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Synthesis of substrates and inhibitors for cell shape-determining proteases Csd4 and Csd6 Soni, Arvind 2021

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Synthesis of substrates and inhibitors for cell shape-determining proteases Csd4 and Csd6  by  Arvind Soni   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  Doctor of Philosophy in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  March 2021  © Arvind Soni, 2021 ii   The following individuals certify that they have read and recommended to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Synthesis of substrates and inhibitors for cell-shape-determining proteases Csd4 and Csd6  submitted by Arvind Soni in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry  Examining Committee: Martin E. Tanner, Professor, Department of Chemistry, UBC Supervisor  Stephen  G. Withers, Professor, Department of Chemistry, UBC Supervisory Committee Member  Michael O. Wolf, Professor, Department of Chemistry, UBC University Examiner John C. Sherman, Professor, Department of Chemistry, UBC University Examiner  Additional Supervisory Committee Members: Laurel L. Schafer, Professor, Department of Chemistry, UBC Supervisory Committee Member Glenn Sammis, Associate Professor, Department of Chemistry, UBC Supervisory Committee Member iii   Abstract Peptidoglycan (PG) is an essential part of the bacterial cell wall. It helps in maintaining the cell shape and protects the bacteria from external osmotic pressure. Recently, genes were found that encode for the proteases necessary for maintaining helical cell shape in the human pathogen, Helicobacter pylori. These proteases were called cell shape-determining proteases (Csds).  Deletion of the genes encoding for these proteases resulted in cell-straightening as well as reduced pathogenicity. Our study focuses on two of these proteases, Csd6 and Csd4.  Csd6 is a carboxypeptidase that converts the uncrosslinked PG tetrapeptide ( PG-L-Ala-D-Glu-meso-Dap-D-Ala)  to a PG tripeptide (PG-L-Ala-D-Glu-meso-Dap). Its homolog was also identified in Campylobacter jejuni and is called Pgp2.  Pgp2 removes terminal D-Ala in both crosslinked and uncrosslinked PG tetrapeptides to give the corresponding PG tripeptides. I synthesized truncated analogs of the uncrosslinked PG tripeptide, N-Ac-D-Glu-meso-oxa-Dap-D-Ala, and the cross-linked PG tetrapeptide, N-Ac-D-Glu-meso-oxa-Dap(L-Ala)-D-Ala. Due to difficulties in preparing meso-Dap, we choose to incorporate an isosteric analog, meso-oxa-Dap. The novelty of our synthetic approach involved the ring-opening of a tripeptide-embedded aziridine with a serine-based nucleophile. This allowed a rapid and efficient preparation of a meso-oxa-Dap-containing tripeptide. These peptides were shown to acts as minimal substrates for Csd6 and Pgp2. Research on PG biosynthesis and modification often relies on obtaining synthetic PG peptides, especially the full-length PG pentapeptide, but this is hampered by difficulties in preparing meso-Dap. Therefore, we used our synthetic procedure to prepare the analogous version of the PG pentapeptide, L-Ala-D-Glu-meso-oxa-Dap-D-Ala-D-Ala. This synthetic procedure was iv   efficient, and large quantities of pentapeptide could easily be prepared. To demonstrate its utility, the pentapeptide was attached to the GlcNAc-1,6-anhydro-MurNAc. The Csd4 enzyme removes the terminal meso-Dap from the uncrosslinked PG tripeptide, PG-L-Ala-D-Glu-meso-Dap, to give the PG dipeptide, PG-L-Ala-D-Glu. The final chapter of the thesis outlines our progress towards making a phosphorus-based inhibitors of Csd4. These compounds are designed to mimic the tetrahedral intermediate formed during catalysis and should act as potent inhibitors of the enzyme. We prepared a phosphonate-containing target compound. This compound is currently being tested for its affinity to Csd4 and its ability to cause cell straightening in live bacteria.   v   Lay Summary In this thesis, I synthesized substrates and inhibitors for the cell-shape-determining enzymes that are necessary for maintaining the helical shape of pathogenic bacteria, H. pylori and C. jejuni, which are found in the human stomach and intestines, respectively. I have shown that these enzymes can utilize small-molecules as substrates instead of the natural macromolecular substrate (the bacterial cell wall). I have also synthesized the truncated version of the bacterial cell wall that could be useful to researchers in the development of new antibiotics. Finally, I prepared a novel inhibitor of a cell shape-determining enzyme that could lead to diminished bacterial colonization.   vi   Preface A version of Chapter 2 has been published and some figures are reproduced with permission from Soni, A. S.; Lin, C. S.-H.; Murphy, M. E. P.; Tanner, M. E. ChemBioChem 2019, 20 (12), 1591–1598. The purification of the Csd6 and Pgp2 enzymes was performed by Chang Sheng-Huei Lin under the supervision of Principal Investigator Michael E. P. Murphy. The synthesis of the compounds was completed by the author of this thesis, under the supervision of principal investigator Martin E. Tanner. A portion of Chapter 3 has been published and some figures are reproduced with permission from: Soni, A.S.; Vacariu, C.M.; Chen, J. Y.; Tanner, M.E. Organic Letters 2020, 22 (6), 2313–2317. The coupling of peptidoglycan pentapeptide to GlcNAc-Anhydro-MurNAc disaccharide was performed by Condarache M. Vacariu under the supervision of principal investigator Martin E. Tanner. The starting material for GlcNAc-Anhydro-MurNAc disaccharide was prepared by Jeff. Y. Chen under supervison of principal investigator Martin E. Tanner.  The synthesis of peptidoglycan pentapeptide was completed by the author of this thesis, under the supervision of Principal investigator Martin E. Tanner. The fourth chapter of this thesis is original unpublished work. All the synthetic experiments were completed by the author of this thesis under the supervision of principal investigator Martin E. Tanner.  vii   Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ................................................................................................................................ v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ............................................................................................................................... xii List of Figures ............................................................................................................................. xiii List of Symbols and Abbreviations ........................................................................................ xxiv Acknowledgements ................................................................................................................ xxviii Dedication ................................................................................................................................. xxix Chapter 1: Introduction ........................................................................................................... 1 1.1 Bacterial cell wall ........................................................................................................... 1 1.2 Peptidoglycan .................................................................................................................. 2 1.2.1 Peptidoglycan backbone variation .......................................................................... 4 1.3 Peptidoglycan biosynthesis ............................................................................................. 4 1.3.1 Biosynthesis of the UDP-MurNAc pentapeptide .................................................... 5 1.3.2 Translocation and polymerization steps of PG biosynthesis .................................. 7 1.3.3 Drugs targeting the peptidoglycan biosynthesis pathway ..................................... 10 1.3.4 PG recycling.......................................................................................................... 12 1.3.5 PG modifications .................................................................................................. 15 1.4 Cell shape determining proteases (Csds) in helical bacteria ......................................... 16 viii   1.4.1 Helicobacter pylori and human disease ................................................................ 17 1.4.2 Campylobacter jejuni and human disease............................................................. 18 1.4.3 Csd enzymes and their cleavage sites in H. pylori ................................................ 19 1.5 Project goals .................................................................................................................. 27 1.5.1 Synthesis and evaluation of minimal linear and branched tripeptide substrates for the cell shape determining proteases Csd6 and Pgp2. ...................................................... 27 1.5.2 Synthesis of meso-oxa-Dap-containing PG pentapeptide and coupling to GlcNAc-anhMurNAc disaccharide ................................................................................................. 30 1.5.3 Synthesis and testing of phosphonamidate and phosphonate inhibitors of the Csd4 enzyme .............................................................................................................................. 32 Chapter 2: Synthesis and evaluation of minimal linear and branched tripeptide substrates for the cell shape-determining proteases Csd6 and Pgp2 ................................. 33 2.1 Previous methods to synthesize orthogonally protected meso-Dap .............................. 33 2.2 Synthesis of tripeptide substrate 1 ................................................................................ 41 2.2.1 Synthesis of tripeptide substrate 1 via orthogonally protected meso-oxa-Dap ..... 41 2.2.2 Synthesis of tripeptide substrate 1 via an embedded aziridine ring-opening ........ 44 2.3 Synthesis of branched tetrapeptide 2 ............................................................................ 50 2.4 Activity test using a mass-spectrometry assay.............................................................. 51 2.5 Conclusions and summary ............................................................................................ 55 2.6 Experimental procedures .............................................................................................. 57 2.6.1 General information .............................................................................................. 57 2.6.2 Synthesis of tripeptide substrate 1 ........................................................................ 57 ix   2.6.3 Synthesis of branched tetrapeptide 2 .................................................................... 64 2.6.4 Cloning, expression and purification of Pgp2 and Csd6....................................... 66 2.6.5 Enzyme Activity test ............................................................................................. 67 Chapter 3: Synthesis of meso-oxa-Dap-containing PG pentapeptide and coupling to GlcNAc-anhMurNAc disaccharide ....................................................................................... 69 3.1 Incorporation of meso-Dap into PG fragments ............................................................. 71 3.2 Synthesis of the meso-oxa-Dap containing PG pentapeptide via pentapeptide-embedded aziridine ring-opening ........................................................................................................... 74 3.3 Synthesis of meso-oxa-Dap containing PG pentapeptide via tripeptide-embedded ring-opening .................................................................................................................................. 76 3.3.1 Ring-opening of aziridine tripeptide using phthalimido serine ............................ 76 3.3.2 Ring-opening of aziridine tripeptide using azido serine ....................................... 80 3.3.3 Synthesis of meso-oxa-Dap pentapeptide using Cbz protected tripeptide ............ 81 3.4 Conclusion and summary .............................................................................................. 84 3.5 Experimental procedures .............................................................................................. 85 3.5.1 General information .............................................................................................. 85 3.5.2 Synthesis of pentapeptide 72 via ring-opening of the aziridine pentapeptide ...... 85 3.5.3 Synthesis of Pentapeptide 61 ................................................................................ 93 3.5.4 Synthesis of pentapeptide 62 .............................................................................. 100 3.5.5 Synthesis of pentapeptide 63 .............................................................................. 101 x   Chapter 4: Progress towards the synthesis and testing of phosphonamidate and phosphonate inhibitors of the Csd4 enzyme ....................................................................... 105 4.1 Design and synthesis of phosphinate (P-C bond) based inhibitor .............................. 106 4.1.1 Kinetic studies of inhibitor 4............................................................................... 108 4.1.2 Structural studies of Csd4 with the phosphinate inhibitor 4 ............................... 109 4.1.3 Studies of inhibitor 4 with H. pylori and C. jejuni.............................................. 110 4.2 Design and synthesis of phosphonamidate and phosphonate inhibitors ..................... 112 4.3 Attempted synthesis of the phosphonamidate inhibitor 6 ........................................... 114 4.3.1 Synthesis of the meso-oxa-Dap fragment ........................................................... 114 4.3.2 Synthesis of the phosphonic acid fragment ........................................................ 115 4.3.3 Coupling of phosphonic acid 114 with meso-oxa-Dap 100 ................................ 119 4.4 Synthesis of phosphonate inhibitors ........................................................................... 125 4.4.1 Synthesis of hydroxy-meso-oxa-Dap .................................................................. 125 4.5 Conclusion and summary ............................................................................................ 132 4.6 Experimental procedures ............................................................................................ 134 4.6.1 General information ............................................................................................ 134 4.6.2 Synthesis of meso-oxa-Dap 100 .......................................................................... 134 4.6.3 Synthesis of mono-protected phosphonate derivative 121 ................................. 137 4.6.4 Synthesis of protected phosphonamidate inhibitors 124 and 132 ....................... 146 4.6.5 Synthesis of hydroxy meso-oxa-Dap 140 ........................................................... 148 4.6.6 Synthesis of phosphonate inhibitor 142 .............................................................. 151 Chapter 5: Conclusions and Future work .......................................................................... 153 xi   5.1 Conclusions for Chapter 2 ............................................................................................ 153 5.1.1 Future work related to Chapter 2 ........................................................................ 154 5.2 Conclusions for Chapter 3 .......................................................................................... 157 5.2.1 Future work related to Chapter 3 ........................................................................ 157 5.3 Conclusions for Chapter 4 .......................................................................................... 159 5.3.1 Future work for Chapter 4................................................................................... 160 Bibliography .............................................................................................................................. 163 Appendix .................................................................................................................................... 179   xii   List of Tables Table 2. 1: Optimization of aziridine ring-opening conditions..................................................... 48  Table 4. 1 Optimization of the coupling reaction for the formation of phosphonate 141 .......... 130                    xiii   List of Figures Figure 1.1: General representation of the bacterial cell envelope .................................................. 1 Figure 1.2: Differences between Gram-negative and Gram-positive bacterial cell walls .............. 