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Synthesis of UDP-6-deoxy-6,6-difluoro-D-GlcNAc : a potential mechanism-based inhibitor of pseB Bassiri, Jackie Mehrnaz 2006

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SYNTHESIS OF UDP-6-DEOXY-6,6-DIFLUORO-D-GLCNAC: A POTENTIAL MECHANISM-BASED INHIBITOR OF PSEB  by Jackie Mehrnaz Bassiri B.Sc, The University of Calgary, 2004  A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF T H E REQUIREMENTS FOR T H E D E G R E E OF  MASTERS OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Chemistry)  THE UNIVERSITY OF BRITISH COLUMBIA December 2006 © Jackie Mehrnaz Bassiri  11  Abstract  Pseudaminic acid is a nine carbon sugar, similar to sialic acid, and has been found to play an important role in the biosynthesis of flagella in pathogenic bacteria such as C. jejuni and H. pylori. For these bacteria the flagella is crucial in motility and colonization of the host. Because this sugar is found only in bacteria, enzymes required for its biosynthesis serve as novel targets for antibiotic development. A UDP-GlcNAc dehydratase, pseB, catalyses the first step of pseudaminic acid biosynthesis in C. jejuni. This enzyme catalyzes the conversion of UDP-GlcNAc to UDP-6-deoxy-4-ketoHexNAc. The proposed mechanism of this conversion is composed of oxidation of the C4 hydroxyl resulting in the formation of a 4-keto group, then a dehydration across the bond between C5 and C6 producing a UDP-4-keto-5,6-ene-hexose, and finally reduction in which a hydride from the enzyme bound NADFt cofactor is transferred to C6, and C5 is reprotonated on the opposite face such that the stereocentre at C5 is inverted during this dehydration reaction. In 1998, the synthesis of CDP-6-deoxy-6,6-difluoro-D-glucose, 9, was published by Chang et al. This compound was the first mechanism-based inactivator known for a 24  dehydratase enzyme, more specifically, CDP-D-glucose 4,6-dehydratase, isolated from Yersinia pseudotuberculosis. Based on this work, a 10-step synthesis of UDP-6-deoxy6,6-difluoro-D-GlcNAc 11 was completed which generated a potential mechanism-based inhibitor for pseB.  Ill  11  The preliminary enzyme kinetics performed did not support 11 as a mechanismbased inactivator of pseB. One possible explanation for this is based on the steric aspect of the inhibitor vs. the substrate. The two fluorine groups on the C-6 positions are possibly too bulky for the inhibitor to bind the enzyme active site and cause inactivation. Another reason might be that the H-bonding of the C-6 hydroxyl group of the substrate to the enzyme is crucial in substrate binding. When it is replaced with two fluorines the lack of this H-bonding does not allow binding of the inhibitor to the active site.  iv TABLE OF CONTENTS Abstract  ii  Table of Contents  iv  L i s t of Figures  <  vii  Abbreviations and Symbols  x  Acknowledgements  xiv  C h a p t e r 1: Introduction  1  1.1  Sugar Nucleotides  2  1.2  Sugar Nucleotide-Modifying Enzymes  4  1.2.1  C-O Bond Cleavage Reactions  6  1.2.2  Catalytic Mechanism of E d with TDP-Hexose Substrate  6  1.2.3  Detailed Stereochemical Studies of the Catalysis of E d Using  0  0  CDP-Hexose 1.2.4 1.3  1.4  C h a p t e r 2:  2.1  9  CDP-6-Deoxy-6,6-difluoro-a-D-glucose: a Mechanism-Based Inhibitor.. 10  Pseudaminic Acid  '  12  1.3.1  Glycosylation in Bacterial Flagella  12  1.3.2  Biosynthesis of Pseudaminic Acid  13  1.3.3  Role of UDP-GlcNAc Dehydratase PseB (CJ1293) in Pse Biosynthesis... 15  Aim of Thesis  17  Synthesis of UDP-6-deoxy-6,6-difluoro-GlcNAc.  .19  Introduction  .20  V  2.2  2.3  Synthetic Strategies for the Proposed Inhibitor  21  2.2.1  Attempted Synthesis of Inhibitor 11 Based on Literature Precedence  21  2.2.2  The Revised Synthetic Plan  29  2.2.3  Remaining Steps in the Synthesis of Compound 11  ....31  Experimental Methods  38  2.3.1  General Method  38  2.3.2  Materials  38  2.3.3  Preparation of methyl 2-acetamido-2-deoxy-a-D-glucopyranoside, 23... .39  2.3.4  Preparation of benzyl 2-acetamido-2-deoxy-a-D-glucopyranoside, 25  2.3.5  Preparation of benzyl 2-acetamido-2-deoxy-6-0-trityl-a-Dglucopyranoside, 26  2.3.6  40  Preparation of benzyl-2-acetamido-3,4-0-benzyl-a-D-glucopyranoside, 27.;  2.3.7  40  41  Preparation of benzyl-2-acetamido-3,4-0-benzyl-6,6'-difluoro-a-Dglucopyranoside, 28  42  2.3.8  Preparation of 2-acetamido-6,6'-difluoro-a-D-glucopyranoside, 29  43  2.3.9  Preparation of acetyl-2-acetamido-3,4-acetyl-6,6'-difluoro-a-Dglucopyranoside, 30  43  2.3.10 Preparation of 2-acetamido-3,4-acety 1-6,6'-difluoro-a-Dglucopyranoside, 31  44  VI  2.3.11 Preparation of dibenzyl 2-acetamido-3,4-acetyl-6,6'-difluoro-a-Dglucopyranosyl phosphate, 32  44  2.3.12 preparation of 2-acetamidp-6,6'-difluoro-a-D-glucopyranoside 1-phosphate, 33  45  2.3.13 Preparation of UDP-2-acetamido-6-deoxy-6,6'-difluoro-a-Dglucopyranoside, 11  Chapter 3: Inhibitor Testing with PseB  46  48  3.1  Overexpression and purification of HiS6-tagged pseB  49  3.2  General Properties of Enzyme Inhibitors  51  3.3  Preliminary Enzyme Kinetics Using UDP-6-deoxy-6,6-difluoro-GlcNAc Inhibitor  52  3.3.1  57  Further Studies  3.4  Conclusion....  59  3.5  Experimental Methods  59  3.5.1  Enzyme Overexpression and Purification  59  3.5.2  Bradford Method for Determining Enzyme Concentration..:  60  3.5.3 , P N M R Kinetic Assay  61  3.5.4  61  3 1  References  ' H N M R Kinetic Assay  .  Appendix: ' H NMR Spectra of Sugar Nucleotide Products  63 67  Vll  LIST OF FIGURES Figure 1.1 Generic scheme showing the structure of sugar nucleotides and the five bases  2  Figure 1.2 One of nature's methods for the biosynthesis of sugars nucleotides  3  Figure 1.3 UDP-galactose 4-epimerase, the best understood nucleotide-modifying enzyme  5  Figure 1.4 Catalytic mechanism of Eod  7  Figure 1.5 Hydride transfer from C-4 of TDP-6-deoxyglucose to the si face of the N A D cofactor  +  :  Figure 1.6 CDP-D-glucose 4,6-dehydratase can reduce its product in the presence of N A D H Figure 1.7 Proposed mechanism of inhibition of Eod by CDP-6-deoxy-6,6-difluoro-a-D-  9 9 11  glucose Figure 1.8 Structures of sialic acid and pseudaminic acid  12  Figure 1.9 Biosynthetic pathway of CMP-pseudaminic acid  .14  Figure 1.10 Potential roles of catalytic residues in the active site of F l a A l  17  Figure 2.1 Structure of UDP-6-deoxy-6,6-difluoro-D-GlcNAc, a potential mechanism-based inhibitor for PseB Figure 2.2 Reported synthesis of CDP-6-deoxy-6,6-difluoro-D-glucose..  21 22  Figure 2.3 Attempted synthesis of UDP-6-deoxy-6,6-difluoro-D-GlcNAc based on literature precedence  23  Figure 2.4 Carbocation formed during tritylation  24  Figure 2.5 Reaction of D M S O with oxalyl chloride in swern oxidation  25  Figure 2.6 The formed intermediate leading to formation of dimethyl sulfide and. the desired oxidized product  26  Vlll Figure 2.7 Swern oxidation reactions at higher temperatures lead to the formation of a different product  26  Figure 2.8 DAST fluorination reaction mechanism  Figure 2.9  1 9  27  F N M R spectrum (^-decoupled) of benzyl-2-acetamido-3,4-0-benzyl-6,6'-  difluoro-a-D-glucopyranoside 23  28  Figure 2.10 Synthetic plan used for the synthesis of inhibitor, 11  30  Figure 2.1.1 Reaction side-product in presence of excess hydrazine acetate  32  Figure 2.12 Molecular structures of 1,2,4-triazole and lH-tetrazole  33  Figure 2.13 Postulated mechanism of anpmeric phosphorylation reaction  ....34  Figure 2.14 The phosphorylation reaction gives only the a-anomer, since the fi-anomer decomposed by forming an oxazoline compound  Figure 2.15 Final coupling in the preparation of UDP-6-deoxy-6,6-difluoro-GlcNAc, 11  34  35  Figure 2.16 F N M R spectrum ('H-coupled) of final product showing splittings betweent F l , 19  F2, H - 5 " , H - 6 "  :  Figure 2.17 P N M R spectrum ('H-decoupled) of the final product 11 3 1  36 37  Figure 3.1 SDS-PAGE gel of crude lysate and purified enzyme: (1= molecular weight markers (Bovine Serum Albumin at 66 kDa and Carbonic Anhydrase at 29 kDa), 2= crude lysate, 3= pure enzyme)  50  ix Figure 3.2 P N M R s of a) UDP-GlcNAc with PseB undergoing dehydration, andb) UDP3 1  GlcNAc undergoing dehydration catalysed by PseB that had been pre-incubated  with  UDP-6-deoxy-6,6-difluoro-GlcNAc for 30 min  53  Figure 3.4 Following the progress of enzymatic reaction by formation of methyl singlet peaks in the H N M R . The peak at 1.46 ppm represents the structure on the right, and the one on !  