycosidases within the same family, the probe design features discussed above may prove insufficient in differentiating such similar enzymes. Therefore, ABPP probes with more unique features are likely needed in order to differentiate glycosidases within the same enzyme family. Our approach for specific probe generation is to create a library of modified inactivators in which a common core structure is decorated with a range of substituents. The hope is that some may prove highly specific for one enzyme over another.  28 These chemical substituents are installed at a remote position (such as C4"!#$!C6" in a cellobioside inactivator) from the reactive group to ensure that the inactivation reaction is not affected.82  1.5 Aims of This Study Sound methodology is needed to efficiently assess and quantitate the actual active enzymes present during the biomass conversion process. These methods will ideally assist in identifying the species with diminishing activity responsible for the “dying off” of overall enzyme activity during the conversion process. This “dying off” phenomenon is a critical problem that compromises effective biofuel conversion. Therefore, a means to effectively measure individual active enzyme concentrations will be addressed within this thesis. Accurate concentration measurements of individual active cellulases in the enzyme mixture can be achieved via an activity-based probe specific to each enzyme component. Our ultimate goal is to generate at least one specific activity-based probe for each of the individual T. reesei cellulases involved in bio-ethanol production conducted at Iogen Corporation. We shall start out with the design and synthesis of a range of inactivators based upon a cellobiose- scaffold. The synthetic inactivators will be subjected to kinetic analysis with four T. reesei cellulases, and the most promising candidates will be identified based on kinetic results. We anticipate one or some of these synthetic inactivators behaving as universal inactivators for all four T. reesei cellulases. This universal inactivator(s) will be selected for elaboration with “specificity-determining” substituents which have been identified along with the discovery of a set of cellulase-specific substrates.  29 The chosen potential inactivator functionalities, located at the anomeric center, are: N- bromoacetyl, O-epoxyalkyl and C-epoxyalkyl reactive groups. Furthermore, 2-deoxy-2-fluoro !- cellobiosides and lactosides are also synthesized as potential inactivators to test the inactivation kinetics of the three retaining cellulases (Figure 1.15).   Figure 1.15 Structures of potential inactivators of cellulases synthesized in this work.          30 Chapter  2: Results and Discussion  2.1 Introduction to T. reesei Cellulases: Cellobiohydrolase I, Cellobiohydrolase II, Endoglucanase I and Endoglucanase II  The main enzyme components that function within the T. reesei cellulolytic machinery are Cellobiohydrolase I (Cel7A, CBHI), Cellobiohydrolase II (Cel6A, CBHII), Endoglucanase I (Cel7B, EGI) and Endoglucanase II (Cel5A, EGII). All of them have been characterized mechanistically, but only CBHI, CBHII, and EGI have been structurally characterized, though structures of homologues of EGII are available. Crystal structures of either the wild type or some mutant forms are available for CBHI, CBHII, and EGI.35-38,83-85 Table 2.1 lists some of the characteristic properties of the four cellulases.  Table 2.1 Properties of the four principal Trichoderma reesei cellulases. Property CBHI (Cel7A) CBHII (Cel6A) EGI (Cel7B) EGII (Cel5A) Family (CAZy)  GH7 GH6 GH7 GH5 Stereochemistry Retaining Inverting Retaining Retaining Endo/exo Exo  Exo  Endo Endo  (reducing end) (non-reducing end) Catalytic nucleophile Glu Asp Glu  Glu Catalytic acid/base Glu Asp Glu  Glu MW (kDa) 54 50 48 44      31 2.1.1 CBHI and CBHII Cellulases The most abundant cellulase produced by T. reesei is cellobiohydrolase I (CBHI, Cel 7A), a GH 7 glycosidase in the CAZy classification.45 It follows a classical double displacement mechanism. CBHI hydrolyzes cellulose from the reducing end, releasing cellobiose as the product. The crystal structure of CBHI reveals a large beta sandwich structure in the catalytic domain with dimensions of 60Å x 50Å x 40Å. The catalytic domain contains a long cellulose- binding tunnel (~50 Å) containing 10 glycosyl binding sites (+3~-7) (Figure 2.1).85  Figure 2.1  Schematic representation of the CBHI of T. reesei catalytic domain with cellulose bound. The sugar binding sites reveal extensive beta sheet structure.  In contrast to the retaining glycoside hydrolases, CBHII (Cel 6A), a family 6 hydrolase, carries out the hydrolysis of cellulose via an inverting mechanism44, and releases cellobiose from the non-reducing end of the cellulose.  The topology of the CBHII catalytic domain reveals a seven-stranded !-barrel, forming an incomplete TIM (triose phosphate isomerase) barrel. CBHII has a shorter binding ‘tunnel’ compared to CBHI with only four subsites (+2 ~ -2) (Figure 2.2).  32 The proposed +3 and +4 glycosyl binding subsites in Figure 2.2 have little interaction with the protein and do not significantly influence substrate binding.84 In addition, it was suggested that some interactions outside of the tunnel might facilitate the binding of cellulose.   Figure 2.2 Catalytic domain of T. reesei CBHII with a bound cellohexaose molecule. The numbers are the glycosyl binding sites. The cleavage occurs between +1/-1 sites.  CBHI and CBHII contain tunnel shaped active sites which complement the long polysaccharide substrates. Both catalytic residues (nucleophile and acid/base carboxylate) have been indentified in CBHI whereas only the acid catalytic residue has been determined in CBHII.35,37,86,87 The cellobiose subunits released by CBHI/CBHII are later hydrolyzed by !- glucosidases to generate glucoses.      33 2.1.2 EGI and EGII Cellulases T. reesei EGI (Cel 7B) makes up 5-10% of the cellulases36 in the cellulolytic mixture. EGI belongs to the same GH family as CBHI (GH 7), and has a catalytic domain which highly resembles CBHI.36 Some major differences between CBHI and EGI are in their active site structures (Figure 2.3), the site of substrate cleavage, and the products that they release. The loops that enclose the active site of CBHI are missing in EGI (Figure 2.3(b)), resulting in a cleft like substrate binding groove, which allows for flexible binding of cellulose. The resulting oligomers are then degraded by the cellobiohydrolases. Four glycosyl binding subsites (+2 to -2) are present in the catalytic domain of EGI.36   Figure 2.3 (a) Crystal structure of catalytic domain of T. reesei EGI.36 (b) The catalytic domain of CBHI. The green loops which enclose the glycosyl binding subsites are missing in EGI of T. reesei.38  EGII (Cel 5A), which was formally known as EGIII, belongs to family 5 glycoside hydrolases and follows a classical double displacement mechanism. EGII also generates a product with retained configuration at the anomeric center. The crystal structure of T. reesei  34 EGII has still not been determined. According to another GH 5 glycosidase, Thermoascus aurantiacus endoglucanase (Cel 5A) which has the catalytic domain structure with a (!/")8- barrel fold(Figure 2.4)88, the catalytic domain has a shallow but long substrate binding groove. Kinetic evidence has shown five substrate binding sites in the catalytic domain of T. reesei EGII, ranging from +2 to -3.89  Figure 2.4 Catalytic domain of Cel 5A of T. aurantiacus.  Synergistic hydrolysis by cellulases secreted from T. reesei allows for the efficient degradation of cellulose. Endoglucanases randomly attack the cellulose chain and produce oligosaccharides of various lengths. Cellobiohydrolases hydrolyze these oligosaccharides, producing cellobiose as the major product. The final step of this synergistic system is hydrolysis of cellobiose by !-glucosidases to yield two molecules of D-glucose (Figure 2.5).90-94   35  Figure 2.5 Synergistic action of endoglucanases, cellobiohydrolases, and !-glucosidase in the conversion of a celloheptaose into glucose. The dotted ovals indicate the configuration of the actual product released after hydrolysis by the cellulases.   2.2 Abg Glycosynthase in Carbohydrate Synthesis Classical chemical synthesis of oligosaccharides is a tedious process, which generally involves a considerable number of protection and deprotection steps. Furthermore, careful control over the stereochemistry and regiochemistry of the sugar product is also challenging. Another problem usually encountered is the difficult purification of multiple compounds with similar chemical properties (such as mixtures of anomers). Chemoenzymatic synthesis, which is a means of glycosidic bond formation facilitated by specific enzymes, could avoid the above  36 mentioned problems and thereby has become a popular alternative for oligosaccharide synthesis.95-101 A glycosynthase is a specific retaining glycosidase mutant which is able to catalyze the formation of a glycosidic bond between an activated glycosyl donor and a suitable acceptor (Figure 2.6). In a glycosynthase, the catalytic nucleophile is replaced with a non-nucleophilic residue, and hence no glycosyl-enzyme intermediate can form102, resulting in an enzyme with no capacity for glycoside hydrolysis. However, the usage of an activated glycosyl donor of inverted stereochemistry at the anomeric center could compensate for the deficient catalytic nucleophile. Within the glycosynthase active site, the acid/base catalytic residue deprotonates the appropriate hydroxyl group of the glycosyl acceptor, and this hydroxyl acts as a nucleophile to attack the activated glycosyl donor in a concerted fashion, promoting the formation of the glycosidic bond (the proposed mechanism of a glycosynthase shown in Figure 2.6). Furthermore, the absence of the catalytic nucleophile prevents the the newly formed oligosaccharides from being hydrolyzed by the same enzyme.   Figure 2.6 Proposed glycosynthase reaction catalyzed by a nucleophile mutant of a specific glucosidase. (D) Glycosyl donor: a glycoside bearing a substituent yielding an electron deficient anomeric center (e.g. "-glycosyl fluoride) usually with the configuration at the anomeric center opposite to the newly forming glycosidic bond. (A) Acceptor: a molecule providing a nucleophilic group for attack at the anomeric center of the glycosyl donor.  37 An Agrobacterium sp !-glucosidase mutant, also known as Abg glycosynthase, has been studied and utilized broadly to facilitate the formation of !-1,4 glycosidic linkages103,104 with a variety of donors and acceptors.102-106 Abg glycosynthase was employed extensively in this work to synthesize cellobioside inactivators and 2,4-dinitrophenyl trisaccharides. Enzymatic coupling avoids the laborious protection/deprotection steps and the newly formed glycosidic bond is solely ! configured.  2.3 Synthesis of Cellobioside Inactivators and 2,4-Dinitrophenyl (2,4-DNP) Substrates 2.3.1 Synthesis of N-Bromoacetyl !-Cellobiosylamine (2.1) One example of an active site-directed inactivator is an N-bromoacetyl glycoside. Such inactivators have been used widely as affinity labels to identify a residue which might be important for enzymatic catalysis or substrate binding.107-113 The inactivation is believed to be caused by the formation of a covalent linkage via an SN2 reaction involving a nucleophilic residue in the enzyme active site and the bromide activated " carbon (Figure 2.7).  Figure 2.7 Potential inactivation of cellulases with N-bromoacetyl !-cellobiosylamine.   38 The synthesis of N-bromoacetyl !-cellobiosylamine (2.1) performed by Black et. al.111 is a simple two step procedure involving the formation of a cellobiosylamine/cellobiose mixture, followed by coupling of the amine with bromoacetic anhydride. The advantage of this synthetic route was that it does not require separation of the cellobiose and cellobiosylamine mixture, and no final deprotection step was needed (Figure 2.8(a)). However, my attempts to couple with bromoacetic anhydride were unsuccessful, even when pure cellobiosylamine was used as starting material. Instead of investigating this route further, an alternative synthetic route which involved the reaction of bromoacetic anhydride with per-O-acetylated cellobiosylamine was performed. Per-O-acetylated "-cellobiose was converted to the "-glycosyl bromide (2.13) upon treatment with hydrogen bromide. Compound 2.13 was then used as an electrophile to couple with an azide anion via a nucleophilic reaction, forming compound 2.14. Hydrogenation of the azido moiety with hydrogen gas, catalyzed by Pd/C, afforded a glycosylamine (2.15). Compound 2.15 was then coupled with bromoacetic anhydride to successfully yield per-O-acetylated N-bromoacetyl !-cellobiosylamine (2.16). Deacetylation was performed in sodium methoxide solution in less than one hour (Figure 2.8(b)).   39  Figure 2.8 Synthesis of N-bromoacetyl !-cellobiosylamine (2.1). (a) Synthetic route performed by Black et. al. (b) i) HBr/AcOH (33%), DCM, trace acetic anhydride 73 %; ii) NaN3, DMF, 80 %; iii) Pd/C, H2(g), MeOH, 62%; iv) bromoacetic anhydride, DMF, 79%; v) MeOH, cat. NaOMe, 58%.  2.3.2 Synthesis of O-Epoxyalkyl !-Cellobiosides (2.2, 2.3, and 2.4) Another class of active site-directed inactivators commonly seen for glycosidase labeling in the literature were epoxide-based glycoside inactivators. Irreversible affinity labels containing epoxide or aziridine functionalities (Figure 2.9(a)) have been used as a popular tool to probe the catalytic residues in many retaining glycosidases, including T. reesei CBHI and EGII.114-122  40 The epoxide moiety can react to form a covalent linkage with the enzyme via protonation of the epoxide oxygen by an acid/base residue followed by an attack from a nucleophilic residue (Figure 2.9(b)). The labeled residue may or may not be the amino acid critical for catalysis reaction owing to the mobility of the alkyl chain which distanced the reactive epoxy group from the sugar moiety. 116 It was also possible that other nucleophilic residues throughout the enzyme may attack the epoxide moiety.  Figure 2.9 (a) Structures of aziridine-based and epoxy-based inactivators. (b)Potential inactivation reaction occurs in cellulases with an O-epoxyalkyl !-cellobioside.  Three O-epoxyalkyl !-cellobiosides, 2.2, 2.3 and 2.4 (Figure 2.10) were synthesized and tested with the four T. reesei cellulases.  The conventional strategy of  synthesis of 1,2 trans O- glycosidic compounds employed the Koenigs/Knorr or similar reactions, which involves glycosidic bond formation between an "-glycosyl halide and an alcohol in the presence of a soft Lewis acid such as silver(I) carbonate (Koenigs/Knorr reaction)123, or mercury(II) salt (Helferich reaction).124 However, these reactions usually generated large quantities of toxic heavy metal  41 wastes when performed on a large scale. As a result, an alternative route was chosen for synthesizing the O-epoxyalkyl !-cellobiosides.   Figure 2.10 O-Epoxyalkyl !-cellobiosides 2.2, 2.3, and 2.4. The three epoxy incorporating cellobiosides differ in alkyl chain length.  Glycoside synthesis with another widely used precursor, the trichloroacetimidate (TCA) offered a less toxic, less expensive, and straightforward approach.125 Utilization of a glycosyl imidate as an electrophile for the formation of the glycosidic linkage was first proposed by Sinay125 but was widely extended later by Schmidt for a range of glycoside syntheses.126,127 Versatility of the configuration at the anomeric center could be achieved by tuning either the configuration of the TCA group or the strength of the promoter used in the alcohol coupling step.128-130 The !-anomer usually forms preferentially when a participating group (acyl group) is present at the C2 position. The synthetic route to 2.2 (EPO3C), 2.3 (EPO4C), and 2.4 (EPO5C), started with per-O-acetylated cellobiose. The anomeric center was selectively deacetylated with hydrazine acetate, forming a hemiacetal (2.17), followed by reaction with trichloroacetonitrile and 1,8-Diazabicycloundec-7-ene (DBU) to form the "-anomer of the trichloroacetimidate (2.18) (Figure 2.11). However, due to the contribution of the C2 participating group, it was not necessary to start with "-trichloroacetimidates to achieve the ! configuration of the product (R,S)-O-epoxypentyl !-cellobioside  (2.4, EPO5C) (R,S)-O-epoxybutyl !-cellobioside     (2.3, EPO4C) (R,S)-O-epoxypropyl !-cellobioside   (2.2, EPO3C)  42 (2.19-2.21). The next coupling step was promoted by boron trifluoride diethyl etherate. Three compounds (2.19, 2.20 and 2.21) were obtained using similar coupling reactions by varying the coupling alcohols. The major side reaction in this approach was the hydrolysis of the TCA group by trace amounts of moisture. Only ~ 50% of TCA sugars were successfully transferred with alcohol acceptors (50% had been hydrolyzed).    Figure 2.11 Synthesis of (R,S)-O-epoxypropyl !-cellobioside (2.2), (R,S)-O-epoxybutyl !- cellobioside (2.3), and (R,S)-O-epoxypentyl !-cellobioside (2.4). i) Hydrazine acetate, DMF, 75%; ii) Cl3CCN, DBU, DCM, 71% iii) boron trifluoride diethyl etherate, DCM, molecular sieves, allyl alcohol (n=1), 40%, 3-buten-1- ol (n=2), 47%, 4-penten-1-nol (n=3), 50%; iv) mCPBA, DCM, (n=1), 52%, (n=2), 75%, (n=3), 30%; v) MeOH, cat. NaOMe (n=1), 90%, (n=2), 90%, (n=3), 90%.  Epoxidation of alkenes using meta-chloroperoxybenzoic acid (mCPBA) afforded a mixture of R and S diastereomers in moderate yields (2.22, 2.23, and 2.24), as reported  43 previously131,132, but the relative ratio of two diastereomers could not be determined from the 1H NMR spectra due to extensive signal overlap. The final deprotection was performed in sodium methoxide solution (concentration of sodium methoxide was less than 75 mM). If the concentration of sodium methoxide solution was too high (over 75 mM), the epoxy moiety was opened up by a methoxide. The mixture of diastereomers (2.2 EPO3C, 2.3 EPO4C, and 2.4 EPO5C) was used in kinetic characterizations of the cellulases without any attempt to separate the diastereomers.  2.3.3 Synthesis of C-Epoxyalkyl !-Cellobiosides (2.5 and 2.6) Another group of inactivators commonly used in mechanistic and structural studies of glycosidases is that of the C-linked epoxyalkyl glycosides. This class of inactivators containing a non-hydrolyzable C-glycosidic linkage should be able to overcome the possible problem of glycosidic bond hydrolysis present for O-linked glycosides during the labeling.115,122,133 Technically compounds 2.5 and 2.6 were not actual glycosides, but for simplicity all of the tetrahydropyrans listed here are referred as “C-linked glycosides”.   Figure 2.12 C-Epoxyalkyl !-cellobiosides 2.5 (EPC4C) and 2.6 (EPC5C). The two epoxy incorporating cellobiosides differ in alkyl chain length.  (R,S)-C-epoxypentyl !-cellobioside (2.6, EPC5C) (R,S)-C-epoxybutyl !-cellobioside    (2.5, EPC4C)  44 The synthesis of C-linked glycosyl epoxides (Figure 2.12) has been studied widely, and considerable research has been done on stereospecific synthesis of C-linked glycosides. Some former approaches to C-linked glycoside syntheses included the employment of the Grignard reaction, using organometallic carbanions and per-O-acetylated glycosyl bromide133, or utilization of alkyltrimethylsilane reacting with per-O-acetylated glycosides.134-136 The limitations of the above methods come from the incompatibility of base labile acetyl groups with the harsh conditions of the Grignard reaction (former) and from the low yield of !-anomer products in the alkyltrimethylsilane reaction (latter). I attempted to synthesize the target C-linked glycosides using allyltrialkylsilanes owing to the advantages of simplicity and economy (Figure 2.13(a)), regardless of the poor yields reported in a previous paper.134 The dominant product turned out to be the "-anomer (~4:1 "/! ratio estimated by TLC), and further recrystallization afforded the pure "-anomers with the mother liquor containing an inseparable mixture of both "- and !-anomers. A similar reaction was also conducted with commercially available per-O-acetyl "-cellobiose and allyltrimethylsilane but the reaction yielded three different products and none of them was the desired compound (Figure 2.13(b)).  Figure 2.13 Attempted synthesis of (a) O-tetraacetyl-C-allyl !-glucoside and (b) O- tetraacetyl-C-allyl !-cellobioside.  45  Stick and his research group carried out numerous efforts towards the synthesis of ! configured C-glycosides. He employed carbon nucleophiles to react with 2,3,4,6-tetra-O-benzyl- D-gluconolactones (2.27) as a means of establishing a linkage to C1.137,138 In this work, lactone substrates were prepared as outlined in Figure 2.14. The resultant crude hemiketal 2.28/2.29 was reduced to the C-glycoside 2.30/2.31 using triethylsilane and boron trifluoride diethyl etherate. At this stage the configuration at C1 was still not obvious for 2.30/2.31, but the  1H NMR spectra indicated that a single anomer had formed, similar to the results reported previously.138 The product (2.30/2.31) was then debenzylated via a Birch reduction with liquid ammonia and sodium to yield deprotected C-glucosides 2.32/2.33.     