2 Figure 1.3: The structure of peptidoglycan showing both crosslinked and non-crosslinked peptide side chains and the reaction catalyzed by transpeptidase. The inset shows the chemical structure of the pentapeptide moiety .............................................................................................................. 3 Figure 1.4: Stepwise assembly of the peptidoglycan chain ............................................................ 5 Figure 1.5: The conversion of UDP-GlcNAc into UDP-MurNAc ................................................. 6 Figure 1.6: The biosynthesis of UDP-MurNAc pentapeptide ........................................................ 7 Figure 1.7: General reaction mechanism of the Mur ligases. ......................................................... 7 Figure 1.8: A) Lipid bound steps of PG biosynthesis  B) An acyl-enzyme mechanism for PG crosslinking ................................................................................................................................... 10 Figure 1.9: The structure of cycloserine and fosfomycin and the natural compounds they structurally resemble ..................................................................................................................... 11 Figure 1.10: A)  Representation of the structure of the vancomycin complex with D-Ala-D-Ala, hydrogen bonding is indicated by green dotted lines. The structural differences between D-Ala-D-Ala and D-Ala-D-Lac are represented by red coloured atoms. B) Structural similarities between a β-lactam antibiotic and D-Ala-D-Ala. C) The inactivation of PBP by β-lactam antibiotics......... 12 Figure 1.11: The PG recycling pathway ....................................................................................... 14 Figure 1.12: Modification of MurNAc and GlcNAc sugars ......................................................... 16 Figure 1.13: Structure of drugs used in combination for the treatment of H. pylori  infections .. 18 Figure 1.14: Structures of drugs used for the treatment of C. jejuni infections ............................ 19 xiv   Figure 1.15: Cell shape determinant peptidases in H. pylori and their cleavage sites. ................. 20 Figure 1.16: A) Schematic representation of the PG trimming process. B) Phase-contrast microscopy images of wild-type and mutant H. pylori (taken from Chan et al., 2015). .............. 22 Figure 1.17: A) Proposed catalytic mechanism of Csd6. B) Crystal structure of Csd6 with 3 domains (taken from Kim et al., 2015) ......................................................................................... 25 Figure 1.18: A) The domain structure of Csd4. B) The crystal structure of Csd4 in complex with N-Ac-L-Ala-iso-D-Glu-meso-Dap ( taken from Chan et al., 2015). C) The proposed mechanism employed by Csd4......................................................................................................................... 27 Figure 1.19: A) Trimming of the uncrosslinked PG tetrapeptide to give PG tripeptide by Csd6/Pgp2. B) Trimming of crosslinked PG tetrapeptide to give crosslinked PG tripeptide by Pgp2........................................................................................................................................................ 28 Figure 1.20: A) Structure of the muramyl tetrapeptide used by Kim et al. to demonstrate Csd6 activity. B) Structures of the linear tripeptide and branched tetrapeptide prepared in Chapter 2.  Red colour represents the structural similarity between molecules. ............................................. 30 Figure 1.21: Structure of the GlcNAc-anhMurNAc pentapeptide containing meso-oxa-Dap. ..... 31 Figure. 1.22: Structures of the phosphorus-based inhibitors of the Csd4 enzyme. ...................... 32 Figure 2.1: Methods developed by Vederas and co-workers to synthesize meso-Dap ................. 36 Figure 2.2: Synthesis of a meso-Dap derivative via a photolytic method .................................... 36 Figure 2.3: Methods developed by various research groups to synthesize orthogonally protected meso-Dap ...................................................................................................................................... 38 Figure 2.4: Methods developed by Fukase and co-workers to synthesize meso-Dap .................. 39 xv   Figure 2.5: Structure of orthogonally protected meso-Dap and an isosteric analogue, meso-oxa-Dap ................................................................................................................................................ 40 Figure 2.6: Structure of substrates for Csd6 /Pgp2. Boxes represent the structural similarity with the natural substrate. ..................................................................................................................... 41 Figure 2.7: The synthesis of the tripeptide substrate 1 using orthogonally-protected meso-oxa-Dap........................................................................................................................................................ 43 Figure 2.8: Mechanism of aziridine ring-opening by nucleophilic hydroxyl group of serine. ..... 43 Figure 2.9: Mechanism of pNZ group removal. ........................................................................... 44 Figure 2.10: Synthesis of peptidyl aziridine 43 ............................................................................ 45 Figure 2.11: H-bonding patterns and effects on nucleophilicity of hydroxyl of protected serine 46 Figure 2.12: Synthesis of meso-oxa-Dap derivative 45 done by the Vederas group .................... 46 Figure 2.13: Synthesis of the tripeptide 46 via embedded tripeptide aziridine ring-opening. ...... 49 Figure 2.14: Plausible mechanism of the Heine rearrangement of tripeptide aziridine 43 ........... 49 Figure 2.15: Deprotection of compound 46 to give tripeptide substrate 1 ................................... 50 Figure. 2.16: Synthesis of branched tetrapeptide 2 ....................................................................... 51 Figure 2.17: Masses of monosodium ion adducts for substrates 1 and 2 and their enzymatic reaction products ......................................................................................................................................... 52 Figure 2.18: MALDI-TOF MS analysis of the reactions catalyzed by Csd6 and Pgp2 with tripeptide 1 .................................................................................................................................... 53 Figure 2.19: MALDI-TOF MS analysis of the reactions catalyzed by Csd6 and Pgp2 with branched tetrapeptide substrate 2 ................................................................................................................. 54 xvi   Figure 3.1: Lytic transglycosylase cleavage leading to the formation of GlcNAc-1,6-anhydro-MurNAc pentapeptide. .................................................................................................................. 70 Figure 3.2: Schematic scheme for the synthesis of the GlcNAc-anhMurNAc pentapeptide by Mobashery and coworkers ............................................................................................................ 72 Figure 3.3: Structure of meso-oxa-Dap containing PG pentapeptide and GlcNAc-anhMurNAc pentapeptide .................................................................................................................................. 74 Figure 3.4: Synthesis of the meso-oxa-Dap containing pentapeptide 72 ...................................... 75 Figure 3.5: Synthesis of protected pentapeptide 29 ...................................................................... 77 Figure 3.6: 1H-NMR spectrum of pentapeptide 61 in deuterated methanol (CD3OD) ................. 78 Figure 3.7: Variable temperature NMR experiment with compound 61 ...................................... 79 Figure 3.8: Ring-opening of aziridine 77 using azido serine 81. .................................................. 80 Figure 3.9: Synthesis of tetrapeptide 83 and a partial 1H-NMR in CD3OD ................................. 81 Figure 3.10: Synthesis of anhydro disaccharide pentapeptide 3 containing meso-oxa-Dap......... 83 Figure 4.1: Comparison between tripeptide substrate (left), tetrahedral intermediate (middle) and dipeptidyl phosphinate inhibitor 4 (right). The scissile peptide bond and the tetrahedral centers are coloured in red ............................................................................................................................ 107 Figure 4.2: Synthesis of the phosphinate inhibitor 4 .................................................................. 108 Figure 4.3: The continuous coupled assay used in the Csd4 kinetic analysis ............................. 109 Figure 4.4: A) Electron density map of inhibitor 4 in the Csd4-Inhibitor complex. The (S) configuration of the inhibitor stereocenter that is close to the phosphinate is shown (left) and Csd4-inhibitor complex (right). B) Structural alignment of the active site of the Csd4-Inhibitor and the Csd4-Tripeptide complex. (images are taken from Liu et al., 2015) .......................................... 110 xvii   Figure 4.5: Phase microscopy images of H. pylori with inhibitor 4 and its histogram ((images are taken from Liu et al., 2015) ........................................................................................................ 111 Figure 4.6: Phase microscopy images of C. jejuni cells (81-176 and ΔkpsM ) with inhibitor 4 and their histograms (images are taken from Liu et al., 2015) .......................................................... 112 Figure 4.7: Structure comparison of phosphinate inhibitor 4 with phosphonamidate inhibitor 5 and phosphonate inhibitor 6............................................................................................................... 113 Figure 4.8: Retrosynthetic analysis for the preparation of inhibitors 5 and 6............................. 114 Figure 4.9: Synthesis of the HCl salt of meso-oxa-Dap 100....................................................... 115 Figure 4.10: Synthesis of alkyl phosphonates 103 and 106 ........................................................ 116 Figure 4.11: A postulated mechanism for the formation of compounds 107 and 108 ................ 117 Figure 4.12: Synthesis of Boc-protected phosphonate 112 ........................................................ 118 Figure 4.13: Synthesis of phosphonic acids ................................................................................ 119 Figure 4.14: Attempted synthesis of phosphonamidate 115 using PyBOP as a coupling reagent..................................................................................................................................................... 119 Figure 4.15: Synthesis of phosphonamidate 115 via phosphochloridate 116 ............................. 120 Figure 4.16: Synthesis of an H-phosphinic acid developed by the Montchamp group .............. 121 Figure 4.17: Synthesis of mono-benzyl phosphonic acid 121 .................................................... 122 Figure 4.18: Possible mechanism of the Hewitt reaction ........................................................... 122 Figure 4.19: Attempted synthesis of inhibitor 5 ......................................................................... 123 Figure 4.20: Attempted synthesis of inhibitor 133 ..................................................................... 124 Figure 4.21: Schematic for the preparation of  a protected phosphonate inhibitor..................... 125 Figure 4.22: Different methods to prepare hydroxy-meso-oxa-Dap ........................................... 126 xviii   Figure 4.23: Synthesis of hydroxy-serine 138 ............................................................................ 127 Figure 4.24: Synthesis of protected hydroxy-meso-oxa-Dap ..................................................... 127 Figure 4.25: Cleavage of the allyl moiety employed by the Chandrasekhar group .................... 128 Figure 4.26: Synthesis of hydroxy-meso-oxa-Dap 140 .............................................................. 128 Figure 4.27: Schematic representation for allyl deprotection mechanism .................................. 129 Figure 4.28: Synthesis of phosphonate inhibitor 142 ................................................................. 132 Figure 5.1: Continuous coupled assay for the Csd6 reaction...................................................... 155 Figure 5.2: Potential inactivation mechanisms of cysteine proteases by halomethyl ketones.... 156 Figure 5.3: Structure of a chloromethyl ketone inhibitor 51 for Csd6........................................ 156 Figure 5.4: Top: Graphical representation of Nod1 protein interaction with PG fragments (taken from Fukase et al., 2011). Bottom: Structure of meso-Dap containing GlcNAc-anhMurNAc peptides prepared by Fukase et al. for stimulatory studies. ........................................................ 158 Figure 5.5: Proposed synthesis of inhibitor 6 ............................................................................. 160 Figure 5.6: Cartoon diagram of Zn-hydroxamate binding in thermolysin and structure of hydroxamate inhibitor for Csd4 (box) ........................................................................................ 161 Figure 5.7: Synthesis of hydroxamate inhibitor 144 ................................................................... 162   Figure A.1: 1H NMR (400 MHz, CDCl3 ) for compound 38 .................................................... 180 Figure A.2: 13C NMR (101 MHz, CDCl3) for compound 38 ..................................................... 180 Figure A.3: 1H NMR (400 MHz, CDCl3 ) for compound 39 ...................................................... 181 Figure A.4: 13C NMR (101 MHz, CDCl3) for compound 39...................................................... 181 xix   Figure A.5: 1H NMR (400 MHz, D2O) for tripeptide 1 .............................................................. 182 Figure A.6: 13C NMR (101 MHz, D2O) for tripeptide 1 ............................................................. 182 Figure A.7: 1H NMR (400 MHz, CDCl3) for compound 43 ....................................................... 183 Figure A.8: 13C NMR (101 MHz, CDCl3) for compound 43...................................................... 183 Figure A.9: 1H NMR (400 MHz, MeOD) for compound 46 ...................................................... 184 Figure A.10: 13C NMR (101 MHz, MeOD) for compound 46 ................................................... 184 Figure A.11: 1H NMR (300 MHz, MeOD) for compound 47 .................................................... 185 Figure A.12: 13C NMR (75 MHz, MeOD) for compound 47 ..................................................... 185 Figure A.13: 1H NMR (400 MHz, CDCl3) for compound 48 ..................................................... 186 Figure A.14: 13C NMR (101 MHz, CDCl3) for compound 48 ................................................... 186 Figure A.15: 1H NMR (400 MHz, D2O) for branched tetrapeptide 2 ......................................... 187 Figure A.16: 13C NMR (101 MHz, D2O) for branched tetrapeptide 2 ....................................... 187 Figure A.17: 1H NMR (300 MHz, CDCl3) for compound 65 ..................................................... 188 Figure A.18: 13C NMR (75 MHz, CDCl3) for compound 65...................................................... 188 Figure A.19: 1H NMR (300 MHz, CDCl3) for compound 66 ..................................................... 189 Figure A.20: 13C NMR (75 MHz, CDCl3) for compound 66...................................................... 189 Figure A.21: 1H NMR (400 MHz, MeOD) for compound 67 .................................................... 190 Figure A.22: 13C NMR (101 MHz, MeOD) for compound 67 ................................................... 190 Figure A.23: 1H NMR (400 MHz, MeOD) for compound 69 .................................................... 191 Figure A.24: 13C NMR (101 MHz, MeOD) for compound 69 ................................................... 191 Figure A.25: 1H NMR (400 MHz, MeOD) for compound 70 .................................................... 192 Figure A.26: 13C NMR (101 MHz, MeOD) for compound 70 ................................................... 192 xx   Figure A.27: 1H NMR (400 MHz, MeOD) for compound 71 .................................................... 193 Figure A.28: 13C NMR (101 MHz, MeOD) for compound 71 ................................................... 193 Figure A.29: 1H NMR (400 MHz, CDCl3) for compound 72 ..................................................... 194 Figure A.30: 13C NMR (101 MHz, CDCl3) for compound 72 ................................................... 194 Figure A.31: 1H NMR (400 MHz, CDCl3) for compound 73 ..................................................... 195 Figure A.32: 13C NMR (101 MHz, CDCl3) for compound 73 ................................................... 195 Figure A.33: 1H NMR (400 MHz, CDCl3) for compound 74 ..................................................... 