1.22 ppm represents the one on the left  55  Figure 3.3 ' H N M R experiments with A)2 hour incubated enzyme added to UDP-GlcNAc (control) and B) incubated enzyme with the inhibitor and added to the substrate. Both control and PseB pre-incubated with UDP-6-deoxy-6,6-difluoro-GlcNAc show similar production of productUDP-6-deoxy-4-keto-HexNAc which can be observed atl.46 and 1.22 ppm (He' signals for unhydrated and hydrated product respectively)  56  Figure 3.5 Biosynthetic pathway of UDP-Bacillosamine found in the bacterial glycoprotein heptasaccharide  58  X  Abbreviations and Symbols  List of abbreviations, most of which are commonly used in the chemical literature, to be employed in this thesis: 5  chemical shift (ppm)  Ac  acetyl  ADP  adenosine 5'-diphosphate  ATP  adenosine 5'-triphosphate  Bn  benzyl  °C  degree Celsius  Cys  cysteine  CDP  cytosine 5'-diphosphate  d  doublet  dd  doublet of doublets  ddd  doublet of doublet of doublets  DMF  A/,A/-dimethylformamide  DMSO  dimethyhlsulfoxide  DNA  deoxyribonucleic acid  DTT  dithiothreitol  E. coli  Escherichia coli  ESI  electrospray ionization  Et  ethyl  XI  g  gram  GlcNAc  N-acetylglucosamine  h  hour  Hz  hertz  IPTG  isopropyl P-thiogalactopyranoside  J  coupling constant  kcat  catalytic rate constant (turnover number)  Ki  inhibition constant  LB  Luria-Bertani  LPS  lipopolysaccharide  Lys  lysine  m  multiplet  M  molar  Me  methyl  mg  milligram  mL  milliliter  mM  millimolar  mmol  millimole  MS  mass spectrometry  NAD  nicotinamide adenine dinucleotide, oxidized form  +  NADH NADP  nicotinamide adenine dinucleotide, reduced form +  nicotinamide adenine dinucleotide phosphate  NADPH  reduced nicotinamide adenine dinucleotide phosphate  NMR  nuclear magnetic resonance  OD600  optical density at 600 nm  ppm  parts per million  psi  pounds per square inch  PCR  polymerase chain reaction  q  quartet  rt  room temperature  s  singlet  SDR  short chain dehydrogenase/reductase  SDS-PAGE  sodium dodecylsulfate polyacrylamide gel electrophoreisis  SN2  second order nucleophilic substitution  t  triplet  THF  tetrahydrofuran  TLC  thin layer chromatography  UDP  uridine 5'-diphosphate  UDP-GlcNAc UDP-N-acetylglucosamine UV  ultraviolet  %  percent  Commonly Occuring Amino Acid Abbreviations A  Ala  Alanine  C  Cys  Cysteine  D  Asp  Aspartate  E  Glu  Glutamate  F  Phe  Phenylalanine  G  Gly  Glycine  H  His  Histidine  1  He  Isoleucine  K  Lys  Lysine  L  Leu  Leucine  M  Met  Methionine  N  Asn  Asparagine  P  Pro  Proline  Q  Gin  Glutamine  R  Arg  Argining  S  Ser  Serine  T  Thr  Threonine  V  Val  Valine  W  Tip  Tryptophan  Y  Tyr  Tyrosine  XIV  Acknowledgements  1 would like to express my sincere gratitude to my supervisor, Dr. Martin E. Tanner, for all of his help, guidance, and patience. 1 would also like to thank the past and present members of the Tanner Group for their friendship, and support during my time at UBC. 1 would especially like to thank James Morrison and Feng Liu for their help with my kinetics and enzyme overexpression. It has been a great pleasure working with all of you. Finally, I would like to thank my family for their unconditional love, with which I am able to grow and prosper in all aspects of my life.  XV  For My Father  Chapter 1  Introduction  2 1.1  Sugar Nucleotides  In recent years, the study of carbohydrates has received much attention. Previously it was believed that these molecules serve pnly as energy sources or structural elements in nature but now it is evident that carbohydrates also play important roles in biochemical recognition events.'' ' In nature, sugar molecules are "activated" as sugar 2 3  nucleotides and thereby made available for biosynthesis (Figure 1 . l ) . Transferase 4  enzymes use these sugar nucleotides to glycosylate a variety of biological nucleophiles. The most common examples of activated sugar nucleotides include: UDP-glucose (UDPGlc), UDP-galactose (UDP-Gal), UDP-glucuronic acid (UDP-GlcUA), UDP-/Vacetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), GDPmannose (GDP-Man), GDP-fucose (GDP-Fuc), and CMP-jV-acetylneuraminic acid (CMP-Neu-5-Ac). There are many more examples of sugar nucleotides found in nature 5  in microorganisms and plants, however these are less common. '  6 7  Base =  Figure 1.1 bases.  Generic scheme showing the structure of sugar nucleotides and the five  .  3  Two methods are used by nature to biosynthesize sugar nucleotides. The first method builds them up from a free sugar and nucleotide triphosphates (Figure 1.2).  8  Examples include UDP-Glc, UDP-GlcNAc, and GDP-Man, where a 6-phosphosugar is generated initially and then is isomerized to a higher energy 1-phosphosugar by a phosphomutase enzyme. In the final step a pyrophosphorylase enzyme catalyzes the reaction of the 1 -phosphosugar with a nucleotide triphosphate to form the sugar Q  nucleotide.  O H  kinase  O  A D P A T P O H  O H  phosphomutase O H  pyrophosphorylase  O  O H  X T P O X D P O P O  PPi  Figure L2  = 3  One of nature's methods for the biosynthesis of sugars nucleotides.  The second method is a more efficient route where enzymes act to simply modify the carbohydrate portion of a pre-existing sugar nucleotide. In this case, the above mentioned pathway can be conveniently avoided.  6,7  4 1.2  Sugar Nucleotide-Modifying Enzymes  Sugar nucleotide-modifying enzymes have been extensively studied and in many cases, their mechanisms are very well known. These enzymes are classified based on the chemical reaction they catalyze and the similarity in their catalytic mechanism. They act on activated carbohydrate substrates and most possess a tightly bound NAD(P) / +  NADH(P) cofactor in their active sites that promotes transient oxidation during catalysis. This cofactor is regenerated after each catalytic cycle and cannot be removed from the active site without denaturing the enzyme. Therefore these enzymes often do not require the addition of exogenous N A D . The best understood of these enzymes in terms of +  mechanism and structure is UDP-galactose 4-epimerase that catalyzes the interconvertion of UDP-Glc and UDP-Gal (Figure 1.3). In the first step, the C-4 hydroxyl of the 9  substrate is oxidized resulting in a bound 4-ketohexopyranosyl intermediate and N A D H . This keto-intermediate is flipped over in the active site of the enzyme so that the carbonyl group is in the proper orientation with respect to the N A D H molecule. This intermediate is then reduced at the C-4 position, forming the other epimer while regenerating N A D . +  An interesting fact to note about this enzyme is that the hydride transfer from its bound N A D H cofactor is non-stereoselective.  NAD  OH  HO"V^^°\  HO-A^-T^A H 0  O-UDP  UDP-Glc  NAD  +  HQ  O-UDP  NADH  Ho-y^^7 HQ O  H  O-UDP  OH  UDP-Gal OH  Figure 1.3  UDP-galactose 4-epimerase, the best understood sugar nucleotide-  modifying enzyme.  UDP-galactose 4-epimerase is a member of the short-chain dehydrogenase/reductase (SDR) family. Members of this family can use the redox 7  reaction at C-4 to catalyze diverse reactions such as C-4 epimerization, C-3/C-5 epimerizations, dehydrations, and substitution reactions.  6  1.2.1  C-O Bond Cleavage Reactions  C-0 bond cleavage reactions form mono- di-, tri-, and even tetradeoxygenated sugars that are found in numerous glycoproteins, glycolipids, and secondary metabolites. These sugars play key roles in the recognition, binding, and/or activity of a diverse set of natural products. Such deoxygenated sugars most often lack a hydroxyl group at C-6 10  and deoxygenation at other positions is rarer." It has been shown that C-6 deoxygenation is the first step in almost all deoxyhexose biosynthethic pathways. These SDR enzymes that catalyze the deoxygenation at C-6 are referred to as sugar nucleotide oxidoreductases (Eod) or sugar nucleotide 4,6-dehydratases .  1.2.2  Catalytic Mechanism of Eod with TDP-Hexose Substrate  Most of the early studies on Eod enzymes focused on TDP-glucoseoxidoreductase that catalyzes intramolecular oxidation-reduction reaction involving a net elimination of water (Figure 1.4). In this reaction, TDP-glucose 1 is converted to TDP-4keto-6-deoxyhexose 4. The product can then act as a precursor for the biosynthesis of other deoxysugars. ' '  12 13 14  7  Figure 1.4  Catalytic mechanism of Eod  In previous studies, substrate 1 was labeled with deuterium or tritium at C-4 and was incubated with E d . It was observed that this reaction led to the production of TDP-40  keto-6-deoxyglucose 4 and the isotope label was detected at the C-6 position. In another 15  experiment, the substrate was incubated with enzyme reconstituted with tritium or deuterium labeled N A D at the 4 position of the molecule. This did not produce tritium +  16  or deuterium labeled TDP-4-keto-6-deoxyglucose. These results suggest that hydride transfer to and from N A D always occurs from the same face of the cofactor. The N A D +  cofactor accepts a hydride from C-4 of substrate 1 and then passes this same hydride to the C-6 of the intermediate 3 as it regenerates N A D . +  Another experiment used to elucidate the mechanism of E reaction in H 0 or H2O. 2  o d  was to carry out the  The product formed from this experiment showed isotope  +  8  incorporation at C-5. This supports the notion that a deprotonation event occurs at C-5 during catalysis. TDP-6-deoxyglucose (unlabeled 5) was incubated with the enzyme and was found to participate in the first oxidation step but could not eliminate water, resulting in accumulation of enzyme bound N A D H (Figure 1.5).  18  When TDP-[4- H]- 6-deoxy-D3  glucose 5 was used, the hydride transfer in the catalytic mechanism of E i was shown to oc  occur onto the si face of the nicotinamide ring. Using these obtained data, the mechanism of catalysis reaction (Figure 1.4) shows that the substrate TDP-D-glucose 1 is oxidized to TDP-4-keto-6-deoxyglucose 2, and N A D is converted to N A D H . In the next step water +  is eliminated across C-5 and C-6, leading to the formation of intermediate 3. Then finally, a reduction step occurs using the hydride from N A D H that produces TDP-4-keto-6deoxyglucose 4.  OTDP  R Figure 1.5  Hydride transfer from C-4 of TDP-6-deoxyglucose to the si face of the  NAD cofactor +  R  9  1.2.3  Detailed Stereochemical Studies of the Catalysis of E d Using CDP-Hexose 0  A related enzyme, CDP-D-glucose 4,6-dehydratase found in Yersinia pseudotuberculosis, has been studied more recently. This enzyme is somewhat different, as it utilizes exogenous N A D for full activity. The same type of stereochemical +  19  experiments were performed as described earlier and results show that the mechanism of these two enzymes are very similar. In one experiment, the apoenzyme (lacking bound cofactor) was incubated with stereospecifically deuterated N A D H and the product of the normal reaction, CDP-4-keto-6-deoxyglucose 7 (Figure 1.6). Results show that this 20  enzyme can convert the CDP-4-keto-6-deoxyglucose 7 into CDP-6-deoxyglucose 8 using N A D H as a source of hydride. Using deuterium-labeled N A D H , incubation of the enzyme with substrate 7 revealed that the pro-S hydrogen of N A D H is being transferred to the 4-keto group of 7.  