46  Figure 2.14 Synthesis of (R,S)-C-epoxybutyl !-cellobioside (2.5) and (R,S)-C-epoxypentyl !-cellobioside (2.6). i) BnBr, NaH, DMF, 86%; ii) 1 M H2SO4, AcOH, 54%; iii) DMSO, Ac2O, 78%; iv) 1-butenylmagnesium bromide (n=2) or 1- pentenylmagnesium bromide (n=3), THF; v) triethylsilane, BF3OEt2, CH3CN, (n=2), 67%, (n=3), 69% yield over two steps;  vi); NH3, THF, Na(s) (n=2), 71%,  (n=3), 66%; vii) "-D-glcF, Abg 2F6 glycosynthase, ammonium acetate, pH 7.0, 100 mM; viii) Ac2O, pyridine, (n=2), 13%, (n=3), 16% yield over two steps; ix) mCPBA, DCM, (n=2), 87%, (n=3), 75%; x) MeOH, cat. NaOMe (n=2), 76%, (n=3), 82%.  Chemoenzymatic glycosylation of the allyl C-glycosides 2.32/2.33 using Abg glycosynthase afforded the saccharides with different degrees of polymerization (di, tri and tetrasaccharides were obtained).139 The reaction mixture was concentrated directly and then acetylated, followed by purification by flash column chromatography. 1H NMR showed that the  47 pure per-O-acetylated C-linked cellobioside 2.34/2.35 had a ! configuration at C1, which agreed with the previous paper.138 Epoxidation and deacetylation was conducted using mCPBA as the oxidant to yield the mixture of diastereomers 2.36/2.37 with R and S epoxide diastereomers, followed by Zemplén deacetylation in sodium methoxide solution at room temperature. Final purification of the deprotected product 2.5/2.6 (EPC4C/EPC5C) was performed via a Waters Sep-Pak tC18 reverse phase cartridge.  2.3.4 Synthesis of 2,4-Dinitrophenyl 2-deoxy-2-fluoro !-cellobioside (2FDNPC, 2.7) and 2,4-Dinitrophenyl 2-deoxy-2-fluoro !-lactoside (2FDNPL, 2.8) 2-Deoxy-2-fluoro sugars are a class of mechanism-based inactivators used in labeling !- retaining glycosidases adopting the classical double displacement mechanism.73,75 The inactivation is the result of accumulation of a stable glycosyl-enzyme intermediate followed by slow deglycosylation. The deglycosylation step is slow enough that the inactivation is often apparently irreversible. The presence of the fluorine group at C2 substantially destabilizes the oxocarbenium ion-like transition states in both the glycosylation and deglycosylation steps.140-143 The destabilization effect comes from the fact that the replacement of the hydroxyl group by fluorine group disrupts the hydrogen bonding interaction between C2 hydroxyl group and the glycone binding site, which might be a key interaction stabilizing the transition state. Furthermore, the fluorine substituent also destabilizes the transition state by an inductive effect. The fluorine has higher electronegativity than a hydroxyl so the 2-fluoro oxocarbenium ion-like transition state is more unstable than that without the fluorine, resulting in reduction of rate in both steps. Incorporation of a reactive leaving group such as a 2,4-dinitrophenyl group or fluoride into a 2-fluoro glycoside at the anomeric center accelerates the glycosylation step, but  48 does not affect the rate of deglycosylation, causing the glycosyl-enzyme intermediate to accumulate. Therefore the compounds behave as essentially irreversible inactivators, although the glycosyl-enzyme intermediate can still be turned over slowly. However, one of the major limitations is that they can only be used for retaining !-glycosidases which follow the classical double displacement mechanism. Herein, 2,4-dinitrophenyl 2-deoxy-2-fluoro !-cellobioside (2FDNPC, 2.7) and 2,4-dinitrophenyl 2-deoxy-2-fluoro !-lactoside (2FDNPL, 2.8) have been synthesized and tested with three of the T. reesei retaining cellulases CBHI, EGI and EGII. Introduction of the fluorine at the C2 position of a glucoside could be accomplished by a nucleophilic glycal reacting with a source of electrophilic fluorine, such as molecular fluorine, acetyl hypofluorite or trifluoromethyl hypofluorite.144-146  Early synthesis of 2FDNPC performed by McCarter utilized acetyl hypofluorite to fluorinate the C2 position of O-acetylated glycal, followed by the coupling of a dinitrophenyl group to C1.145 However, the toxic and explosive hazards of hypofluorite limited large scale synthesis of target compounds. SelectfluorTM (1- chloromethyl-4-fluoro-1,4-diazonia-bicyclo[2,2,2]octane bis(tetrafluoroborate)), an alternative fluorine source, was preferred owing to its ease of handling and low toxicity.140,147-149  49  Figure 2.15 Synthesis of 2,4-dinitrophenyl 2-deoxy-2-fluoro !-cellobioside (2.7) and 2,4 dinitrophenyl 2-deoxy-2-fluoro !-lactoside (2.8). i) SelectfluorTM, CH3NO3/H2O (v/v= 5/1), 54%; ii) DNFB, DABCO, DMF, 22%; iii) AcCl, MeOH, 81%; iv) "-D-glcF, Abg 2F6 glycosynthase, sodium phosphate buffer pH 7.0, 100 mM, 13%; v) "-D-galF, Abg 2F6 glycosynthase, sodium phosphate buffer pH 7.0, 100 mM, 71%.  The synthesis of 2FDNPC (2.7) and 2FDNPL (2.8) was achieved by a modified synthetic route performed by Vocadlo140, as shown in Figure 2.15. We started with the installation of fluorine at the C2 position of a tri-O-acetylated glucal. Fluorination of a monosaccharide bypassed any potential problems that may occur during the separation of diastereomers generated from fluorinating per-O-acetylated cellobioside-based glycal (disaccharide) later on in the synthesis.140 Fluorination of 3,4,6-tri-O-acetyl D-glucal at C2 was carried out using SelectfluorTM as the fluorine source, affording an inseparable mixture of 3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-D- glucose (2.38) 3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-D-mannose (2.39), and a trace amount of  50 3,4,6-tri-O-acetyl-2-deoxy-2-fluoro "-glucosyl/mannosyl fluoride (2.40), as reported in a previous paper .140,149 As shown in a previous paper147-149, the solvent system played an important role in the coupling reaction with SelectfluorTM.  Acetonitrile and H2O (5:1 ratio) resulted in a mixture of 2- deoxy-2-fluoro-mannosyl (2.39) and glucosyl hemiacetals (2.38) (in approximately a 3:2 ratio), whereas pure acetonitrile afforded the mannosyl derivative 2.39 as the major product.  For this reason, a 5:1 ratio of acetonitrile:H2O, was used as a solvent system for the synthesis of compound 2.38 resulting in a 2:3 mixture of 2.38 : 2.39. To the mixture of 2.38 and 2.39 was added 1-fluoro-2,4-dinitrobenzen (DNFB) in the presence of 1,4-diazabicyclo[2,2,2]octane (DABCO) in DMF, to install the dinitrophenyl group at the anomeric center, yielding two diastereomeric products (gluco/manno). At this stage these two diasteromers showed a distinct difference in polarity and were readily separated via flash column chromatography. The assigned gluco configuration of 2.41 was supported by the 1H NMR coupling constant J1,2 (= ~9 Hz). The resulting glycoside 2.41 was deacetylated in MeOH containing 4% (v/v) acetyl chloride, yielding 2.42. This then served as the glycosyl acceptor in the following chemoenzymatic couplings with "-glucosyl fluoride or "-galactosyl fluoride to afford the final products 2.7 (2FDNPC) and 2.8 (2FDNPL). The percentage yields of 2.7 (2FDNPC) and 2.8 (2FDNPL) were comparable to previously reported yields.102 The benefit of introducing the dinitrophenyl group in 2.38 prior to chemoenzymatic glycosyl coupling, was that the hydrophobic interaction from the aryl group promoted binding in Abg glycosynthase.150 In addition, both syntheses of 2.7 (2FDNPC) and 2.8 (2FDNPL) could be  51 done in parallel with the common acceptor 2.42. The use of Abg glycosynthase also avoided tedious protecting/deprotecting steps for this synthetic route.  2.3.5 Synthesis of 2,4-Dinitrophenyl Trisaccharides (2,4-DNP Trisaccharides) Extension of one sugar unit on the potential inactivator could alter the binding affinity of that inactivator to the glycosideases. Herein, Abg glycosynthase was utilized to chemoenzymatically synthesize trisaccharide substrates. Previous papers have shown an aryl cellobioside was an excellent acceptor for enzymatic coupling.100,103,106 Four 2,4-DNP trisaccharides with various non-reducing end sugars (D-galactose, L-arabinose, D-fucose or D- xylose) could be made in parallel by using 2,4-dinitrophenyl !-cellobioside (2,4-DNPC) as the acceptor and corresponding "-glycosyl fluorides as the donors.99 The reactions with "-D- galactosyl ("-galF), !-L-arabinosyl (!-arabF), and "-D-fucosyl fluorides ("-fucF), as glycosyl donors, afforded a single coupling product in each case. Subsequent additions were not observed owing to the lack of an equatorial hydroxyl group at the C4"" position of the trisaccharide product (Figure 2.16). Salt from the buffer and the hydrolyzed donor could be easily removed from the crude reaction mixture using a Waters Sep-Pak tC18 reverse phase cartridge. In contrast, reactions with "-D-xylosyl fluoride ("-xylF) resulted in di, tri, and tetrasaccharide products. Fortunately these products could be purified by silica column chromatography with an appropriate eluent (the lower polarity of the compounds was attributed to the hydrophobic dinitrophenyl group). The yield of desired trisaccharide was low (~ 30%) compared to other trisaccharide substrates.   52  Compounds Other name Group R1 R2 R3 Yield (%) 2.43 "-(OAc)4GalF D-galactosyl OAc H CH2OAc 51 2.44 !-(OAc)3ArabF L-arabinosyl OAc H H 60 2.45 "-(OAc)3FucF D-fucosyl OAc H CH3 87 2.46 "-GalF D-galactosyl OH H CH2OH 82 2.47 !-ArabF L-arabinosyl OH H H 69 2.48 "-FucF D-fucosyl OH H CH3 73 2.49 "-XylF D-xylosyl H OH H N/A 2.9 D-galactosylDNPC D-galactosyl OH H CH2OH 60 2.10 L-arabinosylDNPC L-arabinosyl OH H H 71 2.11 D-fucosylDNPC D-fucosyl OH H CH3 51 2.12 D-xylosylDNPC D-xylosyl H OH H 31  Figure 2.16  Syntheses of 2,4-dinitrophenyl trisaccharides 2.9, 2.10, 2.11 and 2.12. i) HF/pyridine; ii) NH3, MeOH iii) "-glycosyl fluorides (refer to above Table), Abg 2F6 glycosynthase, sodium phosphate buffer pH 7.0, 100 mM.        53 2.4 Kinetic Evaluations of Cellobiose-based Inactivators and Substrates of Cellulases 2.4.1 2,4-Dinitrophenyl !-cellobioside as a Standard Substrate A universal substrate that was able to assay activity for each individual cellulase was necessary to evaluate inactivators. In addition, this substrate can serve as a parent compound to couple with “specificity-determining” substituents, and the enzyme specificity of these groups can be readily evaluated (Section 1.4.3). The initial choices for the standard substrate candidates were p-nitrophenyl !-cellobioside (pNPC), 2,4-dinitrophenyl !-cellobioside (2,4-DNPC), and 3,4-dinitrophenyl !-cellobioside (3,4-DNPC) (Figure 2.17), all of which have been used to study the kinetics of cellulases.151-153 The high molar extinction coefficients of dinitrophenolates and nitrophenolates in the visible light region (Table 2.2) allow hydrolysis to be followed using a UV-Vis spectrophotometer.   Figure 2.17 Chemical structures of three standard substrate candidates: pNPC, 2,4- DNPC and 3,4-DNPC.    54 Table 2.2 Physical properties of, p-nitrophenolate, 2,4-dinitrophenolate and 3,4- dinitrophenolate. Leaving group pKa   (of neutral form) ! (M -1cm-1)(I) pNP 7.15 18000154 2,4-DNP 3.96 12000(II) 3,4-DNP 5.36 5040153 (I) Extinction coefficient ! of absorbance at 400 nm. (II) The value was provided by Dr. Jacqueline Wicki (pH 5.0).  Kinetics parameters for hydrolysis of pNPC and 2,4-DNPC by cellulases from T. reesei were conducted by previous group members Stephenie Leung and Kah-Yee Li 82,155 respectively. Due to possible hydrolysis of 2,4 dinitrophenyl group during the modification of C4"/C6"!with some chemical groups (for improvement of enzyme specificity, see Section 1.4.3), 3,4-DNPC might be a better substrate for this study owing to its higher stability. Therefore, 3,4-DNPC was also tested. The kinetic characterization of 3,4-DNPC is described in this thesis. Incubation of the substrate with the cellulase resulted in a continuous release of 3,4-dinitrophenolate and the initial rate of hydrolysis was determined using the change in absorbance at 400 nm, and the experiment was duplicated three times to minimize the standard error. Michaelis–Menten kinetic parameters (kcat , Km, see Appendix C.1) were determined and are summarized together with the parameters for pNPC and 2,4-DNPC in Table 2.3.     55 Table 2.3 Kinetic parameters for hydrolysis of three !-aryl cellobioside substrates. Systematic errors were determined by GraFit 5.0.13.  CBHI CBHII Substrate Km (µM) kcat (sec-1) kcat/ Km x 10-3 (µM-1sec-1) Km x 10-3 (µM) kcat (sec-1) kcat/ Km x 10-3 (µM-1sec -1) pNPC(I) 13 0.002 0.15 1.43 0.001 0.0007 2,4-DNPC(II) 31 0.032 1.0 1.2 0.45 0.37 3,4-DNPC 20±2 0.013±0.001 0.65±0.10 1.8±0.3 0.0040±0.0001 0.0022±0.0010  EGI EGII Substrate Km x 10-3 (µM) kcat (sec-1) kcat/ Km x 10-3 (µM-1sec-1) Km x 10-3 (µM) kcat (sec-1) kcat/ Km x 10-3 (µM-1sec-1) pNPC(I) 3.25 1.16 0.36 3.81 0.06 0.0015 2,4-DNPC(II) 1.8 38 21 1.5 3.2 2.4 3,4-DNPC 2.4±0.2 1.9±0.1 0.80±0.11 7.2±0.8 0.27±0.01 0.038±0.006 (I)  Data determined by former honour thesis student Stephenie Leung.155 (II) Data determined by former exchange student Kah-Yee Li.82   The Michaelis constant, Km did not vary much between the three substrates with CBHII or EGI. This suggested that the variation in the aryl group did not have a significant effect on the substrate binding to these two enzymes. With EGII, 2,4-DNPC showed slightly better binding affinity than 3,4-DNPC (~5 fold tighter) and pNPC (~2.5 fold tighter). In contrast to the other three enzymes, for which the 2,4-DNPC was the tightest binding substrate, the binding affinity of 2,4-DNPC to CBHI was slightly weaker than that of pNPC or 3,4-DNPC. The first order rate constant kcat varied dramatically with respect to the different substrates.  The kcat values of 2,4- DNPC with the three retaining cellulases (CBHI, EGI and EGII), which operate via a classical double displacement mechanism, were much higher than the kcat values for 3,4-DNPC and pNPC. This implied that the glycosylation step was rate limiting (the rate of which was dependent on the pKa of the leaving group). The first order rate constant kcat should be similar for each substrate if  56 deglycosylation step was the rate determining step, since the glycosyl-enzyme intermediate is the same in all three cases.  The catalytic efficiencies of each enzyme/substrate pair tested here could be compared in terms of their specificity constants (kcat/Km). All four cellulases showed the greatest activity for 2,4-DNPC compared to 3,4-DNPC and pNPC. The apparent second order rate constant (kcat/Km) of 2,4-DNPC with CBHI was 1.5 times and 7 times higher than 3,4-DNPC and pNPC respectively. kcat/Km values also differed considerably between CBHII, EGI and EGII. (e.g. EGII hydrolyzes 2,4-DNPC 1600 times more effectively than pNPC!).  The only products released in the hydrolytic reactions with CBHI, CBHII and EGII were cellobiose and dinitrophenolate during the time of kinetic measurement (Figure 2.18(a)), suggesting there were no side reactions; whereas release of a UV active product with the same Rf as 2,4-dinitrophenyl !-glucoside (2,4-DNPG) was detected with EGI (this phenomenon is discussed later in the same section). However, TLC analysis revealed that direct hydrolysis of the dinitrophenyl group was kinetically favored (i.e the formation of cellobiose was faster than that of glucose/DNPG, Figure 2.18(b)). Therefore, the presence of this side reaction should not severely affect the accuracy of the recorded initial rates of hydrolysis.  57  Figure 2.18 TLC of hydrolytic reactions of 2,4-DNPC with T. reesei cellulases. (a) Samples were taken after 5 minutes of enzyme addition. SM: 2,4-DNPC; lane 2: reaction mixture with CBHI; lane 3: reaction mixture with CBHII; lane 4: reaction mixture with EGI; lane 5: reaction mixture with EGII. (b) Hydrolytic reaction of 2,4-DNPC with EGI. Three sample points were taken during the reaction time between 30 seconds and 2 minutes. Three sample points were also taken during the reaction time between 10 to 30 minutes. Red circles highlight that the major product released during 30 seconds to 2 minutes was cellobiose.  It is worth noting that kcat/Km is not affected by any alternative binding of substrate (Appendix C.4.1). kcat/Km values obtained here therefore are reliable measures of the catalytic efficiency. There were two possibilities for how 2,4-DNPG was released. First, internal glycosidic bond cleavage might occur due to different binding modes in which the dinitrophenyl group binds to the +2 subsite, resulting in the cleavage of the internal glycosidic bond. This alternative cleavage in which the glycosidic bond hydrolysis occurrs between two sugar units (Appendix C.4) has also been observed in the hydrolysis of 4-methylumbelliferyl !-glycosides of cello-  58 oligosaccharides catalyzed by the same enzyme, and is more likely to occur in endoglucanases since they have a wide and kinked cleft catalytic domain, which allows more flexible binding of a sugar.156,157 The second plausible reason could be contamination of the enzyme stock with trace !-glucosidases, which hydrolyze cellobiose and other cello-oligosaccharides to yield D-glucose in Nature.158 This was possible since enzymes used for the assays were purified from a protein mixture secreted by T. reesei. To check the second hypothesis, I conducted a time-dependent inactivation assay with cyclophellitol (0.25 mM), which has been identified as an irreversible inactivator of many !-glucosidases74, to detect possible contamination with !-glucosidase. However, release of 2,4-DNPG was still observed when an aliquot of EGI inactivation mixture was added to 2,4-DNPC, and the hydrolytic activity of EGI remained the same in presence of cyclophellitol. Both results suggest that there is no !-glucosidase contamination in EGI (Appendix B.3). In conclusion, based on the kcat/Km values, 2,4-DNPC is a better substrate than 3,4-DNPC and pNPC in this study. Furthermore, the pKa of dinitrophenol is sufficiently low (4.0) that the 2,4-dinitrophenol is fully ionized once released at pH 5 (optimal pH for T. reesei cellulases), creating a readily observed bright yellow color. In addition, 2,4-DNPC showed good solubility in citrate buffer, while 3,4-DNPC did not. (Dissolving 3,4-DNPC required 10% DMSO in buffer as co-solvent from which it crystallized after a short period).     59 2.4.2 Kinetic Evaluation of Potential Active Site-Directed Inactivators and Mechanism- Based Inactivators for T. reesei Cellulases Kinetic analysis of active site-directed/mechanism-based inactivators usually involves incubation of the enzyme with the inactivators and measuring residual activity of the enzyme over time. Since the inactivation reaction could not be directly monitored continuously, aliquots of the inactivation reaction mixture were taken at different time intervals and quenched into the standard substrate to measure the residual enzyme activity. If inactivation was the outcome of irreversible binding to the active site (i.e. no reactivation of enzyme activity), an exponential loss of enzyme activity over time would be observed. In order to conduct large numbers of assays in an efficient way, the inactivation assays were carried out in 96 well plates using a Beckman Coulter BTX 880 Multimode Detector (a plate reader). The 2,4-DNPC concentration used to measure the enzyme activity was 5 mM (over three times the Km values for the T. reesei cellulases) which was sufficient to give a steady rate reading and to compete with the inactivators, and substantially stop any residual inactivation reaction. The residual enzymatic activity was determined from the rate of substrate hydrolysis by measuring the initial linear rate of the absorbance change at 405 nm, which directly reflected the remaining active enzyme concentration. (Full discussion of irreversible inactivation kinetics is in Appendix C.2).      60 2.4.2.1 Kinetic Evaluation of N-Bromoacetyl !-Cellobiosylamine (2.1) as a Potential Inactivator for T. reesei Cellulases  Assays of the inactivation of T. reesei cellulases by N-bromoacetyl !-cellobiosylamine (2.1) were conducted in a manner similar to other inactivation assays mentioned in Chapter 3 Section 3.3.3.3. Five different concentrations of 2.1 were incubated with each cellulase (0, 0.93, 1.85, 3.70, 4.63, and 5 mM).  The residual activities of the enzyme were then measured at various time points.  However, the results obtained were disappointing since the activity of T. reesei cellulases remained constant when treated with 2.1 within this range of inactivator concentrations. Since it was not a useful inactivator, futher studies of it were abandoned.  2.4.2.2 Kinetic Evaluation of O-linked and C-linked Epoxyalkyl !-Cellobiosides Both O-linked and C-linked epoxyalkyl !-cellobiosides had been used in probing cellulases in the past. The O-epoxyalkyl cellobiosides were chosen first to test with the four T. reesei cellulases owing to their ease of synthesis, although possible hydrolysis of the reducing terminal glycosidic bond (Figure 2.19) was a concern as it had been observed previously in some cases.115,122,133 This approach allowed us to quickly test the inactivation of cellulases by the inactivators carrying epoxide moieties. Unfortunately such enzymatic decomposition of the O- linked versions was observed, and thus it was eventually determined that a non-hydrolyzable linkage was required to prevent the cleavage of aglycone, hence the syntheses and testing of the C-linked epoxyalkyl glycosides.  