196 Figure A.34: 13C NMR (101 MHz, CDCl3) for compound 74 ................................................... 196 Figure A.35: 1H NMR (400 MHz, CDCl3) for compound 75 ..................................................... 197 Figure A.36: 13C NMR (101 MHz, CDCl3) for compound 75 ................................................... 197 Figure A.37: 1H NMR (400 MHz, MeOD) for compound 76 .................................................... 198 Figure A.38: 13C NMR (101 MHz, MeOD) for compound 76 ................................................... 198 Figure A.39: 1H NMR (400 MHz, CDCl3) for compound 77 ..................................................... 199 Figure A.40: 13C NMR (101 MHz, CDCl3) for compound 77 ................................................... 199 Figure A.41: 1H NMR (400 MHz, MeOD) for compound 78 .................................................... 200 Figure A.42: 13C NMR (101 MHz, MeOD) for compound 78 ................................................... 200 Figure A.43: 1H NMR (400 MHz, MeOD) for compound 79 .................................................... 201 Figure A.44: 13C NMR (101 MHz, MeOD) for compound 79 ................................................... 201 Figure A.45: 1H NMR (400 MHz, MeOD) for compound 80 .................................................... 202 Figure A.46: 13C NMR (101 MHz, MeOD) for compound 80 ................................................... 202 Figure A.47: 1H NMR (400 MHz, MeOD) for compound 82 .................................................... 203 Figure A.48: 13C NMR (101 MHz, MeOD) for compound 82 ................................................... 203 xxi   Figure A.49: 1H NMR (400 MHz, MeOD) for compound 84 .................................................... 204 Figure A.50: 13C NMR (101 MHz, MeOD) for compound 84 ................................................... 204 Figure A.51: 1H NMR (400 MHz, MeOD) for compound 85 .................................................... 205 Figure A.52: 13C NMR (101 MHz, MeOD) for compound 85 ................................................... 205 Figure A.53: 1H NMR (400 MHz, MeOD) for compound 63 .................................................... 206 Figure A.54: 13C NMR (101 MHz, MeOD) for compound 63 ................................................... 206 Figure A.55: 1H NMR (400 MHz, CDCl3) for compound 98 ..................................................... 207 Figure A.56: 13C NMR (101 MHz, CDCl3) for compound 98 ................................................... 207 Figure A.57: 1H NMR (400 MHz, CDCl3) for compound 99 ..................................................... 208 Figure A.58: 13C NMR (101 MHz, CDCl3) for compound 99 ................................................... 208 Figure A.59: 1H NMR (400 MHz, MeOD) for compound 100 .................................................. 209 Figure A.60: 13C NMR (101 MHz, MeOD) for compound 100 ................................................. 209 Figure A.61: 1H NMR (400 MHz, CDCl3) for compound 102 ................................................... 210 Figure A.62: 13C NMR (101 MHz, CDCl3) for compound 102 ................................................. 210 Figure A.63: 1H NMR (400 MHz, CDCl3) for compound 103 ................................................... 211 Figure A.64: 13C NMR (101 MHz, CDCl3) for compound 103 ................................................. 211 Figure A.65: 31P NMR (162 MHz, CDCl3) for compound 103 .................................................. 212 Figure A.66: 1H NMR (400 MHz, CDCl3) for compound 108 ................................................... 212 Figure A.67: 13C NMR (101 MHz, CDCl3) for compound 108 ................................................. 213 Figure A.68: 1H NMR (400 MHz, CDCl3) for compound 112 ................................................... 213 Figure A.69: 13C NMR (101 MHz, CDCl3) for compound 112 ................................................. 214 Figure A.70: 31P NMR (162 MHz, CDCl3) for compound 112 .................................................. 214 xxii   Figure A.71: 1H NMR (400 MHz, CDCl3) for compound 117 ................................................... 215 Figure A.72: 13C NMR (101 MHz, CDCl3) for compound 117 ................................................. 215 Figure A.73: 1H NMR (400 MHz, CDCl3) for compound 120 ................................................... 216 Figure A.74: 13C NMR (101 MHz, CDCl3) for compound 120 ................................................. 216 Figure A.75: 31P NMR (162 MHz, CDCl3) for compound 120 .................................................. 217 Figure A.76: 1H NMR (300 MHz, CDCl3) for compound 121 ................................................... 217 Figure A.77: 13C NMR (75 MHz, CDCl3) for compound 121.................................................... 218 Figure A.78: 31P NMR (122 MHz, CDCl3) for compound 121 .................................................. 218 Figure A.79: 1H NMR (300 MHz, CDCl3) for compound 124 ................................................... 219 Figure A.80: 31P NMR (122 MHz, CDCl3) for compound 124 .................................................. 219 Figure A.81: 1H NMR (400 MHz, CDCl3) for compound 132 ................................................... 220 Figure A.82: 31P NMR (162 MHz, CDCl3) for compound 132 .................................................. 220 Figure A.83: 1H NMR (400 MHz, CDCl3) for compound 137 ................................................... 221 Figure A.84: 13C NMR (101 MHz, CDCl3) for compound 137 ................................................. 221 Figure A.85: 1H NMR (300 MHz, CDCl3) for compound 138 ................................................... 222 Figure A.86: 13C NMR (75 MHz, CDCl3) for compound 138.................................................... 222 Figure A.87: 1H NMR (300 MHz, CDCl3) for compound 139 ................................................... 223 Figure A.88: 13C NMR (75 MHz, CDCl3) for compound 139.................................................... 223 Figure A.89: 1H NMR (300 MHz, CDCl3) for compound 140 ................................................... 224 Figure A.90: 13C NMR (75 MHz, CDCl3) for compound 140.................................................... 224 Figure A.91: 1H NMR (300 MHz, CDCl3) for compound 141 ................................................... 225 Figure A.92: 13C NMR (75 MHz, CDCl3) for compound 141.................................................... 225 xxiii   Figure A.93: 31P NMR (122 MHz, CDCl3) for compound 141 .................................................. 226 Figure A.94: 1H NMR (300 MHz, D2O) for compound 142 ...................................................... 226 Figure A.95: 13C NMR (75 MHz, D2O) for compound 142 ....................................................... 227 Figure A.96: 31P NMR (122 MHz, D2O) for compound 142...................................................... 227 xxiv   List of Symbols and Abbreviations Å angstrom δ chemical shift  wavelength Ac acetyl ATP adenosine triphosphate Boc tert-butylcarbonyl Cbz benzyloxycarbonyl C. jejuni Campylobacter jejuni DADPH diaminopimelic acid dehydrogenase DCC N, N-dicyclohexylcarbodiimide DCM dichloromethane DIAD diisopropyl azodicarboxylate DIBAL-H diisobutylaluminum hydride DIPEA N,N-diisopropylethylamine DMAP N,N-dimethyl-4-aminopyridine DMF dimethylformamide E. coli Escherichia coli EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDTA ethylenediaminetetraacetic acid ESI-MS electrospray ionization mass spectrometry Fmoc fluorenylmethyloxycarbonyl xxv   GlcNAc N-acetyl glucosamine HMBC heteronuclear multiple bond correlation HOBt hydroxy benzotriazole H. pylori Helicobacter pylori J coupling constant KI inhibition constant KM Michaelis constant MALDI-TOF matrix-assisted laser desorption/ionization-time of flight MeOD deuterated methanol m-DAP meso-diaminopimelic acid MHz megahertz MS mass spectrometry MurNAc N-acetylmuramic acid MW molecular weight NAD+ nicotinamide adenine dinucleotide, an oxidized form NADH nicotinamide adenine dinucleotide, reduced form NADP+ nicotinamide adenine dinucleotide phosphate, an oxidized form NADPH nicotinamide adenine dinucleotide phosphate, reduced form NHS N-hydroxy succinimide NMR nuclear magnetic resonance PBP penicillin-binding protein PG peptidoglycan xxvi   pNZ p-nitrobenzyl ppm parts per million PyBOP benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate rt room temperature TEA triethylamine THF tetrahydrofuran UDP uridine 5-diphosphate  VT-NMR variable temperature nuclear magnetic resonance  Common Amino Acid Abbreviations A Ala Alanine  C  Cys Cysteine  D Asp Aspartate E Glu Glutamate F Phe Phenylalanine G Gly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine xxvii   P Pro Proline Q Gln Glutamine R Arg Arginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan Y Tyr Tyrosine  xxviii   Acknowledgements First and foremost, I would like to thank my supervisor, Dr. Martin Tanner, for all the guidance and support during my research work. Thank you for always answering my stupid questions and sharing your vast knowledge.  I shall extend my thanks to Dr. Michael Murphy and Angela for sharing their knowledge about microbiology.  I would also like to thank my lab colleagues for their academic support and for ensuring that the work environment is not dull at any moment. I would also like to appreciate the excellent work done by people in the NMR facility as well as the Mass Spectrometry facility during COVID 19; without their technical support and assistance, I wouldn’t be able to finish my thesis work in a timely manner. Finally, I would like to thank my parents, family, friends, and my wife for their constant support and encouragement.   Dedication    xxix   Dedication       Dedicated to my parents and my lovely wife Thanks for all the support, encouragement, love and care   1   Chapter 1: Introduction 1.1 Bacterial cell wall Bacteria are prokaryotic, lack a cytoskeleton and have different morphologies from the spherical shape (cocci) to the straight-rod shape (bacilli). The bacterial cell-envelope acts as a barrier that surrounds the cytoplasm and holds the essential component of the cell, such as the essential organelles and genetic material (Fig.1.1).1, 2 The flagella are hair-like structures that provide locomotion for the bacteria. A key structural component of the cell envelope is the bacterial cell wall, or peptidoglycan (PG), which is an intricate, mesh-like structure that has evolved to protect bacteria from high osmotic pressure. This rigid structure also helps in maintaining the cell shape of bacteria.   Figure 1.1: General representation of the bacterial cell envelope 2   In 1884, Hans Christian Gram developed a staining procedure that allowed him to classify nearly all bacteria into two groups, the one which retains the stain was called Gram-positive and the one which does not was called Gram-negative. There are three essential layers in the Gram-negative cell envelope; the outer membrane (OM), the peptidoglycan cell wall (PG), and the inner membrane (IM) (Fig 1.2). In the case of Gram-positive bacteria, the outer membrane is absent. The PG layer in Gram-negative bacteria is situated in the periplasmic space between the inner membrane and the outer membrane and is relatively thinner than that of  Gram-positive bacteria.2  Figure 1.2: Differences between Gram-negative and Gram-positive bacterial cell walls 1.2 Peptidoglycan As mentioned previously, PG (also called the murien sacculus) is an essential part of the bacterial cell envelope. Its function is to provide rigidity, maintain cellular shape and protect the cell from osmotic pressure.3 The restructuring of PG is also required during the process of cell growth and cell division. As PG is situated outside of the cytoplasmic membrane, it can also act as an anchor for other cellular components such as proteins and teichoic acid. Peptidoglycan consists of glycan strands of alternating N-acetyl glucosamine (GlcNAc) and N-acetylmuramic 3   acid (MurNAc) residues, which are linked to each other by β-(1-4)-glycosidic bonds (Fig. 1.3). A pentapeptide is attached to the MurNAc residues via the lactate moiety at the C-3 position. In most Gram-negative and some Gram-positive species, the peptide chain is comprised of L-Ala1-γ-D-Glu2-meso-Dap3-D-Ala4-D-Ala5 (where meso-Dap corresponds to meso-1,6-diaminopimelic acid and D-Glu is attached via its γ-carboxyl group).3-5 Crosslinking of these peptide chains results in the three-dimensional mesh-like structure that provides strength to bacterial cells (Fig. 1.3). The crosslinking occurs via a transpeptidation reaction in which the amino group of the meso-Dap side chain attacks the D-Ala-D-Ala peptide bond of a neighbouring peptide with the loss of the terminal D-Ala residue.5, 6   Figure 1.3: The structure of peptidoglycan showing both crosslinked and non-crosslinked peptide side chains and the reaction catalyzed by transpeptidase. The inset shows the chemical structure of the pentapeptide moiety 4   1.2.1 Peptidoglycan backbone variation Most bacteria share a similar basic PG structure, but differences in the PG backbone and crosslink can occur from species to species. The GlcNAc-MurNAc disaccharide units are polymerized by a transglycosylase to form glycan strands. Glycan chain length varies in different species, but the normal range lies between 20-40 disaccharide units in E. coli.7 The average length of glycan chain in the PG of genus Bacilli (Bacillus subtilis, Bacillus licheniformis and Bacillus cereus) was found to be between 50 to 250 disaccharide units.8 Staphylococcus aureus, however, has shorter strands with an average chain length of about 18 disaccharide units.9 In most Gram-negative bacteria, the glycan strand terminates with a 1,6-anhydroMurNAc sugar.7, 10 Even greater variations are observed in the peptide stem of peptidoglycan, which is discussed briefly in section 1.3.1. 1.3 Peptidoglycan biosynthesis Peptidoglycan serves as a barrier against external osmotic pressure, and its formation and controlled degradation are crucial for cell growth and cell division. Compounds that interfere with PG biosynthesis may, therefore, serve as antibacterial drugs.11, 12 Peptidoglycan biosynthesis is a two-stage process; the first process involves the assembly of a lipid-linked peptidyl disaccharide unit (lipid II) in the cytoplasm (Fig. 1.4).13 UDP-GlcNAc is first converted to UDP-MurNAc via the action of the enzymes MurA and MurB.14-16 The peptidyl moiety is then added sequentially by the Mur ligases (MurC, D, E, and F).17, 18 Finally, the MurNAc-pentapeptide is transferred to a lipid carrier to give lipid I, which is glycosylated to give the lipid-linked GlcNAc-MurNAc pentapeptide (lipid II) via the action of MurY and MurG (Fig. 1.4). In the second stage of PG 5   biosynthesis, lipid II is translocated across the IM into the periplasm, where glycan polymerization and transpeptidation (crosslinking) occurs.19, 20  Figure 1.4: Stepwise assembly of the peptidoglycan chain 1.3.1 Biosynthesis of the UDP-MurNAc pentapeptide                      UDP-MurNAc synthesis is the first committed step in PG biosynthesis. The MurA enzyme (also called MurZ ) catalyzes the addition of phosphoenolpyruvate (PEP) to the C-3 hydroxyl group of UDP-GlcNAc with the simultaneous release of phosphate (Pi )15, 21 (Fig. 1.5). MurB then reduces the enol pyruvyl moiety to a D-lactyl moiety, giving UDP-MurNAc. MurB is a 6   flavoprotein containing FADH2 as a co-factor. The reducing equivalents are ultimately provided by NADPH.16, 22, 23  Figure 1.5: The conversion of UDP-GlcNAc into UDP-MurNAc As discussed previously, the amino acids are then attached to the D-lactic acid group of UDP-MurNAc to form the primary unit of peptidoglycan biosynthesis (Fig. 1.6).  The ATP-dependent amino acid ligases or Mur ligases catalyze the peptide additions. The first two amino acids, L-Ala and D-Glu, are added by the MurC and MurD ligases, respectively.24 The MurI enzyme, a glutamate racemase, provides a source of D-Glu via the interconversion of L-Glu and D-Glu. 25, 26 A diamino acid is the third to be added by the action of the MurE enzyme. In most Gram-negative bacteria, bacilli and mycobacteria meso-Dap occupies this position. In contrast, most Gram-positive bacteria install L-lysine at this position.27, 28 Finally, the amino acids at positions four and five are incorporated into the peptide chain as D-Ala-D-Ala dipeptide by the action of the MurF ligase.18, 29  7    Figure 1.6: The biosynthesis of UDP-MurNAc pentapeptide All the Mur ligases employ a similar catalytic mechanism, which consists of activation of the carboxyl group on the UDP-linked precursor with ATP, generating an acyl-phosphate intermediate and ADP. Then the nucleophilic amine of the free amino acid or dipeptide attacks to form a tetrahedral intermediate, which collapses to produce phosphate and the peptide bond (Fig. 1.7).30   Figure 1.7: General reaction mechanism of the Mur ligases. 