R  Figure 1.6 NADH.  CDP-D-glucose 4,6-dehydratase can reduce its product in the presence of  10 In another study, a chiral methyl analysis was performed on the 6-deoxy product formed from stereospecifically labeled (6S)-and (6#)-CDP-[4- H,6- H]-D-glucose and the 2  3  Y. pseudotuberculosis enzyme. The overall reaction was found to involve a 21  displacement of the C-6 hydroxyl group by the C-4 hydride with a net inversion of configuration. Assuming that the nicotinamide cofactor in E j suprafacially transfers a 0(  hydride from C-4 to C-6 of the sugar skeleton, one expects that the elimination of water from C5/C6 is a syn-process. This would mean that the reduction of the resulting intermediate must take place with an <3/?//-addition of a hydride and a proton across the double bond. Similar stereochemical observations were obtained in the study of TDP-Dglucose 4,6-dehydratase and GDP-D-mannose 4,6-dehydratase enzymes. '  22 23  1.2.4  CDP-6-Deoxy-6,6-difluoro-a-D-glucose: a Mechanism-Based Inhibitor  Understanding this E d-catalyzed reaction is significant for designing methods to 0  control and/or regulate deoxysugar biosynthesis. A substrate analogue, CDP-6,-deoxy-6,6difluoro-a-D-glucose 9 was synthesized and tested with CDP-D-glucose 4,6dehydratase.  24  It was determined that this compound is a time-dependent, mechanism-  based inactivator for E d (K| = 0.94 m M and k ct = 2.4 x 10" min" ). ESI-MS analysis 2  0  1  ina  showed that the enzyme is inactivated due to covalent binding of an active-site nucleophile by the intermediate 10, trapping the enzyme and destroying its activity (Figure 1.7).. In the first step C-4 is oxidized, followed by the elimination of HF as opposed to water. Next, the transfer of hydride back to the C-6 position results in an  11  enolate which eliminates the second fluoride as it goes to the enone form. At this stage the cofactor is in the oxidized form, so reduction of the enone is not possible and an active site nucleophile may add to form a covalent adduct.  NAD  NAD-H  Eod  OH B:  I  OCDP  OCDP  Nu~ NAD  +  NAD  H  OCDP B-H  1  0  B-H  OCDP  u W W W  Figure 1.7 D-glucose  Proposed mechanism of inhibition of Eod by CDP-6-deoxy-6,6-difluoro-a-  12  1.3  Pseudaminic Acid  1.3.1 Glycosylation in Bacterial Flagella  The flagellated Gram-negative bacteria Campylobacter jejuni and Campylobacter coli are the main causes of bacterial diarrhea throughout the world ' , and Helicobacter 25  26  pylori is responsible for causing duodenal ulcers in humans. It has been estimated that 27  one half to two-third of the world's population is chronically infected with H. pylori, and prevalence among adults in developing countries is around 80-90%. The bacterium can remain in the stomach for decades without producing symptoms, however infections can lead to gastric inflammation and ulceration. In all of these pathogenic organisms, the flagellin proteins are heavily glycosylated with pseudaminic acid (5,7-diacetamido3,5,7,9-tetradeoxy-L-g/vcero-L-ma/7no-nonulosonic acid), which is a nine carbon sugar that is similar to sialic acid (Figure 1.8). '  28 29  Pseudaminic acid was first identified in  1984 in the lipopolysaccharides (LPS) oiPseudomonas aeruginosa. Sialic acid-like sugars have been found in many species of Gram-negative bacteria as components of cell surface glycoconjugates, such as LPS, capsular polysaccharide, poli, and flagella that are crucial for host cell invasion and pathogenesis. " 30  32  NHAc  Sialic acid Figure 1.8  Pseudaminic acid (Pse)  Structures of sialic acid and pseudaminic acid  13 The importance of the production of this a-keto acid is demonstrated by aflagellate C. jejuni  33  and H. pylori  34  mutants that are unable to produce pseudaminic  acid. These results indicate that pseudaminic acid glycosylation of flagellin proteins is necessary for flagellar assembly and function. These bacteria need their flagella for motility, invasion and colonization of the viscous mucus of the stomach and intestine of the host. " 35  37  Because this sugar is found only in bacteria, enzymes required for its  biosynthesis serve as novel targets for antibiotic development.  1.3.2  Biosynthesis of Pseudaminic A c i d  The biosynthesis of pseudaminic acid has been developed based on homology to the known sialic acid biosynthetic pathways and was elucidated using mutational knockout studies and enzyme isolation studies (Figure 1 9) > > > xhe first enzyme in 28  33  38  40  the pathway is an NAD(PJ -dependent UDP-GlcNAc dehydratase/epimerase pseB, which +  catalyzes an oxidation at C-4, dehydration across C-5/C-6 and an inversion of stereochemistry at C-5, producing UDP-6-deoxy-4-keto-L-HexNAc. The inversion of stereochemistry at C-5 produces an L-sugar and is unique among all known sugar nucleotide dehydratases. The pyridoxal phosphate (PLP)-dependent aminotransferase pseC then catalyzes the transfer of an amino group onto the C-4 position, forming UDP4-amino-6-deoxy AltNAc. The third enzyme in the pathway, pseH is responsible for the N-4 acetylation of the substrate forming UDP-6-deoxy-AltdiNAc. The next enzyme, pseG catalyzes the hydrolysis of the glycosidic bond and removes the UDP moiety from  14  C-l position to form 6-deoxy-AltdiNAc. The final steps in the pathway involve the psel and pseF enzymes that catalyze the condensation of 6-deoxy-AltduNAc with pyruvate, and then the activation of Pse with C M P , forming pseudaminic acid and C M P pseudaminic acid, respectively.  OH Q  HO  aminotransferase PscC  dehydratasc/cp unerase Pse B  AcNHl OUDP UDP-GlcNAc  OH  rt0  H 0 2  MoiJDP  L-Glu  a-ketoglutarate  UDP-6-deoxy-4 -keto-HcxNAc  NHAc UDP-4-amino-6-deoxyAltNAc  acetyltransferase PseH  OH H C7>OAcN-  ^OH  3  NHAc  ' r \ UDP  H,0  PEP  COOH  Pseudaminic acid Pse  Figure 1.9  CTP  CoA  OUDP NHAc UDP-6-dcoxy-AltdiNAc  CMP-Psc synthetase PseF AcHNj HO NHAc  Acetyl-CoA  UDP-6-dcoxy- ' AltdiNAc hydrolase PscG  6-deoxy-AltdiNAc Pse synthase Psel  OUDP  H,N  PPi  AcHNj HO NHAc CMP-pseudaminic acid  Biosynthetic pathway of CMP-pseudaminic acid  15 1.3.3  Role of UDP-GlcNAc Dehydratase PseB (CJ1293) in Pse Biosynthesis  In C. jejuni pseB catalyzes the first step in the biosynthetic pathway of pseudaminic acid required for protein glycosylation. The amino acid sequence of this enzyme is 64% identical and 77% similar to that of FlaAl (HP0840) from H. pylori whose crystal structure has been obtained. FlaAl is a novel member of the short-chain 41  dehydrogenase/reductase (SDR) superfamily, and the first UDP-GlcNAc inverting 4,6dehydratase, catalyzing the first step in the biosynthesis of pseudaminic acid. Within 42  this family there are two subfamilies: one contains the short, soluble enzymes that have an S/T-Y-K catalytic triad that promote the hydride transfer steps of the reaction. PseB and F l a A l are members of this subfamily. The other includes the large membrane42  bound subfamily with an altered S-M-K catalytic triad. X-ray studies on FlaAl show that the enzyme possesses a hexameric doughnut-shaped quaternary structure. The enzyme is bilobal in shape—the larger, predominantly N-terminal lobe consists of a ten-stranded mostly parallel P-sheet possessing eight helices on either side. The smaller, predominantly C-terminal lobe has three a-helices and two short two-stranded P-sheets.  42  Crystal packing interactions show that FlaAl contains a tightly bound N A D P / N A D P H +  cofactor within the active site. Amino acid sequences of several 4,6-dehydratases that retain the configuration at C5, such as dTDP-Glc 4,6-dehydratases, GDP-Man 4,6dehydratases, and CDP-Glc 4,6-dehydratases share 18-21% sequence identity and possess a common structural core of ~270 residues. F l a A l , in comparison, shares only 12-16% sequence identity with these enzymes and has a structural common core of -220 residues. In fact, this enzyme is as similar to 4-epimerases as it is to 4,6-dehydratases. 43  16 Crystal structures of FlaAl with the substrate UDP-GlcNAc, and with UDP, UDPglucose (UDP-Glc), and UDP-galactose (UDP-Gal) shows the uracil group of the U D P moiety forming an H-bond to the carbonyl oxygen of Prol97 and the diphosphate group is stabilized by two arginine residues (205 and 258) and a helix dipole moment  42  It is  believed that both FlaAl ofH. pylori and PseB of C. jejuni employ a three step sequential mechanism that is composed of oxidation of the C4 hydroxyl resulting in the formation of a 4-keto group, then a dehydration across the bond between C5 and C6 producing a UDP4-keto-5,6-ene-hexose, and finally reduction in which a hydride from the enzyme bound N A D H cofactor is transferred to C6, and C-5 is reprotonated on the opposite face to form a methyl group with opposite configuration.  The available structural data are not adequate to reveal the mechanism, however, it is suspected that Thrl 31 and Tyrl41 of the S/T-Y-K triad commonly found in SDR enzymes play important roles in the active site of the enzyme. Tyr is thought to play a role in the initial oxidation step by deprotonating the C-4 hydroxyl. A putative mechanism is shown in Figure 1.10 but further work must be done to establish the exact roles of these residues. Other active site residues that presumably play key roles in the dehydration/inversion steps include Lysl33 and Asp 132.  