61  Figure 2.19 The possible hydrolysis of the reducing terminal glycosidic bond.  Herein, three of the O-linked epoxyalkyl !-cellobiosides (2.2 EPO3C, 2.3 EPO4C, and 2.4 EPO5C) and two C-epoxyalkyl !-cellobiosides (2.5 EPC4C and 2.6 EPC5C) were synthesized and tested with four T. reesei cellulases. The inactivation kinetic parameters determined are listed in Table 2.4 and discussed in Section 2.4.2.2.1.             62 Table 2.4 Kinetic parameters for the inactivation of T. reesei cellulase by the O- epoxyalkyl !-cellobiosides and C-epoxyalkyl !-cellobiosides. All of the assays were duplicated two times. Systematic errors were determined by GraFit 5.0.13.    CBHI CBHII Inactivator Ki (mM) ki x 102 (min-1) ki/Ki x 103 (mM-1min-1) Ki (mM) ki x 102 (min-1) ki/Ki x 103 (mM-1min-1) EPO3C n/d(I) n/d n/d 23±6 9.1±1.5 4.0±1.7 EPO4C 4.6±0.4 8.0±0.3 17±2 9.7±0.9 23±9 24±11 EPO5C n/d n/d n/d 21±3 2.9±0.2 1.3±0.3 EPC4C 1.8±0.2 1.6±0.1 9±1. 5.0±0.4 3.1±0.1 6.2±0.7 EPC5C 2.4±0.4 7.0±0.4 29±7 8.3±1.1 16±1 19±3  EGI EGII Inactivator Ki (mM) ki x 102 (min-1) ki/Ki x 103 (mM-1min-1) Ki (mM) ki x 102 (min-1) ki/Ki x 103 (mM-1min-1) EPO3C n/d n/d n/d n/d n/d n/d EPO4C n/d n/d 4.2±0.1 n/d n/d n/d EPO5C 18±7(II) 7.5±1.3(II) 4.1±2.3 14±3 1.5±0.2 1.1±0.3 EPC4C 8.8±0.7(II) 1.7±0.1(II) 1.9±0.2 n/d n/d n/d EPC5C 13±2(II) 7.6±0.5(II) 6.0±1.2 n/d n/d n/d (I) n/d: Not determined. No inactivation occurred. See Appendix A for the inactivator concentrations tested. (II) Due to the alternative cleavage occurring during the inactivation of EGI, the inactivation parameters (numbers italicized) reflect minimum estimates of the actual rate constants.   63 As discussed in Section 2.4.2.2.3, no undesirable internal cleavage was seen for CBHI, CBHII or EGII and only a very small amount for EGI&representing typically less than 10 % decomposition during enzyme inactivation. This alternative cleavage reaction should not therefore significantly affect the kinetic evaluations of ki/Ki for the inactivation of EGI.  2.4.2.2.1 Discussion of Inactivation Results of the Four T. reesei Celluases Inactivation of CBHI (Cel 7A) Three of the five compounds (2.2, 2.3, 2.4, 2.5 and 2.6) tested here were capable of inactivating CBHI in a time dependent manner: EPO4C (2.3), EPC4C (2.5) and EPC5C (2.6). Indeed three of the O-linked epoxyalkyl cellobiosides (2.2, 2.3, and 2.4) have been studied previously115 as CBHI inactivators, and EPO4C (2.3) was the only inactivator, in agreement with our present findings.  However, the Ki (~ 4 mM) value determined here for EPO4C is slightly higher than the reported value (Ki = 1 mM) 115 with the same compound. A possible reason for this deviation was that the conditions used in the epoxidation reaction might have been different, yielding the diastereomers in a different ratio. The other two O-epoxyalkyl cellobiosides, EPO3C (2.2) and EPO5C (2.4) acted as competitive inhibitors with Ki  ~ 0.1 mM. Interestingly, EPC4C (2.5) and EPC5C (2.6) had higher binding affinities (Ki) for CBHI than EPO4C (2.3), while EPC5C (2.6) displayed a higher first order inactivation rate constant (ki) than EPC4C (2.5) (~ 5 fold higher), but a similar ki value to EPO4C (2.3). Overall, EPC5C (2.6) was the most effective inactivator towards CBHI, with the highest ki/Ki.    64 Inactivation of CBHII (Cel 6A) Inactivation of CBHII was seen with all five compounds. According to the kinetic parameters, EPO4C (2.3) and EPC5C (2.6) were the two most effective inactivators with ki/Ki values of 0.024 mM-1min-1 and 0.019 mM-1min-1 respectively, among the five tested&with approximately 6 fold, 20 fold and 3 fold higher inactivation rate constants than EPO3C (2.2), EPO5C (2.4) and EPC4C (2.5) respectively. The ki/Ki values of the CBHII inactivators reflected their alkyl chain lengths. Thus, EPO3C (2.2) and EPC4C (2.5), which have epoxyalkyl chains of similar length, showed comparable inactivation efficiencies toward CBHII, while the EPO4C (2.3) and EPC5C (2.6) pair shared similar inactivation efficiencies. Interestingly the inactivations of CBHII with EPO3C (2.2) and EPC4C (2.5) (which have similar chain lengths), did not follow single exponential decays of enzyme activity, while inactivation by EPO4C (2.3), EPO5C (2.4) and EPC5C (2.6) did&suggesting the presence of two competing inactivation process in these cases. A full discussion of this is presented in Section 2.4.2.2.4.  Inactivation of EGI (Cel 7B) EGI was inactivated by four of the compounds tested, EPO4C (2.3), EPO5C (2.4) EPC4C (2.5) and EPC5C (2.6). In this case, the two O-glycosides EPO4C (2.3) and EPO5C (2.4) shared similar inactivation efficiencies while EPC5C (2.6) inactivated slightly more effectively and EPC4C (2.5) was the most unreactive inactivator.   65 Inactivation of EGII (Cel 5A) Inactivations of EGII by EPO3C (2.2), EPO4C (2.3) and EPO5C (2.4) have been studied previously by Macarron et al..117 Their result showed that EPO5C (2.4) was the most reactive among the three O-linked epoxyalkyl inactivators (Note: EGII was formerly called EGIII). The kinetic results obtained here again showed EPO5C is an inactivator of EGII, albeit with low efficiency. In contrast to the previous result that slow inactivation was observed when EGII was treated with EPO3C and EPO4C, EPO5C was the only compound (among all tested, including EPC4C and EPC5C) that caused time dependent inactivation of EGII (concentrations of inactivators tested are listed in Appendix A. No observable inactivation for EGII was detected in the reactions involving EPO3C (2.2), EPO4C (2.3), EPC4C (2.5) and EPC5C (2.6).  2.4.2.2.2 Summary of the Studies of Epoxide-based Cellulase Inactivators EPO4C (2.3) and EPC5C (2.6) were the overall two most effective inactivators of the four T. reesei cellulases tested (Figure 2.20). As noted before, the length of the O-epoxybutyl moiety of EPO4C (2.3) is similar to that of the C-epoxypentyl moiety of EPC5C (2.6).    66  Figure 2.20 A histogram of inactivator efficiencies of epoxyalkyl cellobiosides with T. reesei cellulases.  The only compound that showed an observable inactivation effect on EGII was EPO5C (2.4); neither of the C-linked inactivators (2.5 and 2.6) proved to react with this enzyme. Therefore, according to our kinetic results which imply the inactivation dependency on alkyl chain length, extension of the C-alkyl chain by one more methylene unit would generate a C- glycoside of length comparable to that of EPO5C (2.4) (i.e. (R,S)-C-epoxypentyl !-cellobioside), so it would likely be an inactivator for EGII.  2.4.2.2.3  Internal Cleavage of Cellobiosides by EGI Alternative cleavage reactions occurred during the inactivation of EGI by the inactivators tested in this work (the example given in Figure 2.21 is the inactivation by EPC5C (2.6)), much as was seen during hydrolysis of 2,4-DNPC (Section 2.4.1). This hydrolysis of the internal glycosidic bond releasing glucose and the monosaccharide epoxide will reduce the inactivator 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 EPO3C EPO4C EPO5C EPC4C EPC5C k i/ K i ( m in -1 m M -1 ) CBHI CBHII EGI EGII  67 concentration (the monosaccharide epoxide is unlikely to function as an inactivator given the inactivity of 2,4-DNPG as a substrate), resulting in error in determination of inactivation rate constants.  However, since the probes ultimately used will incorporate a substituent on either C4" or C6" of the inactivator. It seems probable that this would exclude the alternative binding mode required. As a result, these cellobiose-based inactivators should remain good probes for cellulases.       68  Figure 2.21 (a) TLC results of inactivation reactions of CBHI, CBHII and EGI by EPC5C (2.6). (a.1) 10 min, (a.2) 1 hour, (a.3) 2 hour after the enzyme addition. Lane 1: EPC5C; Lane 2: reaction mixture with CBHI; Lane 3: reaction mixture with CBHII; Lane 4: reaction mixture with EGI; Lane 5: Cellobiose and glucose. The arrows indicate the possible product (right of the TLC plates) released from the alternative cleavage. (b) Mass spectrometric data of inactivation of EGI by EPC5C. (b.1) 1 second after the enzyme addition. (b.2) 1 hour after the enzyme addition.       69 2.4.2.2.4 Inactivation of CBHII  by EPO3C (2.2) and EPC4C (2.5) Did Not Occur According to a Single Exponential Decay The inactivation of CBHII with EPO3C (2.2) did not follow the single exponential decay expected, and the logarithmic plot of relative residual enzyme activity vs. time was a curve rather than the expected straight line (Figure 2.22). The phenomenon was also observed in the inactivation of CBHII with EPC4C (2.5).   Figure 2.22 Natural logarithm (Ln) values of relative residual CBHII activity vs. time for inactivation of CBHII by EPO3C (2.2) and EPC4C (2.5). Concentrations of (a) EPO3C (2.2) were (  ) 15.1 mM, (  ) 20.1 mM; (b) EPC4C (2.5) were ( ) 13.4 mM, ( ) 27.2 mM.  Possible explanations for this behavior are as follows. Alternative binding of the inactivator, leading to internal glycosidic bond cleavage and time-dependent destruction of the -2.25 -1.75 -1.25 -0.75 -0.25 0.25 0.00 50.00 100.00 Ln (V /V o)  Time (min) (a) EPO3C (2.2)-CBHII -2.75 -2.25 -1.75 -1.25 -0.75 -0.25 0.25 0.00 50.00 100.00 Ln (V /V o)  Time (min) (b) EPC4C (2.5)-CBHII  70 inactivator seems unlikely since there was no evidence for such cleavage (TLC result shown in Appendix B.3). Turnover of the covalently modified enzyme (reactivation) following inactivation could provide another explanation though the absence of such behavior with EPC5C (2.6) would require that a different amino acid residue in the enzyme be modified in the two cases. A third possibility is the presence of a minor impurity in the inactivator which acts as a more efficient inactivator than EPO3C/EPC4C to CBHII. The fast loss of enzyme activity in the early stage would be the combined effect of inactivation by both the “contaminant” and the tested epoxide, but once the “contaminant” had been consumed, the inactivation rate would slow. Contamination of CBHII by other cellulases was not considered to be a cause for this phenomenon since this behavior was only observed in the inactivations of CBHII by EPO3C (2.2) and EPC4C (2.5). If the cellulase was contaminated, this phenomenon would be expected in all the inactivation cases. In addition, the effect from the diastereomers mixture should not result in this behavior either; the reason being that even if the diastereomers inactivated CBHII activity at different rates the net inactivation rate should be the combined effect from both inactivations by the two diastereomers, and that should also follow a single exponential decay.     71 2.4.2.3 Kinetic Evaluation of 2,4-Dinitrophenyl 2-deoxy-2-fluoro !-cellobioside (2FDNPC, 2.7) and 2,4-Dinitrophenyl 2-deoxy-2-fluoro !-lactoside (2FDNPL, 2.8) as Mechanism-based Inactivators for T. reesei Retaining Cellulases Kinetic parameters for inactivation of the three retaining T. reesei cellulases, CBHI, EGI, and EGII by 2FDNPC (2.7) and 2FDNPL (2.8) were determined in a similar manner to those of the other inactivators. Table 2.5 Kinetic parameters for the inactivation of EGI by 2FDNPC (2.7) and 2FDNPL (2.8). The assays were duplicated two times. Systematic errors were determined by GraFit 5.0.13.   EGI Inactivator Ki (mM) ki x 102 (min-1) ki/Ki x 103 (mM-1min-1) 2FDNPC 3.1±0.1 2.2±<0.1 7.1±0.5 2FDNPL 10±2 10±1 10±3  Time dependent inactivation of EGI was seen upon treatment of the enzyme with 2FDNPC (2.7) and 2FDNPL (2.8) (Table 2.5). However, unlike the kinetic results typical for irreversible inactivators, the relative residual enzyme activity plots (Ln (V/V0) vs. time) in the reaction with 2FDNPC (2.7) were slightly flattened at later time points (Figure 2.23), possibly due to the turnover of the glycosyl-enzyme intermediate (Figure 2.24(a)). This phenomenon was more obvious when higher concentrations of 2FDNPC (2.7) were studied, possibly due to transglycosylation to a second molecule of 2FDNPC bound in the (+) sites.   72  Figure 2.23 The “flattening-out” phenomenon in the reaction of 2FDNPC (2.7) and 2FDNPL (2.8) with EGI. (a) Concentrations of 2FDNPC (2.7) were ( ) 0 mM, ( ) 1.2 mM, ( ) 5.6 mM, ( ) 9.4 mM, ( ) 23.3 mM. (b) Concentrations of 2FDNPL (2.8) were ( ) 0 mM, ( ) 3.0 mM, ( ) 5.4 mM, ( ) 9.8 mM, and ( ) 20.6 mM. The kobs values were similar in the inactivation of EGI with 5.6 mM, 9.4 mM and 23.3 mM of 2FDNPC. The first three data points at 2 min, 14 min and 26 min of the reaction with 9.4 mM of 2FDNPC were fit into the first order rate equation to show the linear phase of the curve in the early stage of inactivation.  This effect was much less pronounced for inactivation by 2FDNPL (2.8) (Figure 2.23), presumably because the absence of a C4" equatorial hydroxyl group in 2FDNPL (2.8) would prevent transglycosylation of the covalently linked intermediate (Figure 2.24(b)), removing that pathway for reactivation.  -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0 50 100 Ln  (V /V 0)  Time (min) (a) 2FDNPC (2.7)-EGI -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 0 50 100 Ln (V /V 0)  Time (min) (b) 2FDNPL (2.8)-EGI  73   Figure 2.24  (a) Possible transglycosylation of the glycosyl-enzyme intermediate by 2FDNPC (2.7). (b) No transglycosylation of glycosyl-enzyme intermediate by C4" axial hydroxyl group of 2FDNPL (2.8).   The other two retaining cellulases, CBHI and EGII, however, displayed no observable time dependent loss of activity during incubation with either of 2FDNPC (2.7) or 2FDNPL (2.8) consistent with previous reported on CBHI159 (The concentration of inactivators 2.7 and 2.8 tested are listed in Appendix A).  2.4.3 Summary of Inactivation Studies with T. reesei Cellulases  The following histograms (Figure 2.25) sum up the inactivation kinetic results obtained in the current work. The upper figure provides a comparison of inactivator efficiency toward each specific cellulase. The lower one represents the overall performance of each inactivator with the four T. reesei cellulases.  These histograms should allow the reader to quickly assimilate the results. O OO F HO OH OH OH HO HO O O O O OO F HO OHOH OH HO O O NO2 NO2 O OO F HO OH OH OH HO HO O O O O O OO F HO OHOH OH HO HO O NO2 NO2 O (a) (b) H  74   Figure 2.25 Histograms displaying inactivation rate constants (ki/Ki) for each inactivator/cellulase combination grouped by (a) enzyme; (b) inactivator.  The upper histogram shows that CBHI and CBHII displayed diversity in response to the inactivators tested, whereas EGI which is the most reactive enzyme, displayed a moderate selectivity toward different inactivators. EGII was the least reactive enzyme when treated with these inactivators. In addition, EPO4C (2.2) and EPC5C (2.6) were in general the better 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 CBHI CBHII EGI EGII k i/ K i ( m in -1 m M -1 ) EPO3C EPO4C EPO5C EPC4C EPC5C 2FDNPC 2FDNPL (a) 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 k i/ K i ( m in -1 m M -1 ) CBHI CBHII EGI EGII (b)  75 inactivators for CBHI, CBHII and EGI according to both the efficiency and generality of inactivators.  2.4.4 Kinetic Evaluation of 2,4-Dinitrophenyl Trisaccharides with T. reesei Cellulases None of the inactivators tested so far were the universal inactivator we wished to obtain. In addition, the binding affinities and first order rate constants of these inactivators (Ki and ki) still need to be improved to achieve higher efficiency of the probes. Taking as an example the inactivation reaction of CBHII with EPC5C (2.6) (Ki= 8.3 mM, half life ~5 minutes), a concentration greatly exceeding Ki was required to deactivate 50% enzyme activity in 5 minutes. Since some previous studies have shown that binding could be improved by extension of the oligosaccharide chain160, a trisaccharide version of inactivators came to mind as a means to enhance substrate binding. Since the available inactivator was limited, I prepared the equivalent substrates as 2,4-DNP trisaccharides. Owing to its high compatibility with a variety of donors, Abg glycosynthase permitted transfer of a variety of glycosyl units on to 2,4-DNPC at the non- reducing end. In this way 2,4-DNP trisaccharides with D-galactose, L-arabinose, D-fucose and D- xylose at the non-reducing terminus were synthesized from 2,4-DNPC and the corresponding sugar fluorides (Figure 2.26). In addition to enhancing binding efficiency, we anticipated some deviations in binding interactions caused by the structural differences in the non-reducing terminal sugar, and hoped to thereby generate some additional specificity among the different cellulases.   76  Figure 2.26 Structures of 2,4-DNP trisaccharides (2.9, 2.10, 2.11 and 2.12). The conditions employed in the kinetic evaluation of the 2,4-DNP trisaccharides were similar to those for 2,4-DNPC. Seven to eight different substrate concentrations ranging from 0.1 to 7 times Km were incubated with the enzyme at 25 oC and the release of dinitrophenolate was monitored by measuring the absorbance change at 400 nm. Kinetic data are summarized in Table 2.6.          77 Table 2.6  Kinetic parameters for hydrolysis of 2,4-DNP glycoside substrates with T. reesei cellulases determined by absorbance change at 400 nm. All of the assays were duplicated three times. Systematic errors were determined by GraFit 5.0.13.   CBHI CBHII Substrate(III) Km (µM) kcat (sec-1) kcat/ Km x 103 (µM-1sec-1) Km (µM) kcat (sec-1) kcat/ Km x 103 (µM-1sec-1) 2,4-DNPC 31 0.032 1.0 1.2x103 0.45 0.37 GalDNPC 51±7(IV) 0.047±0.002(IV) 0.92±0.17 97±5 0.11±0.01 1.1±0.2 L-ArabDNPC n/d(I) n/d n/d 60±3 0.11±<0.01 1.8±n/d FucDNPC 12±3(II) n/d n/d 51±3 0.086±0.002 1.7±0.1 XylDNPC n/d n/d n/d 34±2 0.18±0.01 5.3±0.6  EGI EGII Substrate Km (µM) kcat (sec-1) kcat/ Km x 103 (µM-1sec-1) Km (µM) kcat (sec-1) kcat/ Km x 103 (µM-1sec-1) 2,4-DNPC 1.8 x103 38 21 1.5 x103 3.2 2.4 GalDNPC 160±20(IV) 0.65±0.04(IV) 4.1±0.7 9.0±1.0 2.8±0.1 310±46 L-ArabDNPC 120±20(IV) 1.1±0.1(IV) 9.2±2.4 14±2 4.4±0.3 310±59 FucDNPC 63±8(IV) 0.32±0.02(IV) 5.1±0.1 5.0±1.0 3.3±0.2 623±143 XylDNPC 98±6(IV) 4.9±0.1(IV) 50±4 14±1 3.2±0.1 229±23 (I) n/d: Not determined. No inactivation occurred. See Appendix A for the inactivator concentrations tested. (II) GalDNPC: D-GalactosylDNPC; ArabDNPC: L-arabinosylDNPC; FucDNPC: D- fucosylDNPC; XylDNPC: D-xylosylDNPC. (III) The number was determined by competitive inhibition assays (Chapter 3 Section 3.3.3.4). (IV) Due to the alternative cleavage occurring during the inactivation of EGI, the inactivation parameters (numbers italicized) reflect minimum estimates of the actual rate constants.   TLC of the enzymatic reaction mixtures showed that there was no cleavage of internal glycosidic bonds in the enzymatic reactions with CBHII and EGII (Figure 2.27). In contrast, alternative cleavage was observed in the reactions of the four 2,4-DNP trisaccharides with CBHI  78 and EGI. Thus the Michaelis-Menten parameters determined with CBHI and EGI represent a minimum estimate of the true parameters.   Figure 2.27 TLC of reaction mixture of 2,4-DNP trisaccharides with T. reesei cellulases. Lane 1:  starting material (SM); Lane 2: reaction mixture with CBHI; Lane 3: reaction mixture with CBHII; Lane 4: reaction mixture with EGI; Lane 5: reaction mixture with EGII. Starting material was (a) L-arabinosylDNPC. (b) FucosylDNPC. (c) XylosylDNPC.  2.4.4.1 Comparison of Kinetic Parameters for 2,4-DNP Substrates on CBHII 2.4.4.1.1 Comparison of kinetic results of 2,4-DNPC and 2,4-DNP trisaccharides with CBHII The extension by one glycosyl unit at the non-reducing terminus improved the substrate binding affinity over 10 times for CBHII relative to 2,4-DNPC, while kcat, which reflected the activation energy barrier of the hydrolytic reaction, dropped approximately 2.5 to 4 fold, depending on the non-reducing terminal sugar. This could be rationalized by the stabilization  79 gained by the additional enzyme-substrate interactions (Figure 2.28(a)). Presumably, the stabilization effected on the ES complex (E#S) provided by the interaction of the non-reducing terminal sugar with CBHII was greater than it was on the oxocarbenium ion-like transition state ([S#Enz]±), and hence an increase in the activation energy barrier was seen (resulting in lower kcat values). However the resultant kcat/Km was improved in each case.  2.4.4.1.2 Comparison of kinetic results of four 2,4-DNP trisaccharides (2.9, 2.10, 2.11, and 2.12) with CBHII No significant variation in kinetic parameters (Km and kcat) was detected among D- galatosylDNPC L-arabinosylDNPC D-fucosylDNPC (2.9, 2.10, and 2.11). This implies that the different terminal glycosyl groups had little effect on the substrate affinity and the first order rate constant of these three substrates (2.9, 2.10, and 2.11). This could be explained by the crystal structure of the CBHII active site determined previously.37,84 The catalytic domain contains six glycosyl binding subsites (-2 to +4) but there was no defined -3 binding site present. Hence the non-reducing terminal sugar likely partially protrudes from active site and therefore no significant difference was observed in the Km values of the three 2,4-DNP trisaccharides.  