1.3.2 Translocation and polymerization steps of PG biosynthesis The formation of the UDP-MurNAc pentapeptide occurs in the cytoplasm involving freely soluble enzymes and substrates. The next step transfers the MurNAc pentapeptide onto a lipid carrier and localizes it to the inner side of the IM ( Fig. 1.18A). MraY is an integral membrane 8   protein that catalyzes the transfer of the UDP-MurNAc-pentapeptide to undecaprenyl phosphate to yield an undecaprenyl-pyrophosphoryl-MurNAc-pentapeptide, known as Lipid I, with the release of uridine monophosphate (UMP). It is an essential step that is reversible and Mg2+ dependent.20, 31, 32 The MurG glycosyltransferase then transfers the N-acetylglucosamine (GlcNAc) from UDP-GlcNAc onto the C-4 hydroxyl of the MurNAc unit of lipid I to form a β-linked disaccharide unit called lipid II.20, 33, 34  Lipid II is the species that is ultimately transported across the IM to position it on the periplasmic side where the final steps of PG biosynthesis occur. The flippase (transporter protein) is responsible for the transport of lipid II from the cytoplasmic membrane to the outer periplasmic space. The enzymes responsible for the “flipping” activity are not yet clearly known, but the integral membrane proteins FtsW and RodA have been implicated as being important.35, 36 Once lipid II is flipped across the cytoplasmic membrane, the polymerization of the disaccharide moiety and crosslinking of the peptide chains take place to form the 3D mesh-like structure of peptidoglycan. The proteins responsible for these steps are called penicillin-binding proteins (PBPs).37, 38 PBPs are classified into two forms; the class A PBP (bifunctional) catalyzes the polymerization of disaccharide as well as the crosslinking of neighbouring stem peptide chain (transpeptidation), while the class B PBP (monofunctional) protein only catalyzes transpeptidation. Class A PBP utilizes lipid II as a substrate and generates free undecaprenyl pyrophosphate (C55-PP). The glucosyltransferase domain belongs to the GT51 family of glycosyltransferases. The C55-PP product is “flipped” back to the cytoplasmic membrane, where it is further hydrolyzed to undecaprenyl phosphate (C55-P) and recycled for peptidoglycan synthesis (Fig 1.8A). Crosslinking of peptide side chains is necessary for the formation of a net-9   like structure of peptidoglycan. The most common form of crosslinking is a type (4→3) crosslinking or D, D-transpeptidation. This involves the formation of a peptide bond between the carboxyl group of  D-alanine in position 4 of an adjacent donor peptide and the amino group of lysine (Gram-positive) or meso-Dap (Gram-negative) in position 3 of an acceptor peptide chain with subsequent release of the terminal D-alanine of the donor peptide chain. The serine nucleophile of the PBP attacks the D-Ala-D-Ala amide bond of the donor peptide and forms an acyl-enzyme intermediate with the release of the terminal D-Ala (Fig. 1.8B). In the second step of catalysis, the amine side chain of the acceptor peptide attacks the acyl-enzyme intermediate resulting in crosslink formation and release of the PBP.  10    Figure 1.8: A) Lipid bound steps of PG biosynthesis  B) An acyl-enzyme mechanism for PG crosslinking  1.3.3 Drugs targeting the peptidoglycan biosynthesis pathway The vital importance of PG biosynthesis to the survival of bacteria has led both nature and humankind to develop antibiotic compounds directed at this process. D-Cycloserine is an antibacterial drug that targets the biosynthesis of the D-Ala-D-Ala peptide (Fig. 1.9)39. It is a structural analog of D-alanine and is used in the treatment of tuberculosis (TB). It also shows irreversible inhibition against the enzyme alanine racemase, which produces the D-alanine used in PG biosynthesis. Fosfomycin inhibits the MurA enzyme that catalyzes the addition of phosphoenolpyruvate to the hydroxyl group of UDP-GlcNAc.14 It is a PEP analog that prevents the formation of UDP-MurNAc by irreversibly binding with MurA.  11    Figure 1.9: The structure of cycloserine and fosfomycin and the natural compounds they structurally resemble Vancomycin inhibits transpeptidation (PBPs) by binding to the D-Ala-D-Ala residue of the uncrosslinked stem peptide and thereby preventing crosslinking (Fig.1.10A). Vancomycin-resistant bacteria mutate their D-Ala-D-Ala moieties to D-Ala-D-Lac to decrease the binding affinity of this interaction. The loss of the key H-bond between the amide NH and the carbonyl of the drug results in a 1000-fold decrease in binding strength.40 The β-lactam antibiotics are very widely used antibiotics that have a β-lactam ring as a core structure. They may be divided into four different categories: penicillins, cephalosporins, monobactams and carbapenem (all of which contain a core four-membered lactam).2 As mentioned previously, PBPs catalyze the transpeptidation reaction between two neighbouring peptide chains attached to the disaccharide unit with the prior formation of an acyl-enzyme intermediate. The β-lactam ring structurally mimics the terminal D-Ala-D-Ala portion of the pentapeptide.  PBPs are inactivated by the reaction of serine nucleophile with the strand β-lactam ring to generate a covalent acyl intermediate, thus resulting in an inactive enzyme (Fig.1.10 C)41. 12    Figure 1.10: A)  Representation of the structure of the vancomycin complex with D-Ala-D-Ala, hydrogen bonding is indicated by green dotted lines. The structural differences between D-Ala-D-Ala and D-Ala-D-Lac are represented by red coloured atoms. B) Structural similarities between a β-lactam antibiotic and D-Ala-D-Ala. C) The inactivation of PBP by β-lactam antibiotics. 1.3.4 PG recycling  In most strains of Gram-negative bacteria, 30-60% of the bacterial cell wall is recycled. The sequence of amidase, transglycosylase and glycosidase, breaks down the muropeptide for further use in PG assembly (Fig. 1.11).42 Amidases first cleave the crosslinks between neighbouring peptide chains. Lytic transglycosylases then cleave the PG glycan chain in the 13   periplasm to give peptide-linked GlcNAc-1,6-anhydroMurNAc (GlcNAc-anhMurNAc) fragments that are transported back into the cytoplasm by the AmpG transporter protein.43 The NagZ glycosidase then hydrolyzes the glycosidic bond between the GlcNAc-anhydro-1,6-MurNAc disaccharide to release the GlcNAc monosaccharide.44 The metal-dependent AmpD cleaves the bond between the lactyl moiety of the anhMurNAc monosaccharide and the attached peptide. The released peptide units are transferred onto UDP-MurNAc by the Mpl ligase to regenerate the UDP-MurNAc pentapeptide unit.45 The free anhMurNac unit is further broken down to give MurNAc-6-phosphate by AnmK, an ATP-dependent enzyme.46 A series of enzymes convert MurNAc-6-phosphate into UDP-GlcNAc that is transformed into the UDP-MurNAc pentapeptide by Mur ligases. Once formed, the UDP-MurNAc pentapeptide is converted into lipid I and lipid II in the usual fashion and then transported to the periplasm for further PG assembly.47 While the process of cell wall recycling is not required for cellular viability; it is thought that this process gives bacteria an advantage in undergrowth conditions with limited nutrients.42, 47-49 14    Figure 1.11: The PG recycling pathway   15   1.3.5 PG modifications While the structure of PG is largely made up of mesh of glycopeptides as described in section 1.2, there are several examples of PG modifications that occur to fulfill various protective and structural roles.3 Lysozyme is a naturally occurring enzyme that hydrolyzes the β-1-4 glycosidic bond between GlcNAc and MurNAc residues, thus resulting in lysis of the cell. To avoid cell lysis, bacteria have devised different strategies to interfere with the lysozyme activity by chemically modifying the GlcNAc and MurNAc sugars (Fig. 1.12).50 The N-deacetylation of GlcNAc and MurNAc at position C-2 helps the bacteria to evade the host immune system and protect cell wall integrity. O-Acetylation at the C-6 hydroxyl group of MurNAc and GlcNAc is also observed. O-Acetylation of MurNAc occurs in a wide variety of Gram-negative and Gram-positive bacteria. This modification increases the steric bulk at C-6, which ultimately causes attenuated lysozyme binding to PG.50, 51 Structural changes also occur in the peptide stem of PG to avoid cell lysis.3 One of the classic examples is the modification of the terminal D-Ala-D-Ala of the PG pentapeptide to D-Ala-D-Lac. This greatly reduces the affinity of vancomycin to PG and results in antibiotic resistance.52  16    Figure 1.12: Modification of MurNAc and GlcNAc sugars 1.4 Cell shape determining proteases (Csds) in helical bacteria Another PG modification that has recently been discovered plays a key role in dictating the cell shape of helical bacteria, such as Helicobacter pylori and Campylobacter jejuni. The cell shape of bacteria is dictated by variations in the elastic peptide crosslinks that bridge the rigid glycan chains of PG.4 A biophysical modelling study by Wingreen et al. showed that changes in PG structure could result in diverse bacterial shapes in Gram-negative bacteria53. Most of the model studies were limited to rod-shaped bacteria (E. coli ) or coccoid bacteria (S. aureus). But the mechanistic pathways of generating cell shape in helical bacteria were not well understood until recently. In 2010, genes were discovered in H. pylori that were responsible for the helical shape of this bacterium. Several of the genes encoded for proteases and the gene products were thus 17   called cell shape-determining proteases (Csds).54-57 Shortly thereafter, a homologous set of genes was identified that control the shape of C. jejuni, and the corresponding gene products were called peptidoglycan peptidases (or Pgps).58-60 These gene products were of interest due to the importance of cell shape in the infectivity of these pathogens. 1.4.1 Helicobacter pylori and human disease H. pylori is a Gram-negative, helical shaped, epsilon proteobacterium that colonizes in the membrane of the stomach lining.61 This allows the bacteria to control the local pH of its colony and escape the acidic environment of the stomach. There are four to six flagella that provide motility via a rotatory movement that generates a torque. The helical cell shape also enhances motility through the viscous mucus layer by a corkscrew mechanism.62 Mutants that have lost their helical shape show attenuated colonization. It is estimated that more than half of the world's population is infected with H. pylori, and the infection is more prevalent in developing countries ( the rate of infection in Canada is around 35%).63 H. pylori infections may result in the development of gastric ulcers and are also the leading factor for gastric cancer. The International Agency for Research on Cancer (IARC) identified H. pylori as a Type I carcinogen due to its association with gastric cancer 64, the second leading cause of cancer-related death worldwide.65 The regimen most commonly recommended for the first-line treatment of H. pylori infection is triple therapy with amoxicillin, clarithromycin and proton pump inhibitors (e.g., lansoprazole, Fig. 1.13).66  Amoxicillin and clarithromycin are antibiotics that inhibit bacterial cell wall biosynthesis and protein synthesis, respectively. Proton pump inhibitors suppress stomach acid secretion and help the stomach wall to heal. 18    Figure 1.13: Structure of drugs used in combination for the treatment of H. pylori  infections 1.4.2 Campylobacter jejuni and human disease C. jejuni is a Gram-negative, helical-shaped bacteria that belongs to the class of epsilon proteobacteria. A leading cause of gastroenteritis worldwide,67 C. jejuni infection is generally acquired by consuming undercooked livestock through drinking contaminated water. Once consumed, the bacteria invade and attach to the epithelial cell lining (mucus layer), inducing an inflammatory response, resulting in moderate to severe diarrhea and fever. C. jejuni infection can also lead to septicemia, arthritis, conjunctivitis, and Guillian-Barrѐ syndrome68. Usually, C. jejuni infection is self-limiting, and treatment with antibiotics only decreases the duration of infection. However, in prolonged cases, patients with an impaired immune system or young children are treated with antibiotics such as macrolide (erythromycin) and quinolone  (ciprofloxacin) classes (Fig 1.14).69, 70 19    Figure 1.14: Structures of drugs used for the treatment of C. jejuni infections 1.4.3 Csd enzymes and their cleavage sites in H. pylori As mentioned previously, the helical shape of H. pylori facilitates movement through the viscous mucus layer of the stomach lining during colonization. H. pylori possess a distinct set of genes called cell shape-determining (csd) genes, which help in maintaining the helical shape of the bacteria. An initial visual screening of libraries of mutant H. pylori cells was used to detect those with shape defects and resulted in the identification of six cell shape determining genes, csd 1-5 and ccmA.4, 55, 57, 71 The Csd1 protein belongs to the M23 family of proteases and shows D, D-endopeptidase activity. It cleaves the tetra-pentapeptide crosslinks in peptidoglycan (Fig. 1.15).4, 72 Csd2 shows homology to the M23 peptidases, is not thought to be catalytically active, but interacts with and stabilizes Csd1.4, 72 Csd3 is a third member of the M23 protease family, a bifunctional enzyme. Csd3 can cleave tetra-pentapeptide crosslinks, which are similarly cleaved by Csd1.57, 72  20    Figure 1.15: Cell shape determinant peptidases in H. pylori and their cleavage sites. Csd3 exhibits D, D-carboxypeptidase activity and can remove the C-terminal D-Ala from either crosslinked or un-crosslinked peptide chains. The process of removing the C-terminal residues from PG peptide chains has been called “trimming.” CcMA does not have structural homology with any known enzyme, but it is important for hydrolytic activity and thought to acts as a membrane-bound scaffold for the other peptidases.4, 73 The Csd4 enzyme is a D, L-carboxypeptidase that trims uncrosslinked tripeptides to dipeptides by hydrolyzing the bond between iso-D-Glu and meso-Dap.54, 56, 58 The Csd5 protein has a transmembrane domain and is thought to acts as a scaffolding protein that interacts with CcMA, MurF and ATP synthase.74  Further high throughput analysis of shape defect mutants using flow-cytometry revealed two more PG hydrolases, Csd6 and Slt, as well as another scaffolding protein, Csd7.57, 75 Csd6 encodes the 21   L, D-carboxypeptidase responsible for trimming of uncrosslinked tetrapeptides to tripeptides by cleaving the bond between D-Ala and meso-Dap.57, 76 The overall process of trimming an uncrosslinked PG pentapeptide to a dipeptide is shown in Fig. 1.16A. It should be noted that once the PG peptide has been trimmed to a dipeptide, it cannot participate in crosslinking as either an acceptor or as a donor. Slt shows lytic transglycosylase activity that trims between the repeating disaccharide units.77 The deletion of most of these genes results in bacteria with curved rod shape; however, the deletion of the genes encoding for the trimming enzymes Csd4 and Csd6 results in a straight rod phenotype and attenuated colonization (Fig.1.16B).54, 56, 57, 76 It is postulated that localized activity of the Csd enzyme during cell growth generates areas of PG with less crosslinking and a looser mesh, thereby prompting curvature of the overall sacculus structure. Csd4 and Csd6 will be the focus of the work described in this thesis. 22    Figure 1.16: A) Schematic representation of the PG trimming process. B) Phase-contrast microscopy images of wild-type and mutant H. pylori (taken from Chan et al., 2015). 