42  17 Lysl33  Lysl33  H,N  H N 2  UDP-O  H  OH  UDP-O  HO 'Q  Asp 132  AcHN-V-«^^»o2-~OH  J  NADP  .1  oxidation •  j \ > O H  \^~0' AcHN-  Tyrl41  OH  O  H O ^  Asp 132  HO. Tyrl41  NADPH  dehydration  Lysl33  Lysl33 HN 3  CH  UDP-O  UDP-Q  3  Asp 132 O H \ NADP  +  HO. Tyrl41  Figure 1.10  1.4  reduction  AcHN  V  H O - ^ ^  ,CH,  Asp 132  O OH NADPH'  'HOTyrl41  Potential roles of catalytic residues in the active site of FlaAl.  A i m of Thesis  Since pseudaminic acid is required for bacterial virulence and is not found in humans, inhibitors developed against the enzymes that are responsible for its biosynthesis could be novel therapeutic targets. There have been many cases of drug resistance and an increase in treatment failure for eliminating the infestation of pathogenic bacteria, therefore the biochemical pathways responsible for their virulence have received much  18 attention. The aim of thesis is to synthesize a potential mechanism-based inhibitor, UDP6-deoxy-6,6-difluoro-GlcNAc, 11, targeting the first enzyme in the biosynthetic pathway of Pse, UDP-GlcNAc dehydratase (pseB). It is hypothesized that this inhibitor will irreversibly bind to the enzyme active site, terminating enzyme activity. The inhibition and inactivation is expected to follow a similar mechanism to that described for the inhibition of Eod by CDP-6-deoxy-6,6-difluoro-D-glucose (Figure 1.7). The enzymeinhibitor complex can then be analyzed by X-ray crystallography to more accurately determine the positioning of the key active site residues during catalysis.  O H  11  O H  Chapter 2  Synthesis of L)DP-6-deoxy-6,6-difluoro-GlcNAc  20  2.1  Introduction  In the first step of the pseudaminic acid biosynthesis pathway a 4,6-dehydratase acts to dehydrate UDP-GlcNAc, generating a ketone at C-4 and a methyl group at C-6. Unlike most known sugar nucleotide dehydratases this enzyme also promotes an inversion of configuration at C-5. To develop methods to control and/or regulate this intriguing enzymatic conversion, synthesis of a potential mechanism-based inhibitor 11 (Figure 2.1) was proposed. It was expected that this inhibitor would serve as a "suicide substrate" and irreversibly attach to an active site residue. Proteolysis studies could then be used to identify the tagged residue. In addition, X-ray studies on the inhibited enzyme could help identify the proximity of the residues with respect to the carbohydrate. The production of flagella in pathogenic bacteria such as C. jejuni and H. pylori is heavily dependent on the presence of pseudaminic acid, and aflagellated bacteria are unable to invade and colonize the host cell. Because the pseudaminic acid biosynthetic pathway is absent in humans, this inhibitor is potentially a novel therapeutic drug. The mechanism of inhibition is expected to resemble that of the 6,6-difluoroglucose analog described in chapter 1 (Figure 1.7).  21  OH  OH  11 Figure 2.1  Structure of UDP-6-deoxy-6,6-difluoro-D-GlcNAc,  a potential mechanism-  based inhibitor for PseB  2.2  Synthetic Strategies for the Proposed Inhibitor  2.2.1  Attempted Synthesis of Inhibitor 11 Based on Literature Precedence  In 1998, the synthesis of CDP-6-deoxy-6,6-difluoro-D-glucose, 9, was published by Chang et al. (Figure 2.2). This compound was the first mechanism-based inactivator 24  known for Eod, more specifically, for CDP-D-glucose 4,6-dehydratase, isolated from Yersinia pseudotuberculosis.  22  1. (COCI) , /-Pr2NEt, DMSO, THF 2  2. DAST, CH CI 2  Quant  25%  Figure 2.2  Reported synthesis of  9  CDP-6-deoxy-6,6-difluoro-D-glucose.  Based on this work, the synthesis of UDP-6-deoxy-6,6-difluoro-D-GlcNAc was proposed using Af-acetyl-D-glucosamine as the starting material (Figure 2.3).  2  23 Dowex 50W ionexchange resin  OH -O  HO HO-  AcHN  OH  .OTr  TrCI, Pyr DMAP, 60°C  MeOH 71%  51% AcHN  AcHN  OMe  20  19  ' GlcNAc  - Q  HO HO-  OMe  BnBr, NaH, DMF 52%  (COCI) , NEt DMSO, THF 2  BnO  „  BnO-V*--**^A AcHN  I OMe  23 Ac 0 H SQ 2  2  4  \ /  OH 3  2. DAST, CH CI 28% 2  O  BnO BnO 2  .OTr  AcHN  TsOH, MeOH 77% OMe  BnO  B n 0  OMe  21  22  <  AcO-\^--*^X AcHN  24  Figure 2.3  I OAc  Attempted synthesis of UDP-6-deoxy-6,6-difluoro-D-GlcNAc based on  literature precedence.  In the first step, a methyl glycoside was prepared to protect the C - l position. This was achieved using methanol and a catalytic amount of acid resin. The next step is the 44  temporary protection of the hydroxyl at the C-6 position so that it can later be selectively deprotected for the installation of fluorines. This was carried out by using a bulky trityl ether protecting group that selectively reacts with the less hindered primary alcohol.  45  The mechanism of this reaction involves generation of a stable tertiary carbocation (Figure 2.4) formed upon departure of the chloride ion. After the formation of the carbocation, the lone pairs on the primary C-6 hydroxyl attack the electrophilic centre.  24 Compound 19 was tritylated to give 20 with 51% yield. The advantage of using trityl substituents for protection of hydroxyls lies in their easy removal by hydrogenolysis or mild acid hydrolysis.  Figure 2.4  Carbocation formed during tritylation.  The remaining free hydroxyls (C-3 and C-4 positions) on compound 20 were then protected as benzyl ethers by a common benzylation procedure using benzyl bromide and sodium hydride in D M F  4 6  to give 21 with 52% yield. This reaction is highly exothermic,  therefore the flask containing the reaction mixture was cooled to 0°C in an ice bath before adding benzyl bromide into the reaction mixture.  In the next step, the cleavage of the trityl group was achieved at room temperature with /^-toluene sulfonic acid to give 77% of compound 22. With the only unprotected 47  hydroxyl residing on the C-6 position of the carbohydrate, the molecule is ready to undergo a fluorination reaction at C-6. If the installation of the fluoro moiety using diethylaminosulfur trifluoride (DAST) was carried out at this stage, the resulting product would incorporate only one fluorine group. Since two fluorine groups are required on the  25 desired inhibitor, the hydroxyl group was oxidized to an aldehyde using the swern oxidation.  48  The Swern oxidation is useful for oxidizing primary and secondary alcohols. This oxidation technique avoids the use of toxic metals, such as chromium. Also the reactions are carried out under very mild conditions, and further oxidation from aldehyde to carboxylic acid is not possible. However, a drawback of this reaction is the production of the unpleasant smelling side product, dimethyl sulphide (Figure 2.6). In the Swern oxidation, only dimethyl sulfoxide (DMSO) and oxalyl chloride are initially added into a flask. These reagents react and the intermediate quickly decomposes giving carbon dioxide, carbon monoxide, and dimethylchlorosulfonium chloride (Figure 2.5).  Figure 2.5  Reaction of DMSO with oxalyl chloride in Swern oxidation  Next, compound 22, deprotected at C-6, is added into the reaction mixture. After the addition of the alcohol and elimination of HC1 the key alkoxysulfonium intermediate is formed (Figure 2.6). At this stage triethylamine is added to deprotonate the alkoxysulfonium ion and give the sulphur ylide which then undergoes an intramolecular elimination via a five-membered ring intermediate. Swern oxidations must be performed  26 at low temperatures (-78°C), otherwise during the rearrangement of the intermediate, the undesired byproduct thioacetal will form (Figure 2.7). The ' H - N M R spectrum (300 MHz, CDCI3) of the product formed from oxidation of 22 showed an aldehyde peak at 9.53 ppm, confirming the formation of the desired aldehyde. This aldehyde was not purified and was immediately carried on to the next step.  R 7 V^CH . K  R  Figure 2.6  R  3  "  The formed intermediate leading to formation of dimethyl sulfide and the  desired oxidized product.  i  2  H  R"  Figure 2.7  Swern oxidation reactions at higher temperatures lead to the formation of  a different product.  27  The conversion of the C-6 aldehyde into a difluoro-functionality was accomplished by using D A S T , as depicted in Figure 2.8. First, an N-S double bond is formed via 49  elimination of a fluoride ion which in turn undergoes a nucleophilic attack on the carbonyl carbon. The resulting hydroxyl then attacks the sulfur, and loss of fluoride activates it as a leaving group. A second fluoride then displaces the oxygen at C-6 and gives the difluoro product. DAST was introduced into the reaction flask via a plastic syringe since fluoride forms very strong bonds with the silica in the glass. The combined yield of compound 23 was 2 8 % .  23  Figure 2.8  DASTfluorination reaction mechanism  The F N M R spectrum (H-decoupled) of compound 23 shows two doublets 19  resulting from the two diastereotopic fluorines that couple to one another (JFF - 2 8 4 . 7 Hz) (Figure 2.9 ).  28  •. i,.'11,'..i,.' i|.. • i .."i,.'. i  ppm (fiP  1S0  Figure 2.9  ;  i  | . I...".i.  , .ii. 'i  |  i  . r i. i .  i. i . |' . i  i  i  i  |.'. .1'..  i . . ' : i . . . i , . ' 11.'. .i..' i .  ^-t32M  F NMR spectrum ('H-decoupled) of  19  benzyl-2-acetamido-3,4-0-benzyl-  6,6 '-difluoro-a-D-glucopyranoside 23  The next step in the planned sequence was to deprotect the benzyl ethers at C-3 and C-4, as well as the methyl glycoside at the C - l position using strongly acidic conditions. Also in the same step, acetic anhydride is added so that deprotection and peracetylation can be achieved in one pot. This had previously been successful on the gluco- analog as reported by Chang et al. (Figure 2.2). Using strong acid in cleaving 24  ethers is well known in organic chemistry. However, in carbohydrate synthesis this is not  29 a common method for removing ethers at a non-anomeric centre due to sensitivity of these molecules to strongly acidic conditions required for such reactions. Initially the same reaction conditions as reported were used. This resulted in a partially deprotected product with hydroxyl groups on C-3 and C-4. The methyl ether remained intact as observed by mass spectroscopy and ' H N M R . Attempts to increase the concentration of H2SO4  in the reaction mixture led to the decomposition of the starting material. Use of  greater amounts of a weaker acid, C H 3 C O O H , also led to the production of C-3 and C-4 deprotected compound without affecting the C - l position. Its seemed as if the strong acidic conditions required to hydrolyze the methyl glycosidic linkage were too harsh, thus a more versatile mean of protecting the hydroxyl at C - l was required.  2.2.2  The Revised Synthetic Plan  With the problem encountered during the deprotection of the methyl glycoside, a revised synthetic plan (Figure 2.10) was designed which began with the preparation of a benzyl glycoside, 25. This scheme was used to obtain the final desired product.  30  B n O H , HCI  P H  TrCI, P y r P  DMAP, 60^C *~  HO  x  x  A  C  H  OBn  N  .F „0  H , Pd(OH) MeOH 2  \ AcHN  UH  9  6  1. ( C O C I ) , N E t DMSO.THF 2  2  BnO' BnO-  3  2. D A S T , C H C I  %  2  BnO B  n  2  38%  29 A c 0 , Pyr DMAP 2  98%  NH NH .acetate DMF *~ 98% 2  2  AcO' AcOEt NP(OBn) , 1 H-Tetrazole CH CI 2. H 0 , T H F 1.  2  2  2  2  2  2  30%  F  •YZA^O \  >-\  UMP-morpholidate, 1 H-Tetrazole,  \  **V^i "* A«-MM  I  '  pyr^o'^ HO-  21%  AcHN  ^  32  Figure 2.10  °  „OBn 0  -h)Bn  Synthetic plan usedfor the synthesis of inhibitor, 11.  A^-acetyl-D-glucosamine was treated with BnOH and HCI and the desired abenzylated sugar, 25, was recrystalized from ethanol. The next four steps leading up to, 50  and including the D A S T fluorination reaction were analogous to the reagents and conditions described earlier. Compound 25 was protected to give 26 in 51% yield. C-3/C-4 Benzylation, and C-6 deprotection of 26 gave 61% yield of the product 27. This  31 was then subjected to swern oxidation and DAST fluorination reaction to give compound 28 in 38% yield. Deprotection of the benzyl ethers of compound 28 was achieved using Pd(OH)2 as the heterogenous catalyst at 50 Psi H2 atmosphere, giving quantitative yields of 29. Attempts to achieve this hydrogenation reaction under milder conditions such as using Pd/C (10% w/w) at 1 atm H2 resulted in deprotection of C-3 and C-4 positions, however, it did not deprotect the anomeric benzyl substituent.  2.2.3  Remaining Steps in the Synthesis of Compound 11  The problem of deprotection at C - l was overcome with the revised strategy of introducing a benzyl glycoside instead of a methyl glycoside. The next step in the synthetic route to compound 11 is peracetylation of the free hydroxyl groups. For reasons unknown to us, this step required harsh and vigorous reaction conditions. The peracetylation of 29 was attempted with published literature procedures. However with 51  common procedures only yields of 40-50% were achieved. After greatly increasing the amount of acetic anhydride and D M A P in the reaction mixture, as well as increasing reaction time and temperature (55°C), the reaction went to completion after 24 hours and a 98% yield of 30 was achieved after purification.  The next step involved another anomeric deprotection, this time of the peracetylated compound 30. Commonly, these deprotections are achieved by using a nucleophilic amine such as benzyl amine or dimethyl amine. When such reactions were  32 carried out on a peracetylated GlcNAc itself, the reaction proceeded cleanly and gave good yields of product. When the same reaction was attempted with compound 30, however, no desired product was obtained, and as the reaction was left for longer periods, decomposition of the starting material was detected.  A more reactive amine nucleophile,  hydrazine acetate was then utilized and it was found that the reaction goes to 52  completion cleanly with near quantitative yields. It was found that exactly one equivalence of hydrazine acetate had to be used in this reaction. Excess amounts of hydrazine acetate lead to the formation of deprotected C-3/C-4 acetyl groups. Also, since the interconversion between the open chain and the closed chain is in equilibrium in aqueous solution, the presence of excess hydrazine formed a Schiff-base with the carbonyl group of the open chain form, leading to the production of another unwanted side product (Figure 2.11). Anomeric deacetylation of 30 using this nucleophile gave 98% product which required no further purification.  AcO'  NH2NH2.acet.ate (excess)  AcO'  AcO' N-NH  O  f  Figure 2.11  Reaction side-product in presence of excess hydrazine acetate  2  33 Phosphorylation of 31 using dibenzyl A^A^-diethylphosphoramidite and 1,2,4triazole and subsequent oxidization of the resulting phosphite gave a 7% yield of 53  compound 32. The same reaction using 1 H-tetrazole (Figure 2.12) instead of 1,2,4triazole resulted in a 30% yield of the desired compound. A general mechanism of this reaction is shown in Figure 2.13. The lone pair of the phosphoramidite amine is protonated by lH-tetrazole, making the resulting ammonium ion a better leaving group. With the attack of the hydroxyl group at phosphorus, diisopropyl amine is released into the solution. Presumably the pKa of 1 H-tetrazole is just right for its role in the reaction, resulting in much better yields. The second step of adding H2O2 oxidized the phosphite and gave compound 32 in 30% overall yield.  N  1,2,4-triazole  Figure 2.12  N  1 H-tetrazole'  Molecular structures of 1,2,4-triazole and 1 H-tetrazole  34  Figure 2.13  Postulated mechanism of anomeric phosphorylation reaction  It is noteworthy to mention that the phosphorylation reaction gave only the aproduct. This stereoselectivity of the phosphate incorporation depends on the presence of the acetamido group acting as a neighboring participating group at the C-2 position of the sugar. While the p-anomer presumably forms during the reaction, the participating 2acetamido group generates an oxazoline intermediate due to a backside attack at the anomeric center, and the phosphate is not isolated (Figure 2.14).  Figure 2:14  The phosphorylation reaction gives only the a-anomer, since the p-anomer  decomposed by forming an oxazoline compound.  35 In the next step, debenzylation of the phosphate group was achieved using 10% w/w Pd/C catalyst under H2 (1 atm), and deprotection of acetyl groups gave near quantitative yields of 33 in a one pot reaction. The final step includes the coupling of the monophosphate sugar with 4-morpholine-MA -dicyclohexylcarboxamidinium uridine 5'/  monophosphomorpholidate (Figure 2.15). The product was purified using anion 54  exchange chromatography eluted with a linear gradient of triethylammonium bicarbonate buffer, giving 11 as white, hydroscopic powder in 20% yield. o  Figure 2.15  Final coupling in the preparation of  UDP-6-deoxy-6,6-difluoro-GlcNAc,  11  The F NMR spectrum ( H-coupled) of the final product (Figure 2.16) shows a 19  ]  doublet of doublet of doublets for each of the fluorines (Fl and F2): the smallest coupling of Fl at -133.95 ppm is due coupling to Fl and H-5" of the pyranoside (J(HS-)-FI Hz). The next largest coupling is between fluorine and H-6" of the pyranoside = 54.29 Hz. The largest coupling occurs from fluorine-fluorine coupling with  = 8.81 J(H-6-)-FI  JFI-F2~  284.70 Hz. The second fluorine (F2) shows a similar coupling pattern at -136.06 ppm : 17.23 Hz,  J(H-6-)-FI  = 53.77, and J j-F2= F  284.73 Hz.  36  1  T  1  r •734.0  Figure 2.16  i  1  1  r  i  •135.0  1  1  r  T  ~\  •136,0  1  1  r  —  1  •137.0  F NMR spectrum ('H-coupled) of final product showing splittings  19  betweentFl, F2, H-5", H-6"  The P N M R spectrum ('H-decoupled) shows two doublets, one for each 31  phosphorous, with UDP-GlcNAc-a-P being more deshielded than the UDP-GlcNAc-P-P.  37  NH AcHN  O—P-  -o-. '  N1  4' \  V  5  3'  \  I  OH  ppm (11)  Figure 2.17  •10,0  2'  OH  • 15.0  P NMR spectrum (' H-decoupled) of the final product 11.  31  °  38  2.3  Experimental Methods  2.3.1  General Method  A l l reactions involving air-sensitive reagents were performed under argon using syringe-septum cap techniques. A l l glassware was flame-dried before use. Flash chromatography was performed using silica gel (230-400 mesh, BDH). A l l N M R spectra were recorded on the automated Bruker 300 MHz or 400 MHz. A l l chemical shifts were recorded using the 5 scale in ppm. The molecular masses of synthesised compounds were determined on a Waters Micromass L C T mass spectrometer using electrospray ionization-mass spectroscopy (ESI-MS).  2.3.2 Materials  A l l reagents were purchased from Aldrich Chemical Co. unless otherwise stated. D2O and CDCI3 were purchased from Cambridge Isotope Laboratories.  