On the other hand, D-xylosylDNPC (2.12) displayed slightly higher binding affinity to CBHII, possibly due to its C4"" equatorial hydroxyl group mimicking that of the natural substrate (i.e. cellulose).  80  Figure 2.28 (a) Reaction coordination diagram of classical mechanism of an inverting glycosidase. $G± is the activation energy of the reaction. S#Enz is Michealis- complex; P#Enz is product and enzyme non-covalent complex. (b) Energy diagram of classical double displacement mechanism of a retaining glycosidase. $G1± and $G2± are the activation energies of the glycosylation/deglycosylation steps. Covalent Int. is the covalently linked glycosyl-enzyme intermediate.       81 2.4.4.2 Comparison of Kinetic Parameters for 2,4-DNP Substrates on EGII: 2.4.4.2.1 Comparison of kinetic results of 2,4-DNPC and 2,4-DNP trisaccharides with EGII 2,4-DNP trisaccharide substrates had much higher binding affinity than 2,4-DNPC (>100 fold) for EGII, but kcat was not significantly different among the 2,4-DNP substrates. The invariant kcat values indicate that the activation energy of the rate determining step of hydrolysis was largely unchanged (%G1± if glycosylation is rate determining, or %G2± if deglycosylation is rate determining), presumably because the interaction of the additional sugar with the -3 subsite of the catalytic domain stabilized both the enzyme-substrate complex (E#S or the covalent int.) and ([S#Enz]± or [P#Enz]±) in the rate determining step equally well (Figure 2.28(b)). This improved binding affinity considerably increases the hydrolytic efficiency of EGII toward the 2,4-DNP trisaccharides relative to 2,4-DNPC.  2.4.4.2.2 Comparison of kinetic results of four 2,4-DNP trisaccharides (2.9, 2.10, 2.11, and 2.12) with EGII Since there was no crystal structure of T. reesei EGII available, I was unable to directly correlate the kinetic data with substrate binding as I did for CBHII. Based on the classification of glucoside hydrolases by Henrissat, which groups the glycosidases into families according to their primary sequence similarity, it was reasonable to assume that the structure of the catalytic domain of EGII of T. reesei resembled other members in the same family (GH5).  82 The following discussion was based on the crystal structure determined for endoglucanase Cel 5A of Bacillus agaradherans, under the assumption that the two enzymes have high structural similarity. The crystal structure (Figure 2.29) of B. agaradherans161 solved with cellobiose bound to the -2 and -3 subsites revealed the weak interaction between the glycosyl unit in the -3 pocket and the enzyme. The C6 hydroxyl group of the non-reducing glucosyl terminus was the only hydroxyl forming direct hydrogen bonding interaction with the enzyme residues. This explains the insensitivity to configurational difference at the C4 hydroxyl group of the non-reducing terminal sugar (L-arabinosylDNPC (2.11) and D-xylosylDNPC (2.12)). D-GalactosylDNPC (2.9) displayed a slightly higher affinity than L-arabinosylDNPC (2.10), presumably due to the extra hydrogen bonding interaction between the C6"" hydroxyl group of D-galactosylDNPC with the residues (Tyr 40 and Lys 267 in B. agaradherans) stabilizing the Michaelis complex. Moreover, D-fucosylDNPC (2.11) had ~ 3 times higher affinity than L-arabinosylDNPC (2.10), presumably due to the additional hydrophobic interaction formed between the C5"" methyl group and the -3 binding site in which the hydrophobic interaction (with aromatic residues) played the main role on binding -3 subsites with the glycosyl unit in the crystallographic study on Cel5A of B. agaradherans.The kcat values of these four trisaccharides, in contrast, were invariant, suggesting that these intermediates maintained their same strength at the transition state.  83  Figure 2.29 Protein interactions with cellobiose in the active site (-3 and -2 subsites) of Cel 5A of B. agaradherans. (a) A schematic representation of Cel 5A- cellobiose interaction. (b) Electron density figure for cellobiose bound in the active site.  2.4.4.3 Comparison of Kinetic Parameters for 2,4-DNP Trisaccharide Substrates  on CBHI and EGI Cleavage of glycosidic bond between first and second sugars from reducing end was the predominant enzymatic reaction. Hydrolytic reactions monitored by TLC revealed that EGI and CBHI cleaved the substrate internally with release of 2,4-DNPG. Further, the rate of 2,4-DNPG formation (Figure 2.27) was greater than that of dinitrophenolate formation, resulting in difficulty in determination of Michaelis–Menten parameters. The individual kinetic parameters Km or kcat, determined based on the rate of dinitrophenolate release, were thus not meaningful. On the other hand, the second  84 order rate constant (kcat/Km) can still be determined in the presence of alternative substrate binding mode.  Substrate depletion assays at low substrate concentration were attempted (Details of substrate depletion assays referred to Chapter 3 Section 3.3.3.5) and the kcat/ Km values were obtained by fitting the progress curve to a first order rate equation. However, the rate of hydrolysis of dinitrophenyl group became nearly zero before the total dinitrophenolate had been released, presumably due to the alternative cleavage, resulting in depletion of the substrate (2,4- DNPG was not a substrate for any of the cellulase). As a result, regular initial rate assays were employed to evaluate the kinetic parameters of the substrates with CBHI and EGI. Unfortunately, the initial hydrolytic rates of dinitrophenyl group of D-fucosylDNPC (2.11), L-arabinosylDNPC (2.10) and D-xylosylDNPC (2.12) with CBHI were rarely detectable and thus the kinetic parameters could not be determined using the UV-Vis spectrophotometer. The Km value of fucosylDNPC with CBHI was estimated using a competitive inhibition method. The Dixon plot was graphed and extrapolation of the curves gave the Ki (~0.01 mM) for D-fucosylDNPC (2.11).  EGI exhibited lower hydrolytic efficiency with D-galactosylDNPC (2.9), L- arabinosylDNPC (2.10), and D-fucosylDNPC (2.11) compared to 2,4-DNPC. In contrast, D- xylosylDNPC (2.12) was a more effecient substrate with EGI (kcat/Km~2 fold higher relative to 2,4-DNPC), implying that the configuration of the C4"" hydroxyl group might play an important role in the substrate specificity. In summary, the kinetic results of the 2,4-DNP trisaccharides varied from one cellulase to another. Great improvements in hydrolytic efficiency were seen in EGII but not in the other three  85 cellulases. The cleavage of internal glycosidic bonds by CBHI and EGI limits the utility of trisaccharide based affinity labels for these two cellulases.  2.5 Other Work Done to Probe Substrate Specificity As mentioned in Chapter 1, the ABPP probes that we were attempting to make must be specific to each cellulase for the measurement of each individual active enzyme concentration. The specificity could be from a specific inactivator (e.g. different reactive group employed) differential binding affinity of the multiple glycosyl units with glycosyl binding subsites (e.g. trisaccharide v.s disaccharide), or a remote chemical group which formed specific interactions with cellulases (discussed in this section). Our attempts to improve the catalytic efficiency by adding a sugar unit onto 2,4-DNP substrates did not work with CBHI and EGI since the cleavage of the first and second sugar (from the reducing end) was pronounced (Section 2.4.4.3). One of the other possible ways to improve the substrate specificity of each enzyme was to incorporate a non-sugar moiety at the C6" position of the cellobioside, and this work was performed by others in parallel. Installation of substituents at C6" might enhance the specificity. The 2,4-DNP cellobiosides could readily be modified by employing an azide group at the C6" in a reaction with various chemical groups bearing an alkyne functionality via “Click” chemistry.162 Ideally, specific inactivators with respect to each cellulase could be generated by incorporation of a universal inactivating moiety and the C6" functional group (this specific functional group enhances specificity towards specific cellulases in a cellobioside scaffold). Therefore, it was important to include some results obtained from the kinetic characterization of the synthetic  86 substrates with C6" modifications for the four cellulases in this thesis. The synthesis and kinetic characterization were both conducted by Kah-Yee Li (Table 2.7).82 The following table only shows a part of the results derived from Kah-Yee Li’s report. The catalytic efficiency of EGI shows considerable improvement with two of the modified substrates (compounds 15 and 19). In contrast, the catalytic efficiency drops dramatically in EGII, and only moderate effects were observed for CBHI and CBHII. One of the exciting results was that compounds 15 and 19 exhibit much higher specificity toward cellulase EGI than the other three cellulases. The results showed the plausibility of tuning substrate specificity to the cellulases by the incorporation of different chemical moieties into 2,4-DNPC. In light of this promising study, a larger library of modified compounds is being assembled in parallel with further investigation of a universal inactivator. Table 2.7 Michaelis-Menten parameters for the hydrolysis of DNPC and its derivatives by T. reesei. 82     87  Compound Km (mM) CBHI CBHII EGI EGII DNPC 0.0312 1.21 1.82 1.35 15 0.724 0.213 0.226 0.330 19 0.748 0.651 0.215 0.509 21 0.299 0.377 0.150 0.556   kcat/Km (sec-1 mM-1) Compound CBHI CBHII EGI EGII DNPC 0.0312 0.373 20.7 2.38 15 0.639 0.273 209 0.0471 19 3.34 0.482 160 0.143 21 1.89 0.339 78.3 0.061  2.6 Summary of the Kinetic Studies from the Synthetic Compounds with T. reesei Cellulases The kinetic study performed with the inactivators and substrates provided valuable information for future research. It showed that inactivation by exo-alkyl epoxide cellobiosides was alkyl chain length dependent. One might be able to obtain a universal inactivator for the four cellulases by further tuning the length of the epoxyalkyl chain. Furthermore, review of the kinetic data collected up to this stage revealed that C-linked epoxide cellobiosides as the most ideal class of compounds to carry on our ABPP probe design, owing to their non-hydrolysable aglycone. We also showed that the hydrolysis of an internal glycosidic bond in trisaccharide versions of substrates, presumably due to alternative cleavage defined in this thesis (Appendix  88 C.4) might be a problem for our ABPP probe design since the cleavage will result in detachment of the reporter group. More studies were required to support the hypothesis of alternative cleavage. Nonetheless this class of compounds would not be applicable to our study with T. reesei cellulases and would not be carried on for further investigation. Another problem that remained unsolved was that EGI hydrolyzes the internal glycosidic bond in disaccharides, greatly reducing the efficiency of inactivators. Our best hope relies on the modification of the C4" and/or C6" position with a heterogeneous group which would provide cellulase specificity, and ideally would also prohibit the “alternative cleavage” and thereby prevented internal glycosidic bond hydrolysis. Further, it will prohibit glucosidase cleavage in “real life” applications. This is currently under investigation by others. One further study I intended to perform was to investigate the labeled nucleophilic residue at the active site via comparative liquid chromatography (LC) followed by electrospray ionization mass spectrometry (ESI MS) of proteolytic digested peptides. This experiment would confirm the formation of a covalent linkage between the enzyme and inactivator, and provide insight into the site of the inactivation reaction with the synthetic compounds in the enzyme active site. In summary, according to the kinetic study of all the inactivators and 2,4-DNP derivatives, our plans for the future study could be focused on the following points. First, enlargement of the library of specific substrates for T. reesei cellulases would be performed in rapidly since “Click” chemistry has proved useful in coupling the chemical moiety to the cellobiose scaffold. In addition, more potential inactivators for T. reesei cellulases such as cyclophellitol (Figure 1.14(c)) and C-linked epoxides with various lengths of linkers would be  89 investigated. Afterward, a compound with both a specific binding moiety (at C4"%C6", Section 2.5) and an effective inactivating group (at C1, the main focus of this thesis) would be synthesized and tested with the cellulases of interest in order to validate our proposal.                90 Chapter  3: Materials and Methods  3.1 Generous Gifts Trichoderma reesei cellulases, 2,4-DNPC, 3,4-DNPC, per-O-acetylated α-glucosyl fluoride and α-glucosyl bromide and α-xylosyl fluoride were generous gifts from Iogen Corporation (enzymes) and Dr. Hong-Ming Chen (chemical compounds). Expression vectors cloned with Abg 2F6 gene and E. coli BL21 (DE3) were obtained from Dr. Emily Kwan and Dr. David Kwan respectively.  3.2 General Synthesis  All reagents used were obtained from commercial suppliers (Aldrich, Fluka, and Sigma) and were used directly without further treatment unless otherwise noted. Methylene chloride (DCM) and pyridine were dried over CaH2 and distilled before use. DMF was dried with 4 Å molecular sieves for at least two days before use. MeOH was dried with Mg and distilled before use. THF was dry over sodium and benzophenone. Dionized water was prepared with a Millipore-Directed QTM 5 Utrapure Water System filter. TLC was performed on E. Merck pre- coated 0.2 mm aluminum-backed sheets of Silica Gel 60F254 and was visualized using UV 254 nm and/or further stained with 10% ammonium molybdate in 2M H2SO4, followed by charring. Flash column chromatography was performed using Silicycle silica gel 230-400 mesh. All NMR spectra were recorded with Bruker AV-300 (300 MHz), AV-400 (400 MHz) or AV-600 (600 MHz) spectrometers. Chemical shifts were presented on the δ scale in parts per million referenced to deuterium solvents such as CDCl3 (7.27 ppm), D2O (4.75 ppm) or MeOD (3.31  91 ppm).  Low and high resolution mass spectra were carried out with PE-Sciex API 300 triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) ion source in mass spectrometry laboratory of the Department of Chemistry at the University of British Columbia.  3.2.1 General Methods 3.2.1.1 Acetylation of Free Sugars  Acetylation of free sugars was carried out in a mixture of dry pyridine and acetic anhydride (in the ratio of Vpyridine:VAc2O = 3:2) at RT overnight. Upon completion, a small volume of cold water was added to hydrolyze the unreacted acetic anhydride followed by the evaporation of solvent. The crude product was redissolved in ethyl acetate and washed with water, 10% HCl, saturated NaHCO3 and brine successively. The organic layer was dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography or by crystallization with proper solvent.  3.2.1.2  General Deacetylation 3.2.1.2.1 Deacetylation by Sodium Methoxide  The acetylated sugar was suspended in dry MeOH (in a ratio of 10 mg protected sugar to 1 mL of methanol) at RT and a catalytic amount of sodium methoxide was added. In cases where the protected sugar carries a reactive functional group such as an epoxide or acetyl bromide, the sodium methoxide concentration was carefully controlled. The reaction mixture was stirred at RT until the reaction was completed (monitored by TLC), then neutralized with  92 Amberlite® IR120 H+, filtered, and concentrated under reduced pressure. Purification was carried out by flash column chromatography and/or Waters Sep-Pak tC18 reverse phase cartridge.  3.2.1.2.2 Deacetylation with Ammonia  The acetylated sugar was suspended in dry MeOH (2 g protected sugar in ~30 mL MeOH) and cooled down to 0 oC in ice water bath. Ammonia gas was bubbled into the solution for 10 minutes, and the reaction mixture was stirred at 4 oC overnight. After the reaction was complete (checked by TLC), solvent was evaporated under reduced pressure and the resulting residue was crystallized from an appropriate solvent or purified via flash column chromatography.  3.2.1.2.3  Deacetylation in Acidic Condition  The per-O-acetylated sugar was suspended in dry MeOH at 0 oC (in a ratio of 100 mg protected sugar in 10 mL of methanol), and AcCl was added slowly until the concentration of AcCl reached 4% (v/v in MeOH). The reaction mixture was then stirred at 4 oC until deprotection was complete (monitored by TLC). Solvent was removed under reduced pressure and purification was carried out by flash column chromatography and/or Waters Sep-Pak tC18 reverse phase cartridge.  3.2.1.3 De-benzylation of C-linked Glucosides  A dry three necked flask was charged with a stir bar and cooled down to -70 oC in a dry ice/acetone bath. NH3 gas was slowly flowed in until the desired volume (in the ratio of 1.2 g  93 sugar in 70 mL liquid ammonia) had been reached. Pieces of sodium (~ 10 eqv.) were added by portions until a dark blue color developed and persisted. The benzylated sugar dissolved in THF (in the ratio of 1.2 g dissolved in 25 mL dry THF) was added dropwise into the flask via an addition funnel and the reaction mixture was stirred at -70 oC. Upon completion of the reaction (monitored by TLC), saturated NH4Cl solution was slowly added until the blue color was discharged, and the solvent was allowed to evaporate overnight. The resulting white residue was dissolved in EtOH and filtered, followed by purification via a Waters Sep-Pak tC18 reverse phase cartridge.  3.2.1.4 General Procedure for Synthesis of "-Glycosyl Fluorides Per-O-acetylated sugar was added to 70%/30% (w/w) HF/pyridine (in a ratio of 200 mg sugar in 1 mL solvent) at 0 oC in a plastic bottle with vigorous stirring. The reaction mixture was stirred overnight at 4 oC. Upon completion of reaction, the unreacted HF was slowly quenched with ice-cold saturated NaHCO3 (the crude product precipitated out as a white solid) until there was no release of gas bubbles. The mixture was then extracted with ethyl acetate and this was washed successively with water and brine. The organic layer was dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. Further purification was performed by flash column chromatography with the eluent composed of an appropriate ratio of petroleum ether and ethyl acetate.  3.2.1.5 General Procedure for Synthesis of "-Glycosyl Bromides The per-O-acetylated sugar was dissolved in dry DCM under argon (in a ratio of 4 g sugar in 40 mL solvent), and the mixture was cooled down to 0 oC, followed by the addition of  94 an appropriate volume of 33% (w/w) HBr in AcOH (in a ratio of 100 mg of starting material to 0.5 mL of HBr/AcOH solution). The reaction mixture was allowed to warm up to RT and stirred overnight. Upon completion, the reaction mixture was extracted with DCM (5 times reaction volume), and successively washed with H2O, saturated NaHCO3, brine. The organic layer was dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The resulting compound was dried in vacuo.  3.2.1.6 General Procedure for Alkenyl Alcohol Coupling with "-Cellobiosyl Trichloroacetimidates To molecular sieves and trichloroacetimidate (2.18) were added dry DCM (in a ratio of 1.6 g sugar in 12 mL DCM) under an argon atmosphere. Alkenyl alcohol (> 40 eqv.), which had been dried under molecular sieves overnight, was added via syringe. The reaction mixture was stirred for 10 minutes, and then cooled down to -30 oC, followed by the addition of a catalytic amount of boron trifluoride diethyl etherate. Upon completion, the reaction mixture was quenched with triethylamine, filtered through Celite, and rinsed with DCM. The DCM solution was washed with H2O, saturated NaHCO3 then brine sucessively. The organic layer was dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. Purification of crude product was performed by flash column chromatography with the eluent composed of ethyl acetate and toluene in proper ratio.  3.2.1.7 General Procedure for Epoxidation of Alkenyl Glycosides  A mixture of alkenyl cellobioside and mCPBA (4~5eqv.) in dry DCM (in a ratio of 100 mg sugar in 5 mL solvent) was refluxed at 60 oC until the reaction was complete (checked by  95 TLC). After cooling down to RT, the reaction mixture was washed successively with H2O, saturated NaHCO3 and brine. The organic layer was dried over anhydrous MgSO4, filtered, and then concentrated under reduced pressure. The crude residue was purified by flash column chromatography with the eluent composed of ethyl acetate and petroleum ether in proper ratio. All of the epoxidation reactions performed in this thesis using mCPBA yielded a mixture of diastereomers carry R and S configured carbons in the epoxide moiety. The ratio of the two diastereomers could not be determined based on 1H NMR spectra due to the extensive signal overlap.  3.2.1.8 General Procedure for Oligosaccharide Synthesis with Abg 2F6 Glycosynthase A mixture of the glycosyl acceptor and glycosyl donor (1.2~2 eqv. to glycosyl acceptor) was dissolved in 50-100 mM ammonium acetate buffer (pH 7.0) or sodium phosphate buffer (pH 7.0) in a Falcon® tube (in a ratio of 200 mg glycosyl acceptor in 20 mL buffer). Abg 2F6 glycosynthase was added to the reaction mixture (0.1 mg/mL to 1.7 mg/mL), and the solution was incubated at RT for 1 hour to three days. Upon completion (monitored by TLC), reaction mixture was lyophilized and desalted via a Waters Sep-Pak tC18 reverse phase cartridge.       