23   1.4.3.1 The cell shape-determining protease Csd6 Csd6 structurally resembles an L, D-transpeptidase (L, D-Tpase) of the YkuD family, enzymes that catalyze the 3→4 crosslinking of peptidoglycan. Instead of transpeptidation, Csd6 functions as an L, D-carboxypeptidase (L, D-Cpase) and of a PG tetrapeptide to give a PG tripeptide.76 In Campylobacter jejuni, the homolog of Csd6 is known as peptidoglycan peptidase (Pgp2), which also trims uncrosslinked PG tetrapeptides to tripeptides, but also has been reported to remove the terminal D-Ala residue from crosslinked tetrapeptides (not shown in Fig. 1.15).59, 78 The evolutionary relationship between Csd6 and YkuD strongly suggests that the Csd6 mechanism involves covalent catalysis employing an active site cysteine (Figure 1.17A). The cysteine first attacks the amide carbonyl located between the meso-Dap and D-Ala residues to form a tetrahedral intermediate. The collapse of the intermediate and release of D-Ala gives an acyl-enzyme intermediate. Hydrolysis of the thioester linkage releases the PG tripeptide and the free enzyme. The crystal structure of Csd6 consists of three domains: the NTD (N-terminal domain), the middle L, D carboxypeptidase domain and the C-terminal NTF2- like domain (Fig 1.17B).76  Csd6 exists as a dimer in the solution. After structural characterization, the N-terminal domain has been found to play a dominant role in dimerization. The middle L, D-CPase domain contains a deep pocket as the active site and a correctly positioned catalytic triad, as in a typical transpeptidase. The catalytic triad of Csd6 consists of His-160, Gly-161, and Cys-176.76 His-160 residue serves as a base during the formation of the tetrahedral intermediates and as an acid during the breakdown of the tetrahedral intermediates. To further analyze the importance of the active site cysteine, Kim et al. mutated Cys-176 into alanine and used a mass spectral analysis of the reaction with a muropeptide substrate to demonstrate a complete loss of activity. Therefore, it was determined that C176 plays 24   an important role in catalysis.76 Upon treatment with EDTA, there was no decrease in CPase activity, which shows that Csd6 is metal independent.76 The role of the C-terminal NTF2-domain has not been established. It has been proposed to act as a pseudaminidase, but this is largely speculative to date.79  25    Figure 1.17: A) Proposed catalytic mechanism of Csd6. B) Crystal structure of Csd6 with 3 domains (taken from Kim et al., 2015)  1.4.3.2 The cell shape-determining protease Csd4 Csd4 is a D, L-carboxypeptidase and is related to the M14 family of metalloprotease that includes carboxypeptidase A.54, 58 Csd4 shows the highest activity at around pH 6 as expected for an enzyme operating in an acidic environment. The structure of Csd4 consists of three domains: an N-terminal M14-like carboxypeptidase domain, a central β- barrel domain and a C-terminal immunoglobulin-like domain (Fig. 1.18A).54 The function of the C-terminal and central domains is unclear, but they have been postulated to be involved in recognition of the other shape-determining proteins or PG strands. Csd4 hydrolyzes the amide bond between iso-D-Glu and meso-Dap of the PG tripeptide to release free meso-Dap and the PG-dipeptide.54, 55 Chan et al. showed that the tripeptide N-Ac-L-Ala-iso-D-Glu-meso-Dap served as a Csd4 substrate and that it was possible to solve a structure of the Csd4-tripeptide complex (Fig 1.18B).54 A structure of Csd4 in complex with a muramyl tripeptide and meso-Dap has also been reported by Kim et al.56 The active 26   site Zn+2 is coordinated to Gln-46, Glu-49 and His-128. The use of Gln as a metal-binding residue is atypical of M14 family members indicating that Csd4 and its homologs constitute a distinct M14 sub-family. Replacement of this glutamine with histidine resulted in the loss of enzyme activity.54 In the first step of catalysis, the Zn ion plays an important role by delivering hydroxide to the amide carbonyl with the assistance of Glu-222 that acts as a catalytic base (Fig 1.18C). It also serves to electronically stabilize the tetrahedral intermediate with the help of Arg-86 and Glu-222. In the second step of catalysis, Glu-222 acts as an acid and protonates the departing meso-Dap. 54   27    Figure 1.18: A) The domain structure of Csd4. B) The crystal structure of Csd4 in complex with N-Ac-L-Ala-iso-D-Glu-meso-Dap ( taken from Chan et al., 2015). C) The proposed mechanism employed by Csd4. 1.5 Project goals 1.5.1 Synthesis and evaluation of minimal linear and branched tripeptide substrates for the cell shape determining proteases Csd6 and Pgp2. Recently, the cell shape determining proteins Csd6 and Pgp2 were found to be required in maintaining the helical cell shape of H. pylori and C. jejuni, respectively.  Studies have shown that loss of either gene resulted in straight-rod phenotype and reduced pathogenicity.57, 59 Therefore, these peptidases serve as attractive targets for the development of species-selective antibacterial 28   agents. As these enzymes are only found in helical bacteria, such compounds should not be detrimental to other commensal gut bacteria. As discussed in section 1.4.2.1, the Csd6 and Pgp2 enzymes hydrolyze the bond between meso-Dap and D-Ala of the uncrosslinked PG tetrapeptide to provide the PG tripeptide.76 It has also been reported that Pgp2 shows similar activity with crosslinked tetrapeptide (Fig. 1.19).59    Figure 1.19: A) Trimming of the uncrosslinked PG tetrapeptide to give PG tripeptide by Csd6/Pgp2. B) Trimming of crosslinked PG tetrapeptide to give crosslinked PG tripeptide by Pgp2. 29   Previous studies relied primarily on the use of isolated sacculi (intact bacterial cell walls) as substrates for Csd6 and Pgp2.57, 59 Treatment with a muramidase and HPLC analysis of the resulting fragments was used to uncover the substrate specificity of these enzymes. Kim et al. utilized a synthetic muramyl tetrapeptide, β-methyl MurNAc-L-Ala-iso-D-Glu-meso-Dap-D-Ala, and showed that it acts as a substrate for Csd6 from H. pylori (Fig. 1.20A).76 This provides key evidence that small molecules can act as substrates for these enzymes. The difficulty in obtaining sufficient quantities of the muramyl tetrapeptide for enzymatic studies is prohibitive. Therefore, the goal of Chapter 2 is to design and test minimal Csd6/Pgp2 substrate, Ac-iso-D-Glu-meso-oxa-Dap-D-Ala, compound 1 (Fig.1.20B). For ease of synthesis, meso-Dap has been replaced by the isostere meso-oxa-Dap (this will be discussed further in chapter 2). To confirm that Pgp2 can act on crosslinked tetrapeptides and to analyze whether Csd6 also has this activity, a minimal “branched” tetrapeptide (Ac-iso-D-Glu-(N-Ac-D-Ala)-meso-oxa-Dap-D-Ala, compound 2, will also be prepared (Fig. 1.20B). The N-Ac-D-Ala residue attached to the (R)-stereocenter of meso-oxa-Dap is designed to mimic the peptide of a neighbouring PG strand. Chapter 2 will describe the synthesis of these compounds and their testing as substrates of Csd6 and Pgp2. The enzyme is provided by our collaborator Chang Shang-Huei Lin in the lab of Prof. Michael Murphy, Department of Microbiology, University of British Columbia. 30    Figure 1.20: A) Structure of the muramyl tetrapeptide used by Kim et al. to demonstrate Csd6 activity. B) Structures of the linear tripeptide and branched tetrapeptide prepared in Chapter 2.  Red colour represents the structural similarity between molecules. 1.5.2 Synthesis of meso-oxa-Dap-containing PG pentapeptide and coupling to GlcNAc-anhMurNAc disaccharide Research into the biosynthesis, degradation/ recycling, and modification of PG, as well as the effect of PG fragments on the immune system, often requires access to synthetic PG fragments. In the case of Gram-negative bacteria, the synthesis of such fragments is difficult as the (S)-stereocenter of meso-Dap, and not the (R)-stereocenter, must be linked between the iso-D-Glu and L-Ala residues (Fig. 1.21). Therefore, it is necessary to synthesize an orthogonally protected version of meso-Dap in which the (R) and (S)-stereocenters are differentiated. In Chapter 2, we outline an efficient approach to inserting the isosteric analog meso-oxa-Dap into the tripeptide. In 31   Chapter 3, we expand this methodology to prepare the full-length meso-oxa-Dap-containing PG pentapeptide with suitable protecting groups for attachment to MurNAc carboxylates. To demonstrate the utility of this compound, we couple it to the GlcNAc-anhMurNAc disaccharide to produce compound 3. Compound 3 is a close analog of the natural substrate for the AmpG pore protein and will be used in our ongoing studies of this system (Fig. 1.21). This work was done in conjunction with my colleague Condurache M. Vacariu who prepared the disaccharide portion of compound 3.  Figure 1.21: Structure of the GlcNAc-anhMurNAc pentapeptide containing meso-oxa-Dap.   32   1.5.3 Synthesis and testing of phosphonamidate and phosphonate inhibitors of the Csd4 enzyme A practical approach to inhibit metalloproteases involves using phosphorus-based compounds that are designed to mimic the tetrahedral intermediate. The tetrahedral geometry and negative charge are the key factors that allow the inhibitor to chelate tightly to the active site metal.80, 81 Previously, in the Tanner lab, Dr. Yanjie Liu synthesized a phosphonic acid (X=CH2)-based Csd4 inhibitor, compound 4, which showed activity in the micromolar range (Fig.1.22).58 A more detailed discussion of the design and testing of this inhibitor will be presented in Chapter 4. In studies on the inhibition of related metalloproteases, it has been shown that the replacement of phosphonic P-CH2 bond in an inhibitor with a P-NH or P-O bond results in tighter binding due to additional hydrogen bonding.82 In Chapter 4 of this thesis, our progress towards the synthesis and testing of meso-oxa-Dap containing phosphonamidate (compound 5 and 5a ) and phosphonate (compound 6 and 6a)  inhibitors of Csd4 will be presented (Fig.1.22). This work was done in collaboration with Dr. Anson Chan in the laboratory of Prof. Michael Murphy, Department of Microbiology, University of British Columbia.  Figure. 1.22: Structures of the phosphorus-based inhibitors of the Csd4 enzyme. 33   Chapter 2: Synthesis and evaluation of minimal linear and branched tripeptide substrates for the cell shape-determining proteases Csd6 and Pgp2 In most Gram-negative bacteria, meso-Dap plays a vital role in PG synthesis as it forms crosslinks between neighbouring PG glycan chains resulting in a mesh-like structure.5, 45 This crosslinking event is a crucial target for many antibacterial drugs.83 Research in the biosynthesis, degradation, and modification of PG often requires access to synthetic PG peptides.84-86 However, in the studies on the PG of Gram-negative bacteria, access to these peptides is complicated due to the involvement of meso-Dap, as the (S)-stereocenter of this residue must be linked between the iso-D-Glu and L-Ala residues. For this reason, it is necessary to prepare an orthogonally protected meso-Dap in which the two ends can be distinguished.  2.1 Previous methods to synthesize orthogonally protected meso-Dap The Vederas group has developed several different strategies to synthesize orthogonally protected meso-Dap (Fig. 2.1). The first strategy involved an ene-reaction strategy that employs 2-phenyl cyclohexyl glyoxylate as a chiral auxiliary.87 The condensation of 2-phenycyclohexyl glyoxylate with N-Cbz-L-allylglycine methyl ester, compound 7,-using tin tetrachloride afforded the corresponding functionalized alcohol 8 with an isomeric ratio of 86:14. This alcohol could ultimately be converted into an isomeric mixture of meso-Dap and L, L-Dap (86:14); however, due to poor diastereoselectivity in the ene reaction, this approach was not investigated further.  In a subsequent approach, they employed an ene reaction between the allyl glycine derivative 7 and methyl glyoxylate to give the ene adduct 9 as a 50: 50 mixture of stereoisomers. The double bond was reduced, and the hydroxyl group was oxidized to give ketone 10. An attempt 34   at enantioselective hydrogenation of the keto group using a Binap-ruthenium catalyst afforded only an isomeric mixture of the hydroxy derivative 11 (79:21). The hydroxy derivative was then further transformed into meso-Dap. Although the route was short, it gave an inseparable isomeric mixture of meso-Dap and L, L-Dap (79:21).87 Due to the poor stereoselectivity in these approaches, they then prepared a vinyl sulphide derivative of L-allylglycine methyl ester, compound 13.  To prepare compound 13, a commercially available Schöllkopf bis-lactim ether was reacted with the known 2-(phenylthio)-3-chloropropene to give a dihydropyrazine derivative 12, which was hydrolyzed to compound 13. This was reacted with methyl glyoxylate in the presence of bis-(oxazoline)-copper complex to give alcohol 14 in modest yield. The diastereomeric excess was not determined due to the unstable nature of the molecule. Then, the olefinic bond and the phenylthio group were simultaneously removed with a NiCl2-sodium borohydride mixture to generate the saturated alcohol 15. This could eventually be converted into an isomeric mixture of meso-Dap and L, L-Dap (94:6).87 The same group also prepared meso-Dap by conjugate addition of a radical derived from diacyloxyiodo-benzene with a dehydroamino acid. Thermolysis of glutamate-derived diacyloxyiodobenzene 16 in the presence of benzene as the solvent and 1,4-cyclohexadiene as the hydrogen donor generates a primary radical, which then undergoes conjugate addition with dehydroalanine 17 to give an unsaturated derivative 18. This was subjected to stereoselective hydrogenation, and removal of the trityl group gave selectively protected meso-Dap derivative in a 9:1 mixture.88  35    36    Figure 2.1: Methods developed by Vederas and co-workers to synthesize meso-Dap A final report from the Vederas group outlined a photolytic method to form meso-Dap (Fig. 2.2). Glutamic acid derivative 19 is coupled to hydroperoxide 20 to give a perester, which is then deprotected to give a peracid. This peracid is then coupled to Boc-Asp-OtBu to form diacyl peroxide 21. This compound is irradiated with UV light to generate carboxyl radicals that decarboxylate and couple to generate a meso-Dap derivative (albeit in modest yield).89  Figure 2.2: Synthesis of a meso-Dap derivative via a photolytic method  37   Martin and co-workers synthesized meso-Dap by the opening of epoxy alcohol with azide (Fig. 2.3). The ω-semialdehyde derived from aspartic acid 22 was subjected to Wittig homologation with methyl (triphenylphosphoranylidene)acetate providing the corresponding unsaturated ester 23. The reduction of this ester with DIBAL-H gave the allylic alcohol derivative 24, and a Katsuki-Sharpless asymmetric epoxidation was performed to give corresponding epoxy alcohol 25. The ring-opening of this epoxy alcohol with azide gave diol 26. Oxidation of diol generated a carboxylate, and reduction of the azide produced a protected version meso-Dap.90 Mobashery and co-workers used the same strategy to prepare diol 26, which was ultimately converted in a meso-Dap-containing PG pentapeptide.91 Boons et al. employed a strategy of cross-metathesis between an allyl glycine derivative 27 and vinyl glycine derivative 28  to give alkene 29. Reduction of the double bond gave orthogonally protected meso-Dap.92  38     Figure 2.3: Methods developed by various research groups to synthesize orthogonally protected meso-Dap Fukase et al. used a Kocienski-modified Julia olefination reaction between Garner’s aldehyde and sulfone 30 (both derived from D-serine) to generate alkene 31. This could ultimately be converted into an orthogonally protected meso-Dap (Fig. 2.4).93 A chemoenzymatic approach 39   has also been reported by Fukase et al. for the synthesis of meso-Dap that uses a prohibitively expensive D-aminoacylase enzyme.94    Figure 2.4: Methods developed by Fukase and co-workers to synthesize meso-Dap The drawback of the above-mentioned methods is that many of them involve lengthy multi-step syntheses or contain low-yielding steps that are not amenable to scale up. In addition, many involve the use of expensive catalysts, such as the Grubbs catalyst or expensive enzymes, and may produce mixtures of stereoisomers that are difficult to separate. In order to avoid the difficulties in working with peptides containing meso-Dap, the Csd6/Pgp2 substrate prepared in this study incorporates the isosteric analogue meso-oxa-Dap, in which the central methylene unit is replaced 40   by an oxygen atom (Fig. 2.5). Vederas and co-workers pioneered the use of this analogue and developed synthetic methods involving aziridine ring-opening reactions with serine-derived nucleophiles to prepare orthogonally protected versions.95   Figure 2.5: Structure of orthogonally protected meso-Dap and an isosteric analogue, meso-oxa-Dap Our goal in this chapter was to prepare minimal peptide substrates for the cell-shape determining enzymes Csd6/Pgp2. It has been reported that both enzymes can cleave the terminal D-Ala from a non-crosslinked tetrapeptide and that Pgp2 can also act on crosslinked PG tetrapeptides.57, 59 As the normal substrate is a complex polymeric molecule, kinetic and inhibition studies are extremely difficult to perform. By identifying a small molecule that may serve as a substrate, such studies become possible. Given the difficulties in preparing meso-Dap-containing peptides (vide supra), we planned to use meso-oxa-Dap as an isosteric analog.  Our goal was to prepare the “minimal” tripeptide substrate, Ac-iso-D-Glu-meso-oxa-Dap-D-Ala, compound 1, which mimics an uncrosslinked PG tetrapeptide. We also planned to prepare the branched analogue, Ac-iso-D-Glu-(N-Ac-D-Ala)-meso-oxa-Dap-D-Ala, compound 2, which serves as a mimic for crosslinked PG tetrapeptide (Fig. 2.6) 41    Figure 2.6: Structure of substrates for Csd6 /Pgp2. Boxes represent the structural similarity with the natural substrate. 