39 2.3.3  Preparation of methyl 2-acetamido-2-deoxy-a-D-glucopyranoside, 23  A flask containing D M S O (0.20 mL, 2.8 mmol) and anhydrous THF was cooled to -60°C (acetone/dry ice bath). Oxalyl chloride (0.12 mL, 1.3 mmol) was added dropwise and reaction was stirred for 30 mins. A solution of sugar 22 (0.459 g, 1.1 mmol) in 49  THF was canulated in the mixture and reaction was stirred for 30 mins. Triethylamine (0.77 mL, 5.5 mmol) was added and the reaction was allowed to warm to room temperature while stirring for 45 mins. The reaction mixture was diluted with C H C L and washed with water. The aqueous layer was extracted three more times with CHCI3 . The organic layers were combined and washed with brine, dried over MgS04 and concentrated to yield the product aldehyde which was taken to the next step without further purification. This aldehyde (0.301 g , 0.7 mmol) was dissolved in CH C1 (5 mL) 2  2  and D A S T (0.23 mL, 1.5 mmol) was added drop-wise while stirring. The reaction was allowed to stir at room temperature for 24 hours. Water was added (5 mL) and the organic layer was separated, washed with saturated NaHC03 and H 0 . The organic layer 2  was dried over MgS04 and concentrated in vacuo. The solid was recrystalized from ethanol to give 23 (0.089 g, 28%) as white powder. H N M R (400 MHz, CDC1 ), Sppm: ]  3  1.85 (s, 3H, OAc), 3.36 (s, 3H, OMe), 3.72-3.76 (m, 2H, H-3 and H-5), 3.79-3.90 (m, 1H, H-4), 4.22-4.26 (m, 1H, H-2), 4.61-4.90 (m, 5H, 0-CH -Ph, H - l ) , 5.19 (d, 1H, J=8.80 2  Hz, NH), 5.90 (t, 1H, .7=53.56 Hz, H-6), 7.29-7.36 (m, 10H, Ph); ESI M S m/z 458.1 [M-t-Naf  40 2.3.4  Preparation of benzyl 2-acetamido-2-deoxy-a-D-glucopyranoside, 25  This compound was prepared following the published procedure outlined in Shulman, et al. which lacked spectroscopic data. To a suspension of 2-acetamido-250  deoxy-D-glucose (12 g, 54 mmol) in benzyl alcohol (200 mL) was added concentrated aqueous hydrogen chloride (2.5g) drop-wise, and the mixture was stirred at 75°C overnight. The reaction mixture was cooled and precipitated with diethyl ether (1.5 L) and filtered. The filtrate was washed with cold diethyl ether and recrystalized from 2propanol (300 mL) to give 9.91 g (58%) of 25 as white solid. ' H N M R (400 MHz, CD OD,) 5: 1.95 (s, 3H), 3.37 (t, 1H, J= 9.2 Hz), 3.68 (m, 3H), 3.82 (d, 1H, 7=10.8 Hz), 3  3.89 (dd, 1H, J=3A, 10.8 Hz), 4.61 (dd, 2H, J=l 1.8, 98.4 Hz), 4.85 (s, 1H), 7.24-7.37 (m,  5H); ESI MS m/z 334.2 [M+Na]  +  !  2.3.5  Preparation of benzyl 2-acetamido-2-deoxy-6-0-trityl-a-Dglucopyranoside, 26  This compound was prepared following the published procedure outlined in Chaudhary, et al. , which lacked spectroscopic data. Trityl chloride (16.14 g, 58 mmol) 45  was added to a solution of 25 (11.35 g, 48 mmol) in pyridine (130 mL). D M A P (300 mg) was added and the resulting mixture was stirred at 60°C for 10 hours. Pyridine was removed by evaporation under reduced pressure. Flash chromatography (5% MeOH/EtOAc) gave 15.50 g (51%) of product 26. T L C (5% petroleum ether/ EtOAc), R : 0.18; ' H N M R (300 MHz, CDC1 ) 5: 1.98 (s, 3H), 3:31-3.43 (m, 2H), 3.56 (t, 1H), f  3  41 3.69 (t, 1H), 3.74-3.80 (m, 1H), 4.07-4.14 (m, 1H), 4.62 (dd, 2H, J=11.8 Hz, 98.4 Hz), 4.91 (d, 1H, J=3.80 Hz), 5.89 (d, 1H, .7=8.66 Hz), 7.22-7.48 (m, 20H); ESI M S m/z 514.2 [M+Na]  2.3.6  +  Preparation of benzyl-2-acetamido-3,4-(?-benzyl-a-D-glucopyranoside 27  This compound was prepared following the published procedure outlined in Fukuzawa, et al. and Ichihara et al. , which lacked spectroscopic data. Compound 26 46  47  (12.51 g, 22 mmol) was dissolved in anhydrous D M F (150 mL). This solution was added via syringe to a solution of sodium hydride (60% dispersion in mineral oil, 2.94 g, 73 mmol). At 0 °C benzyl bromide (8.75 mL, 73 mmol) was added drop-wise over 1 min and the resulting solution was stirred for 40 hours at room temperature. The reaction mixture was poured into cold water (50 mL) and extracted with EtOAc (3x100 mL). The Organic layer was then washed with brine and dried over MgSC^ and concentrated in vacuo to give 13.25 g crude product as a syrup, (ESI MS m/z 756.3 [M+Na] ). This was dissolved +  in methanol (200 mL), and TsOH (400 mg) was added to the solution. This was stirred at room temperature for 24 hours. Triethylamine (1 mL) was added and the reaction mixture was concentrated in vacuo. The residue was dissolved in methylene chloride and washed with water, dried over MgSC^ and concentrated. The resulting solid was recrystalized from ethanol to give compound 27 (5.05 g, 61%) as white powder. TLC (10% petroleum ether/ EtOAc), R : 0.26; *H N M R (300 MHz, CDC1 ) 5: 1.81 (s, 3H, OAc), 3.41-3.49 (m, f  3  2H), 3.60 (t, 1H, J= 9.92 Hz), 3.62-3.90 (m, 2H), 4.07 (t, 1H, J= 8.81 Hz), 4.57-4.90 (m, 6H), 5.37 ( d , l H , / = 8.00 Hz), 7.25-7.31 (m, 15H); ESI M S m/z 514.1 [M+Na]  +  42  2.3.7  Preparation of benzyl-2-acetamido-3,4-0-benzyl-6,6'-difluoro-a-D-  glucopyranoside 28 A flask containing D M S O (1.80 mL, 25 mmol) and anhydrous THF was cooled to -60°C (acetone/dry ice bath). Oxalyl chloride (1.05 mL, 12 mmol) was added drop-wise and reaction was stirred for 30 mins. A solution of sugar 27 (5.00 g, 10 mmol) in THF was canulated in the mixture and reaction was stirred for 30 mins. Triethylamine (7.05 mL, 50 mmol) was added and the reaction was allowed to warm to room temperature while stirring for 45 mins. The reaction mixture was diluted with C H C I 3 and washed with water. The aqueous layer was extracted three more times with C H C I 3 . The organic layers t  were combined and washed with brine, dried over MgSC»4 and concentrated to yield the product aldehyde with near quantitative yields. This compound was taken to the next step without further purification. This aldehyde (4.29g , 8.7 mmol) was dissolved in C H 2 C I 2 (100 mL) and DAST (2.43 mL, 18 mmol) was added drop-wise while stirring. The reaction was allowed to stir at room temperature for 24 hours. Water was added (75 mL) and the organic layer was separated, washed with saturated NaHCCh and H2O. The organic layer was dried over MgSCM and concentrated in vacuo. The solid was recrystalized from ethanol to give 28 (1.68 g, 38%) as white powder. H N M R (400 MHz, ]  CDCI3), 5ppm: 1.80 (s, 3H, OAc), 3.74-3.77 (m, 2H, H-3 and H-5), 3.89-3.98 (m, 1H, H4), 4.26-4.32 (m, 1H, H-2), 4.64-4^93 (m, 6H, 0-CH -Ph), 4.94 (d, 1H, .7=3.51 Hz, H - l ) , 2  5.25 (d, 1H, 7=8.83 Hz, NH), 5.92 (t, 1H, .7=53.86 Hz, H-6), 7.32-7.36 (m, 15H, Ph); ESI MS m/z 534.2 [M+Na]  +  43 2.3.8  Preparation of 2-acetamido-6,6'-difluoro-a-D-glucopyranoside 29  Compound 28 (1.68 g, 3.2 mmol) was dissolved in MeOH (300 mL) and palladium hydroxide (1.00 g, 96%) was added. The mixture was introduced into a Parr hydrogenator and shaken at room temperature under 50 Psi of H2 for 24 hours. Filtration through Celite and removal of solvent gave 29 (0.81 g, 96%). H N M R (400 MHz, !  CD3OD), Sppm: 1.98 (s, 3H, OAc), 3.43 (t, 1H, 7=9.47 Hz), 3.69 (t, 1H, 7=9.39 Hz), 3.83 (dd, 1H, J= 3.37 Hz, 10.61 Hz, H-2), 3.91-3.99 (m, 1H), 5.11 (d, 1H, 7=3.32 Hz, H - l ) , 6.08 (t, 1H, 7=54.0 Hz, H-6); ESI M S m/z 264.2 [M+Na]  2.3.9  +  Preparation of acetyl-2-acetamido-3,4-acetyl-6,6'-difluoro-a-Dglucopyranoside 30  Compound 29 (l.OOg, 4.1 mmol) was dissolved in pyridine (10 mL) and D M A P (500mg) was added. Under A r atmosphere, acetic anhydride (15 mL) was added via syringe and the reaction mixture was stirred at 50°C for 2 days. The solution was poured into water (50 mL) and stirred for one hour. This was then extracted with CH2CI2. The organic layer was dried over anhydrous MgSCM and concentrated in vacuo. The resulting solid was chromatographed with silica (10% EtOAc/petroleum ether) to give 30(1.50 g, 98%). T L C (20% petroleum ether/ EtO Ac), R : 0.23; ' H N M R (300 MHz, CDCI3), 8ppm: f  1.93 (s, 3H, OAc), 2.04 (s, 3H, OAc), 2.06 (s, 3H, OAc), 2.21 (s, 3H, OAc), 3.93-4.05 (m, 1H, H-5), 4.41-4.49 (m, 1H, H-2), 5.22-5.39 (m, 2H, H-3 and H-4), 5.64 (d, 1H,  44 J=8.79 Hz, NH), 5.79 (t, 1H, J=50.93 Hz, H-6), 6.21 (d, 1H, .7=3.58 Hz, H - l ) ; ESI M S m/z 390.0 [M+Na]  +  2.3.10 Preparation of 2-acetamido-3,4-acetyl-6,6'-difluoro-a-D-glucopyranoside 31  To a solution of sugar 30 (1.52 g, 4.1 mmol) in D M F (25 mL) was added hydrazine acetate (0.38 g, 4.1 mmol) and the mixture was stirred at 50°C for 5 mins until all hydrazine acetate dissolved. The solution was cooled to room temperature and stirred for 5 hours. Mixture was diluted with EtOAc (15 mL), washed with brine, dried over anhydrous MgS04 and concentrated in vacuo to give 31 (1.32 g, 98%) as a colourless syrup. ' H N M R (400 MHz, CDCh), bppm: 1.97 (s, 3H, OAc), 2.04 (s, 3H, OAc), 2.05 (s, 3H, OAc), 4.21-4.32 (m, 2H, H-2 and H-5), 5.22-5.35 (m, 3H, H - l , H-3, and H-4), 5.80 (dt,lH, J= 3.27 Hz, 54.34 Hz, H-6), 5.96 (d, /= 8.80 Hz, NH); ESI M S m/z 348.1 [M+NaJ'  2.3.11 Preparation of dibenzyl 2-acetamido-3,4-acetyl-6,6'-difluoro-a-Dglucopyranosyl phosphate 32  Compound 31 (0.15 g, 0.46 mmol) was dissolved in anhydrous CH2CI2 (10 mL). This was added via syringe to a vigorously stirred suspension of lH-tetrazole (0.16 g, 2.3 mmol) and dibenzyl N,N'-diethylphosphoramidite (0.6 mL, 1.82 mmol) in dry CH2CI2 (5 mL) under argon atmosphere at room temperature. The reaction was completed in 2  45 hours. The mixture was diluted with  CH2CI2,  washed with saturated NaHC03, water, and  brine. The organic layer was dried over M g S 0 4 and concentrated in vacuo. The resulting syrup was then dissolved in THF (10 mL) and cooled to -78°C. H20 (aq) (30%, 2 mL) 2  was added drop-wise via syringe to the vigorously stirred solution. After addition was complete, the bath was removed and the mixture was allowed to warm to room temperature over 2 hours. The mixture was diluted with cold saturated  Na2S 03, 2  followed  