96 3.2.2 Synthesis and Characterization (2,3,4,6-Tetra-O-acetyl-!-D-glucopyranosyl)-(1%4)-O-2,3,6-tri-O-acetyl-"-D-glucopyranosyl bromide (2.13)163   Per-O-acetylated cellobiose (3.0 g, 4.4 mmol) was brominated according to General Procedure for Synthesis of "-Glycosyl Bromides. The crude product was crystallized from Et2O to yield hepta-O-acetyl "-D-cellobiosyl bromide 2.13 (2.27 g, 73%). 1H NMR is consistent with the previous reported data. ESI MS m/z calcd. for C26H35BrO17: 698.1, Found: 721.5/723.3 [M+Na]+.  (2,3,4,6-Tetra-O-acetyl-!-D-glucopyranosyl)-(1%4)-O-2,3,6-tri-O-acetyl-!-D-glucopyranosyl azide (2.14)164   Protected cellobiosyl bromide 2.13 (1.2 g, 1.6 mmol) was dissolved in 10 mL dry DMF under Ar followed by the addition of NaN3 (143 mg, 2.2 mmol, 1.4 eqv.). The reaction was heated up to 70 oC and stirred for 6 hours. After the reaction was complete, the residue was extracted with 200 mL ethyl acetate and washed successively with H2O, saturated NaHCO3 and brine. The organic phase was dried over anhydrous MgSO4, filtered and concentrated under reduced  97 pressure. Purification of the resulting residue by flash column chromatography (petroleum ether:ethyl acetate = 2:1) afforded 2.14 as a white solid (860 mg, 1.3 mmol, 80%). ESI MS m/z calcd. for C26H35N3O17: 661.2, Found: 684.2 [M+Na]+. 1H-NMR (CDCl3, 300 MHz): # 5.18 (dd, 1H, J2, 3 9.6, J3, 4 8.9 Hz, H3), 5.14 (dd, 1H, J2’, 3’  9.1, J3’, 4’  9.4 Hz, H3’), 5.06 (dd, 1H, J3’, 4’ 9.4, J4’, 5’ 9.6 Hz, H4’), 4.92 (dd, 1H, J1’, 2’ 8.0, J2’, 3’ 9.1 Hz, H2’), 4.86 (dd, 1H, J1, 2 8.9, J2, 3 9.6 Hz, H2), 4.61 (d, 1H, J1, 2 8.9 Hz, H1), 4.53 (dd, 1H, J5, 6a 1.8, J6a, 6b 12.1 Hz, H6a), 4.51 (d, 1H, J1’, 2’ 8.0 Hz, H1’), 4.37 (dd, 1H, J5’, 6’a 4.3, J6’a, 6’b 12.6 Hz, H6’a), 4.11 (dd, 1H, J5, 6b 4.8, J6a, 6b 12.1 Hz, H6b), 4.03 (dd, 1H, J5’, 6’b 2.3, J6’a, 6’b 12.6 Hz, H6’b), 3.79 (dd, 1H, J3, 4 8.9, J4, 5 9.6 Hz, H4), 3.67 (ddd, 1H, J4, 5 9.6, J5, 6a 1.8, J5, 6b 4.8, Hz, H5), 3.66 (ddd, 1H, J4’, 5’  9.6, J5’, 6’a 4.3, J5’, 6’b 2.3 Hz, H5’), 2.13 (s, 3H, COCH3), 2.08 (s, 3H, COCH3), 2.06 (s, 3H, COCH3), 2.03 (s, 3H, COCH3),  2.02 (s, 3H, COCH3),  2.00 (s, 3H, COCH3),  1.98 (s, 3H, COCH3).  (2,3,4,6-Tetra-O-acetyl-!-D-glucopyranosyl)-(1%4)-O-2,3,6-tri-O-acetyl-!-D- glucopyranosylamine (2.15)164   A mixture of protected cellobiosyl azide 2.14 (500 mg, 0.8 mmol) and Pd/C (10%, 100 mg), in HPLC grade ethyl acetate (30 mL) was stirred under H2 overnight. After completion, the reaction mixture was filtered through Celite and washed with ethyl acetate, and then concentrated under reduced pressure. The crude product was purified by flash column chromatography (petroleum ether:ethyl acetate= 1:1) to afford a white solid 2.15 (300 mg, 0.5 mmol, 62%). ESI MS m/z calcd. for C26H37N3O17: 635.2, Found: 658.4 [M+Na]+.  1H-NMR (CDCl3, 300 MHz): # 5.19  98 (dd, 1H, J2, 3 8.9, J3, 4 9.1 Hz, H3), 5.13 (dd, 1H, J2’, 3’  8.9, J3’, 4’ 9.4 Hz, H3’), 5.04 (dd, 1H, J3’, 4’ 9.4, J4’, 5’ 9.6 Hz, H4’), 4.90 (dd, 1H, J1’, 2’ 8.0, J2’, 3’ 8.9 Hz, H2’), 4.71 (dd, 1H, J1, 2 9.1, J2, 3 8.9 Hz, H2), 4.49 (d, 1H, J1’, 2’ 8.0 Hz, H1’), 4.46 (dd, 1H, J5, 6a 2.1, J6a, 6b 12.1 Hz, H6a), 4.35 (dd, 1H, J5’, 6’a 4.3, J6’a, 6’b 12.3 Hz, H6’a), 4.06 (dd, 1H, J5, 6b 4.1, J6a, 6b 12.1 Hz, H6b), 4.16-4.02 (br, 1H, H1), 4.03 (dd, 1H, J5’, 6’b 2.3, J6’a, 6’b 12.3 Hz, H6’b), 3.70 (dd, 1H, J3, 4 9.1, J4, 5 9.8 Hz, H4), 3.64 (ddd, 1H, J4, 5 9.8, J5, 6a 2.1, J5, 6b 4.1 Hz, H5), 3.57 (m, 1H, H5’), 2.11 (s, 3H, COCH3), 2.07 (s, 3H, COCH3), 2.04(s, 3H, COCH3), 2.01 (s, 3H, COCH3), 2.00 (s, 3H, COCH3), 1.99 (s, 3H, COCH3), 1.96 (s, 3H, COCH3), 1.78-1.89 (m, 2H, NH2).  N-Bromoacetyl-(2,3,4,6-tetra-O-acetyl-!-D-glucopyranosyl)-(1%4)-O-2,3,6-tri-O-acetyl-!-D- glucopyranosylamine (2.16)   Protected cellobiosylamine 2.15 (290 mg, 0.5 mmol) was dissolved in dry DMF (6 mL) under Ar atmosphere, followed by the addition of bromoaceic anhydride (300 mg, 1.2 mmol, 2.5 eqv.) and the reaction mixture was stirred at RT for 4 days. Upon completion of the reaction, the solvent was removed under reduced pressure, and the resulting residue was extracted with ethyl acetate, washed with H2O, saturated NaHCO3 and brine successively. The organic layer was dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. Purification of the resulting residue by flash column chromatography (petroleum ether:ethyl acetate= 1:1) afforded a white solid 2.16. (275 mg, 0.4 mmol, 79%). ESI MS m/z calcd. for C28H38BrNO18: 755.1, Found: 778.4/780.3 [M+Na]+. 1H-NMR (CDCl3, 300 MHz): # 7.06 (d, 1H, J1, NH 9.1 Hz, NH), 5.30 (dd,  99 1H, J2, 3 9.0, J3, 4 9.4 Hz, H3), 5.15 (dd, 1H, J1, NH 9.1, J1, 2 9.1 Hz, H1), 5.13 (d, 1H, J2’, 3’ 9.1, J3’, 4’ 9.1 Hz, H3’), 5.06 (dd, 1H, J3’, 4’ 9.4, J4’, 5’ 9.6 Hz, H4’), 4.93 (dd, 1H, J1’, 2’ 8.0, J2’, 3’ 9.1 Hz, H2’), 4.92 (dd, 1H, J1, 2 9.1, J2, 3  9.1 Hz, H2), 4.50 (d, 1H, J1’, 2’ 8.0 Hz, H1’), 4.45 (m, 1H, H6a), 4.38 (dd, 1H, J5’, 6’a  4.6, J6’a, 6’b  12.6 Hz, H6’a), 4.13 (dd, 1H, J5, 6b  4.1, J6a, 6b  12.1 Hz, H6b), 4.05 (dd, 1H, J5’,6’b  2.1, J6a’,6’b  12.6 Hz, H6’b), 3.83 (d, 2H, J 7.3, CH2BrCO), 3.77-3.75 (m, 2H, H4, H5), 3.66 (m, 1H, H5’), 2.13 (s, 3H, COCH3), 2.10 (s, 3H, COCH3),  2.06 (s, 3H, COCH3), 2.04 (s, 3H, COCH3), 2.03 (s, 3H, COCH3), 2.01 (s, 3H, COCH3), 1.99 (s, 3H, COCH3).13C- NMR (CDCl3, 75 MHz): # 170.9, 170.4, 170.2, 170.1, 169.3, 169.2, 168.9, 166.7 (NCOCH2Br), 100.5, 99.9, 78.5, 76.1, 74.6, 72.8, 72.0,71.5, 70.4, 67.8, 61.7, 61.6 , 28.1 (NCOCH2Br), 20.8, 20.6 (2 C), 20.5 (3 C), 20.4.  N-Bromoacetyl-(!-D-glucopyranosyl)-(1%4)-O-!-D-glucopyranosylamine (2.1)111   To a solution of the protected N-bromoacetyl derivative 2.16 (150 mg, 0.2 mmol) in dry MeOH (30 mL) was added Mg(OMe)2 (final concentration was 0.8 mM in MeOH), and the reaction mixture was allowed to stir at 4 oC  for a day. The basic mixture was neutralized with Amberlite 120 H+ resin, filtered, and the residue was concentrated under reduced pressure. The crude product was purified by flash chromatography (ethyl acetate:MeOH:H2O = 8:2:1) to yield a white solid 2.1 (54 mg, 0.1 mmol, 58%). Compound has approximate 87% purity based on 1H NMR spectrum. ESI MS m/z calcd. for C14H24BrNO11: 461.1, Found: 486.2 [M+Na]+. 1H-NMR (D2O, 300 MHz): # 4.97 (d, 1H, J1, 2 9.2 Hz, H1), 4.49 (d, 1H, J1’, 2’ 8.0 Hz, H1’), 3.95 (s, 2H,  100 CH2BrCO), 3.44-3.98 (m, 10H, H3-H6, H3’-H6’), 3.39 (dd, 1H, J1, 2 9.2, J2, 3 9.2 Hz, H2), 3.29 (dd, 1H, J1’, 2’ 8.0, J2’, 3’ 8.9 Hz, H2’).  (2,3,4,6-Tetra-O-acetyl-!-D-glucopyranosyl)-(1%4)-O-2,3,6-tri-O-acetyl-"/!-D- glucopyranose (2.17)165   Per-O-acetylated cellobiose (3.0 g, 4.4 mmol) was dissolved in dry DMF (33 mL) followed by the addition of hydrazine acetate (0.5 g, 4.9 mmol, 1.2 eqv.). The reaction mixture was stirred at RT overnight. Upon completion, the solvent was removed under reduced pressure. The resulting residue was redissoved in ethyl acetate (250 mL), washed with H2O, saturated NaHCO3 and brine successively. The organic phase was dried over anhydrous MgSO4, filtered, evaporated. The crude product was recrystallized with EtOH to give the resulting hemiacetal 2.17 (2.1 g, 3.3 mmol, 75%). ESI MS m/z calcd. for C26H36O18: 636.2, Found: 659.3 [M+Na]+.         101 (2,3,4,6-Tetra-O-acetyl-!-D-glucopyranosyl)-(1%4)-O-2,3,6-tri-O-acetyl-"-D-glucopyranosyl trichloroacetimidate (2.18)   A mixture of hemiacetal 2.17 (4.0 g, 6.3 mmol) and trichloroacetonitrile (12 mL, 120 mmol, 19 eqv.) in dry DCM (30 mL) was stirred under N2 at 0 oC. A catalytic amount of 1,8- Diazabicyclo[5.4.0]undec-7-ene (0.3 mL) was added to the solution and the reaction mixture was warmed up to RT. The solution was allowed to stir for three hours, and the residue was washed with H2O, saturated NaHCO3, and brine successively. The organic layer was dried over anhydrous MgSO4 and filtered. Solvent was then removed under reduced pressure to yield a white solid 2.18 (3.5 g, 4.5 mmol, 71%). ESI MS m/z calcd. for C28H36ClNO18: 779.1, Found: 802.5 [M+Na]+.  2-Propenyl (2,3,4,6-tetra-O-acetyl-!-D-glucopyranosyl)-(1%4)-O-2,3,6-tri-O-acetyl-!-D- glucopyranoside (2.19)166   The reaction was performed according to the General Procedure for Alkenyl Alcohol Coupling with "-Cellobiosyl Trichloroimidates; Protected trichloroacetimidate 2.18 (1.6 g, 2.0 mmol), allyl alcohol (7 mL, 102 mmol, 51 eqv.), molecular sieve (20 mg), boron trifluoride  102 diethyl etherate (2 mL), and dry DCM (12 mL) were used. The reaction was completed in 5 hours and crude product was purified by flash column chromatography (petroleum ether:ethyl acetate = 2:1) to yield a white solid 2.19 (541 mg, 0.8 mmol, 40%). ESI MS m/z calcd. for C29H40O18: 676.1, Found: 699.4 [M+Na]+. 1H-NMR (CDCl3, 300 MHz): # 5.84 (m, 1H, H2), 5.25 (m, 2H, H1), 5.19 (dd, 1H, J2’, 3’ 9.1, J3’, 4’ 9.6 Hz, H3’), 5.15 (dd, 1H, J2’’, 3’’ 9.6, J3’’, 4’’ 9.4 Hz, H3’’), 5.06 (dd, 1H, J3’’, 4’’ 9.4, J4’’, 5’’ 9.8 Hz, H4’’), 4.93 (dd, 1H, J1’’, 2’’ 7.9, J2’’, 3’’ 9.6 Hz, H2’’), 4.92 (dd, 1H, J1’, 2’ 7.9, J2’, 3’ 9.1 Hz, H2’), 4.53 (dd, 1H, J5’, 6’a  2.1 Hz, H6’a), 4.52 (d, 2H, J1’, 2’ = J1’’, 2’’  7.9 Hz, H1’, H1’’), 4.37 (dd, 1H, J5’’, 6’’a 4.3, J6’’a, 6’’b 12.6 Hz, H6’’a), 4.30 (dd, 1H, J1a, 1b 13.2, J1a, 2 4.8 Hz, H3a), 4.00-4.15 (m, 3H , H3b, H6b’, H6b’’), 3.79 (t, 1H, J3’, 4’ 9.6, J4’, 5’ 9.6 Hz, H4’), 3.66 (ddd, 1H, J4’’, 5’’ 9.8, J5’’, 6’’a 4.3, J5’’, 6’’b 2.1 Hz, H5’’), 3.58 (ddd, 1H, J4’, 5’ 9.6, J5’, 6’a 2.1, J5’, 6’b 4.6 Hz, H5’), 2.13 (s, 3H, COCH3), 2.09 (s, 6H, 2x COCH3), 2.04 (s, 3H, COCH3), 2.02 (s, 6H, 2x COCH3), 1.99 (s, 3H, COCH3).  3-Butenyl (2,3,4,6-tetra-O-acetyl-!-D-glucopyranosyl)-(1%4)-O-2,3,6-tri-O-acetyl-!-D- glucopyranoside (2.20) 132   The reaction was performed according to the General Procedure for Alkenyl Alcohol Coupling with "-Cellobiosyl Trichloroimidates. Protected trichloroacetimidate 2.18 (1.3 g, 1.7 mmol), 3-buten-1-ol (1.4 mL, 16.1 mmol, 10 eqv.), molecular sieve (20 mg), boron trifluoride diethyl etherate (2 mL), and dry DCM (10 mL) were used. The crude product was purified by flash column chromatography (petroleum ether:ethyl acetate= 2:1) to yield a white solid 2.20  103 (550 mg, 0.8 mmol, 47%). ESI MS m/z calcd. for C30H42O18: 690.6, Found: 713.4 [M+Na]+. 1H-NMR (CDCl3, 300 MHz): # 5.75 (m, 1H, H2), 5.18 (dd, 1H, J2’, 3’ 9.6, J3’, 4’ 9.6 Hz, H3’), 5.15 (dd, 1H,  J2’’, 3’’ 9.1, J3’’, 4’’ 9.4 Hz, H3’’), 5.07 (dd, 1H, J3’’, 4’’ 9.4, J4’’, 5’’ 9.6 Hz, H4’’), 5.02-5.11 (m, 2H, H1), 4.93 (dd, 1H, J1’’, 2’’ 7.8, J2’’, 3’’ 9.1 Hz, H2’’), 4.90 (dd, 1H, J1’, 2’ 7.9, J2’, 3’ 9.6 Hz, H2’), 4.52 (d, 1H, J1’’, 2’’ 7.8 Hz, H1’’), 4.52 (dd, 1H, J5’, 6’a  2.1, J6’a, 6’b 12.7 Hz, H6’a), 4.47 (d, 1H, J1’, 2’ 7.9 Hz, H1’), 4.37 (dd, 1H, J5’’, 6’’a 4.6, J6’’a, 6’’b 12.6 Hz, H6’’a), 4.10 (dd, 1H, J5’, 6’b 5.0, J6’a, 6’b 12.6 Hz, H6’b), 4.05 (dd, 1H, J5’’, 6’’b 2.3, J6’’a, 6’’b 12.6 Hz, H6’’b), 3.90 (ddd, 1H, J1a, 1b 9.6, J1a, 2a = J1a, 2b 6.6 Hz, H4a) , 3.78 (dd, 1H, J3’, 4’ 9.6, J4’, 5’ 9.4 Hz, H4’), 3.65 (ddd, 1H, J4’’, 5’’ 9.6, J5’’, 6’’a 4.6, J5’’, 6’’b 2.3 Hz, H5’’), 3.60 (ddd, 1H, J4’, 5’ 9.4, J5’, 6’a 2.1, J5’, 6’b 5.0 Hz, H5’), 3.51 (ddd, 1H, J1a, 1b 9.8, J1b, 2a = J1b, 2b 6.9 Hz , H4b), 2.32 (m, 2H, H3), 2.13 (s, 3H, COCH3), 2.10 (s, 6H, 2xCOCH3), 2.04 (s, 3H, COCH3), 2.03 (s, 3H, COCH3), 2.02 (s, 3H, COCH3), 1.99 (s, 3H, COCH3).  4-Pentenyl (2,3,4,6-tetra-O-acetyl-!-D-glucopyranosyl)-(1%4)-O-2,3,6-tri-O-acetyl-!-D- glucopyranoside (2.21)132   The reaction was performed according to the General Procedure for Alkenyl Alcohol with "- Cellobiosyl Trichloroimidates. Protected trichloroacetimidate 2.18 (600 mg, 0.8 mmol), 4- pente-1-nol (1.8 mL, 17.3 mmol, 22 eqv.), molecular sieve (20 mg), boron trifluoride diethyl etherate (2.5 mL), and dry DCM (30 mL) were used. The reaction was completed in 6 hours and crude product was purified by flash column chromatography (petroleum ether:ethyl acetate = 2:1)  104 to yield a white solid 2.21 (275 mg, 0.4 mmol, 50%). ESI MS m/z calc C31H44O18: 704.3, Found: 727.6 [M+Na]+. 1H-NMR (CDCl3, 300 MHz): # 5.75 (m, 1H, H2), 5.15 (dd, 1H, J2’, 3’ 9.1, J3’, 4’ 9.1 Hz, H3’), 5.12 (dd, 1H, J2’’, 3’’ 9.1, J3’’, 4’’ 9.6 Hz, H3’’), 5.04 (dd, 1H, J3’’, 4’’ 9.6, J4’’, 5’’ 9.6 Hz, H4’’), 5.01-5.03 (m, 2H, H1), 4.90 (dd, 1H, J1’’, 2’’ 8.0, J2’’, 3’’ 9.1 Hz, H2’’), 4.87 (dd, 1H, J1’, 2’ 8.0, J2’, 3’ 9.1 Hz, H2’), 4.48 (d, 1H, J1’’, 2’’ 8.0 Hz, H1’’), 4.42 (d, 1H, J1’, 2’ 8.0 Hz, H1’), 4.49 (m, 1H, H6’a), 4.35 (dd, 1H, J5’’, 6’’a 4.3, J6’’a, 6’’b 12.3 Hz, H6’’a), 4.07 (dd, 1H, J5’, 6’b 5.0, J6’a, 6’b 12.1 Hz, H6’b), 4.03 (dd, 1H, J5’’, 6’’b 1.4, J6’’a, 6’’b 12.1 Hz, H6’’b), 3.82 (dt, 1H, J4a, 5a = J4b, 5a 6.2, J5a, 5b 9.6 Hz, H5a), 3.75 (dd, 1H, J3’, 4’ 9.1, J4’, 5’ 9.6 Hz, H4’), 3.64 (ddd, 1H, J4’’, 5’’ 9.6, J5’’, 6’’a 4.1, J5’’, 6’’b 2.1 Hz, H5’’), 3.57 (ddd, 1H, J4’, 5’ 9.6, J5’, 6’a 2.1, J5’, 6’b 4.8 Hz, H5’), 3.45 (dt, 1H, J4a, 5b = J4b, 5b 6.9, J5a, 5b 9.6 Hz, H5b), 2.10 (s, 3H, COCH3), 2.06 (s, 3H, COCH3), 2.04 (m, 2H, H3), 2.00 (s, 3H, COCH3), 2.01 (s, 6H, 2x COCH3), 1.99 (s, 3H, COCH3), 1.96 (s, 3H, COCH3), 1.63 (m, 2H, H4).  (R, S)-2,3-Epoxypropyl (2,3,4,6-tetra-O-acetyl-!-D-glucopyranosyl)-(1%4)-O-2,3,6-tri-O- acetyl-!-D-glucopyranoside (2.22)167,132   The reaction was performed according to the General Procedure for Epoxidation of Alkenyl Glycoside. Propenyl glycoside 2.19 (150 mg, 0.2 mmol), mCPBA (153 mg, 0.9 mmol, 4 eqv.) and dry DCM (8 mL) were used. The reaction was complete overnight, and the crude product was purified by flash column chromatography (petroleum ether:ethyl acetate= 1:1) to yield a white solid 2.22 (80 mg, 0.1 mmol, 52%). ESI MS m/z calcd. for C29H40O19: 692.2, Found:  105 715.5 [M+Na]+. 1H-NMR (CDCl3, 300 MHz): # 5.19, 5.18 (2xdd, 1H, J2’, 3’ 9.2, J3’, 4’ 9.2 Hz, H3’), 5.15 (dd, 1H, J2’’, 3’’ 9.2, J3’’, 4’’ 9.6 Hz, H3’’), 5.06 (dd, 1H, J3’’, 4’’ 9.2, J4’’, 5’’ 9.6 Hz, H4’’), 4.93 (dd, 1H, J1’’, 2’’ 8.9, J2’’, 3’’ 9.2 Hz, H2’’), 4.90 (dd, 1H, J1’, 2’ 7.9, J2’, 3’ 9.2 Hz, H2’), 4.57 (m, 1H, H6’a), 4.51 (d, 2H, J1’, 2’ = J1’’, 2’’ 7.9 Hz, H1’, H1’’), 4.36 (dd, 1H, J5’’, 6’’a  4.4, J6’’a, 6’’b  12.3 Hz, H6’’a), 4.10 (m, 1H, H6’b), 4.05 (dd, 1H, J5’, 6’’b  2.7, J6’’a, 6’’b 11.9 Hz, H6’’b), 3.79 (dd, 1H, J3’,4’ 9.2 Hz, H4’), 3.67 (m, 1H, H5’’), 3.63 (m, 1H, H5’), 3.47 (dd, 1H, J2, 3a 6.5, J3a, 3b 11.6 Hz, H3a), 3.12 (m, 1H, H2), 2.78 (m, 1H, H3b), 2.62 (m, 1H, H1a), 2.54 (dd, 1H, 2.39, 4.78, H1b), 2.12 (s, 3H, COCH3), 2.09 (s, 3H, COCH3), 2.05 (s, 3H, COCH3), 2.03 (s, 3H, COCH3), 2.02 (s, 3H, COCH3), 2.01 (s, 3H, COCH3), 1.98 (s, 3H, COCH3).  (R, S)-3,4-Epoxybutyl (2,3,4,6-tetra-O-acetyl-!-D-glucopyranosyl)-(1%4)-O-2,3,6-tri-O- acetyl-!-D-glucopyranoside (2.23)132   The reaction was performed according to the General Procedure for Epoxidation of Alkenyl Glycoside. Butenyl glycoside 2.20 (250 mg, 0.4 mmol), mCPBA (312 mg, 1.9 mmol, 5.2 eqv.) and dry DCM (15 mL) were used. The reaction was stirred overnight, and the crude product was purified by flash column chromatography (petroleum ether:ethyl acetate= 2:1) to yield a white solid 2.23 (204 mg, 0.3 mmol, 75%).  ESI MS m/z calcd. for C30H42O19: 706.2, Found 729.7 [M+Na]+. 1H-NMR (CDCl3, 300 MHz): # 5.19, 5.18 (2xdd, 1H, J2’, 3’ 9.2, J3’, 4’ 9.6 Hz, H3’), 5.15 (d, 1H, J2’’, 3’’ 9.2, J3’’, 4’’ 9.2 Hz, H3’’), 5.06 (dd, 1H, J3’’, 4’’ 9.2, J4’’,5’’ 9.6 Hz, H4’’), 4.93 (dd, 1H, J1’’, 2’’ 8.9, J2’’, 3’’ 9.2 Hz, H2’’), 4.88-4.94 (2xdd, 1H, H2’), 4.49 (dd, 1H, J5’, 6’a 3.75, J6’a, 6’b  106 11.9 Hz, H6’a), 4.52 (d, 1H, J1’’, 2’’  8.9 Hz, H1’’), 4.50 (d, 1H, J1’, 2’ 7.5 Hz, H1’), 4.37 (dd, 1H, J5’’, 6’’a  4.4, J6’’a, 6’’b  12.6 Hz, H6’’a), 4.10 (dd, 1H, J5’, 6’b 4.8, J6a’, 6’b 11.9 Hz, H6’b), 4.05 (d, 1H, J6a’’, 6’’b  11.9, H6’’b), 3.97 (m, 1H, H4a), 3.77 (dd, 1H, J3’, 4’ 9.6, J4’, 5’ 9.2 Hz, H4’), 3.56-3.67 (m, 3H, H5’, H5’’, H4b), 2.88 (m, 1H, H2), 2.76 (m, 1H, H1a), 2.44 (dd, 1H, J3, 4b 4.8, J4a, 4b 2.4 Hz, H1b) , 2.13 (s, 3H, COCH3), 2.09 (s, 3H, COCH3), 2.03 (s, 3H, COCH3), 2.02 (s, 3H, COCH3), 2.01 (s, 3H, COCH3), 1.99 (s, 3H, COCH3), 2.12 (s, 3H, COCH3), 1.65-1.95 (m, 2H, H3).  (R, S)-4,5-Epoxypentyl (2,3,4,6-tetra-O-acetyl-!-D-glucopyranosyl)-(1%4)-O-2,3,6-tri-O- acetyl-!-D-glucopyranoside (2.24)132   The reaction was performed according to the General Procedure for Epoxidation of Alkenyl Glycoside. Pentenyl glycoside 2.21 (250 mg, 0.4 mmol), mCPBA (305 mg, 1.8 mmol, 5 eqv.) and dry DCM (15 mL) were used. The reaction was allowed to stir overnight, and the crude product was purified by flash column chromatography (petroleum ether:ethyl acetate = 2.5:1) to yield white solid 2.24 (80 mg, 0.1 mmol, 30%). ESI MS m/z calcd. for C31H44O19: 720.3, Found: 743.7 [M+Na]+. 1H-NMR (CDCl3, 300 MHz): # 5.17 (dd, 1H, J2’, 3’ 9.2, J3’, 4’ 9.6 Hz, H3’), 5.14 (dd, 1H, J2’’, 3’’ 9.2, J3’’, 4’’ 9.2 Hz, H3’’), 5.06 (dd, 1H, J3’’, 4’’ 9.2, J4’’, 5’’ 9.6 Hz, H4’’), 4.92 (dd, 1H, J1’’, 2’’ 8.2, J2’’, 3’’ 9.2 Hz, H2’’), 4.89 (2xdd, 1H, 8.2, 8.5, H2’), 4.53 (m, 1H, H6’a), 4.51 (d, 1H, J1’’, 2’’  8.2, H1’’), 4.46 (d, 1H, J1’, 2’, 7.8, H1’), 4.36 (dd, 1H, J5’’, 6’’a  4.4, J6’’a, 6’’b 12.3, H6’’a), 4.09 (m, 1H, J5’, 6’b  5.1, J6’a, 6’b  11.9, H6’b), 4.05 (dd, 1H, J5’’, 6’’b  2.1, J6a’’, 6’’b  12.6,  107 H6’’b), 3.88 (m, 1H, H5a), 3.77 (dd, 1H, J3’, 4’ 9.6, J4’, 5’ 9.2 Hz, H4’), 3.65 (ddd, 1H, J4’’, 5’’ 9.6, J5’’, 6’’a 4.4, J5’’, 6’’b  2.1 Hz, H5’’), 3.58 (m, 1H, H5’), 3.52 (m, 1H, H5b), 2.81 (m, 1H, H2), 2.74 (m, 1H, H1a), 2.40 (dd, 1H, J4, 5b 5.4, J5a, 5b 2.7 Hz, H1b) , 1.98 (s, 3H, COCH3), 2.01 (s, 3H, COCH3), 2.02 (s, 6H, 2xCOCH3), 2.03 (s, 3H, COCH3), 2.09 (s, 3H, COCH3), 2.13 (s, 3H, COCH3), 1.45-1.78 (m, 4H, H3, H4).   (R, S)-2,3-Epoxypropyl (!-D-glucopyranosyl)-(1%4)-O-!-D-glucopyranoside (2.2)132   The deacetylation was conducted according to the general procedure Deacetylation by Sodium Methoxide.  A suspension of protected epoxypropyl glycoside 2.22 (100 mg, 0.1 mmol) in 50 mM sodium methoxide/methanol solution (20 mL) was stirred at RT for an hour, and the crude product was purified by flash column chromatography (ethyl acetate:MeOH:H2O = 6:2:1) to yield a white solid 2.2 (50 mg, 0.1 mmol, 90%). Compound has approximate 90% purity based on 1H NMR spectrum. ESI MS m/z calcd. for C15H26O12: 398.1, Found: 421.2 [M+Na]+. 1H- NMR (D2O, 300 MHz): # 4.47-4.53 (m, 2H, H1’, H1’’), 4.07-4.23 (m, 1H, H3a), 3.25-3.97 (m, 14H, H2’-H6’, H2’’-H6’’, H3b, H2), 2.95 (m, 1H, H1a), 2.67 (m, 1H, H1b).      108 (R, S)-3,4-Epoxybutyl (!-D-glucopyranosyl)-(1%4)-O-!-D-glucopyranoside (2.3)132   The deacetylation was conducted according to the general procedure Deacetylation by Sodium Methoxide. A suspension of protected epoxybutyl glycoside 2.23 (100 mg, 0.1 mmol) in 50 mM sodium methoxide/methanol solution (20 mL) was stirred at RT for an hour, and the resulting syrup was purified by flash column chromatography (ethyl acetate:MeOH:H2O = 8:2:1), to afford 2.3 as a white solid (52 mg, 0.1 mmol, 90%). Compound has approximate 90% purity based on 1H NMR spectrum. ESI MS m/z calcd. for C16H28O12: 412.1, Found: 435.3 [M+Na]+. 1H-NMR (D2O, 300 MHz): # 4.49 (d, 2H, J1’, 2’ = J1’’, 2’’ = 7.8 Hz, H1’, H1’’), 4.01-4.09 (m, 1H, H4a), 3.21-3.98 (m, 14H, H2’-H6’, H2’’-H6’’, H4b, H2), 2.91 (m, 1H, J1a, 2 4.1 Hz, H1a), 2.70 (m, 1H, J1b, 2 3.7 Hz, H1b), 1.94-2.05 (m, 1H, H3a), 1.68-1.79 (m, 1H, H3b).  (R, S)-4,5-Epoxypentyl (!-D-glucopyranosyl)-(1%4)-O-!-D-glucopyranoside (2.4)132   The deacetylation was conducted according to the general procedure Deacetylation by Sodium Methoxide. A suspension of protected epoxypentyl glycoside 2.24 (90 mg, 0.1 mmol) in 50 mM sodium methoxide/methanol solution (15 mL) was stirred at RT for 30 minutes, and the resulting product was purified byflash column chromatography (ethyl acetate:MeOH:H2O = 10:2:1) to afford a white soild 2.4 (46 mg, 0.1 mmol, 90%). Compound has approximate 85%~90% purity  109 based on 1H NMR spectrum. ESI MS m/z calcd. for C17H30O112: 426.2, Found: 449.4 [M+Na]+. 1H-NMR (D2O, 300 MHz): # 4.47-4.49 (t, 2H, J1’, 2’ = J1’’, 2’’ = 7.0 Hz, H1’, H1’’), 3.13-3.97 (m, 15H, H2’-H6’, H2’’-H6’’, H5, H2), 2.90 (m, 1H, J1a, 2 4.3 Hz, H1a), 2.67 (m, 1H, J1b, 2 3.9 Hz, H1b), 1.52-1.81 (m, 4H, H3, H4).  Methyl 2,3,4,6-tetra-O-benzyl-"-D-glucopyranoside (2.25)168   Methyl-"-D-glucoside (30 g, 54.2 mmol) was dissolved in dry DMF (350 mL) under N2 at 0 oC. NaH (10 g, 60% in mineral oil, 250 mmol, ~ 4.6 eqv.) was added in portions over 15 minutes. Benzyl bromide (110 mL, 630 mmol, 11.6 eqv.) was added via an addition funnel equipped with a drying tube while the solution was vigorously stirred at 0 oC. The reaction mixture was then allowed to warm up to RT and stirred overnight. After completion, the reaction was quenched by slow addition of MeOH (over 40 minutes) and the solvent was removed under reduced pressure. The residue was redissolved in ethyl acetate, and washed with H2O and brine. The organic phase was dried over anhydrous MgSO4, filtered, and concentrated. The yellow oil product was purified by flash column chromatography (petroleum ether:ethyl acetate = 10:1 ~ 3:1) to give a white solid 2.25 (74 g, 133 mmol, 86%). ESI MS m/z calcd. for C35H38O6: 554.3, Found: 577.4 [M+Na]+. 