2.2 Synthesis of tripeptide substrate 1 2.2.1 Synthesis of tripeptide substrate 1 via orthogonally protected meso-oxa-Dap  Our initial approach towards the synthesis of Ac-iso-D-Glu-meso-oxa-Dap-D-Ala, compound 1, utilized the orthogonally protected meso-oxa-Dap, compound 37, that could be obtained with the methodology developed by the Vederas group (Fig. 2.7).95  The aziridine 35 was prepared from the N-trityl-serine 32. The carboxylate group of serine 32 was protected using allyl bromide to give serine 33. This compound was reacted with MsCl and then refluxed to give aziridine 34. The aziridine 34 was then treated with TFA to give the free amine, which was protected subsequently with pNZ-Cl (p-nitrobenzyl chloroformate) to give protected aziridine 35.  Compound 37 was then prepared via a ring-opening reaction of protected aziridine 35 with the hydroxyl nucleophile of Fmoc-Ser-Ot-Bu, compound 36. The Lewis acid BF3 coordinates with the carbamate carbonyl and activates the aziridine (Fig. 2.8). The hydroxyl group of serine 36 is then able to attack the β-position carbon of the aziridine to give orthogonally protected meso-oxa-Dap 42   37. The allyl group of compound 37 was then removed using Pd(PPh3)4 in conjunction with barbituric acid as a scavenger, and the free acid obtained was then coupled to D-Ala-Ot-Bu to give dipeptide 38. Removal of the pNZ protecting group using SnCl2/HCl gave the free amine, which was coupled to N-Ac-D-Glu-α-Ot-Bu to give the fully protected tripeptide, 39, with the D-glutamic acid residue linked via its -carboxylate. In the pNZ removal step, the nitro group is reduced by SnCl2/HCl to give the free p-amino-benzyloxy carbonyl derivative, which spontaneously eliminates out the carbamic acid and generates a quinonimine methide (Fig. 2.9). The carbamic acid spontaneously decarboxylates to give the free amine.96   43    Figure 2.7: The synthesis of the tripeptide substrate 1 using orthogonally-protected meso-oxa-Dap.  Figure 2.8: Mechanism of aziridine ring-opening by nucleophilic hydroxyl group of serine. The global deprotection of the Fmoc group and t-butyl ester group using piperidine and TFA, respectively, gave substrate 1, which was purified by ion-exchange chromatography. While 44   this route yielded the desired target tripeptide, the synthesis was lengthy (eleven linear steps), and several of the yields were low (aziridine opening and peptide couplings).  Figure 2.9: Mechanism of pNZ group removal. Attempts were made to increase the yield of peptide coupling reactions using different coupling reagents, but no improvement was observed. Therefore, we decided to develop an improved synthesis that could provide larger amounts of tripeptide in fewer steps. 2.2.2 Synthesis of tripeptide substrate 1 via an embedded aziridine ring-opening In this approach, the aziridine is first embedded into a tripeptide and then used a serine hydroxyl-based nucleophile is added to generate meso-oxa-Dap in a late stage of synthesis. This avoids several steps of protection and deprotection that were present in the previous route as it would not be necessary to prepare meso-oxa-Dap with four orthogonal protecting groups. The strategy of nucleophilic ring-opening of aziridine-containing peptides has been employed by several research groups using strong nucleophiles such as thiols, amines, and azides.97-99 However, the use of alcohol as a nucleophile for the ring-opening of a peptide embedded aziridine has not 45   been reported. A report of the opening of an aziridine carboxamide by alcohol, albeit in modest yields, however, suggested that this might be possible.100 Aziridine 42 was prepared using a 4-step literature known method (Fig 2.10).97  A trityl protected serine 32 was coupled with tosylate salt of D-Ala-OBn to give dipeptide 40. This dipeptide was then treated with MsCl, subsequently refluxed in the presence of a base to give aziridine 41. Treatment of aziridine 41 under acidic condition gave free amine 42. This free amine was then coupled to N-Ac-D-Glu-OBn to give the tripeptide precursor 43 with the D-glutamic acid residue linked through its γ-carboxylate moiety.  Figure 2.10: Synthesis of peptidyl aziridine 43 In the Vederas method to prepare meso-oxa-Dap, aziridine ring-opening yields were optimized using BF3.OEt2 as a  Lewis acid catalyst.95 Therefore, we decided to use it as a catalyst for all future aziridine ring-opening reactions. To enhance the nucleophilicity of the hydroxyl group on the serine, an amino group needs to be bis-protected (Fig. 2.11).  46    Figure 2.11: H-bonding patterns and effects on nucleophilicity of hydroxyl of protected serine In a mono-protected serine, H-bonding between the NH group and the alcohol could decrease the nucleophilicity of the hydroxyl group. Bisprotection of the amino group could lead to a reversal of hydrogen bonding that will increase the electron density on the oxygen of the serine hydroxyl, thereby improving its nucleophilicity. Therefore, the phthalimide group was chosen as a protecting group for the amino group of serine.95  The initial aziridine-opening reaction was done at a higher temperature with the reaction conditions developed by the Vederas group for the synthesis of  N-phthalimido protected meso-oxa-Dap 45 from aziridine 44 (Fig 2.12).95  Figure 2.12: Synthesis of meso-oxa-Dap derivative 45 done by the Vederas group Treatment of aziridine 43 with 2 equivalents of N-Pht-D-Ser-OBn in the presence of 0.5 equivalents of BF3.OEt2 in refluxing toluene did not give the protected tripeptide 46 (Table 1, entry 47   1). Therefore, the ring-opening reactions were carried at different temperatures, and the amount of catalyst was also varied (Table 1, entry 2-6).  Changing the solvent to CH2Cl2 and increasing the amount of  BF3.OEt2 to 1.5 equivalents yielded product in a modest 10 % yield. The best yield was obtained when 2.5 equivalents of BF3.OEt2 was used at a temperature of -78 °C in CH2Cl2 (Table 1, entry 3), further increasing the amount of BF3.OEt2 did not improve the yield. Attempts to increase the yield by changing the serine protecting groups were also unsuccessful (Table 1, entry 7-8). Similarly, the use of THF (-78 °C) or CH3CN (-30 °C) as solvent gave no product (Table 1,9-10). Finally, an attempt under basic conditions (NaH/ THF) was unsuccessful in promoting the reaction.              48   Table 2. 1: Optimization of aziridine ring-opening conditions    49   Treatment of aziridine 43 with 2 equivalents of N‐phthalimido‐Ser‐OBn in the presence of 2.5 equivalents of BF3.OEt2 was therefore carried out in CH2Cl2 at a temperature of −78 °C with gradual warming to 0 °C (Fig. 2.13). This gave the protected tripeptide 46 in a 30 % yield. No isomeric product resulting from attack at the more substituted aziridine α-carbon was observed.95 However, a significant amount (38 %) of rearranged starting material, tentatively assigned to be oxazoline 47, and resulting from the competing Heine rearrangement, was observed.101-103   Figure 2.13: Synthesis of the tripeptide 46 via embedded tripeptide aziridine ring-opening. It is well known that N-acyl aziridines undergo ring-expansion to form oxazolines in the presence of Lewis acids. The Lewis acid, BF3, coordinates with the N-atom of aziridine 43 that makes α-carbon more electrophilic, which is the driving force for the rearrangement (Fig. 2.14). The formation of oxazoline 47 is highly stereoselective.101-103  Figure 2.14: Plausible mechanism of the Heine rearrangement of tripeptide aziridine 43 50   It was difficult to monitor the ring-opening of aziridine by mass spectroscopy because both aziridine 43 and oxazoline 47 had the same molecular weight. Also, both the compounds had the same rf value on thin-layer chromatography (TLC). Therefore, it was difficult to calculate the yield of oxazoline 47 during the optimization reaction of aziridine ring-opening. Both protected tripeptide 46 and oxazoline 47 were readily separable by silica gel chromatography. Then protected tripeptide 46 was deprotected through hydrogenolysis followed by treatment with hydrazine hydrate, and the product was purified by ion-exchange chromatography (Fig. 2.15).  Figure 2.15: Deprotection of compound 46 to give tripeptide substrate 1 The NMR spectra of the resulting tripeptide 1 (Ac‐iso‐D‐Glu‐meso‐oxa‐Dap‐D‐Ala) were identical to those of the same compound prepared in the first approach. Despite the 30 % yield of the aziridine‐opening reaction, the second approach required four fewer steps and gave a substantially higher overall yield of 3.9 % than previous route (overall yield: 0.63%) 2.3 Synthesis of branched tetrapeptide 2 To synthesize the branched tetrapeptide 2, the protected tripeptide 46 was used as a precursor (Fig. 2.16).  The phthalimide group of tripeptide 46 was removed using hydrazine hydrate to give a free amine, which was immediately coupled to N-Ac-D-Ala to give the protected 51   tetrapeptide 48. Then the benzyl groups were deprotected through hydrogenolysis to give the target branched tetrapeptide 2, which was purified by ion-exchange chromatography.   Figure. 2.16: Synthesis of branched tetrapeptide 2 2.4 Activity test using a mass-spectrometry assay In studies with intact PG sacculi, the groups of Salama and Gaynor provided compelling evidence that Csd6/Pgp2 cleaves the terminal D-alanine from uncrosslinked PG tetrapeptide to generate PG tripeptides.57, 59 In addition, Pgp2, but not Csd6, showed a small amount of activity with crosslinked PG tetrapeptides to give PG tripeptides.57, 59 To show that the “minimal” meso-oxa-Dap containing tripeptide 1 and tetrapeptide 2 can act as a substrate for both H. pylori Csd6 and with C. jejuni Pgp2, a mass spectral assay was employed (Fig. 2.17). 52    Figure 2.17: Masses of monosodium ion adducts for substrates 1 and 2 and their enzymatic reaction products The tripeptide substrate 1 (5 mM ) was incubated for 2h at 37 °C with each enzyme (5 µM), which was dissolved in 20 mM Tris-HCl buffer, pH 7.0, and 150 mM sodium chloride. The reactions were analyzed by MALDI-TOF MS (Fig. 2.18). Mass spectra were obtained in a positive ion mode. In each enzymatic reaction, a substantial peak at m/z 386 was observed; this peak corresponds to the mass of the monosodium adduct of the product dipeptide Ac-iso-D-Glu-meso-oxa-Dap 49. The mass spectral results clearly show that the tripeptide 1 can serve as a small-molecule substrate for both Csd6/Pgp2 and that meso-oxa-Dap is accepted as an analogue of meso-Dap.    53    Figure 2.18: MALDI-TOF MS analysis of the reactions catalyzed by Csd6 and Pgp2 with tripeptide 1  54    Figure 2.19: MALDI-TOF MS analysis of the reactions catalyzed by Csd6 and Pgp2 with branched tetrapeptide substrate 2 55   A similar experiment was run with the branched tetrapeptide 2 that serves as a model of the crosslinked PG tetrapeptide. The tetrapeptide 2 was incubated with both enzymes, and the reactions were analyzed by MALDI-TOF MS. In the case of the enzymatic reaction with Csd6, no significant peak of product 50 (m/z 499 for the monosodium adduct ) was observed even after 24 h of incubation (Fig. 2.19 left). In the case of Pgp2, however, a significant product peak was observed after 24 h of incubation (Fig. 2.19 right). These results mirror those reported in studies using intact PG sacculi by the Salama group and Gaynor groups.57, 59 The reactions with both of the enzymes were faster with the linear peptide substrate, which suggests that the primary role of these enzymes in nature is to catalyze the trimming of uncrosslinked PG tetrapeptides. In the case of Pgp2, the branched peptide also showed activity, indicating that the enzyme active site can accommodate significant steric bulk attached to the “side-chain” amino group of meso-Dap. With Csd6, no such activity was observed with branched peptide, indicating that this enzyme is less tolerant of substitution at this position. Whether the ability of Pgp2 to trim crosslinked tetrapeptide is physiologically relevant remains to be determined. 2.5 Conclusions and summary In this chapter, we have reported two approaches to synthesize meso-oxa-Dap-containing tripeptide substrates for Csd6/Pgp2. The first approach involves the initial synthesis of orthogonally protected meso-oxa-Dap and then attachment of other amino acids to it. The synthesis of substrate 1 required eleven linear steps, and many were low yielding or not amenable to scale-up.  The second approach was to embed an aziridine into a tripeptide and then ring-open the aziridine at a late stage to give a meso-oxa-Dap-containing tripeptide. This required only seven 56   linear steps. This approach could be used to prepare sufficient quantities of substrates that would allow for the testing of inhibitors against Csd6 and Pgp2, despite the low yield for the tripeptidyl aziridine ring-opening. We have also shown that both Csd6 and Pgp2 can accept the "minimal" meso-oxa-Dap-containing tripeptide as a substrate in the enzymatic reaction.  However, in the case of a branched tetrapeptide, only Pgp2 showed a lower but detectable level of activity. These results mirror those reported with intact PG and suggests that the shortened form of this natural substrate and the replacement of meso-Dap with meso-oxa-Dap, does not dramatically perturb the positioning of these compounds within the active site of the enzymes.           57   2.6 Experimental procedures 2.6.1 General information  All reactions were performed in flame-dried glassware. Dry solvents (CH2Cl2 and MeOH) and Et3N were obtained by distillation under Ar from CaH2. All other solvents and reagents were used without further purification. NMR were recorded on a Bruker AV400 spectrometer or a Bruker AV300 spectrometer at a field strength of 400 MHz or 300 MHz for 1H NMR and 101 MHz or 75 MHz for 13C NMR. Column chromatography was done on SiliaFlash Silica Gel F60, 40 - 63 µm purchased from Silicycle. Thin-layer chromatography was done on silica gel 60 F254 purchased from Merck and visualized under short wave UV light or KMnO4 stain. High-resolution spectra were recorded on a Waters/Micromass LCT TOF spectrometer equipped with electrospray (ESI) ionization. 2.6.2 Synthesis of tripeptide substrate 1 2.6.2.1 Dipeptide 38   To a solution of Pd(PPh3)4 (62 mg, 0.05 mmol) and 1,3-dimethylbarbituric acid (84 mg, 0.05 mmol) in N,N-dimethylformamide (5 mL) , a solution of ester 37 (370 mg, 0.54 mmol) in N,N-dimethylformamide (5 mL) was added dropwise. The reaction mixture was stirred for 18 h in the dark at rt. The solvent was removed by evaporation under reduced pressure and the residue was dissolved in ethyl acetate (20 mL) and washed three times with 20 mL of 0.1 N HCl. The 58   organic layer was then washed with water (20 mL). The organic layer was dried over Na2SO4 and concentrated under reduced pressure to give crude acid (200 mg) which was carried on further without purification. A solution of the crude acid (200 mg, 0.308 mmol) in N,N-dimethylformamide (10 mL) was treated with HOBt (51 mg, 0.40 mmol) and HBTU (130 mg, 0.401 mmol), and the mixture was stirred for 30 min at rt. D-Alanine t-butyl ester (56 mg, 0.31 mmol) and DIPEA (80 mg, 0.62 mmol) were added to the reaction mixture, which was stirred for another 2 h at rt. Distilled water (20 mL) was then added to the reaction mixture and the resulting mixture was extracted with ethyl acetate (3x20 mL). The organic layer was collected and washed with 50 mL brine solution. The organic layer was separated, dried over Na2SO4, filtered and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (2% MeOH in DCM) to give dipeptide 38 as yellow solid (150 mg, 35% yield). 1H NMR (400 MHz, CDCl3 ) δ 8.16 (d, J = 8.3 Hz, 2H), 7.75 (d, J = 7.5 Hz, 2H), 7.60 (d, J = 7.5 Hz, 2H), 7.47 (d, J = 8.3 Hz, 2H), 7.38 (t, J = 7.4 Hz, 2H), 7.32 – 7.24 (m, 2H), 6.98 (d, J = 6.9 Hz, 1H), 5.98 (d, J = 8.1 Hz, 1H), 5.90 (d, J = 6.8 Hz, 1H), 5.28 – 5.04 (m, 2H), 4.52 – 4.34 (m, 3H), 4.34 – 4.26 (m, 2H), 4.21 (t, J = 7.1 Hz, 1H), 3.98 – 3.83 (m, 2H), 3.84 – 3.72 (m, 1H), 3.61 (t, J = 7.9 Hz, 1H), 1.48 (s, 9H), 1.45 (s, 9H), 1.38 (d, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 172.0, 168.9, 168.6, 156.1, 155.7, 147.6, 143.9, 143.7, 143.5, 141.3, 128.0, 127.7, 127.1, 125.1, 123.8, 120.0, 82.8, 82.4, 71.9, 70.6, 67.2, 65.6, 54.8, 54.3, 49.0, 47.1, 28.0, 18.4. HRMS (ESI): m/z calcd for C40H48N4O12Na [M+Na]+ 799.3166, found 799.3156  59   2.6.2.2 Triester 39  To a solution of dipeptide 38 (150 mg, 0.193 mmol) in DMF (1 mL), a solution of 6M SnCl2 (5 mL) and 160 mM HCl in dioxane (50 μL)  was added dropwise. The reaction was stirred for 1 h at rt. The volatiles removed under reduced pressure and 20 mL ethyl acetate was added to the reduced volume. A saturated solution of NaHCO3 was added to the mixture until the pH was approx. 