by EtO Ac and stirred for 5 mins. The organic layer was dried over MgS04 and concentrated in vacuo. The product was chromatographed on silica using 20% acetone/CHCL as the eluting solvent to give product 32 (0.88 g, 30%). T L C (20% acetone/CHCh), R : 0.15; H N M R (300 MHz, CDC1 ), 8ppm: 1.68 (s, 3H, OAc), 1.99 (s, ]  f  3  3H, OAc), 2.00 (s, 3H, OAc), 4.04-4.29 (m, 1H), 4.31-4.34 (m, 1H), 5.02-5.17 (m, 4H), 5.21-5.26 (m, 2H), 5.60 (t,lH, 7=53.7 Hz), 5.66 (dd, 1H, 7=3.26 Hz), 5.70 (d, 1H, NH); ESI M S m/z 608.2 [M+Na]  +  2.3.12 preparation of 2-acetamido-6,6'-difluoro-a-D-glucopyranoside 1-phosphate 33  Compound 32 (0.80g, 1.36 mmol) was dissolved in methanol (10 mL) and w/w 10% Pd/C (0.30 g) was added. The reaction was flushed with H (3x) and stirred under 2  H (1 arm) for 24 hours. Triethylamine (0.04 mL) was added and the mixture was filtered 2  through Celite, and rinsed with methanol. Removal of solvent under reduced pressure gave a pale yellow syrup. This syrup was then brought to pH ~11 using 1M NaOMe in methanol, and the mixture was stirred at room temperature for 2 hours. The reaction was  '  46  quenched by stirring the mixture with resin 8X (pyridinium form) for 15 mins. The resin was filtered off and solvent removed to give compound 33 in 95% yield. *H N M R (300 MHz, D 0 ) , 5ppm: 1.81 (s, 3H, OAc), 3.32 (t, 1H, J = 9.48 Hz), 3.56 (t, 1H), 3.73-3.93 2  (m, 2H), 5.38 (dd, 1H, J= 2.91 Hz, 6.80 Hz), 5.84 (t, 1H, .7=53.90 Hz). ESI M S m/z 320.4 [M+H]  +  2.3.13 Preparation of UDP-2-acetamido-6-deoxy-6,6'-difluoro-a-D-glucopyranoside 11 Sugar 33 (0.050 g, 0.12 mmol) was dissolved in pyridine and coevaporated (two times). Tri-«-octylamine (0.05 mL, 0.12 mmol) was added and the mixture was coevaporated with dry pyridine (two times). 4-Morpholine-A^ Ndicyclohexylcarboxamidinium uridine 5'-monophosphomorpholidate (0.137 g, 0.20 mmol) was added and the mixture was coevaporated with dry pyridine (two times). This was dissolved in pyridine, 1 H-tetrazole (0.026 g, 0.37 mmol) was added, and the solution was stirred at room temperature for four days. The mixture was diluted with water, extracted with diethyl ether and the aqueous layer concentrated to 1 mL. This was loaded onto a 220 mL column of DEAE-cellulose (DE52, Whatman Inc.) and eluted with a linear gradient of 0.1-0.5 M triethylammonium bicarbonate buffer. The eluant was monitored at A 2 5 4 using a SPECTRUM Spectra/Chrom Flow Thru U V detector, and UVactive fractions were analyzed by ESI-MS. Those containing the product 11 were lyophilized to dryness, giving a 21% yield of white powder. H N M R (300 MHz, D 0 ) , ]  2  Sppm: 2.06 (s, 3H, OAc), 3.68 (t, 1H, J = 9.48 Hz, H-4"), 3.91 (t, 1H, .7=9.48 Hz, H-3"),  47 4.00 (m.; 1H, H-2"), 4.11-4.33 (m, 6H, H-5", H-2', H-3', H-4', H-5'a, H-5'b), 5.53 (m, 1H, H - l " ) , 5.91 (d, 1H, 7=7.71 Hz, H-5), 5.98 (s, 1H, H - l ' ) , 6.15 (t, 1H, 7= 53.88 Hz, H 6"), 7.90 (d, 1H, 7=7.81 Hz, H-6). F N M R (CD OD) 5ppm: -133.95 (ddd,7=8.81, ] 9  3  54.29, 284.70 Hz), -136.05 (ddd, 7= 17.23, 53.77, 284.73 Hz). P N M R (CD OD) 31  3  5ppm: -10.93 (d, 7=24.02 Hz), -13.31 (d, 7=23.39 Hz). ESI M S m/z 626.2 [M-H]  +  Chapter 3  Inhibitor Testing with PseB  49  3.1  Overexpression and purification of His6-tagged pseB  The cloning of genes and overexpression of gene products or recombinant proteins are well known molecular biology techniques which allow the production of large amounts of a protein by taking advantage of the genetic and metabolic machinery of a microorganism. To clone a gene, the genomic D N A of certain organisms are cut into small fragments at specific sites using specialized enzymes. These fragments contain 42  the intact gene, which are then inserted into a 'vector', which is a double-strand D N A molecule that incorporates fragments of foreign D N A .  42  Bacterial 'plasmids' are the most  common used vectors. These are small, circular D N A molecules that can exist and replicate in a cell without the help of the cell's D N A . The vector with its added D N A fragment can be introduced into an appropriate bacterial host, such as Escherichia coli for the expression of the foreign protein. This technique allows production of the protein at levels much higher than endogenous proteins. Addition of a tag to a protein' gives the protein a unique binding affinity that makes its purification easier. The most common tag is the His-tag, which usually consists of six histidine residues at either the N - or Cterminus of a protein. His-tag proteins have affinity towards electron deficient transition metals. Immobilized metal affinity chromatography (IMAC) is a useful purification method for tagged proteins, as the resin in these columns contains a transition state metal ion which can bind to the electron rich imidazole on the histidines. For example, a nickel column has immobilized nickel ions (Ni ) stationed inside it, allowing binding of the 2+  protein of interest to the nickel resin while all other proteins will pass through the column. The protein is then eluted from the column by adding imidazole, which competes  50 with the His-tags for nickel binding. The protein of interest is now the only protein component in the eluted mixture and can be even further purified from any minor unwanted contaminants by a second step of purification, such as size exclusion chromatography. A n SDS-PAGE (sodium dodecyl sulfate polyacrylamide) gel is used to check the purity of the protein.  Overexpression and purification of the enzyme pseB showed a band corresponding to the pure protein of approximately 38 kDa after purification (Figure 3.1).  1 Figure 3.1  2  3  SDS-PA GE gel of crude lysate and purified enzyme: (1= molecular weight  markers (Bovine Serum Albumin at 66 kDa and Carbonic Anhydrase at 29 kDa), 2= crude lysate, 3- pure enzyme)  51 3.2  General Properties of Enzyme Inhibitors  Although inhibition patterns can be complex, inhibition can generally be divided into four categories: competitive inhibition, uncompetitive inhibition, noncompetitive inhibition, and irreversible inhibition. In competitive inhibition, the inhibitor simply competes with the substrate to bind to the same active site of the enzyme. Uncompetitive inhibition occurs when an inhibitor binds only to the enzyme-substrate complex, blocking the enzyme's catalytic activity. The inhibitor itself does not bind the free enzyme. Noncompetitive inhibition occurs if an inhibitor binds to both the free enzyme and the enzyme-substrate complex. Often this inhibitor binds to a site on the enzyme other than the active site to change the enzyme's catalytic ability. If an inhibitor inactivates the enzyme via formation of a covalent bond, irreversible inhibition occurs. In this case, the enzyme is simply considered to be 'dead'.  UDP-6-deoxy-6,6-difluoro-GlcNAc is a potential irreversible inhibitor which is expected to covalently bind to the enzyme active site and destroy its activity. Addition of the natural substrate after the incubation of 11 with pseB should not result in formation of dehydrated product.  52  3.3  Preliminary Enzyme Kinetics Using UDP-6-deoxy-6,6-difluoro-GlcNAc  Inhibitor  To determine if the enzyme is active, pseB was incubated with UDP-GlcNAc and the reaction progress was followed using P N M R spectroscopy (Figure 3.2a). The 31  doublet at -12.5 ppm corresponding to the P-phosphate can be seen to gradually convert into a new doublet at -12.8 ppm. This indicates that the enzyme is active and catalysis is taking place. After 14 hours of incubation, ESI-MS analysis shows a peak at 588 m/z [M-H ]" corresponding to the expected 6-deoxy product. In a second experiment, +  inhibitor 11 was incubated with pseB to see if it serves as a substrate and reaction progress was followed with P N M R and F N M R spectroscopy. The N M R spectra 31  19  were unchanged throughout the 24 hour incubation time, suggesting no reaction was occurring (data not shown).  T o determine whether the inhibitor caused irreversible inactivation of the enzyme a pre-incubation experiment was performed. In an eppindorff tube, 0.75 m M of inhibitor was incubated with 0.21 m M of enzyme. The concentration of the inhibitor is 3.6 times greater than concentration of the enzyme. This ensures all the enzyme active sites come in contact with the inhibitor. After a 30 min incubation time, a sample of this mixture was diluted into a buffer containing UDP-GlcNAc, such that the final concentration of 11 and UDP-GlcNAc were 0.06 m M and 5 m M , respectively. At this point, since the substrate concentration is much higher than the concentration of the inhibitor, there will be no interfering N M R signals from the inhibitor in the experiment. The reaction progress of  53 this experiment, along with a control reaction with equal amounts of pseB and substrate, were followed by P N M R spectroscopy (Figure 3.2). As shown, pre-incubation of the 31  inhibitor with the enzyme for 30 min did not lead to inactivation of the enzyme catalysis.  54B)  —i—i—i—i—i—|—i—i—i—i—|—i—i—i—i | i i 70.50 -7 7.00 -11.50 -12.00  Figure 3.2  i  i  p  1  -12.50  • • • i  1  • •  -13.00  1  i  1  -13.50  P NMRs of a) UDP-GlcNAc with PseB undergoing dehydration, and b)  Si  UDP-GlcNAc undergoing dehydration catalysed by PseB that had been pre-incubated with UDP-6-deoxy-6,6-difluoro-GlcNAcfor  30 min  It could be possible that the inactivation is time-dependent and the 30 min incubation time was not long enough for inhibition to occur. Thus, in a second experiment the enzyme catalyzed reactions were incubated for 2 h before adding the substrate and the reaction progress was followed by ' H N M R spectroscopy (Figure 3.3). This experiment required that enzyme and the phosphate buffer to be exchanged with D2O prior to the kinetics assay.  