1H-NMR (CDCl3, 300 MHz): # 7.33-7.09 (m, 20H, Ar), 4.98-4.40 (m, 8H, CH2ph), 4.61 (d, 1H, J1, 2 3.6 Hz, H1), 3.97 (dd, 1H, J2, 3 9.4, J3, 4 8.9 Hz, H3), 3.75 (m, 1H, H5), 3.71-3.66 (m, 2H, H6), 3.63 (dd, 1H, J3, 4 , 8.9  J4, 5 9.6 Hz, H4), 3.53 (dd, 1H, J1, 2 3.6 Hz, J2, 3 9.4, H2), 3.34 (s, 3H, OMe).  110 2,3,4,6-Tetra-O-benzyl-"-D-glucopyranose (2.26) 166   2.25 (50 g, 90 mmol) was dissolved in AcOH (456 mL), and the solution was allowed to heat up to 80 oC, followed by the addition of pre-warmed 1 M H2SO4 (200 mL). The solution was stirred at 80 oC for 5 days. After cooling to RT, the reaction mixture was poured into deionized H2O (4.5 L) and was allowed to sit for 5 days at RT. The resulting precipitate was filtered and purified by flash column chromatography (petroleum ether:ethyl acetate = 5:1 ~ 1:1) followed by crystallization from MeOH to afford 2.26 as a white solid (26.5 g, 49 mmol, 54%).  ESI MS m/z calcd. for C34H36O6: 540.3, Found: 563.1 [M+Na]+.  2,3,4,6-Tetra-O-benzyl-D-glucono-1,5-lactone (2.27)169   A solution of 2.26 (4 g, 7.4 mmol) in a mixture of DMSO and acetic anhydride (DMSO:Ac2O = 3:2) was stirred at RT overnight. Upon completion of the reaction, the unreacted acetic anhydride was quenched with H2O (200 mL). The crude product was extracted with ethyl acetate (2x 400 mL), washed successively with H2O, saturated NaHCO3 and brine. The organic layer was dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. A yellow syrup was obtained and purified via flash column chromatography (petroleum ether:ethyl acetate = 10:1 ~ 7:1) to yield 2.27 as a syrup (3.1 g, 5.8 mmol, 78%). ESI MS m/z calcd. for C34H34O6: 538.2,  111 Found: 561.3 [M+Na]+. 1H-NMR (CDCl3, 400 MHz): # 7.14-7.36 (m, 20H, Ar), 4.95 (d, 1H, J 11.2, CH2ph), 4.71-4.46 (m, 7H, CH2ph), 4.43-4.36 (m, 1H, H5), 4.08 (d, 1H, J2, 3 6.5 Hz, H2), 3.91 (dd, 1H, J3, 4 6.9, J4, 5 8.2 Hz, H4), 3.87 (dd, 1H, J2, 3 6.5, J3, 4 6.9 Hz, H3), 3.69 (dd, 1H, J5, 6a 2.4, J6a, 6b 11.0 Hz, H6a), 3.63 (dd, 1H, J5, 6b 3.1, J6a, 6b 11.0 Hz, H6b).  But-3-en-1-ylmagnesium bromide   A dry three necked round bottom flask was charged with activated Mg powder (1.3 g, 54.6 mmol, 1.1 eqv.) and dry THF (~ 20 mL) under an argon atmosphere. A solution of 4-bromo-1- butene (5 mL, 50 mmol) in dry THF (16 mL) was added dropwise until gentle reflux (total addition time was over 3 hours). After addition, the reaction mixture was stirred at RT for another hour. The final concentration was 0.9 M in THF (the concentration of Grignard reagent was measured by titration with 0.1M HCl containing phenolphthalein (0.1% in H2O/EtOH = 30/70 as indicator).  Pent-4-en-1-ylmagnesium bromide   The synthesis of Grignard reagent of magnesium bromo pentene followed the essentially samemethod. Final concentration was 0.7 M.    112  4-(1-Hydroxyl-2,3,4,6-tetra-O-benzyl-!-D-glucopyranosyl)-but-1-ene (2.28)   A dry three necked flask was charged with benzylated lactone 2.27 (1.7 g, 3.1 mmol) in dry THF (18.5 mL) under a N2 atmosphere and the reaction mixture was cooled down to -78 oC using an acetone and dry ice bath. Freshly made but-3-enyl magnesium bromide (0.9 M, 19 mL, 4.4 eqv.) was added dropwise into the flask, and the reaction mixture was allowed to stir at -78 oC for another hour. Upon completion, the reaction mixture was warmed up to 0 oC followed by the addition of ammonium chloride (250 mg), and stirred for another 5 minutes. The mixture was then filtered, and the solution was concentrated under reduced pressure. The resulting residue was redissoved in DCM and washed successively with H2O, 1 M HCl, and brine. The organic phase was dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The crude product 2.28 was used in the next step without further purification. ESI MS m/z calcd. for C38H42O6: 594.3, Found: 617.5 [M+Na]+.  5-(1-Hydroxyl-2,3,4,6-tetra-O-benzyl-!-D-glucopyranosyl)-pent-1-ene (2.29)   A dry three necked flask was charged with benzylated lactone 2.27 (1.6 g, 2.9 mmol) in dry THF (22 mL) under a N2 atmosphere and was cooled down to -78 oC using an acetone and dry ice bath. Freshly made pentenyl Grignard reagent (0.7 M, 25mL, 4.5 eqv.) was added dropwise into  113 the flask, and the reaction mixture was stirred at -78 oC for another hour. The reaction was warmed up to 0 oC after completion, quenched with ammonium chloride (250 mg), and stirred for another 5 minutes. After filtration, the solution was concentrated under reduced pressure. The resulting residue was redissolved in DCM and washed successively with H2O, 1 M HCl and brine. The organic layer was dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The crude product 2.29 was used in the next step without further purification. ESI MS m/z calcd. for C39H44O6: 608.3, Found:  [M+Na]+.  4-(2,3,4,6-Tetra-O-benzyl-!-D-glucopyranosyl)-but-1-ene (2.30)138   To the crude product of hemiketal 2.28 was added acetonitrile (35 mL), and the solution was cooled down to -40 oC using an acetone and dry ice bath. After the addition of triethylsilane (3.1 mL) and boron trifluoride diethyl etherate (2.6 mL), the reaction mixture was stirred at -40 oC for 40 minutes. Upon completion, the reaction mixture was warmed up to 0 oC followed by the addition of saturated NaHCO3 (40 mL), and extraction with ethyl acetate (80 mL). The organic phase was washed with H2O and brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. Purification of the resulting residue by flash column chromotagraphy (petroleum ether: ethyl acetate= 18:1) afforded 2.30 as a white solid (1.2 g, 2.1 mmol, 67% yield over two steps). ESI MS m/z calcd. for C38H42O5: 578.3 Found: 601.4 [M+Na]+. 1H-NMR (CDCl3, 400 MHz): # 7.19-7.39 (m, 20H, Ar), 5.85 (m, 1H, H2), 5.04 (m, 1H, H1a), 4.98 (m, 1H, H1b), 4.86-4.50 (m, 8H, CH2ph), 3.77-3.70 (m, 3H, H2’, H3’, H6’a), 3.65 (dd, 1H, J3’, 4’ 9.1, J4’,  114 5’ 9.1 Hz, H4’), 3.41 (ddd, 1H, J4’, 5’ 9.1, J5’, 6’a 2.1, J5’, 6’b 4.0 Hz, H5’), 3.33 (m, 1H, H1’), 3.27 (dd, 1H, J5’, 6’b 4.0, J6’a, 6’b 9.4 Hz, H6’b), 2.32 (m, 1H, H3a), 2.16 (m, 1H, H3b), 1.95 (m, 1H, H4a), 1.59 (m, 1H, H4b).  5-(2,3,4,6-Tetra-O-benzyl-!-D-glucopyranosyl)-pent-1-ene (2.31)138   To the crude product of hemiketal 2.29 was added acetonitrile (30 mL), and the solution was cooled down to -40 oC using acetone and dry ice bath. After the addition of triethylsilane (2.8 mL) and boron trifluoride diethyl etherate (2.3 mL), the reaction was stirred at -40 oC for 40 minutes. Upon completion, the reaction mixture was warmed up to 0 oC followed by the addition of saturated NaHCO3 (30 mL), and extraction with ethyl acetate (60 mL). The organic phase was washed with H2O and brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. Purification of the resulting residue by flash column chromatography (petroleum ether: ethyl acetate= 20:1) afforded 2.31 as a white solid (1.2 g, 2.0 mmol, 69% yield over two steps).  ESI MS m/z calcd. for:  Found C39H44O5: 592.3, Found: 615.4 [M+Na]+. 1H-NMR (CDCl3400 MHz): # 7.39-7.19 (m, 20H, Ar), 5.85 (m, 1H, H2), 5.04 (m, 1H, H1a), 4.98 (m, 1H, H1b), 4.97- 4.57 (m, 8H, CH2ph), 3.77-3.69 (m, 3H, H2’, H3’, H6’a), 3.64 (dd, 1H, J3’, 4’ 9.1, J4’, 5’ 9.4 Hz, H4’), 3.42 (ddd, 1H, J4’,5’ 9.4, J5’, 6’a 2.1, J5’, 6’b 4.3 Hz, H5’), 3.33-3.24 (m, 2H, H1’, H6’b), 2.10 (m, 2H, H3), 1.87 (m, 1H, H5a), 1.68 (m, 1H, H4a), 1.50 (m, 2H, H4b, H5b).   115  4-(!-D-Glucopyranosyl)-but-1-ene (2.32)   Deprotection was performed according to the general method De-benzylation of C-linked Glucoside. Protected butene derivative 2.30 (1.2 g, 2.1 mmol) was deprotected with liquid NH3 (70 mL) and Na(s) (522 mg) in dry THF (75 mL). The reaction was completed in 30 minutes and 2.32 was obtained as a white solid (326 mg, 1.5 mmol, 71%). ESI MS m/z calcd. for C10H18O5: 218.1, Found: 241.3 [M+Na]+.  5-(!-D-Glucopyranosyl)-pent-1-ene (2.33)   Deprotection was performed according to the general method De-benzylation of C-linked Glucoside. Protected pentene derivative 2.31 (1.1 g, 1.9 mmol) was deprotected with liquid NH3 (70 mL) and Na(s) (600 mg) in dry THF (30 mL). The reaction was completed in 30 minutes and 2.33 was obtained as a white solid (290 mg, 1.2 mmol, 66%). ESI MS m/z calcd. for C11H20O5: 232.1, Found: 255.4 [M+Na]+.      116 4-[(2,3,4,6-Tetra-O-acetyl-!-D-glucopyranosyl)-(1%4)-O-2,3,6-tri-O-acetyl-!-D- glucopyranosyl]-but-1-ene (2.34)137   Synthesis of 2.34 was performed according to General Procedure for Oligosaccharide Synthesis with Abg 2F6 Glycosynthase. C-!-butenyl glucoside 2.32 (390 mg, 1.79 mmol) and "-GluF (421 mg, 2.3 mmol, 1.3 eqv.) were dissolved in 100 mM pH 7.0 ammonium acetate buffer (35 mL), followed by the addition of of Abg 2F6 glycosynthase (8 mg), and the reaction mixture was incubated at RT for two days. After the solvent was removed by lyophilization, the crude product was acetylated according to General Acetylation of Free Sugars (5 mL pyridine and 1.5 mL acetic anhydride). Purification of acetylated disaccharide by flash column chromatography (petroleum ether:ethyl acetate = 2:1) afforded a white solid 2.34 (160 mg, 0.2 mmol, 13% yield over two steps). ESI MS m/z calcd. for C30H42O17: 674.2, Found: 697.4 [M+Na]+. 1H-NMR (CDCl3, 400 MHz): # 5.76 (ddt, 1H, J2, 3 6.9, J1a, 2 9.9, J1b, 2 16.3 Hz, H2), 5.15 (dd, 1H, J2’, 3’ 9.4, J3’, 4’ 9.4 Hz, H3’), 5.14 (dd, 1H, J2’’, 3’’ 9.1, J3’’, 4’’ 9.5 Hz, H3’’), 5.06 (dd, 1H, J3’’, 4’’ 9.5, J4’’, 5’’ 9.4 Hz, H4’’), 4.97-5.04 (m, 2H, H1), 4.94 (dd, 1H, J1’’, 2’’ 8.0, J2’’, 3’’ 9.1 Hz, H2’’), 4.80 (t, 1H, J1’, 2’ 9.4, J2’, 3’ 9.4 Hz, H2’), 4.51 (d, 1H, J1’’, 2’’ 8.0 Hz, H1’’), 4.47 (dd, 1H, J5’, 6’a 1.8, J6’a, 6’b 11.9 Hz, H6’a), 4.38 (dd, 1H, J5’’, 6’’a 4.3, J6’’a, 6’’b 12.5 Hz, H6’’a), 4.09 (dd, 1H, J5’, 6’b 5.5, J6’a, 6’b 11.9 Hz, H6’b), 4.05 (dd, 1H, J5’’, 6’’b 2.1, J6’’a, 6’’b 12.5 Hz, H6’’b), 3.72 (t, 1H, J3’, 4’ 9.4, J4’, 5’ 9.4 Hz, H4’), 3.65 (ddd, 1H, J4’’, 5’’ 9.4, J5’’, 6’’a 4.3, J5’’, 6’’b 2.1 Hz, H5’’), 3.51 (ddd, 1H, J4’, 5’ 9.4, J5’, 6’a 1.8, J5’, 6’b 5.5 Hz, H5’), 3.38 (m, 1H, J1’, 2’ 9.4 Hz, H1’), 2.18-2.26 (m,  117 2H, H3), 1.99 (s, 3H, COCH3), 2.02 (s, 6H, 2xCOCH3), 2.04 (s, 6H, 2xCOCH3), 2.09 (s, 3H, COCH3), 2.13 (s, 3H, COCH3), 1.41-1.63 (m, 2H, H4).  5-[(2,3,4,6-Tetra-O-acetyl-!-D-glucopyranosyl)-(1%4)-O-2,3,6-tri-O-acetyl-!-D- glucopyranosyl]-pent-1-ene (2.35)137   Synthesis of 2.35 was performed according to General Procedure for Oligosaccharide Synthesis with Abg 2F6 Glycosynthase. C-!-Pentenyl glucoside 2.33 (400 mg, 1.7 mmol) and "-GluF (405 mg, 2.2 mmol, 1.3 eqv.) were dissolved in 100 mM pH 7.0 ammonium acetate buffer (26 mL), followed by the addition of of Abg 2F6 glycosynthase (8 mg), and the reaction mixture was incubated at RT for 1.5 days. After the solvent was removed by lyophilization, the crude product was acetylated according to General Acetylation of Free Sugar (5 mL pyridine and 1.5 mL acetic anhydride). Purification of desired acetylated disaccharide by flash column chromatography (petroleum ether:ethyl acetate= 3:1) afforded a white solid 2.35 (185 mg, 0.27 mmol, 16% yield over two steps). ESI MS m/z calcd. for C31H44O17: 688.3, Found: 711.4 [M+Na]+. 1H-NMR (CDCl3, 300 MHz): # 5.76 (ddt, 1H, J2,3 6.9, J1a, 2 9.9, J1b, 2 16.3 Hz, H2), 5.15 (t, 1H, J2’, 3’ 9.4, J3’, 4’ 9.4 Hz, H3’), 5.14 (dd, 1H, J2’’, 3’’ 9.1, J3’’, 4’’ 9.4 Hz, H3’’), 5.07 (t, 1H, J3’’, 4’’ 9.4, J4’’, 5’’ 9.4 Hz, H4’’), 4.90-5.02 (m, 3H, H1, H2’’), 4.79 (t, 1H, J1’, 2’ 9.4, J2’, 3’ 9.4 Hz, H2’), 4.48 (d, 1H, J1’’, 2’’ 8.0 Hz, H1’’), 4.45 (dd, 1H, J5’, 6’a 1.6, J6’a, 6’b 11.9 Hz, H6’a), 4.37 (dd, 1H, J5’’, 6’’a 4.1, J6’’a, 6’’b 12.6 Hz, H6’’a), 4.09 (dd, 1H, J5’, 6’b 5.5, J6’a, 6’b 11.9 Hz, H6’b), 4.04 (dd, 1H, J5’’, 6’’b 2.3, J6’’a, 6’’b 12.6 Hz, H6’’b), 3.71 (t, 1H, J3’, 4’ 9.4, J4’, 5’ 9.4 Hz, H4’), 3.64 (ddd,  118 1H, J4’’, 5’’ 9.4, J5’’, 6’’a 4.1, J5’’, 6’’b 2.3 Hz, H5’’), 3.52 (ddd, 1H, J4’, 5’ 9.4, J5’, 6’a 1.6, J5’, 6’b 5.5 Hz, H5’), 3.33-3.39 (m, 1H, H1’), 1.98-2.08 (m, 2H, H3), 1.99 (s, 3H, COCH3), 2.01 (s, 6H, 2xCOCH3), 2.03 (s, 3H, COCH3), 2.04 (s, 3H, COCH3), 2.09 (s, 3H, COCH3), 2.12 (s, 3H, COCH3), 1.37-1.61 (m, 4H, H4, H5).  (R, S)-1,2-Epoxy-4-[(2,3,4,6-tetra-O-acetyl-!-D-glucopyranosyl)-(1%4)-O-2,3,6-tri-O-acetyl- !-D-glucopyranosyl] butane (2.36)137   The procedure followed the General Procedure for Epoxidation of Alkenyl Glycosides; A solution of C-butenyl cellobioside 2.34 (130 mg, 0.2 mmol) and mCPBA (156 mg, 0.9 mmol, 5 eqv.) in dry DCM (15 mL) was stirred for 6 hours, and the crude product was purified by flash column chromatography (petroleum ether:ethyl acetate= 1:1 ~ 1:1.5) to yield a white solid 2.36 (120 mg, 0.2 mmol, 87%). ESI MS m/z calcd. for C30H42O18: 690.2, Found: 713.5 [M+Na]+. 1H-NMR (CDCl3, 400 MHz): # 5.15 (2xt, 2H, J2’, 3’ = J2’’, 3’’ = J3’, 4’ = J3’’, 4’’ 9.4 Hz, H3’, H3’’), 5.08 (t, 1H, J3’’, 4’’ 9.4, J4’’, 5’’ 9.4 Hz, H4’’), 4.94 (dd, 1H, J1’’, 2’’ 7.9, J2’’, 3’’ 9.4 Hz, H2’’), 4.80, 4.81 (2xdd, 1H, J1’, 2’ 7.9, J2’, 3’ 9.4 Hz, H2’), 4.51 (d, 1H, J1’’, 2’’ 7.9 Hz, H1’’), 4.78 (dd, 1H, J5’, 6’a 2.1, J6’a, 6’b 12.2 Hz, H6’a), 4.47 (m, 1H, H6’’a), 4.09, 4.07 (2xdd, 1H, J5’, 6’b 5.2, J6’a, 6’b 12.2 Hz, H6’b), 4.05 (dd, 1H, J5’’, 6’’b 2.4, J6’’a, 6’’b 12.2 Hz, H6’’b), 3.73, 3.72 (2xt, 1H, J3’, 4’ 9.1, J4’, 5’ 9.1 Hz, H4’), 3.66 (ddd, 1H, J4’’, 5’’ 9.4, J5’’, 6’’a 4.0, J5’’, 6’’b 2.4 Hz, H5’’), 3.53 (m, 1H, H5’), 3.38-3.45 (m, 1H, H1’), 2.88-2.92 (m, 1H, H2), 2.75 (dd, 1H, J1a, 1b 4.6, J1a, 2 4.3, H1a), 2.47 (m, 1H, H1b), 1.99 (s, 3H, COCH3), 2.02 (s, 6H, 2x COCH3), 2.04 (s, 3H, COCH3), 2.05 (s, 3H,  119 COCH3), 2.10 (s, 3H, COCH3), 2.12 (s, 3H, COCH3), 2.10-2.19 (m, 1H, H3a), 1.35-1.90 (m, 3H, H3b, H4).  (R, S)-1,2-Epoxy-5-[(2,3,4,6-tetra-O-acetyl-!-D-glucopyranosyl)-(1%4)-O-2,3,6-tri-O-acetyl- !-D-glucopyranosyl] pentane (2.37)137   The procedure followed the General Procedure for Epoxidation of Alkenyl Glycosides; A solution of C-pentenyl cellobioside 2.35 (114 mg, 0.2 mmol) and mCPBA (117 mg, 0.7 mmol, 4 eqv.) in dry DCM (14 mL) was stirred for 4 hours, and the crude product was purified by flash column chromatography (petroleum ether:ethyl acetate= 1.5:1 ~ 1:1.5) to yield a white solid 2.37 (90 mg, 0.1 mmol, 75%). ESI MS m/z calcd. for C31H44O18: 704.3, Found: 727.5 [M+Na]+. 1H- NMR (CDCl3, 400 MHz): # 5.15 (2xt, 2H, H3’, H3’’), 5.07 (t, 1H, J3’’, 4’’ 9.4, J4’’, 5’’ 9.4 Hz, H4’’), 4.93 (dd, 1H, J1’’, 2’’ 7.9, J2’’, 3’’ 9.1 Hz, H2’’), 4.80, 4.81 (2xdd, 1H, J1’, 2’ 9.4, J2’, 3’ 9.7 Hz, H2’), 4.51 (d, 1H, J1’’, 2’’ 7.9 Hz, H1’’), 4.50 (m, 1H, H6’a), 4.38 (dd, 1H, J5’’, 6’’a 4.3, J6’’a, 6’’b 12.5 Hz, H6’’a), 4.09, 4.07 (2xdd, 1H, J5’, 6’b 2.4, J6’a, 6’b 12.2 Hz, H6’b), 4.05 (dd, 1H, J 5’’, 6’’b 1.9, J6’’a, 6’’b 12.5 Hz, H6’’b), 3.72 (dd, 1H, J3’, 4’ 9.4, J4’, 5’ 9.4 Hz, H4’), 3.65 (ddd, 1H, J4’’, 5’’ 9.4, J5’’, 6’’a 4.3, J5’’, 6’’b 1.9 Hz, H5’’), 3.53 (m, 1H,  H5’), 3.35-3.39 (m, 1H, H1’), 2.88-2.92 (m, 1H, H2), 2.74 (m, 1H, H1a), 2.46 (m, 1H, J1a, 1b 5.0, J1b, 2 2.74, H1b), 1.99 (s, 3H, COCH3), 2.01 (s, 6H, 2xCOCH3), 2.04 (s, 6H, 2xCOCH3), 2.09 (s, 3H, COCH3), 2.13 (s, 3H, COCH3), 2.00-2.11 (m, 1H, H3a), 1.40-1.65 (m, 5H, H3b, H4,H5).   120 (R, S)-1,2-Epoxy-4-[(!-D-glucopyranosyl)-(1%4)-O-!-D-glucopyranosyl] butane (2.5)139   The general procedure Deacetylation by Sodium Methoxide was followed. A suspension of acetylated epoxybutyl derivative 2.36 (120 mg, 0.17 mmol) in 50 mM sodium methoxide solution (30 mL) was stirred at RT for an hour, and the resuting crude product was purified on a Waters Sep-Pak tC18 reverse phase cartridge to afford the final product 2.5 (52 mg, 0.1 mmol, 76%). Compound has approximate 90% purity based on 1H NMR spectrum. ESI MS m/z calcd. for C16H28O11: 396.18, Found:  419.2 [M+Na]+. 1H-NMR (D2O, 400 MHz): # 4.48 (d, 1H, J1’’, 2’’ 7.9 Hz, H1’’), 2.87-3.94 (m, 2H, H6a’, H6a’’), 3.64-3.69 (m, 2H, J5’, 6’b = J5’’,6’’b = 5.8, J6’a, 6’b = J6’’a, 6’’b = 12.8 Hz, H6’b, H6’’b), 3.27-3.51 (m, 9H, H1’-H5’, H2’’-H5’’), 3.12-3.17 (m, 1H, H2), 2.74 (m, 1H, H1a), 2.67 (m, 1H, H1b), 1.94-2.01, 1.80-1.87, 1.68-1.73, 1.50-1.62 (m, 4H, H3, H4).  (R, S)-1,2-Epoxy-5-[(!-D-glucopyranosyl)-(1%4)-O-!-D-glucopyranosyl] pentane (2.6)139   The general procedure Deacetylation by Sodium Methoxide was followed. A suspension of acetylated epoxypentyl derivative 2.37 (75 mg, 0.1 mmol) in 50 mM sodium methoxide solution (15 mL) was stirred at RT for an hour, and the crude product was purified with Waters Sep-Pak tC18 reverse phase cartridge to afford the final product 2.6 (35 mg, 0.1 mmol, 82%). Compound  121 has approximate 90% purity based on 1H NMR spectrum. ESI MS m/z calcd. for C17H30O11: 410.2, Found: 433.3 [M+Na]+. 1H-NMR (MeOD, 400 MHz): # 4.40 (d, 1H, J1’’, 2’’ 7.7 Hz, H1’’), 2.83-3.90, 3.64-3.69 (m, 4H, H6’, H6’’), 3.09-3.54 (m, 9H, H1’-H5’, H2’’-H5’’), 2.92-2.96 (m, 1H, H2), 2.74 (t, 1H, J1a, 1b = J1a, 2 4.5 Hz, H1a), 2.48 (dd, 1H, J1a, 1b 4.5, J1b, 2 2.6 Hz, H1b), 1.95- 1.87, 1.70-1.79, 1.63-1.43 (m, 6H, H3, H4, H5).  2,3,4,6-Tetra-O-acetyl-"-D-galactopyranosyl fluoride (2.43)170   Fluorination at the anomeric center was performed according to the General Procedure for Synthesis of "-Glycosyl Fluorides. A mixture of per-O-acetylated galactose (1.5 g, 3.9 mmol) and 70%/30% (w/w) HF/ pyridine (10 mL, 350 mmol, 90 eqv.) was stirred overnight at 0 oC. Upon completion, the crude product was purified by flash column chromatography (petroleum ether:ethyl acetate= 4:1) to yield a white powder 2.43 (700 mg, 2.0 mmol, 51%). 1H-NMR (CDCl3, 300 MHz): # 5.80 (dd, 1H, J1, F 53.4, J1, 2 2.7 Hz, H1), 5.52 (m, 1H, J3, 4 3.3, J4, 5 1.2 Hz, H4), 5.36 (dd, 1H, J2, 3 10.8, J3, 4 3.3 Hz, H3), 5.18 (ddd, 1H, J2, F 24.3, J1, 2 2.7, J2, 3 10.8 Hz, H2), 4.35-4.10 (m, 3H, H5, H6), 2.15 (s, 3H, COCH3), 2.11 (s, 3H, COCH3), 2.06 (s, 3H, COCH3), 2.19 (s, 3H, COCH3).      122 "-D-Galactopyranosyl fluoride (2.46)171    The deacetylation of 2.43 followed the General Procedure for Deacetylation with Ammonia. Into a suspension of acetylated galactosyl fluoride (700 mg 2.0 mmol) in dry methanol (50 mL) was bubbled ammonia gas and the reaction mixture stirred at 0 oC for three days. The crude product was purified by flash column chromatography (ethyl acetate:MeOH:H2O= 17:2:1) to yield a white solid of 2.46 (300 mg, 1.6 mmol, 82%). ESI MS m/z calcd. for C6H11FO5: 182.1, Found: 181.3 [M+Na]+. 1H-NMR (D2O, 400 MHz): # 5.69 (dd, 1H, J1, F 53.4, J1, 2 2.5 Hz, H1), 3.73-4.09 (m, 6H, H2-H6).  3,4,6-Tri-O-acetyl-2-deoxy-2-fluoro-D-gluco/mannopyranose (2.38, 2.39)   To solution of D-glucal (6.2 g, 22.7 mmol) in nitromethane and H2O (90 mL, v/v =5/1) was added SelectFluorTM (9.8 g, 27.3 mmol, 1.2 eqv.) in portions, and the reaction mixture was allowed to stir at RT overnight. After the starting material was all consumed (monitored by TLC), the mixture was refluxed for an additional three hours and then concentrated under reduced pressure. The resulting residue was redissolved in DCM, washed with H2O, saturated NaHCO3, and brine. The organic layer was then dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The resulting mixture contained 2.38 and 2.39 based on 1H  123 NMR; 3.8 g, 12.3 mmol, 54% calculated from a mixture of 2.38 (40%) and 2.39 (60%). ESI MS m/z calcd. for C12H17FO8: 308.1, Found: 331.3 [M+Na]+. The mixture was subjected to coupling with 2,4-DNFB without further purification.  2,4-Dinitropheny 3,4,6-Tri-O-acetyl-2-deoxy-2-fluoro-!-D-glucopyranoside (2.41)172   To a mixture of the 2-fluoro-gluco and manno hemiacetals 2.38 and 2.39 (2.0 g, 6.5 mmol) and 2,4-DNFB (1.8 g, 9.7 mmol, 1.5 eqv.) in dry DMF (10 mL) was added DABCO (300 mg, 2.7 mmol) and the reaction mixture was stirred at RT for three days. The solvent was removed under reduced pressure, and the resulting residue was diluted with ethyl acetate, and washed with H2O, saturated NaHCO3 and brine successively. The organic layer was dried over anhydrous MgSO4, filtered and concentrated, and the crude mixture was partially purified by flash column chromatography (petroleum ether:ethyl acetate = 4:1 to 1:1) to afford an anomeric mixture of the two 2-fluoro glucoside derivatives. Two fractions were collected. The first fraction (containing primarily "-anomer of glucosyl derivatives) was crystallized from ethyl acetate to yield pure "- anomer of glycosyl derivatives, and the mother liquid was crystallized again from EtOH to afford the !-anomer of glucoside derivative 2.41. The second fraction (containing a higher proportion of !-anomer of glycosyl derivatives) was recrystallized with ethyl acetate and petroleum ether co-solvent to afford 2.41 as a white needle-like crystal (670 mg in total, 1.4 mmol, 22% calculated from the mixture of 2.38 and 2.39). ESI MS m/z calcd. for C18H19FN2O12: 474.1, Found 497.2 [M+Na]+. 1H-NMR (CDCl3, 300 MHz): # 8.78 (m, 1H, Ar), 8.45 (m, 1H,  124 Ar), 7.41 (m, 1H, Ar), 5.50-5.39 (m, 2H, H1, H3), 5.15 (dd, 1H, J3, 4 9.5, J4, 5 9.6 Hz, H4), 4.73 (ddd, 1H, J2, F 50.0, J1, 2 9.0, J2, 3 9.0 Hz, H2), 4.28 (dd, 1H, J5, 6b 5.0, J6a, 6b 12.6 Hz, H6a), 4.20 (dd, 1H, J5, 6b 2.7, J6a, 6b 12.6 Hz, H6b), 3.99 (ddd, 1H, J4, 5 9.6, J5, 6a 5.0, J5, 6b 2.7 Hz, H5), 2.13 (s, 3H, COCH3), 2.08 (s, 3H, COCH3), 2.07 (s, 3H, COCH3).  2,4-Dinitrophenyl-2-deoxy-2-fluoro-!