8. The resulting precipitate was removed by filtration. The aqueous layer was extracted with 2x10 mL ethyl acetate. The organic layers were combined and washed with 20 mL of water and then with brine solution. The organic layer was separated, dried over Na2SO4, filtered and concentrated under reduced pressure to give crude amine product (75 mg). In a separate flask, a  solution containing N-Ac-D-Glu-α-OtBu (31 mg, 0.13 mmol) in N, N-dimethylformamide (10 mL) was treated with HOBt (21 mg, 0.14 mmol) and HBTU (53 mg, 0.14 mmol), and stirred for 30 min at rt, treated with a solution of crude amine (75 mg, 0.13 mmol) and DIPEA (33 mg, 0.25 mmol) in DMF (5 mL), and stirred for another 3 h at rt. Distilled water (30 mL) was then added to the reaction mixture which was extracted with ethyl acetate (3x30 mL). The organic layer was collected and washed with 25 mL of brine solution. The organic layer was separated, dried over Na2SO4, filtered and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (2% MeOH in DCM) to give triester 39 as white solid (45 mg, 30% yield). 1H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 7.3 Hz, 2H), 7.62 (t, J = 7.0 Hz, 2H), 7.39 60   (t, J = 6.7 Hz, 2H), 7.34 – 7.27 (m, 3H), 7.22 (d, J = 7.7 Hz, 1H), 6.48 (d, J = 8.0 Hz, 1H), 6.45 (d, J = 7.6 Hz, 1H), 4.61 – 4.49 (m, 2H), 4.47 – 4.41 (m, 2H), 4.40 – 4.27 (m, 2H), 4.23 (t, J = 7.1 Hz, 1H), 3.96 (dd, J = 9.1, 3.8 Hz, 1H), 3.92 (dd, J = 9.6, 3.8 Hz, 1H), 3.71 (dd, J = 9.6, 2.8 Hz, 1H), 3.56 (dd, J = 9.1, 5.6 Hz, 1H), 2.40 – 2.22 (m, 3H), 1.94 (s, 3H), 1.87 – 1.78 (m, 1H), 1.46 (s, 9H), 1.45 (s, 9H), 1.43 (s, 9H), 1.36 (d, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 172.4, 172.0, 171.4, 170.8, 169.2, 169.1, 156.3, 144.0, 143.8, 141.3, 127.7, 127.1, 125.2, 120.0, 82.7, 82.5, 81.8, 71.8, 70.5, 67.0, 55.1, 52.9, 52.2, 49.0, 47.2, 32.4, 29.2, 28.0, 27.9, 23.1, 18.3. HRMS (ESI): m/z calcd for C43H60N4O12Na [M+Na]+ 847.4105, found 847.4102 2.6.2.3 Tripeptide substrate 1  To a solution of triester 39 (45 mg, 0.05 mmol) in DCM (2.5 mL) at 0 °C, TFA (2.5 mL) was added dropwise. The mixture was stirred for 2 h at rt. The volatiles were co-evaporated with diethyl ether (3x 10 mL). The residue obtained was dissolved in 20 % piperidine in DMF (5 mL) and stirred for 1 h at rt. The volatiles were removed under vacuum and the residue was dissolved in H2O (2 mL). The pH of the solution was adjusted to pH 8 by adding NaHCO3 (0.5 M). The pH8 solution was loaded onto a column of AG 1-X8 resin (formate form, 100−200 mesh, 5 mL). The column was washed with water (50 mL) and formic acid (0.1 M, 50 mL; 0.5 M, 50 mL). The triacid was eluted with formic acid (4 M, 100 mL). The collected fractions were combined and evaporated to dryness in vacuo to give tripeptide substrate 1 as a white solid ( 17 mg, 70 %). 1H 61   NMR (400 MHz, D2O) δ 4.46 (t, J = 4.8 Hz, 1H), 4.34 – 4.21 (m, 2H), 3.93 (dd, J = 6.1, 3.4 Hz, 1H), 3.90 – 3.70 (m, 4H), 2.38 (t, J = 7.3 Hz, 2H), 2.21 – 2.08 (m, 1H), 1.97 (s, 3H), 1.96 – 1.87 (m, 1H), 1.33 (d, J = 7.4 Hz, 3H). 13C NMR (101 MHz, D2O) δ 176.7, 175.8, 175.4, 174.0,171.4 171.3, 70.0, 68.8, 54.3, 53.8, 52.5, 49.2, 31.5, 26.8, 21.7, 16.3. HRMS (ESI): m/z calcd for C16H27N4O10 [M+H]+ 435.1649, found 435.1642. 2.6.2.4 N-acyl aziridine 43   N,N-Diisopropylethylamine (2.17 mL, 12.4 mmol) was added dropwise to a solution of  N-Ac-D-Glu-α-OBn (2.61g, 9.33 mmol, 1.5 equiv),  aziridine 42 (1.54 g, 6.22 mmol, 1.0 equiv), and PyBOP (4.85 g, 9.33 mmol, 1.5 equiv) in dichloromethane (100 mL) at rt. The resulting solution was stirred overnight, washed with 10% aqueous citric acid (2x100 mL), saturated aqueous sodium bicarbonate (2x100 mL), saturated aqueous sodium chloride solution (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chomatography (50% ethyl acetate in petroleum ether) to give N-acyl aziridine 43 as a white solid (2.4 g, 71%).1H NMR (400 MHz, CDCl3) δ 7.44 – 7.28 (m, 11H), 7.09 (d, J = 7.6 Hz, 1H), 5.25 – 5.08 (m, 4H), 4.66 (dd, J = 9.0, 4.5 Hz, 1H), 4.63 – 4.57 (m, 1H), 3.11 (dd, J = 6.1, 3.1 Hz, 1H), 2.52 – 2.44 (m, 3H), 2.37 (dd, J = 3.1, 1.3 Hz, 1H), 2.32 – 2.24 (m, 1H), 2.04 – 1.92 (m, 4H), 1.47 (d, J = 7.3 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 183.3, 172.5, 171.8, 62   170.7, 167.2, 167.2, 135.2, 135.2, 128.7, 128.7, 128.6, 128.5, 128.3, 128.1, 67.3, 51.7, 48.1, 35.9, 32.8, 30.8, 27.3, 23.0, 17.7. HRMS (ESI): m/z calcd for C27H31N3O7Na [M+Na]+ 532.2060, found 532.2058 2.6.2.5 Oxa-Dap 46  A flame dried roundbottomed flask containing oven dried 4 °A molecular sieves was cooled under an Ar atmosphere , charged with N-acyl aziridine 43 (0.500 g, 0.98 mmol) followed by  a solution of  N-Pht-D-Ser(OH)-OBn ( 0.68 g, 1.96 mmol) in DCM (10 mL), cooled to -78 °C and treated dropwise with BF3.OEt2 ( 0.31 mL, 2.45 mmol) over 15 min. After stirring for 1 h , the reaction mixture was filtered and the filtrate evaporated under reduced pressure. The residue was purified by silica gel column chomatography (100 % ethyl acetate ) to give oxa-Dap 46 as white solid (0.248 g, 30 % ). 1H NMR (400 MHz, MeOD) δ 7.91 – 7.84 (m, 2H), 7.83 – 7.78 (m, 2H), 7.39 – 7.26 (m, 15H), 5.22 (dd, J = 9.6, 5.3 Hz, 1H), 5.19 (s, 2H), 5.16 (s, 2H), 5.06 (dd, J = 12.5, 5.8 Hz, 2H), 4.51 (t, J = 4.9 Hz, 1H), 4.44 (dd, J = 9.2, 4.9 Hz, 1H), 4.23 – 4.16 (m, 1H), 4.16 – 4.07 (m, 2H), 3.74 – 3.62 (m, 2H), 2.30 – 2.25 (m, 2H), 2.23 – 2.11 (m, 1H), 1.97 (s, 3H), 1.94 – 1.81 (m, 1H), 1.27 (d, J = 7.3 Hz, 3H). 13C NMR (101 MHz, MeOD) δ 171.8, 170.8, 170.5, 170.2, 168.7, 166.1, 165.9, 134.4, 134.4, 133.9, 133.0, 130.2, 126.8, 126.8, 126.6, 126.6, 126.5, 126.5, 126.5, 126.4, 126.3, 121.7, 68.8, 65.9, 65.8, 65.2, 65.0, 51.5, 50.5, 49.9, 29.9, 25.5, 19.6, 14.4. HRMS (ESI): m/z calcd for C45H46N4O12Na [M+Na]+ 857.3010, found 857.3002 63   2.6.2.6 Oxazoline 47  Oxazoline 47 eluted after oxa-Dap 46 using 3% MeOH in ethyl acetate. Evaporation of collected fraction gave an oily liquid (0.200 g, 38 % ). 1H NMR (300 MHz, MeOD) δ 7.40 – 7.29 (m, 10H), 5.23 – 5.07 (m, 4H), 4.73 – 4.53 (m, 2H), 4.52 – 4.40 (m, 2H), 4.29 (dd, J = 8.6, 7.7 Hz, 1H), 2.41 (t, J = 7.8 Hz, 2H), 2.31 – 2.16 (m, 1H), 2.08 – 1.99 (m, 1H), 1.98 (s, 3H), 1.43 (d, J = 7.3 Hz, 3H). 13C NMR (75 MHz, MeOD) δ 172.3, 172.0, 171.6, 170.7, 135.8, 135.8, 128.2, 128.2, 127.9, 127.9, 127.9, 127.8, 127.8, 70.2, 68.0, 66.6, 66.6, 51.7, 48.2, 26.9, 23.7, 20.9, 15.9. HRMS (ESI): m/z calcd for C27H31N3O7Na [M+Na]+ 532.2060, found 532.2055 2.6.2.7 Tripeptide substrate 1  Oxa-Dap 46 (150 mg, 0.18 mmol) was dissolved in ethanol (5 mL), treated with Pd/C (15 mg), placed under an atmosphere of H2 gas and stirred for 12 h.  The reaction mixture was filtered over celiteTM and the filtrate was evaporated under reduced pressure to give a residue which was used directly in the next step. This residue was dissolved in ethanol (5 mL) and the treated dropwise with hydrazine hydrate ( 0.06 mL, 1.80 mmol), and stirred for 3 h. The volatiles was evaporated 64   under reduced pressure. The residue was dissolved in water (2 mL) and loaded onto a column of AG 1-X8 resin (formate form, 100−200 mesh, 10 mL). The column was washed with water (50 mL) and formic acid (0.1 M, 50 mL; 0.5 M, 50 mL). Elution with formic acid (4.0 M, 100 mL). and evaporation of the collected fractions in vacuo gave tripeptide substrate 1 as a white solid ( 40 mg, 51 %), which exhibited identical spectral properties as the material described above. 2.6.3 Synthesis of branched tetrapeptide 2 2.6.3.1 Protected tetrapeptide 48  Protected oxa-Dap 46 (90 mg, 0.11 mmol) was dissolved in ethanol (2 mL) cooled to 0°C, treated with hydrazine hydrate (50 % hydrazine, 7.5 µL, 0.12 mmol), stirred for 45 min, treated with acetic acid (1 mL), heated to 50 °C, stirred for 10 min and concentrated in vacuo. The residue was digested with ethyl acetate and filtered. The filtrate was concentrated in vacuo to give crude amine product which was used directly in the next step. In a separate flask, N-Ac-D-Ala-OH (21.2 mg, 0.16 mmol) was dissolved in DCM/DMF (9:1, 10 mL), cooled to 0°C, treated with EDC (31 mg, 0.16 mmol) followed by HOBt (24.8 mg, 0.16 mmol), stirred for 30 min at 0 °C, and treated with solution of crude amine (40 mg) and DIPEA (56 µL, 0.32 mmol) in DCM (3 mL). The ice bath was removed, and the reaction mixture warmed to room temperature with stirring over 4 h. The reaction mixture was diluted with DCM (20 mL), washed 3 times with 20 mL H2O, dried over 65   Na2SO4, filtered and evaporated under reduced pressure. The residue was purified by silica gel chromatography (3.5% MeOH in DCM) to give protected tetrapeptide 48 as white solid (40 mg, 45%)1H NMR (400 MHz, CDCl3) δ 7.88 – 7.74 (m, 2H), 7.43 – 7.27 (m, 15H), 6.89 (d, J = 8.4 Hz, 1H), 6.42 (d, J = 8.0 Hz, 1H), 6.28 (d, J = 7.6 Hz, 1H), 5.30 – 5.09 (m, 6H), 4.71 – 4.63 (m, 2H), 4.62 – 4.54 (m, 2H), 4.21 – 4.09 (m, 1H), 3.97 (dd, J = 9.2, 2.6 Hz, 1H), 3.91 (dd, J = 9.7, 2.7 Hz, 1H), 3.73 (dd, J = 9.8, 3.1 Hz, 1H), 3.41 (dd, J = 9.1, 3.9 Hz, 1H), 2.48 – 2.34 (m, 2H), 2.33 – 2.17 (m, 2H), 2.01 (s, 3H), 1.95 (s, 3H), 1.39 (d, J = 7.7 Hz, 3H), 1.36 (d, J = 7.3 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 173.2, 173.1, 172.1, 172.1, 171.0, 170.3, 170.2, 169.9, 135.6, 135.5, 128.8, 128.8, 128.7, 128.7, 128.6, 128.5, 128.4, 128.3, 128.1, 128.1, 77.3, 70.8, 67.7, 67.3, 53.6, 53.0, 51.4, 48.8, 48.6, 32.1, 29.2, 23.4, 23.2, 19.0, 17.2, 16.9. HRMS (ESI): m/z calcd for C42H52N5O12 [M+H]+ 818.3612, found 818.3619 2.6.3.2 Branched tetrapeptide 2  Protected tetrapeptide 48 (13 mg, 15.9 mmol) was dissolved in EtOH (3 mL) and Pd/C ( 5 mg) was added to it. The reaction mixture was stirred under an atmosphere of H2 gas for 3 h. The reaction was filtered over celite and the solvent was evaporated under reduced pressure to give the residue which was dissolved in H2O (2 mL). The pH of the solution was adjusted to 8 by adding NaHCO3 (0.5 M). This was loaded onto a column of AG 1-X8 resin (formate form, 100−200 mesh, 5 mL). The column was washed with distilled water (50 mL) and formic acid (0.1 M, 50 mL; 0.5 66   M, 50 mL) and then was eluted by formic acid (4.0 M, 100 mL). The fractions containing the compound were combined and evaporated to dryness in vacuo to give branched tetrapeptide 2 as a white solid ( 3.6 mg, 42 %). 1H NMR (400 MHz, D2O) δ 4.60 – 4.55 (m, 1H), 4.51 (t, J = 4.8 Hz, 1H), 4.40 – 4.31 (m, 3H), 3.94 (dd, J = 10.3, 4.7 Hz, 1H), 3.86 – 3.80 (m, 2H), 3.80 – 3.74 (m, 1H), 2.46 (t, J = 7.5 Hz, 2H), 2.26 – 2.15 (m, 1H), 2.04 (s, 3H), 2.03 (s, 3H), 2.02 – 1.94 (m, 1H), 1.42 (d, J = 7.2 Hz, 3H), 1.38 (d, J = 7.4 Hz, 3H).  13C NMR (101 MHz, D2O) δ 176.7, 175.8, 175.5, 175.3, 174.4, 174.2, 171.6, 171.4, 70.6, 70.1, 53.9, 52.7, 49.8, 31.7, 26.8, 21.9, 21.8, 17.0, 16.8, 16.5, 16.4. HRMS (ESI): m/z calcd for C21H34N5O12 [M+H]+ 548.2126, found 548.2121 2.6.4 Cloning, expression and purification of Pgp2 and Csd6  (Chang S.-H. Lin)  Recombinant Pgp2 and Csd6 were produced in Escherichia coli. The coding region of the pgp2 gene corresponding to amino acids 43-325 was cloned from C. jejuni strain 81-176 into the plasmid pET-15b using the NdeI and XhoI restriction sites.  Pgp2 protein is produced with an N-terminal His6-tag and a thrombin recognition site.  A Csd6 expression plasmid was a gift from Dr. Nina R. Salama.  The csd6 gene coding for amino acid 18-330 (without the predicted signal peptide) was cloned from H. pylori strain G27 into pET-15b.  Pgp2 and Csd6 enzymes were produced and purified as previously described with modification.  Briefly, E. coli BL21(DE3) cells carrying the expression plasmid were cultured in 1 L of LB media supplemented with 100 g mL-1 ampicillin at 37 °C with shaking to an optical density of 0.6-0.8 before adding 0.25 mM isopropyl-β-D-thiogalactopyranoside to induce expression for an additional 16 hours at 25°C.  Cultures were pelleted by centrifugation and stored at -80 °C. For Pgp2 purification, cells were 67   thawed in binding buffer (50 mM Tris-HCl, pH 7.0, 150 mM NaCl, and 1 mM DTT), supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF), treated with DNase, and then lysed at 4 °C using a homogenizer.  The lysate was centrifuged and loaded onto a HisTrap HP 5 ml column (GE Healthcare) and washed with 15-column volumes of 20 mM imidazole in binding buffer. Pgp2 was then eluted with increasing imidazole in binding buffer. The protein were digested with thrombin (250:1 w/w Pgp2: thrombin ratio) overnight at 4 °C to remove the His6-tag.  Thrombin was removed with p-aminobenzamidine agarose beads (5 mg thrombin: 1ml beads).  Pgp2 protein was reloaded onto HisTrap HP 5 ml column to remove uncleaved protein.  The protein without the His6-tag was further purified by gel-filtration chromatography on a Superdex 200 16/60 column (GE Healthcare) in binding buffer.  Protein fractions were pooled and concentrated to 150-200 M and flash-frozen in liquid nitrogen.  Csd6 purification was performed as described for Pgp2 except the binding buffer was adjusted to pH 7.5. Protein purity for both enzymes was assessed by SDS-PAGE. 2.6.5 Enzyme Activity test Tripeptide 1 (5 mM) was incubated for 2 h at 37 °C with either Csd6 (5 µM) or Pgp2 (5 µM) in 20 mM Tris-HCl buffer, pH 7.0 containing 150 mM sodium chloride.  The samples were quenched by heating to 80 °C for 10 min.  Before analysis, a sample solution (1 µl ) was mixed on the MALDI target with a matrix solution of 2,5-dihydroxy benzoic acid  (DHB) dissolved in 0.1% (v/v) trifluoroacetic acid and 50% (v/v) acetonitrile/ H2O.  For sample deposition, a 384-position stainless steel sample plate was used.  Mass spectra were acquired on a matrix-assisted laser desorption/ionization-time of flight mass spectrometer (MALDI-TOF MS, Bruker Autoflex 68   MALDI-TOF ).  Mass spectra were obtained in a positive ion mode. Acquisition and data processing were controlled by the Flex analysis software.  Experiments with branched tetrapeptide 2 were run in an identical fashion, but with a 24 h incubation time.  As a control, substrates were incubated in the absence of enzymes.             69   Chapter 3: Synthesis of meso-oxa-Dap-containing PG pentapeptide and coupling to GlcNAc-anhMurNAc disaccharide The major component of the bacterial cell wall is the peptidoglycan (PG). PG consists of a glycan chain with an appended peptide that is crosslinked to the peptide of a neighbouring glycan chain to form a mesh-like structure.5, 45 In gram-negative bacteria, the pentapeptide chain is comprised of L-Ala1-γ-D-Glu2-meso-Dap3-D-Ala4-D-Ala5 (where D-Glu is attached to meso-Dap via its γ-carboxyl group). It is often difficult to isolate PG degradation products from the bacterial cell because after digestion, the mixture is usually heterogeneous, and the purification is extremely challenging. Therefore, synthetic PG fragments are often used in research on the biosynthesis, degradation, recycling, and modification of PG, as well as on the effects of PG fragments on the immune system.84-86  In the case of Gram-negative bacteria, the synthesis of PG pentapeptide becomes difficult as the iso-D-Glu and L-Ala residues need to be attached to the (S)-stereocenter of the meso-Dap. Therefore, it is necessary to synthesize a PG pentapeptide from an orthogonally protected version of meso-Dap in which the (R) and (S)-stereocenters are differentiated. Rapid synthesis of this pentapeptide, or an isosteric analog, could greatly aid studies on PG utilizing enzymes and proteins.  One example in which such compounds could be useful is on studies of PG recycling, a process whereby the PG is broke down, internalized, and then reincorporated into growing PG strands.47-49 During PG recycling, lytic transglycosylase cleaves the glycosidic bond between the GlcNAc and MurNAc residues via intramolecular cyclization of the MurNAc moiety to yield a GlcNAc-1,6-anhydroMurNAc pentapeptide product (Fig. 3.1).43 AmpG is a pore protein that 70   transports the GlcNAc-1,6-anhydroMurNAc pentapeptide from the periplasm to the cytoplasm, where it is further broken down and reincorporated into PG biosynthesis.104, 105 One of the breakdown products, 1,6-anhydroMurNAc pentapeptide, acts as an inducer of the expression of the β-lactamase AmpC, which confers resistance of the β-lactam antibiotics.106-108 Therefore, the inhibition of the AmpG pore protein could potentially help combat antibiotic-resistant organisms.109, 110 Studies on AmpG are hampered by a lack of the availability of the GlcNAc-1,6-anhydroMurNAc pentapeptide substrate. The goal of this chapter is to develop a short synthesis of the PG pentapeptide containing a meso-oxa-Dap residue and to attach it to the GlcNAc-1,6-anhydroMurNAc disaccharide as evidence of its utility.  Figure 3.1: Lytic transglycosylase cleavage leading to the formation of GlcNAc-1,6-anhydro-MurNAc pentapeptide. 