Reaction progress was followed by the appearance of the methyl signals at l .46 ppm which corresponds to the product of the reaction, and at 1.22 ppm which  •  •  1  r  • 55 corresponds to the diol formed from hydration of the ketone at C-4 (Figure 3.4). Incorporation of a deuterium at C-5 can be inferred from the lack of coupling to the C-6 proton signals.  OH  Figure 3.4  Following the progress of enzymatic reaction by formation of methyl  singlet peaks in the 'H NMR. The peak at 1.46 ppm represents the structure on the right, and the one on 1.22 ppm represents the one on the left.  56  t=0 min  t=30 min  it  • p  t=60 min  irfit  AcNH  Figure 3.3  OUDP  AsNHl OUDP  'H NMR experiments with A) 2 hour incubated enzyme added to UDP-  GlcNAc (control) and B) incubated enzyme with the inhibitor and added to the substrate. Both control and PseBpre-incubated with UDP-6-deoxy-6,6-difluoro-GlcNAc show similar production of product UDP-6-deoxy-4-keto-HexNAc which can be observed at L 4 6'and 1.22 ppm (He" signals for unhydrated and hydrated product respectively).  57  From these experiments it is obvious that the inhibitor is not inactivating the enzyme even after 2 h incubation time. One possible explanation for this is based on the steric aspect of the inhibitor vs. the substrate. The two fluorine groups on the C-6 positions are possibly too bulky for the inhibitor to bind tightly in the enzyme active site and cause inactivation. Another reason might be that the H-bonding of the C-6 hydroxyl group of the substrate to the enzyme is crucial in substrate binding so that when it is replaced with two fluorines, the lack of this H-bonding does not allow binding of the inhibitor to the active site.  3.3.1  Further Studies  In the pathogenic bacteria Campylobacter jejuni 2,4-diacetamido-2,4,6-trideoxya-D-glucopyranose, termed A^/V'-diacetylbacillosamine, is the first carbohydrate in the glycoprotein N-linked heptasaccharide. As with pseudaminic acid, this compound is also believed to play a major part in the production of surface-associated virulence factors. Figure 3.5 shows a scheme for the biosynthesis of UDP-7V,A^'-diacetylbacillosamine, 36, in C. jejuni starting with UDP-GlcNAc. In this scheme, Cjl 120c (PglF) performs a 58  cofactor-dependent hydride transfer from the C4 position of UDP-GlcNAc to C6, with elimination of water across the C5-C6 bond, forming UDP-2-acetamido-2,6-dideoxy-a-D4-ketohexulose 34. The mechanism of C j l 120c is similar to that of pseB, however with Cjl 120c the inversion of stereochemistry at C5 position does not occur. The remaining  58 steps include C j l 121c (PglE) which catalyzes the pyridoxal-dependent transfer of an amino group from L-glutamate to the C4 position of 34 to form compound 35; and Cjl 123c (PglD) which achieves the N-acetylation at the C4 position of compound 35.  58  The. enzyme C j l 120c also belongs to the SDR enzyme family. As mentioned previously, enzymes in this family are divided into two subfamilies: the short soluble ones with an S Y K catalytic triad (such as PseB), and the large membrane-bound enzymes with an altered S M K catalytic triad (Cj 1120c). The activity of Cj 1120c is more similar to the successful experiment performed with CDP-D-glucose 4,6-dehydratase. For future 22  studies, the synthesized inhibitor 11 can be tested with Cjl 120c to determine if any irreversible inactivation of this enzyme is observed.  .OH Cj 1120c  -°v  HO HO-  Cj1121c H  AcHN  OUDP  AcHN 34  2  H o \ - - ^ \ ArHN  OUDP  A  C  M  N  35  UDP-a-D-GlcNAc  OUDP  Cj 1123c  AcHN HO • . AcHN 36  Figure 3.5  Biosynthetic pathway of UDP-Bacillosamine found in the bacterial  glycoprotein heptasaccharide  j OUDP  59  3.4  Conclusion  The experiments described in this thesis illustrate important stages of drug discovery, which include designing, synthesizing and testing a newly developed compound to determine its efficacy. From the results of the experiments, the synthesis of UDP-6-deoxy-6,6-difluoro-GlcNAc, 11, was successful, but contrary to our expectations, the compound did not inhibit pseB. Nevertheless, this compound could be more useful for the study of Cjl 120c.  3.5  Experimental Methods  3.5.1  Enzyme Overexpression and Purification  Expression plasmids containing the pseB gene were obtained from the National Research Council of Canada. This recombinant pseB plasmid was transformed into E. coli BL21 (DE3) competent cells which were incubated in 10 mL L B medium containing 50 mg/liter kanamycin at 37°C/225 rpm until an A6oo of 0.6 had been reached. The culture was allowed to continue to grow for 5 h after 70 mg/liter of isopropyl P-Z)galactopyranoside (IPTG) was added. Cells were harvested by centrifuging at 6000 X g for 30 min and then resuspended in 10 mL of phosphate buffer (20 m M , pH 8.0) containing 2 m M dithiothreitol, 1 mg/liter of aprotinin, and 1 mg/ liter pepstatin A. The  60 cells were lysed by passage through a French pressure cell at 20,000 psi. The lysate was centrifuged at 6000 X g for 1 h and passed through a 0.22 um filter.  A column containing 10 mL of chelating Sepharose Fast Flow resin (GE Healthcare) was charged with 20 mL of 100 m M NiSC^, washed with 20 mL of distilled H2O and 30 mL of sodium phosphate buffer (20 m M , pH 8.0, containing 0.5 M NaCl and 5 m M of imidazole). The lysate was loaded onto the column and eluted with the same buffer containing increasing amounts of imidazole in a stepwise fashion (5 m M , 125 m M , and 500 mM). Eluate fractions that were eluted with 500 m M imidazole and showed absorbance at 280 nm were collected. These fractions were concentrated using Amicon Ultra Centricons (Millipore) before flash freezing with liquid N in the presence of 10% 2  glycerol and 2 m M dithiothreitol.  3.5.2  Bradford Method for Determining Enzyme Concentration  The concentration of the purified enzyme was determined using a standard Bradford assay. This method quantitates the binding of Coomassie brilliant blue to an 43  unknown protein and compares this binding to that of different amounts of a standard protein, in this case bovine serum albumin. Using the standard curve obtained from this protocol, the yield of the enzyme was quantified to be 32 mg/mL.  61 3.5.3  3 1  P N M R Kinetic Assay  A control sample containing 0.21 m M enzyme was incubated for 30 min at 21 °C in 120 p L phosphate buffer (10 m M N a H P 0 , pH 8.0). In a separate tube, 0.21 m M 2  4  enzyme and 0.75 m M inhibitor in buffer solution was also incubated at 21 °C for 30 min. Aliquots (60 pL) of these pre-incubated solutions were then added into two separate N M R tubes containing 600 p L phosphate buffer (10 m M N a H P 0 , pH 8.0), 200 p L 2  4  D2O, and 5 m M UDP-GlcNAc substrate. The reactions were monitored as a function of time by P spectroscopy. 3 1  3.5.4  *H N M R Kinetic Assay  The dehydratase pseB was prepared for N M R assay by exchanging to deuterated phosphate buffer (10 m M N a H P 0 , pH 8.0, reconstituted in D 0 ) by spinning a 10-100 2  4  2  p L sample of enzyme (32 mg/mL) with 1 mL of phosphate buffer through a membrane filter (Amicon Ultra-4, 10 000 MWCO) at 5000 rpm to a final volume of 250 pL. This was repeated twice more to completely exchange the buffer. Then, 0.21 m M of this enzyme solution was inclubated with 0.75 m M UDP-6-deoxy-6,6-difluoro-GlcNAc for 2 hours at 21 °C. After this incubation period, 60 p L of this mixture was added to 5 m M of UDP-GlcNAc substrate in 200 p L D 0 and 600 pL deutorated phosphate buffer (10 m M 2  NaH PC>4, pH 8.0), resulting in a final concentration of 0.06 m M of UDP-6-deoxy-6,62  difluoro-GlcNAc. A control sample without the UDP-6-deoxy-6,6-difluoro-GlcNAc was  62 also prepared in the same manner. The reactions were followed as a function of time by ' H N M R spectroscopy.  63 References: 1. Dwek, R. A . Chem. Rev. 1996, 96, 683. 2. Sharon, N . ; Lis, H. Essay Biochem. 1995, 30, 5.9. 3. Rademacher, T.W.; Parekh, R.B.; Dwek, R.A. Ann. Rev. Biochem. 1998, 57, 785 4. Leloir, L. F. Science. 1971, 172, 1299. 5. Heidlas, J. E. W., K.W.; Whitesides, G.M. (1992). Acc. Chem. Res 25: 307.  6. Liu, H.-w.; Thorson, J.S. Annu. Rev. Microbiol. 1994, 48, 223. 7. Feingold, D. S.; Avigad, G. In The Biochemistry of Plants; Academic Press, 1980; Vol. 3, pp 101-170 8. Heidlas, J. E. W., K.W.; Whitesides, G.M. J. Org. Chem. 1992, 57, 152. 9. Frey, P. A . FASEB J. 1996, 10, 461. 10. Johnson, D. A . ; Liu, H.-w. In Comprehensive Natural Products Chemistry Barton, D.; Nakanishi, K.; Eds.; Elsevier: New York, 1999; Vol. 3, p 311. 11. Glycosciences: Status and Prospectives; Gabius, H.-J.; Gabius, S., Eds.; Chapman & H a l l : Weinhein, 1997. 12. Glaser, L ; Zarkowsky, R. 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Biochemistry. 2006, 45, 13659-13669  Appendix: ' H N M R Spectra of Sugar Nucleotide Products  68  69  U T  1  1  r—|  6.0  1  1  1  1  1  5.0  1  1  1  1  |  4.0  i  i  i  i  |  3.0  i  i  i  i  |  2.0  r  JLIAJ  -JL  i  1  1  1  1  ppm (fl}°  1  1  1  6.0  1  1  1  _A___JJL  1  1  5.0  1  1  1  1  1  4.0  1  1  1  1  1  3.0  i  i  i  i  I  2.0  r  71  72  1  JLA  T  1  (ffy0  1  1  1  1  1  6.0  1  1  1  1  r-—i  5.0  1  1  1  1  4.0  1  1  1  1  |  3.0  i  r—i  i  |  2,0  i  r  73  74  i—i—i—i—I—i—i—i—i—|—i—i—i—i—|—i—i—i—i—|—i—i  7.0  ppm (11)  6.0  5.0  4.0  i  i  |  3.0  i  i  i  i  |  2,0  i  i  i  r  

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