-D-glucopyranoside (2.42)172   The deacetylation of acetylated 2-fluoroglucoside 2.41 was performed according to the General Procedure for Deacetylation in Acidic Condition. To a suspension of 2.41 (293 mg, 0.62 mmol) in dry MeOH (35 mL) was added AcCl (1.5 mL) at 0 oC, and the reaction mixture was allowed to stir at 4 oC overnight. Upon completion of reaction, the crude product was crystallized from acetone and hexane to yield a white solid of 2.42 (175 mg, 0.5 mmol, 81%). ESI MS m/z calcd. for: C12H13FN2O9: 348.3, Found: 372.2 [M+Na]+. 1H-NMR (MeOD, 300 MHz): # 8.47 (m, 1H, Ar), 8.49 (m, 1H, Ar), 7.65 (m, 1H, Ar), 5.58 (dd, 1H, J1,F 4.2, J1,2 7.8 Hz, H1), 4.34 (ddd, 1H, J2,F 51.8, J1,2 7.8, J2,3 9.0 Hz, H2), 3.93 (dd, 1H, J5, 6a 2.0, J6a, 6b 12.1 Hz, H6a), 3.80- 3.66 (m, 2H, H3, H6b), 3.66-3.62 (m, 1H, H5), 3.46 (t, 1H, J3, 4 9.3, J4, 5 9.3 Hz, H4).     125 2,4-Dinitrophenyl (!-D-glucopyranosyl)-(1%4)-O-2-deoxy-2-fluoro-!-D-glucopyranoside (2.7)102,145   The synthesis of 2.7 was conducted according to the General Procedure for Oligosaccharide Synthesis with Abg 2F6 Glycosynthase. The dinitrophenyl 2-fluoroglucoside glycosyl acceptor 2.42 (104 mg, 0.3 mmol) and glycosyl donor "-D-glcF (66 mg, 0.4 mmol, 1.2 eqv.) were dissolved in 100 mM pH 7.0 sodium phosphate buffer (11 mL), followed by the addition of Abg 2F6 glycosynthase (5.5 mg). The reaction mixture was incubated at RT until the glycosyl donor was consumed. After the buffer was removed, the crude product was purified using a Waters Sep-Pak tC18 reverse phase cartridge followed by acetylation with pyridine and acetic anhydride (procedure followed the General Acetylation of Free Sugars). The desired disaccharide was then purified by flash column chromatography (ethyl acetate:petroleum ether= 1:2). Acetylated disaccharides were deprotected with AcCl (the procedure of deacetylation followed the general procedure for Deacetylation in Acid Conditions) and was purified by flash column chromatography (DCM:MeOH = 5:1). After the solvent was removed under reduced pressure, the resulting residue was precipitated with acetone and ether to yield a white solid 2.7 (20 mg, 0.04 mmol, 13%). Compound has approximate 90% purity based on 1H NMR spectrum. ESI MS m/z calcd. for C18H23FN2O14: 510.1, Found 533.1 [M+Na]+. ESI MS m/z calc. for C18H23FN2O14: 510.1, Found 533.1 [M+Na]+. 1H-NMR (MeOD, 400 MHz): # 8.76 (m, 1H, Ar), 8.51 (m, 1H, Ar), 7.67(m, 1H, Ar), 5.65 (dd, 1H, J1, F 3.1, J1, 2 7.6 Hz, H1), 4.48 (d, 1H, J1’, 2’ 7.9 Hz, H1’), 4.41 (ddd, 1H, J2, F 50.9, J1, 2 7.6, J2, 3 8.2 Hz, H2), 3.25-4.00 (m, 11H, H3-6, H2’-6’).  126 The configuration of the linkage between two sugar units was comfired by the 1H NMR of the acetylated dinitrophenyl 2-fluorocellobioside. 2,4-Dinitrophenyl (2,3,4,6-tetra-O-acetyl-!-D- glucopyranosyl)-(1%4)-O-2,6-bi-O-acetyl-2-deoxy-2-fluoro-!-D-glucopyranoside, 1H-NMR (CDCl3, 400 MHz): # 8.75 (m, 1H, Ar), 8.44 (m, 1H, Ar), 7.39 (m, 1H, Ar), 5.38-5.47 (m, 2H, H3, H1), 5.17 (t, 1H, J2’, 3’ 9.3, J3’, 4’ 9.3 Hz, H3’), 5.09 (dd, 1H, J3’, 4’ 9.3, J4’, 5’ 9.2 Hz, H4’), 4.95 (dd, 1H, J1’, 2’ 8.4, J2’, 3’ 9.3 Hz, H2’), 4.66 (ddd, 1H, J2,F 49.1, J1, 2 6.4, J2, 3 7.3 Hz, H2), 4.57 (d, 1H, J1’, 2’ 8.4 Hz, H1’), 4.56 (m, 1H, H6a), 4.38 (dd, 1H, J5’, 6’a 4.3, J6’a, 6’b 12.5 Hz, H6’a), 4.12-4.07 (m, 2H, H6b, H6b’), 3.97-3.91 (m, 2H, H4, H5), 3.70 (ddd, 1H, J4’,5’ 9.2, J5’,6’a 4.3, J5’,6’b 2.1 Hz, H5’), 2.15 (s, 3H, COCH3), 2.12 (s, 3H, COCH3), 2.04 (s, 6H, 2xCOCH3), 2.03 (s, 3H, COCH3), 2.00 (s, 3H, COCH3).  2,4-Dinitrophenyl (!-D-galactopyranosyl)-(1%4)-O-2-deoxy-2-fluoro-!-D-glucopyranoside (2.8)   Synthesis followed the General Procedure for Oligosaccharide Synthesis with Abg 2F6 Glycosynthase. The dinitrophenyl 2-fluoroglucoside glycosyl acceptor 2.42 (52 mg, 0.1 mmol) and glycosyl donor 2.46 (40 mg, 0.2 mmol, 1.5 eqv.) were dissolved in 100 mM pH 7.0 sodium phosphate buffer (7.5 mL), followed by the addition of Abg 2F6 glycosynthase (2 mg). Upon completion of reaction, the resulting crude product was purified with a Waters Sep-Pak tC18 reverse phase cartridge to yield pure compound 2.8 (50 mg, 0.1 mmol, 71%). Compound has around 90% purity based on 1H NMR spectrum. ESI MS m/z calcd. for C18H23FN2O14: 510.1,  127 Found: 533.0 [M+Na]+. 1H-NMR (MeOD, 400 MHz): # 8.76 (m, 1H, Ar), 8.50 (m, 1H, Ar), 7.67(m, 1H, Ar), 5.65 (dd, 1H, J1, F 3.1, J1, 2 7.5 Hz, H1), 4.43 (ddd, 1H, J2, F 51.4, J1, 2 7.5, J2, 3 8.7 Hz, H2), 4.42 (d, 1H, J1’, 2’ 7.5 Hz, H1’), 4.02-3.50 (m, 11H, H3-H6, H2’-H6’).  2,3,4-Tri-O-acetyl-!-L-arabinopyranosyl fluoride (2.44)170   Fluorination at the anomeric center was performed according to the General Procedure for Synthesis of "-Glycosyl Fluorides. A mixture of per-O-acetylated L-arabinopyranose (1.5 g, 4.7 mmol) and 70%/30% (w/w) HF/ pyridine (13 mL, 455 mmol, 97 eqv.) was stirred overnight at 0 oC. Upon completion of reaction, the crude product was purified by flash column chromatography (petroleum ether:ethyl acetate = 4:1) to yield a white powder of 2.44 (789 mg, 2.7 mmol, 60%). 1H-NMR (CDCl3, 300MHz): # 5.79 (dd, 1H, J1, F 53.7, J1, 2 2.74 Hz, H1), 5.41- 5.42 (m, 1H, H4), 5.37 (dd, 1H, J2, 3 10.5, J3, 4 3.2 Hz, H3), 5.23 (ddd, 1H, J2, F 23.3, J1, 2 2.7, J2, 3 10.5 Hz, H2), 4.14 (br. d, 1H, J5a, 5b 13.2 Hz, H5a), 3.88 (dd, 1H, J4, 5b 1.8, J5a, 5b 13.2 Hz, H5b), 2.15 (s, 3H, COCH3), 2.13 (s, 3H, COCH3), 2.04 (s, 3H, COCH3).        128 !-L-Arabinopyranosyl fluoride (2.47)171   The deacetylation of 2.44 followed the General Procedure for Deacetylation with Ammonia. A suspension of acetylated L-arabinosyl fluoride 2.44 (371 mg, 1.3 mmol) in dry methanol (30 mL) was bubbled ammonia gas at 0 oC and stirred at 4 oC for three days. The crude product was purified by flash column chromatography (DCM:MeOH = 10:1) to yield a white solid of 2.47 (139  mg, 0.9 mmol, 69%).  1H-NMR (MeOD, 400 MHz): # 5.53 (dd, 1H, J1, F 54.1, J1, 2 2.5 Hz, H1), 3.72-3.98 (m, 5H, H2-H5).  2,3,4-Tri-O-acetyl-"-D-fucopyranosyl fluoride (2.45)170   Fluorination at anomeric center was performed according to the General Procedure for Synthesis of "-Glycosyl Fluorides. A mixture of per-O-acetylated D-fucospyranose (846 mg, 2.5 mmol) and 70%/30% HF/ pyridine (7 mL, 245 mmol, 52 eqv.) at 0 oC was stirred overnight at 4 oC. Upon completion of reaction, the crude product was purified by flash column chromatography (petroleum ether:ethyl acetate = 8.5:1) to yield a white powder of 2.45 (647 mg, 2.2 mmol, 87%). 1H-NMR (CDCl3, 400 MHz): # 5.75 (dd, 1H, J1, F 53.6, J1, 2 2.7 Hz, H1), 5.34-  129 5.37 (m, 2H, H3, H4), 5.17 (ddd, 1H, J2, F 23.0, J1, 2 2.7,  J2, 3 10.5 Hz, H2), 4.34 (br, 1H, H5), 2.15 (s, 3H, COCH3), 2.11 (s, 3H, COCH3), 2.13 (s, 3H, COCH3), 1.20 (d, 3H, J5, 6 6.4 Hz, H6).  "-D-Fucopyranosyl fluoride (2.48)171   The deacetylation of 2.45 followed the General Procedure for Deacetylation with Ammonia. Into a suspension of acetylated D-fucosyl fluoride 2.45 (647 mg, 2.2 mmol) in dry methanol (30 mL) was bubbled ammonia gas at 0 oC and the reaction stirred at 4 oC for three days. The crude product was purified by flash column chromatography (DCM:Acetone = 1.5:1) followed by crystallization from MeOH and ethyl acetateto yield the deprotected compound 2.48 as a white solid (258 mg, 1.6 mmol, 73%).1H-NMR (MeOD, 400 MHz): # 5.53 (dd, 1H, J1, F 54.5, J1, 2 2.1 Hz, H1), 4.09 (br. 1H, H5), 3.72-3.78 (m, 3H, H2-H4), 1.24 (d, 3H, J5, 6 6.7 Hz, H6).  2,4-Dinitrophenyl [(!-D-galactopyranosyl)-(1%4)-O-!-D-glucopyranosyl]-(1%4)-O-!-D- glucopyranoside (2.9)102   Synthesis of the trisaccharide was based on the method General Procedure for Oligosaccharide Synthesis with Abg 2F6 Glycosynthase. DNPC 2.50 (250 mg, 0.5 mmol), "- galactosyl fluoride 2.46 (160 mg, 0.9 mmol, 1.8 eqv.), and 150 mM phosphate buffer (pH 7.0, 40  130 mL) were added to a Falcon® tube, followed by the addition of Abg 2F6 glycosynthase (4.6 mg). The reaction mixture was incubated at RT for three hours and the solvent was removed via lyophilisation. A large volume of MeOH was added to the residue to precipitate buffer salts, and the solution was filtered and concentrated. Purification of crude product by flash column chromatography (ethyl acetate:MeOH:H2O = 7:2:1) yielded 2.9 as a white powder (200 mg, 0.3 mmol, 60%). Compound has approximate 85% purity based on 1H NMR spectrum.ESI MS m/z calcd. for C24H34N2O20: 670.2, Found: 693.3 [M+Na]+.  1H-NMR (MeOD, 400 MHz): 8.74 (m, 1H, Ar), 8.48 (m, 1H, Ar), 7.64 (m, 1H, Ar), 5.32 (d, 1H, J1, 2 7.3 Hz, H1), 4.49 (d, 1H, J1’, 2’ 7.9 Hz, H1’), 4.36 (d, 1H, J1’’, 2’’ 7.6 Hz, H1’’), 3.47-3.95 (18H, H2-H6, H2’-H6’, H2’’-H6’’).  2,4-Dinitrophenyl [("-L-arabinopyranosyl)-(1→4)-O-!-D-glucopyranosyl]-(1→4)-O-!-D- glucopyranoside (2.10)   Synthesis of the trisaccharide was based on the method General Procedure for Oligosaccharide Synthesis with Abg 2F6 Glycosynthase. DNPC 2.50 (45 mg, 0.09 mmol), "- L-arabinopyranosyl fluoride 2.47 (38 mg, 0.2 mmol, 2.8 eqv.), and 100 mM phosphate buffer (pH 7.0, 10 mL) were added to a Falcon® tube, followed by the addition of Abg 2F6 glycosynthase (4.5 mg). The reaction mixture was incubated at RT for three hours and the solvent was removed via lyophilisation. The resulting residue was desalted on a Waters Sek-Pak tC18 reversed phase cartridge, and then crystallized from MeOH and diethyl ether to yield white  131 powder 2.10 (40 mg, 0.06 mmol, 71%). Compound has approximate 90% purity based on 1H NMR spectrum. HRMS m/z calcd. for C23H32N2O19: 640.2, Found: 663.1512 [M+Na]+. 1H- NMR (D2O, 400 MHz): 8.87 (m, 1H, Ar), 8.51 (m, 1H, Ar), 7.58 (m, 1H, Ar), 5.42 (d, 1H, J1, 2 7.3 Hz, H1), 4.54(d, 1H, J1’, 2’ 7.9 Hz, H1’), 4.34 (d, 1H, J1’’, 2’’ 7.6 Hz, H1’’), 3.32-4.00 (18H, H2-H6, H2’-H6’, H2’’-H6’’). The configuration of the glycosidic linkage between non-reducing terminal sugar and middle sugar unit was comfired by the 1H NMR of the acetylated trisaccharide. 2,4-Dinitrophenyl [(2,3,4-tri-O-acetyl-"-L-arabinopyranosyl)-(1$4)-O-2,3,6-tri-O-acetyl-!-D-glucopyranosyl]- (1$4)-O-2,3,6-tri-O-acetyl-!-D-glucopyranoside, 1H-NMR (CDCl3, 600 MHz): # 8.71 (m, 1H, Ar), 8.42 (m, 1H, Ar), 7.44 (m, 1H, Ar), 5.33 (d, 1H, J1, 2 7.2 Hz, H1), 5.28 (dd, 1H, J2, 3 8.2, J3, 4 8.2 Hz, H3), 5.22 (dd, 1H, J1, 2 7.2, J2, 3 8.2 Hz, H2), 5.21-5.20 (m, 1H, H4’’), 5.17 (dd, 1H, J2’, 3’ 9.2, J3’, 4’ 9.2 Hz, H3’), 5.04 (dd, 1H, J1’’, 2’’ 6.1, J2’’, 3’’ 8.7 Hz, H2’’), 5.01 (dd, 1H, J2’’, 3’’ 8.7, J3’’, 4’’ 3.1 Hz, H3’’), 4.86 (dd, 1H, J1’, 2’ 8.2, J2’, 3’ 9.2 Hz, H2’), 4.62 (dd, 1H, J5, 6a 2.1, J6a, 6b 12.3 Hz, H6a), 4.56 (d, 1H, J1’, 2’ 8.2 Hz, H1’), 4.43 (d, 1H, J1’’, 2’’ 6.1 Hz, H1’’), 4.40 (dd, 1H, J5’, 6a 1.5, J6’a, 6’b 11.7 Hz, H6’a), 4.15 (dd, 1H, J5, 6b 4.6, J6a, 6b 12.3 Hz, H6b), 4.09 (dd, 1H, J5’, 6’b 5.1, J6’a, 6’b 12.3 Hz, H6’b), 3.96 (dd, 1H, J3, 4 8.2, J4, 5 9.7 Hz, H4), 3.93 (dd, 1H, J4’’, 5’’a 4.1, J5’’a, 5’’b 12.1 Hz, H5’’a), 3.91-3.88 (m, 1H, H5), 3.79 (dd, 1H, J3’, 4’ 9.2, J4’, 5’ 9.7 Hz, H4’), 3.63-3.60 (m, 1H, H5’), 3.57 (dd, 1H, J5’’, 6’’b 2.6, J6’’a’, 6’’b 12.8 Hz, H6’’b), 2.15-2.01 (8x s, 27H, 9x COCH3). 13C NMR (CDCl3, 150 Hz): # 170.4, 170.2, 170.0, 169.8 (2 C), 169.7, 169.3 (3 C) (C=O), 153.5, 142.1, 128.6 (2 C), 121.3, 118.4 (Ar-C), 101.0, 100.5, 98.7, 76.4, 75.5, 73.3, 73.2, 72.8, 71.8 (2 C), 70.6, 70.0, 69.4, 67.0, 62.6, 62.1, 61.3; 20.9, 20.8 (2 C), 20.72 (3 C), 20.6, 20.5 (2 C) (CH3CO).   132 2,4-Dinitrophenyl [(!-D-fucopyranosyl)-(1$4)-O-!-D-glucopyranosyl]-(1$4)-O-!-D- glucopyranoside (2.11)   Synthesis of the trisaccharide was based on the method General Procedure for Oligosaccharide Synthesis with Abg 2F6 Glycosynthase. DNPC 2.50 (150 mg, 0.3 mmol), "- fucopyranosyl fluoride 2.48 (97 mg, 0.6 mmol, 2 eqv.), and 150 mM phosphate buffer (pH 7.0, 20 mL) were added to a Falcon® tube, followed by the addition of Abg 2F6 glycosynthase (6.0 mg). The reaction mixture was incubated at RT for three hours and the solvent was removed via lyophilisation. The crude product was crystallized from MeOH and diethyl ether to yield 2.11 aswhite crystals (100 mg, 0.15 mmol, 51%). Compound has approximate 90% purity based on 1H NMR spectrum. HRMS m/z calcd. for C24H34N2O19: 654.2, Found: 677.1661 [M+Na]+. 1H- NMR (D2O, 400 MHz): 8.87 (m, 1H, Ar), 8.52 (m, 1H, Ar), 7.59 (m, 1H, Ar), 5.42 (d, 1H, J1, 2 7.3 Hz, H1), 4.55 (d, 1H, J1’, 2’ 7.9 Hz, H1’), 4.38 (d, 1H, J1’’, 2’’ 7.6 Hz, H1’’), 3.32-4.00 (18H, H2-H6, H2’-H6’, H2’’-H6’’). The configuration of the glycosidic linkage between non-reducing terminal sugar and middle sugar unit was comfired by the 1H NMR of the acetylated trisaccharide. 2,4-Dinitrophenyl [(2,3,4-tri-O-acetyl-!-D-fucopyranosyl)-(1$4)-O-2,3,6-tri-O-acetyl-!-D-glucopyranosyl]- (1$4)-O-2,3,6-tri-O-acetyl-!-D-glucopyranoside, 1H-NMR (CDCl3, 600 MHz): # 8.71 (m, 1H, Ar), 8.42 (m, 1H, Ar), 7.44 (m, 1H, Ar), 5.32 (d, 1H, J1, 2 6.7 Hz, H1), 5.28 (dd, 1H, J2, 3 8.2, J3, 4 8.7 Hz, H3), 5.22 (dd, 1H, J1, 2 6.7, J2, 3 8.2 Hz, H2), 5.19-5.20 (m, 1H, H4’’), 5.17 (dd, 1H, J2’, 3’  133 9.2, J3’, 4’ 9.2 Hz, H3’), 5.08 (dd, 1H, J1’’, 2’’ 7.7, J2’’, 3’’ 10.2 Hz, H2’’), 4.95 (dd, 1H, J2’’, 3’’ 10.2, J3’’, 4’’ 3.1 Hz, H3’’), 4.88 (dd, 1H, J1’, 2’ 8.2, J2’, 3’ 9.2 Hz, H2’), 4.62 (dd, 1H, J5, 6a 2.6, J6a, 6b 13.0 Hz, H6a), 4.55 (d, 1H, J1’, 2’ 8.2 Hz, H1’), 4.43 (dd, 1H, J5’, 6’a  2.1, J6’a, 6’b 12.3 Hz, H6’a), 4.42 (d, 1H, J1’’, 2’’  7.7 Hz, H1’’), 4.15-4.08 (m, 2H, H6’b, H6b), 3.98 (dd, 1H, J3, 4 8.7, J4, 5 9.7 Hz, H4), 3.91-3.88 (m, 1H, H5), 3.80 (dd, 1H, J3’, 4’ 9.2, J4’, 5’  9.7 Hz, H4’), 3.75-3.72 (m, 1H, H5’’), 3.64-3.61 (m, 1H, H5’), 2.16-1.97 (9x s, 27H, 9x COCH3), 1.21 (d, 3H, H6’’). 13C NMR (CDCl3, 150 Hz): # 170.5 (2 C), 170.3, 170.1 (2 C), 170.0, 169.7 (2 C), 169.4 (C=O), 153.5, 142.2 (2 C), 128.6, 121.3, 118.4 (Ar-C); 101.1, 100.4, 98.7, 75.8, 75.4, 73.3, 73.1, 72.9, 71.8, 71.7, 71.3, 70.6, 69.8, 69.1, 62.1, 61.2, 60.4; 20.9, 20.8, 20.7 (3 C), 20.6 (2 C), 20.5 (2 C) (CH3CO), 16.1.  2,4-Dinitrophenyl [(!-D-xylopyranosyl)-(1$4)-O-!-D-glucopyranosyl]-(1$4)-O-!-D- glucopyranoside (2.12)   Synthesis of the trisaccharide was based on the method General Procedure for Oligosaccharide Synthesis with Abg 2F6 Glycosynthase. DNPC 2.50 (100 mg, 0.2 mmol), "- xylosyl fluoride 2.49 (71 mg, 0.5 mmol, 2.4 eqv.), and 150 mM phosphate buffer (pH 7.0, 10 mL) were added to a Falcon® tube, followed by the addition of Abg 2F6 glycosynthase (17 mg). The reaction mixture was incubated at RT for three days (a mixture of di, tri and tetrasaccharides was formed) and solvent was removed via lyophilisation. The resulting residue was desalted on a Waters Sek-Pak tC18 reversed phase cartridge. The crude product was purified by flash column  134 chromatography (ethyl acetate:MeOH:H2O = 7:2:1) to yield 2.12 as a white solid (40 mg, 0.06 mmol, 31%). Compound has approximate 90% purity based on 1H NMR spectrum. HRMS m/z calcd. for C23H32N2O19: 640.2, Found: 663.1512 [M+Na]+. 1H-NMR (D2O, 400 MHz): 8.87 (m, 1H, Ar), 8.51 (m, 1H, Ar), 7.58 (m, 1H, Ar), 5.42 (d, 1H, J1, 2 7.3 Hz, H1), 4.53 (d, 1H, J1’, 2’ 7.9 Hz, H1’), 4.30 (d, 1H, J1’’, 2’’ 7.6 Hz, H1’’), 3.24-4.00 (18H, H2-H6, H2’-H6’, H2’’-H6’’). The configuration of the glycosidic linkage between non-reducing terminal sugar and middle sugar unit was comfired by the 1H NMR of the acetylated trisaccharide. 2,4-Dinitrophenyl [(2,3,4-tri-O-acetyl-!-D-xylopyranosyl)-(1$4)-O-2,3,6-tri-O-acetyl-!-D-glucopyranosyl]- (1$4)-O-2,3,6-tri-O-acetyl-!-D-glucopyranoside, 1H-NMR (CDCl3, 600 MHz): # 8.70 (m, 1H, Ar), 8.42 (m, 1H, Ar), 7.44 (m, 1H, Ar), 5.31 (d, 1H, J1, 2 6.7 Hz, H1), 5.27 (dd, 1H, J2, 3 8.2, J3, 4 8.2 Hz, H3), 5.21 (dd, 1H, J1, 2 6.7, J2, 3 8.2 Hz, H2), 5.14 (dd, 1H, J2’, 3’ 9.2, J3’, 4’9.2 Hz, H3’), 5.11 (dd, 1H, J2’’, 3’’ 8.7, J3’’, 4’’ 8.7 Hz, H3’’), 4.85 (ddd, 1H, J3’’, 4’’ 8.7, J4’’, 5’’a 5.1,  J4’’, 5’’b 8.2 Hz, H4’’), 4.86 (dd, 1H, J1’’ ,2’’ 7.7, J2’’, 3’’ 8.7 Hz, H2’), 4.84 (dd, 1H, J1’’, 2’’ 6.7, J2’’, 3’’  8.7 Hz, H2’’), 4.62 (dd, 1H, J5,6a  2.1, J6a,6b  12.3 Hz, H6a), 4.55 (d, 1H, J1’, 2’ 7.7 Hz, H1’), 4.48 (d, 1H, J1’’, 2’’ 6.7 Hz, H1’’), 4.39 (dd, 1H, J5’, 6’a 1.5, J6’a, 6’b 12.3 Hz, H6’a), 4.12 (dd, 1H, J5’, 6’b 4.6, J6’a, 6’b  12.3 Hz, H6’b), 4.08 (dd, 1H, J5, 6b 5.1, J6a, 6b 12.2 Hz, H6b), 4.05 (dd, 1H, J4’, 5’’a 5.1, J5’’a, 5’’b 11.8 Hz, H5’’a), 3.97 (dd, 1H, J3, 4 8.2, J4, 5 9.7 Hz, H4), 3.90-3.87 (m, 1H, H5), 3.77 (dd, 1H, J3’, 4’ 9.2, J4’, 5’ 9.2 Hz, H4’), 3.61-3.59 (m, 1H, H5’), 3.32 (dd, 1H, J4’’, 5’’b  8.1, J5’’a, 5’’b  11.8 Hz, H5’’b), 2.13-2.01 (8x s, 27H, 9x CH3CO), 1.21 (d, 3H, COCH3). 13C NMR (CDCl3, 150 Hz): # 170.3, 170.0, 169.8, 169.6, 169.3 (2 C), 169.2, 153.5, 142.1, 140.2, 128.6, 121.3, 118.4 (Ar-C), 101.4, 100.5, 98.7, 76.8, 75.5, 73.3, 73.1, 72.8, 71.7, 71.3, 71.2, 71.0, 70.6, 68.6, 62.2, 62.0, 61.2; 20.8 (2 C), 20.7 (2 C), 20.6 (2 C), 20.5 (2 C) (CH3CO).   135 3.3 Enzymology 3.3.1 Production of Abg 2F6 Glycosynthase The procedure for producing Abg 2F6 mutant was similar to that previously described.105,103 The expression vector that carries the gene for Abg 2F6, designated pET29abgE358S, was transformed into electrocompetent E. coli BL21 (DE3) by electronic shock.  The transformants were plated on LB kanamycin agar (30-50 'g/mL kanamycin) and incubated at 30 oC overnight.  The cells were incubated overnight at 30 oC in 10 mL LB medium containing 30-50 'g/mL kanamycin. The overnight cultures were poured into 6 L LB kanamycin liquid media, and then incubated with shaking at 200 rpm overnight at 30 oC.  The cells were collected by centrifugation at 5000 rpm for 30 minutes at 4 oC.  The cell pellet was suspended in lysis buffer at RT for an hour and the debris was removed by centrifugation. The protein was purified from the soluble cell extract on a Ni2+ affinity column (1 mL HisTrip (CE Healthcare)) with Tris (20 mM) buffer containing NaCl (500 mM) and imidazole. The enzyme was gradually eluted with imidazole (20 mM to 500 mM) over 40 minutes, and concentrated. The purity of the protein was verified by SDS-PAGE and visualized by staining with Coomassie Blue. Final concentration of proteins was estimated by absorbance at 280 nm (!!"#!!!" ! !!!").103 3.3.2 Purification of T. reesei Cellulases by Iogen Corp. CBHI, CBHII, EGI, and EGII Cellulase purifications were performed by Iogen Corp. Cel 6A (CBHII) and Cel 7A (CBHI) were purified as described previously.173,174 The enzymes were first purified by anion exhange and then by affinity chromatography. The affinity ligand used was 4-aminophenyl 1- thio-!-D-cellobioside, which binds the active site of CBHI and CBHII with high affinity. These enzymes were eluted from the column using 10 mM cellobiose. CBHII was purified from  136 enzyme mixture from a strain of T. reesei that does not produce CBHI. Similarly, CBHI was purified from enzyme mixture from another strain of T. reesei that does not contain CBHII to ensure zero contamination by the other cellobiohydrolase. EGI and EGII were purified by anion exchange chromatography, which follows a procedure similar to that previously reported175. The two endoglucanases were purified from enzyme mixture from a strain of T. reesei that does not produce two cellobiohydrolases. This ensured that neither of these endoglucanases have any contaminating cellobiohydrolase activity. The enzyme purity was assessed by SDS-PAGE prior to use (Appendix B.3).  3.3.3 Enzyme Kinetics 3.3.3.1 General Methods All kinetic assays with dinitrophenyl glycoside substrates were carried out either in 1 cm pathlength matched quartz or 1 cm pathlength disposable acryl cuvettes with a Cary 300 UV-Vis spectrophotometer equipped with a circulating water bath for temperature control, or Cary 4000 UV-Vis with a Cary Temperature Controller. Michaelis-Menten parameters were determined from the GraFit 5.0.13 program by fitting the data to the Michaelis-Menten expression. In time dependent inactivation assays, all absorbance measurements were recorded on a Beckman Coulter BTX 880 Multimode Detector (a plate reader). All buffer chemicals used were obtained from Sigma Chemical Co. Enzyme concentration was determined by Iogen Corporation. All kinetic studies for were performed in 50 mM citrate buffer, pH 5.0, containing 1% w/v bovine serum albumin (BSA) at 25 oC.  137 Buffer systems used in various kinds of assays: -  Assays carried out with T. reesei cellulases: 50 mM sodium citrate, 0.1% (w/v) BSA at pH 5.0. -  Expression and storage of Abg 2F6 glycosynthase: 50 mM sodium phosphate at pH 7.0 -   Enzymatic reaction with T. reesei cellulases (for TLC): 50 mM ammonium acetate, 0.1% (w/v) BSA at pH 5.0.  3.3.3.2 Steady-State Kinetic Measurements All substrate kinetic studies were conducted by monitoring the continuous release of dinitrophenolate via the measurement of the absorbance change at 400 nm wavelength in pH 5.0 buffer as reported previously150 (the extinction coefficients were reported in Chapter 2 Table 2.1).  Initial rates of enzyme-catalyzed hydrolysis of aryl glycosides were determined by incubating a solution of the appropriate substrate concentration in quartz cuvettes or polystyrene cuvettes within the spectrophotometer until thermally equilibrated (~ 5 minutes incubation time). Reactions were initiated by the addition of enzyme, and the release of phenol product was monitored at 400 nm. To ensure kinetic linearity, and to obtain a sufficient absorbance change for accurate calculation of the reaction rates, the concentration of enzyme added and the range of time that the initial rate of hydrolysis recorded was selected such that no more than 10% of the total substrate was hydrolyzed. Enzymatic hydrolysis rates of each substrate were measured at 6 to 8 different substrate concentrations ranging from about 0.1 to 7 Km. Values for Michaelis-Menten parameters (Km and  138 kcat) were determined by fitting the initial rates of hydrolysis versus substrate concentrations to the appropriate non-linear regression equation using the computer program GraFit 5.0.13..176  3.3.3.3 Irreversible Inactivation Kinetics The kinetic parameters for the inactivation of T. reesei CBHI, CBHII, EGI and EGII by the inactivators were determined as follows. T. reesei cellulases were incubated at 30 oC in the presence of varying concentrations of inactivators in 50 mM pH 5.0 citrate buffer containing 0.1% w/v BSA. The inactivation was initiated by the addition of the appropriate volume of enzyme solution. At different time intervals, aliquots of the inactivation mixture (20 'l) were removed and added into 180 'L 2,4-DNPC to halt the inactivation reaction by dilution of inactivator and competition with high concentration of substrate (5 mM final concentraion, preincubated at 30 oC). The residual enzyme activity was estimated by the initial rate of hydrolysis of 2,4-DNPC (dinitrophenolate released, i.e. absorbance change per second at 405 nm), which is directly proportional to the amount of active enzyme. The inactivation was monitored until 80 to 90% of enzyme activity was lost. 96 well plates and a Beckman Coulter BTX 880 Multimode Detector were used to achieve medium throughput measurements. Data collected was exported and analyzed using MS Excel. Pseudo-first order rate constants (kobs) for each inactivator concentration were estimated from the slope of  Ln (V/Vo) v.s. time in which V is the initial rate at different time interval and Vo is the rate of the control reaction (no inativator added). The values of Ki and ki were determined by fitting the kobs values to the equation describing inactivation, using the GraFit 5.0.13 program.  139 kobs= ki [I] Ki+[I]    Standard and systematic errors of all the assays conducted in this work were determined by averaging the duplicated result and by GraFit 5.0.13.  3.3.3.4 Competitive Inhibition Studies To Determine Inhibition Constant Ki The Ki value for fucosylDNPC with CBHI was determined by using a “range-finding” method. At two fixed substrate (2,4-DNPC) concentrations the enzyme was incubated with a range of fucosylDNPC concentrations (0.2 to 6 Ki) which bracketed the final determined Ki value. Initial absorbance change at 400 nm was recorded after the addition of CBHI. The assay was repeated with 2 different concentrations of 2,4 DNPC (1 Km and 30 Km). The observed rates were plotted in a Dixon plot (1/V0 v.s [inhibitor]) and Ki was determined (assuming competitive inhibition) by an intersection of these two lines (1 Km and 30 Km). The assay was performed at 25 oC in 50 mM citrate buffer (pH 5.0) containing 0.1% w/v BSA.  3.3.3.5 Substrate Depletion Assay for Rapid Determination of kcat/Km The second order rate constant of enzymatic reaction (kcat/Km) can be estimated with a substrate depletion assay. The substrate, at a concentration less than 0.2 Km, was incubated in buffer and thermally equilibrated in a UV/Vis spectrophotometer. Reactions were initiated by the addition of the enzyme, and 2,4 dinitrophenolate released was monitored by recording the absorbance change at 400 nm wavelength over 30 minutes or until complete substrate depletion was observed. The progress curve (absorbance v.s time) was fitted to a first-order rate equation  140 using the Cary 4000 UV/Vis. At low substrate concentrations, the Michaelis-Menten equation for reaction rates can be rewritten as: ! ! !!"#!! !!!!!!! The pseudo first-order rate constants !!"#!!  can therefore be conveniently obtained by deriving the observed rate constate by the enzyme concentration.               141 References 1) Hahn-Haegerdal, B.; Galbe, M. Gorwa-Grauslund, M. F.; Liden, G.; Zacchi, G. Trends Biotechnol. 2006, 24, 549-556. 2) Lin, Y.; Tanaka, S. Appl. Microbiol. Biotechnol. 2006, 69, 627-642. 3) Sun, Y.; Cheng, J. Bioresour. Technol. 2002, 83, 1-11. 4) Brumberg, L.; Cohn, D. R. Effective octane and efficiency advantages of direct injection alcohol engine; MIT Laboratory for Energy and the EnvironmentReport LFEE: Cambridge, MA, January 2008.  5) Cohn, D. R.; Bromberg, L.; Heywood, J. B. 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Z. 1913, 49, 333-369.  151 Appendix A  CONCENTRATIONS OF COMPOUNDS USED IN INACTIVATION ASSAYS (PARTIAL DATA)   CBHI Inactivator Concentration tested (mM) EPO3C 0, 1.7, 6.3, 8.4, 13.4, 20.1 EPO5C 0, 1.2, 3.2, 6.5, 13.4, 19.1 NCOCH2Br 0, 0.9, 1.8, 3.7, 4.1,5.0 2FDNPC 0, 0.01, 0.03, 0.05, 0.2, 0.4 2FDNPL 0, 0.04, 0.1, 0.2, 0.4, 0.9   CBHII Inactivator Concentration tested (mM) CH2Br 0, 0.9, 1.8, 3.7, 4.1, 5.0    EGI Inactivator Concentration tested (mM) EPO3C 0, 1.7, 6.3, 8.4, 13.4, 20.1 NCOCH2Br 0, 0.9, 1.8, 3.7, 4.1, 5.0    EGII Inactivator Concentration tested (mM) EPO3C 0, 1.7, 6.3, 8.4, 13.4, 20.1 EPO4C 0, 0.8, 2.0, 4.1, 8.1, 18.2 NCOCH2Br 0, 0.9, 1.8, 3.7, 4.1, 5.0 2FDNPC 0, 0.6, 1.3, 2.0, 6.2, 12.4 2FDNPL 0, 2.9, 5.2, 10.4, 15.6, 19.8 EPC4C 0, 1.5, 2.8, 4.9, 15.0, 22.5 EPC5C 0, 1.4, 2.7, 4.7, 14.1, 27.2      152  Appendix B  GRAPHICAL PRESENTATION OF DATA B.1 Steady State Kinetics for the Hydrolysis of 3,4-DNP cellobioside and 2,4-DNP trisaccharides   Michaelis-Menten plot for the hydrolysis of 3,4-DNPC catalyzed by CBHI    Michaelis-Menten plot for the hydrolysis of 3,4-DNPC catalyzed by CBHII   Michaelis-Menten plot for the hydrolysis of 3,4-DNPC catalyzed by EGI   Michaelis-Menten plot for the hydrolysis of 3,4-DNPC catalyzed by EGII   [3,4 DNPC] (mM) 0 0.03 0.06 0.09 0.12 0.15 0.18 R at e x1 0 -̂ 5 (m M s- 1) 0 0.5 1 1.5 2 2.5 3,4 DNPC-CBHI 1 / [Substrate] 0 20 40 60 80 1 / R at e 0 0.2 0.4 0.6 0.8 1 1.2 [3,4 DNPC] (mM) 0 4 8 12 16 20 R at e x1 0 -̂ 5 (m M s- 1) 0 2 4 6 8 3,4 DNPC-CBHII 1 / [Substrate] 00.20.40.60.811.21.41.61.82 1 / R at e 0 0.2 0.4 0.6 0.8 [3,4 DNPC] (mM) 0 4 8 12 16 20 R at e x1 0 -̂ 5 (m M s- 1) 0 5 10 15 20 25 3,4 DNPC-EGI 1 / [Substrate] 0 0.20.40.60.8 1 1.21.41.61.8 1 / R at e 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 [3,4 DNPC] (mM) 0 5 10 15 20 25 R at e x1 0 -̂ 5 (m M s- 1) 0 5 10 15 20 25 30 35 40 3,4 DNPC-EGII 1 / [Substrate] 0 0.2 0.4 0.6 1 / R at e 0 0.02 0.04 0.06 0.08 0.1  153  Michaelis-Menten plot for the hydrolysis of Arabdnpc catalyzed by CBHII   Michaelis-Menten plot for the hydrolysis of Arabdnpc catalyzed by EGI    Michaelis-Menten plot for the hydrolysis of Arabdnpc catalyzed by EGII   Michaelis-Menten plot for the hydrolysis of Fucdnpc catalyzed by CBHII      154  Michaelis-Menten plot for the hydrolysis of Fucdnpc catalyzed by EGI   Michaelis-Menten plot for the hydrolysis of Fucdnpc catalyzed by EGII                 Michaelis-Menten plot for the hydrolysis of Xydnpc catalyzed by CBHI   Michaelis-Menten plot for the hydrolysis of Xydnpc catalyzed by CBHII  155   Michaelis-Menten plot for the hydrolysis of Xydnpc catalyzed by EGI   Michaelis-Menten plot for the hydrolysis of Xydnpc catalyzed by EGII                 Michaelis-Menten plot for the hydrolysis of Galdnpc catalyzed by CBHI   Michaelis-Menten plot for the hydrolysis of Galdnpc catalyzed by CBHII   156  Michaelis-Menten plot for the hydrolysis of Galdnpc catalyzed by EGI   Michaelis-Menten plot for the hydrolysis of Galdnpc catalyzed by EGII [Fucdnpc] (mM) -0.02 0 0.02 0.04 0.06 1/ V o (A u- 1 m in -1 ) 0 100 200 300 1 mM DNPC 0.03 mM DNPC Fucdnpc-CBHI Dixon plot: inhibition of CBHI by Fucdnpc.     157 B.2 Time Dependent Inactivation Kinetics of T.reesei Cellulases with the Inactivators   Inactivation of EGI by 2FDNPC  Inactivation of EGI by 2FDNPL &'()*! &'(+*! &'('*! &*(,*! &*(-*! &*()*! &*(+*! &*('*! *('*! *(+*! *()*! *(**! '**(**! .**(**! +**(**! /**(**! 01 23 %3 #4 ! 561! 2FDNPC-EGI *(.'!57! *().!57! .(*8!57! '(.)!57! )(9/!57! -().!57! *!57! &+()*! &+(**! &.()*! &.(**! &'()*! &'(**! &*()*! *(**! *()*! *(**! )*(**! '**(**! ')*(**! .**(**! 01 23 %3 #4 ! 561! 2FDNPL-EGI +()-!57! )(+9!57! ,(8.!57! '+(+,!57! ',(9/!57! *(9+!57! '(*/!57! *!57! [2FDNPC] (mM) 0 2 4 6 8 k i 0 0.002 0.004 0.006 0.008 0.01 0.012 0.014 0.016 Parameter Value Std. Error Vmax 0.0221 0.0003 Km 3.1015 0.1075 1 / [Substrate] 0 2 4 1 / R at e 0 200 400 600 2FDNPC-EGI [2FDNPL] (mM) 0 2 4 6 8 10 12 14 16 18 20 k i 0 0.02 0.04 0.06 Parameter Value Std. Error Vmax 0.1018 0.0080 Km 10.0256 1.6568 2FDNPL-EGI 1 / [Substrate] 0 0.20.40.60.8 1 1.21.41.6 1 / R at e 0 20 40 60 80 100 120  158    Inactivation of CBHI by EPC4C  Inactivation of CBHII by EPC4C  &'(9*! &'(/*! &'(.*! &'(**! &*(8*! &*(9*! &*(/*! &*(.*! *(**! *(.*! *(**! )*(**! '**(**! ')*(**! 01 23 %3 #4 ! 561! EPC4C-CBHI *(-)!57! '()!57!! .()!57! -()!57! ')!57! *!57! &+(**! &.()*! &.(**! &'()*! &'(**! &*()*! *(**! *()*! *(**! )*(**! '**(**! ')*(**! .**(**! 01 23 %3 #4 ! 561! EPC4C-CBHII .(-.!57! '(+/!57! .(-.!57! '+(//!57! .-(',!57! *!57! [EPC4C] (mM) 0 2 4 6 8 10 12 14 16 k i 0 0.002 0.004 0.006 0.008 0.01 0.012 0.014 Parameter Value Std. Error Vmax 0.0155 0.0004 Km 1.7777 0.1616 1 / [Substrate] 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1 / R at e 0 20 40 60 80 100 120 140 160 180 200 220 EPC4C-CBHI [EPC4C](mM) 0 5 10 15 20 25 k i 0 0.005 0.01 0.015 0.02 0.025 Parameter Value Std. Error Vmax 0.0307 0.0009 Km 4.9517 0.4326 1 / [EPC4C] 0 0.2 0.4 0.6 0.8 1 / R at e 0 20 40 60 80 100 120 140 160 EPC4C-CBHII  159  Inactivation of EGI by EPC4C   Inactivation of CBHI by EPC5C  &+(**! &.()*! &.(**! &'()*! &'(**! &*()*! *(**! *()*! *(**! '**(**! .**(**! 01 23 %3 #4 ! 561! EPC4C-EGI ),(+8!57! .(,-!57! 9(.)!57! '/(*9!57! .,(9,!57! *!57! &+()*! &+(**! &.()*! &.(**! &'()*! &'(**! &*()*! *(**! *()*! '(**! *(**! .*(**! /*(**! 9*(**! 8*(**! !" #$% &% '( # 561! EPC5C-CBHI .(,-!57! /()!57! '()!75! ')!57! .,(-!57! *!57! [EPC4C] (mM) 0 20 40 60 k i 0 0.002 0.004 0.006 0.008 0.01 0.012 0.014 Parameter Value Std. Error Vmax 0.0166 0.0004 Km 8.7861 0.6675 EPC4C-EGI 1 / [Substrate] 0 0.2 0.4 1 / R at e 0 20 40 60 80 100 120 140 160 180 200 220 240 260 [EPC5C] (mM) 0 20 40 k i 0 0.02 0.04 0.06 Parameter Value Std. Error Vmax 0.0704 0.0037 Km 2.3978 0.4528 EPC5C-CBHI 1 / [Substrate] 0 0.2 0.4 0.6 1 / R at e 0 20 40  160  Inactivation of CBHII by EPC5C  Inactivation of EGI by EPC5C &+(**! &.()*! &.(**! &'()*! &'(**! &*()*! *(**! *()*! '(**! *(**! .*(**! /*(**! 9*(**! 8*(**! 01 23 %3 #4 ! 561! EPC5C-CBHII .*(/8!57! .(.'!57! /(-+!57! -(.)!57! +'()'!57! *!57! &/(**! &+()*! &+(**! &.()*! &.(**! &'()*! &'(**! &*()*! *(**! *()*! *(**! )*(**! '**(**! ')*(**! .**(**! 01 !23 %3 #4 ! 561! EPC5C-EGI +.(8'!57! '8(-)!57! -(8'!57! +(-)!57! +(-)!57! .(',!57! *!57! [EPC5C] (mM) 0 20 40 k i 0 0.02 0.04 0.06 0.08 0.1 0.12 Parameter Value Std. Error Vmax 0.1567 0.0076 Km 8.2847 1.0731 EPC5C-CBHII 1 / [Substrate] 0 0.2 0.4 1 / R at e 0 20 40 [EPC5C] (mM) 0 20 40 k i 0 0.02 0.04 0.06 Parameter Value Std. Error Vmax 0.0760 0.0046 Km 12.7705 1.7740 EPC5C-EGI 1 / [Substrate] 0 0.2 0.4 0.6 0.8 1 1 / R at e 0 20 40 60 80 100 120 140 160 180 200  161  [EPO3C] (mM) 0 2 4 6 8 10 12 14 16 18 20 22 K i 0 0.02 0.04 Parameter Value Std. Error Vmax 0.0911 0.0152 Km 22.6377 6.1011 EPO3C-CBHII 1 / [Substrate] 0 0.2 0.4 0.6 1 / R at e 0 20 40 60 80 100 120  Inactivation of CBHII by EPO3C  [EPO4C] (mM) 0 2 4 6 8 10 12 14 K i 0 0.02 0.04 0.06 Parameter Value Std. Error Vmax 0.0800 0.0026 Km 4.6385 0.3683 EPO4C-CBHI 1 / [Substrate] 0 0.2 0.4 0.6 0.8 1 1.2 1 / R at e 0 20 40 60 80 100  Inactivation of CBHII by EPO4C &.()*! &.(**! &'()*! &'(**! &*()*! *(**! *()*! *(**! )*(**! '**(**! ')*(**! 01 23 %3 #4 ! 561! EPO3C-CBHII '(98!57! 9(.8!57! 8(+8!57! ')(*8!57! .*('*!57! *!57! &/()*! &+(-)! &+(**! &.(.)! &'()*! &*(-)! *(**! *(-)! *(**! .*(**! /*(**! 9*(**! 8*(**! 01 23 %3 #4 ! 561! EPO4C-CBHI *(8'!57! .(*.!57! +(./!57! 8(*,!57! '/('9!57! *!57!  162  [EPO4C] (mM) 0 2 4 6 8 10 12 14 16 18 20 K i 0 0.02 0.04 0.06 0.08 0.1 Parameter Value Std. Error Vmax 1.2618 0.9398 Km 242.5611 192.2394 EPO4C-EGI 1 / [Substrate] 0 0.2 0.4 0.6 0.8 1 1.2 1 / R at e 0 20 40 60 80 100 120 140 160 180 200 220 240  Inactivation of EGI by EPO4C  [EPO5C] (mM) 0 2 4 6 8 10 12 14 16 18 20 k i 0 0.002 0.004 0.006 0.008 0.01 0.012 0.014 Parameter Value Std. Error Vmax 0.0285 0.0022 Km 21.4475 2.7670 EPO5C-CBHII 1 / [Substrate] 0 0.2 0.4 0.6 0.8 1 / R at e 0 200 400 600  Inactivation of CBHII by EPO5C &+()*! &+(**! &.()*! &.(**! &'()*! &'(**! &*()*! *(**! *()*! *(**! )*(**! '**(**! 01 !23 %3 #4 ! 561! EPO4C-EGI 8(*,!57! /(*)!57! '8(.*!57! *(8'!57! .(*.!57! *!57! &+()! &+! &.()! &.! &'()! &'! &*()! *! *()! *(**! '**(**! .**(**! +**(**! /**(**! 01 !23 %3 #4 ! 561! EPO5C-CBHII '(..! 57! +(.)! 57! 9(*)! 57!  163  [EPO5C] (mM) 0 20 40 k i 0 0.02 0.04 0.06 Parameter Value Std. Error Vmax 0.0752 0.0134 Km 17.5808 7.2016 EPO5C-EGI 1 / [Substrate] 0 0.2 0.4 1 / R at e 0 20 40 60 80 100 120 140 160 180 200 220 240  Inactivation of EGI by EPO5C  [EPO5C] (mM) 0 2 4 6 8 10 12 14 16 18 20 k i 0 0.002 0.004 0.006 0.008 0.01 Parameter Value Std. Error Vmax 0.0153 0.0016 Km 13.9983 2.7156 EPO5C-EGII 1 / [Substrate] 0 0.2 0.4 0.6 0.8 1 / R at e 0 200 400 600  Inactivation of EGII by EPO5C      &/()! &/! &+()! &+! &.()! &.! &'()! &'! &*()! *! *()! *(**! )*(**! '**(**! ')*(**! .**(**! 01 !23 %3 #4 ! 561! EPO5C-EGI .()*!57! 9(9-!57! '+(++!57! .-()*!57! +,('-!57! *!57! &.()! &.! &'()! &'! &*()! *! *()! *(**! '**(**! .**(**! +**(**! 01 !23 %3 #4 ! 561! EPO5C-EGII '(..!57! +(.)!57! 9()!57! '+(/!57! ',(*,!57! *!57!  164 B.3 TLC and Gel Results  \ Figure B.3.1 TLC of (a) inactivation mixture of CBHI, CBHII and EGI with EPC4C. SM: EPC4C; (b) inactivation mixture of CBHI, CBHII and EGI with EPO3C. SM: EPO3C; (c) reaction mixture of DNPC and EGI with cyclophellitol. SM: DNPC, C: control (DNPC, EGI and buffer only); Rxn: reaction (DNPC, EGI, cyclophellitol, and buffer); Ref1: cellobiose; Ref2: glucose and DNPG. (d) Electrophoresis gel of the four T. reesei cellulases. C1: CBHI; C2: CBHII; E1: EGI; E2: EGII.  165 Appendix C  BASIC ENZYME KINETICS C.1 Fundamentals of Enzyme Kinetics  L. Michaelis and M. L. Menten177 proposed that steady-state enzymatic reactions could be accounted for by the following scheme (1):    They proposed a two step enzyme-catalyzed reaction. First the substrate binds reversibly at the catalytic domain of the enzyme, forming an enzyme substrate non-covalently linked complex ES, also known as the Michaelis complex. The complex sequentially turns into product and free enzyme in the second step with the first order rate constant kcat.  The Michaelis-Menten equation describes the initial rate v of the above enzyme- catalyzed reaction. [E]o is the total enzyme concentration; [S] is the substrate concentration at any point; kcat is the apparent first-order rate constant; and Km  is the Michaelis constant.   ES  
  S   There are two assumptions made in deriving the Michaelis-Menten equation. First, the substrate concentration vastly exceeds the enzyme concentration. This is generally true because an enzyme catalyzes its reaction in a very efficient way and only small amount are added. Second, the reverse reaction can be ignored since no significant accumulation of product when the initial rate is recorded.  166  The Michaelis-Menten mechanism assumes that the ES complex is in rapid equilibrium with the free enzyme and substrate, which holds when k2 << k1/k-1 in the scheme (2).  As a result, in the simple case of Michaelis-Menten kinetics, the expression of Km = (k-1 + k2)/k1 can be simplified to k-1/k1. Km thus provides a measure of the affinity of substrate to enzyme. The lower the Km, the higher the affinity is. Km is also the substrate concentration where the reaction rate is half of the maximum (v=Vmax/2). At low substrate concentration ([S]<< Km), the Michaelis-Menten equation can be rewritten as ! ! !!!!!!!!!"#!! Therefore, the Michaelis-Menten expression can be graphed as Figure C.1.1  Figure C.1.1 Plot of initial rate (v) versus substrate concentration [S] for a typical Michaelis-Menten kinetics.   167 with [S]>> Km, the initial rate can be expressed as the equation below.!! ! !!!!!!"#! ! !!!"# The apparent second order rate constant kcat/Km describes the reaction rate relating to the concentration of free enzyme and free substrate. It is also referred to as a specificity constant, which is an indication of the catalytic efficiency of the enzyme to that substrate. kcat/Km is commonly used for comparing the kinetic results among different substrates for the same enzyme.  C.2 Irreversible Inactivation and Mechanism-based Inhibition Kinetics Most of the enzymatic reactions studied in this thesis focus on the irreversible inactivation of cellulases by active site-directed or mechanism-based inactivators. The kinetic analysis of this irreversible inactivation is discussed in this section. Similar to Michaelis- Menten kinetic analysis with substrates, the reaction involves the initial binding of the inactivator followed by the formation of a covalent inactivator species (scheme (3)). However, in the reaction with active site-directed inactivators, the covalently linked species E-I will not be turned over, though, with some classes of mechanism-based inactivator studies in this thesis, slow reactivation is indeed  observed (scheme (4)), making these very slow substrates.  If kreactivation is negligible compared with ki then inactivation ensures.  168 Kinetic studies of inactivation were conducted under the conditions [I]>>[E], thus the kinetic equation resembles that of the Michaelis-Menten kinetics, and thus pseudo first-order kinetics with respect to [E] would be observed. ! ! !! ! !!!!! ! ! !!! ! !!"#!!! !!"# ! !! !!!!!! ! !!! where ki is the rate constant for inactivation, and Ki is an apparent dissociation constant for all the enzyme-bound species. In the above equation, kobs is the observed rate constant for the time-dependent loss of enzyme acitivty. Ki and ki can be determined from the plot of kobs against different concentrations of [I] (the concentrations bracketed the Ki value ultimately determined) used to inactivate the enzyme activity. These two values serve as a good indication of the quality of inactivators. In some cases, either due to very rapid inactivation or to insolubility or scarcity of inacitivator, a [I]>Ki can not be studied. In that case, the equation can be rewritten as !!"# ! !! !!!!!! and only the second order inactivation rate constant ki/Ki can be determined.  C.3 Competitive Inhibition Kinetics An inhibitor which binds to the active site of the enzyme reversibly and thereby prevents the substrate S from binding to the same site and vice versa (scheme (5)) is referred as a competitive inhibitor.  169  Another equilibrium is established in the presence of a competitive inhibitor I. Km and Ki are the Michaelis and inhibitionconstants of substrate and inhibitor respectively. In the presence of a competitive inhibitor, Km has a higher apparent value by the factor of (1+[I]/Ki), and the kinetic equation can be rewritten as the following: ! ! !!!!!!!!!!"#! ! !!!!! !!!!!!! Vmax of the enzymatic reaction does not change, and thus the presence of the competitive inhibitor causes the substrate an apparent worse-binding substrate. In other words, the competitive inhibitor does not affect the turnover number (kcat) of the enzyme to that substrate.  C.4 Alternative Cleavage in This Thesis In our cases, it is likely that the tested saccharide binds to the catalytic domain of the cellulase in more than one way within the extensive glycosyl binding sites. These multiple binding modes can result in the cleavage of different glycosidic bonds within the glycosyl groups or result in competitive inhibition if no reaction occurs. Herein, the terms “alternative cleavage” in this thesis refer to this internal glycosidic bond cleavage (Figure C.4.1).   170  Figure C.4.1 Alternative cleavage enzymatic reaction with an (a) inactivator or (b) aryl substrate.  Alternative cleavage of inactivator (Figure C.4.1(a)) will decrease the concentration of reagent. In addition, alternative cleavage of dinitrophenyl substrates releases the products which are not detectable using a UV-Vis spectrometer (Figure C.4.1(b)) and also results in the reduction of substrate concentration.  C.4.1 Multiple Binding Modes Does Not Affect kcat/Km (or ki/Ki) A substrate might bind to the active site of the enzyme in a non-productive binding mode, in competition with the productive mode of binding (Scheme (6) and Figure C.4.2).   171 Where [E] is the concentration of free enzyme. The presence of any non-productive binding mode results in decrease of kcat and Km.  The rate of the productive reaction becomes ! ! !!"#!!!!!!!!!! ! ! !!!!!! !! !!!!!! Thereby the apparent kcat’ =kcat /(1+Km/Ks)    Km’= Km /(1+Km/Ks)    (kcat’ and Km’ are the apparent values) Interestingly therefore the apparent second order rate constant kcat/Km is not affected by non-productive binding. The specificity of the substrate is determined by the ratio of kcat/Km which is not affected by the other binding modes, as long as they do not result in significant changes on [S] or [E].  Figure C.4.2 Productive and non-productive binding. The red arrow indicates the glycosidic bond that is hydrolyzed.   

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