71   3.1 Incorporation of meso-Dap into PG fragments In Chapter 2, we discussed the different ways research groups had synthesized meso-Dap in a suitably protected form for incorporation into peptides. Mobashery and co-workers have published the only known synthesis of the GlcNAc-1,6-anhydroMurNAc pentapeptide bearing a meso-Dap residue (Fig. 3.2).91 They utilized the method developed by Hernàndez and Martin to synthesize the meso-Dap derivative 52 in overall 12 steps starting from L-aspartic acid.90 The methyl ester group was deprotected using Ba(OH)2, and the 1,2-diol was converted immediately into an acetonide 53. The carboxylate group of octanoate 53 was then activated with N-hydroxysuccinimide (NHS) and coupled with D-Ala-D-Ala-OBn to provide tripeptide 54. Acidic treatment of tripeptide 54 deprotected the diol, which was immediately transformed to a benzyl ester 55 in two steps (first oxidative cleavage of the diol and then protection of the resulting carboxylate using benzyl bromide). The Boc group of compound 55 was then removed using TFA, and the free amine was coupled with Boc-L-Ala-iso-D-Glu(ONHS)-OBn to give the protected pentapeptide 56. Treatment of pentapeptide 56 with TFA gave amine 57.  This amine 57 was coupled to the NHS ester of the disaccharide 58 to give the protected GlcNAc-anhMurNAc pentapeptide 59. The two-step global deprotection using acetic acid followed by catalytic hydrogenation gave GlcNAc-anhMurNAc pentapeptide 60. The synthesis of GlcNAc-anhMurNAc pentapeptide 60 was lengthy as it requires the preparation of orthogonally protected meso-Dap followed by its incorporation into the peptide chain. Several of the steps required for preparing meso-Dap were also low-yielding and not amenable to scale up. Therefore, as an alternative, we chose to use an isosteric analog of meso-Dap, meso-oxa-Dap.    72    Figure 3.2: Schematic scheme for the synthesis of the GlcNAc-anhMurNAc pentapeptide by Mobashery and coworkers 73    In chapter 2, we outlined an efficient approach to insert meso-oxa-Dap into a tripeptide during the preparation of substrates for the cell-shape determining proteases of H. pylori and C. jejuni. The meso-oxa-Dap residue was generated from an aziridine embedded within a tripeptide precursor by an attack with a protected serine derivative. In this chapter, we expand this methodology to prepare the full-length meso-oxa-Dap-containing PG pentapeptide with suitable protecting groups for attachment to MurNAc carboxylates (Fig. 3.3). To demonstrate the utility of meso-oxa-Dap containing PG pentapeptide, we couple it to the GlcNAc-anhMurNAc disaccharide to produce compound 3.  Compound 3 is a close analog of the natural substrate for the AmpG pore protein.  74    Figure 3.3: Structure of meso-oxa-Dap containing PG pentapeptide and GlcNAc-anhMurNAc pentapeptide 3.2 Synthesis of the meso-oxa-Dap containing PG pentapeptide via pentapeptide-embedded aziridine ring-opening The first approach towards the synthesis of the meso-oxa-Dap-containing pentapeptide involved preparing the aziridine-containing pentapeptide and then ring-opening of the aziridine using serine as a nucleophile (Fig. 3.4). The success of this approach would expand the usefulness of our previous work by demonstrating that the ring-opening reaction could be performed on longer peptides.  Literature known D-Ala-D-Ala dipeptide 64 was treated with 4M HCl to remove the N-terminal Boc group,111 and the free amine obtained was coupled with N-trityl-L-Ser to give tripeptide 65. The hydroxyl group of tripeptide 65 was activated using MsCl in the presence of a base, and then the mixture was refluxed to give tritylated aziridine 66. The trityl group was removed under acidic conditions to give aziridine tripeptide 67. The precursor of dipeptide 70 was 75   synthesized from NHS ester of Fmoc-L-Ala 68 and free amine of D-Glu-1-OBn-5-OtBu 69. Subsequent removal of t-butyl ester under acidic conditions gave dipeptide 70. The carboxylate of dipeptide 70 was coupled to the tripeptide aziridine 67 to provide pentapeptide-embedded aziridine 71 that is then subjected to ring-opening with N-Pht-D-Ser-OBn.95 The ring-opening was performed under previously optimized conditions in Chapter two, which involves 2.5 equivalents BF3.OEt2 in DCM at -78 °C. The ring-opening product 72 was formed, but in a low yield of 9%.   Figure 3.4: Synthesis of the meso-oxa-Dap containing pentapeptide 72 The primary product in this reaction was oxazoline 73 (30%), formed from an intramolecular Heine rearrangement of starting material 71.101-103 The ring-opening reaction was 76   done in the presence of molecular sieves to avoid the attack of water on the aziridine. Still, a small amount (10%) of hydrolyzed aziridine was observed.95 An attempt to increase the yield of the compound by 72 using 5 equivalents of BF3.OEt2 was unsuccessful. The overall yield was 2.3% starting from dipeptide 64. Therefore, it appears that the peptide-embedded aziridine ring-opening is not necessarily useful with longer peptides. The possibility of multiple sites for Lewis acid coordination may result in unreactive conformers and nonproductive sequestration of the catalyst. Therefore, we decided to change our strategy and carry out the aziridine ring-opening at a tripeptide stage.   3.3 Synthesis of meso-oxa-Dap containing PG pentapeptide via tripeptide-embedded ring-opening 3.3.1 Ring-opening of aziridine tripeptide using phthalimido serine The synthesis of tripeptide-embedded aziridine 77 began with the coupling of O-allyl protected D-Ala to N-trityl protected L-Ser to give dipeptide 74 (Fig. 3.5).  The dipeptide 74 was converted into aziridine 75 by treatment with MsCl and base.  Removal of the trityl group using TFA gave the unprotected aziridine 76.  This aziridine 76 was then coupled to N-Fmoc-D-Glu-α-OBn to provide tripeptide 77.  The aziridine-opening reaction was performed using 2.5 equivalents of catalyst as previously described to give the fully protected meso-oxa-Dap-containing tripeptide 78 in a 33% yield.  The Heine rearrangement by-product 79 was also obtained in a 37% yield.  These yields were similar to those obtained previously for the synthesis of the tripeptide substrates of Csd6/Pgp2 (Chapter 2). Since compound 77 can readily be prepared on a gram scale, this methodology can be used to rapidly prepare meso-oxa-Dap containing tripeptides on a multi-77   hundred-milligram scale.  It should be noted that the highest yields of 78 were obtained when the ring-opening was run on larger scales (> 400 mg), which most likely allows the intermolecular coupling to compete more effectively with the intramolecular rearrangement.  Figure 3.5: Synthesis of protected pentapeptide 29 The next goal was to elaborate tripeptide 78 into the full-length pentapeptide 61. The allyl group was removed using Pd(PPh3)4 and morpholine, and the resulting free carboxylic acid was 78   coupled to D-Ala-OBn to give the tetrapeptide 80. Treatment of tetrapeptide 80 with piperidine followed by coupling with N-Boc-L-Ala gave protected pentapeptide 61. Theoretically, the proton NMR signals of the alanine methyl groups should appear as distinct doublets; in the case of pentapeptide 61, one would expect to see three doublets having slightly different chemical shifts. However, the proton NMR of pentapeptide 61 in CD3OD showed at least five doublets corresponding to the alanine CH3 groups (Fig. 3.6). Since the proton NMR spectrum of the tetrapeptide 80 did not show such a splitting pattern, this data suggested epimerization may be occurring during the deprotection or coupling steps.   Figure 3.6: 1H-NMR spectrum of pentapeptide 61 in deuterated methanol (CD3OD) 79   Attempts to synthesize pentapeptide 61 using different coupling reagents did not affect the stereochemical outcome of the reaction. To rule out the possibility that the extra doublets might be due to aggregation or the presence of conformers with restricted rotation, we decided to perform a variable temperature NMR (VT-NMR) experiment (Fig. 3.7). The VT-NMR experiment was performed using deuterated dimethyl sulfoxide (DMSO-d6) as the solvent. If the presence of the additional peaks was due to rotamers, one would expect the spectrum to simplify at the higher temperatures since the rotamers would interconvert rapidly on the NMR time scale. The proton NMR was recorded at three different temperatures of 35, 50 and 75 °C. As the temperature increases, the peaks due to the alanine methyl groups shifted slightly but did not broaden and were still individually detected. These results suggested that the most likely cause was epimerization during coupling.  Figure 3.7: Variable temperature NMR experiment with compound 61  80   3.3.2 Ring-opening of aziridine tripeptide using azido serine At the same time, as we were working on the preparation of compound 61, we were experimenting with different protecting groups on the serine nucleophile. One option was to use a sterically less bulky azido serine derivative. This will actually decrease the number of steps since the azide will be reduced during the removal of the benzyl protecting groups.  Azido serine 81 was prepared using a literature known procedure from the HCl salt of D-Ser-OBn-NH2.112 The ring-opening of aziridine 77 was done using 2.5 equivalents of BF3 etherate and 2 equivalents of azido serine 81 (Fig 3.8). Despite the lower steric bulk, the overall yield of tripeptide 82 formation was modest due to the competing Heine rearrangement.   Figure 3.8: Ring-opening of aziridine 77 using azido serine 81. After obtaining the tripeptide 82, the Fmoc group was removed using piperidine as a base. The resulting amine was then coupled with N-Boc-L-Ala to give tetrapeptide 83 in good yield. However, 1H-NMR showed a similar splitting pattern for both alanine CH3 groups, as seen before in the synthesis of pentapeptide 61, indicating that epimerization had occurred (Fig. 3.9).  81    Figure 3.9: Synthesis of tetrapeptide 83 and a partial 1H-NMR in CD3OD 3.3.3 Synthesis of meso-oxa-Dap pentapeptide using Cbz protected tripeptide A final approach we were taking was to synthesize the pentapeptide via the Pht-protected tripeptide 78 but to replace the Pht group with a Cbz group prior to further couplings. This would have the advantage of removing the Pht group at an earlier stage and using one-step hydrogenolysis as the final deprotection step. Tripeptide 78 was treated with hydrazine and then protected with Cbz-Cl to give compound 84 (Fig 3.10). Treatment of compound 84 with piperidine followed by coupling with N-Boc-L-Ala gave tetrapeptide 85. The allyl group was removed using Pd(PPh3)4 and morpholine. The resulting carboxylate was then coupled to D-Ala-OBn to give the protected pentapeptide 63. The 1H-NMR spectrum showed only three doublets for all alanine CH3 groups indicating that epimerization had not occurred during these deprotection/coupling steps. The 82   overall yield was 3.9%. It is not clear why the Cbz group altered the stereochemical outcome of these coupling reactions, but it could be related to the NH functionality that is not present in the Pht or azide protecting groups. This pentapeptide 63 is suitably protected for attachment to the carboxylic acid of MurNAc residues. The synthesis of the GlcNAc-anhMurNAc disaccharide and the coupling with the pentapeptide was done by the graduate student, Condurache Vacariu, in our lab. The N-Boc protecting group was removed under acidic conditions to generate the free amine 86, which was then coupled to the carboxylate 87 to give protected disaccharide pentapeptide 88. Total deprotection using hydrogenolysis to remove benzyl/Cbz protecting group on the peptides followed by saponification using K2CO3 in H2O/MeOH to remove the acetate groups on the sugar gave the GlcNAc-AnhMurNAc pentapeptide 3. Overall, this methodology can be used to prepare multi-hundred milligram quantities of a peptidoglycan pentapeptide containing the isosteric meso-oxa-Dap instead of meso-Dap. GlcNAc-AnhMurNAc pentapeptide is reported to be a substrate for the pore protein Amp G,104, 105 and compound 3 will be used in studies of the specificity of transport by this protein. 83    Figure 3.10: Synthesis of anhydro disaccharide pentapeptide 3 containing meso-oxa-Dap. 84   3.4 Conclusion and summary In summary, this study outlines a short synthetic route into a close analog of the peptidoglycan pentapeptide in which meso-oxa-Dap replaces meso-Dap. Initially, the ring-opening of pentapeptide-embedded aziridine 71 resulted in a significantly lower yield. Therefore, we decided to use tripeptide-embedded aziridine 77 for the ring-opening with an appropriately protected serine derivative. When the nitrogen protecting group on the meso-oxa-Dap side chain was either Pht or azide, problems with epimerization were encountered. Fortuitously, when the protecting group was changed to Cbz, the epimerization problem was resolved. While the overall yield in the synthesis is modest due to the aziridine ring-opening reaction that suffers from the competing intramolecular Heine rearrangement, the dramatically shorter route still makes this an attractive alternative to Mobashery’s approach. Therefore, preparing an isosteric analog to the PG pentapeptide containing meso-oxa-Dap presents a useful option to the researchers in this field.       85   3.5 Experimental procedures 3.5.1 General information The reaction was performed using flame-dried glassware. All reagents were purchased from Sigma-Aldrich, AK Scientific and Ark pharm. Solvents ( DCM and triethylamine) were distilled under a Argon from calcium hydride. All other solvents and reagents were used without further purification. Molecular sieves, Type 3 Å, were dried in an oven at 150 °C overnight. NMR spectra were recorded on a Bruker AV400 spectrometer or a Bruker AV300 spectrometer at a field strength of 400 MHz or 300 MHz for 1H NMR and 101 MHz or 75 MHz for 13C NMR. Column chromatography was performed using. SiliaFlash silica gel F60, 40 - 63 µm purchased from Silicycle. Thin-layer chromatography was performed using silica gel 60 F254 purchased from Merck and visualized under short wave UV or KMnO4 stain. High-resolution mass spectra were recorded on a Waters/Micro mass LCT TOF spectrometer equipped with electrospray (ESI) ionization. 3.5.2 Synthesis of pentapeptide 72 via ring-opening of the aziridine pentapeptide 3.5.2.1 Tripeptide 65  To a solution of 4M HCl in dioxane (20 mL) at 0 °C, N-Boc-D-Ala-D-Ala-OBn 64 (3.4 g, 9.7 mmol) was added in one portion with stirring. The ice-bath was removed. The mixture was stirred for 2 h, and evaporated to dryness under a vacuum. The residue was then triturated with diethyl ether (3x10 mL) and placed under high vacuum for 1 h to give a white foam, which was 86   used without purification. The free amine was dissolved in DCM (50 mL) treated with PyBOP (5.56 g, 10.7 mmol) and trityl L-serine triethylammonium salt (4.78 g, 10.7 mmol) followed by DIPEA (2.54 mL, 14.60 mmol), which was added dropwise. After stirring for 18 h, the mixture was diluted with DCM (200 mL). The organic layer was washed successively with 10% aq. citric acid (2x100 mL), aq. sodium bicarbonate (2x100 mL) and then with brine (100 mL). The organic layer was dried over sodium sulfate, filtered and evaporated under reduced pressure to give a residue that was purified with silica gel column chromatography (30 % ethyl acetate in petroleum ether) to give alcohol 65 as a white solid (5.1 g, 90%). 1H NMR (300 MHz, CDCl3) δ 7.86 (d, J = 7.7 Hz, 1H), 7.50 – 7.38 (m, 5H), 7.38 – 7.16 (m, 15H), 5.15 (dd, J = 12.2, 9.8 Hz, 2H), 4.72 – 4.51 (m, 1H), 4.44 – 4.28 (m, 1H), 3.72 (d, J = 10.6 Hz, 1H), 3.51 – 3.17 (m, 3H), 2.82 – 2.67 (m, 1H), 1.39 (d, J = 7.2 Hz, 3H), 1.33 (d, J = 7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 174.3, 173.1, 172.2, 145.6, 135.2, 128.7, 128.7, 128.5, 128.2, 128.2, 126.9, 71.7, 67.3, 63.9, 59.5, 48.9, 48.3, 18.1, 18.0. HRMS (ESI): m/z calcd for C35H37N3O5Na [M+Na]+ 602.2631, found 602.2629 3.5.2.2 N-trityl aziridine 66  Triethylamine (2.16 mL, 15.5 mmol) was added dropwise to a solution of alcohol 65 (6 g, 10.4 mmol) and methanesulfonyl chloride (0.97 mL, 12.4 mmol) at 0 °C. The resulting solution was stirred at 0°C temperature for 2 h, then diluted with DCM (100 mL) and washed subsequently with 10% aq. citric acid (2x50 mL), aq. sodium bicarbonate (2x50 mL) and brine (50 mL). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The 87   residue was dissolved in THF (60 mL), treated dropwise with triethylamine (2.89 mL, 20.7 mmol),  heated at reflux (bath temperature 65 °C) and stirred for 24 h.  The solution was cooled to rt, diluted with DCM (50 mL), and washed subsequently with 10% aq. citric acid (50 mL), saturated aqueous sodium bicarbonate (50 mL), and brine (50 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (5% ethyl acetate in petroleum ether) to give N-trityl aziridine 66 as a white solid (5.3 g, 90%). 1H NMR (300 MHz, CDCl3) δ 7.47 – 7.42 (m, 5H), 7.41 – 7.21 (m, 15H), 6.85 (d, J = 7.3 Hz, 1H), 5.20 (dd, J = 12.6, 10.1 Hz, 2H), 4.70 – 4.53 (m, 2H), 2.07 (dd, J = 2.8, 0.9 Hz, 1H), 2.01 (dd, J = 6.6, 2.7 Hz, 1H), 1.54 – 1.47 (m, 4H), 1.43 (d, J = 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 172.4, 171.7, 170.8, 143.2, 135.3, 129.3, 128.6, 128.5, 128.2, 127.8, 127.2, 74.6, 67.2, 48.3, 48.1, 33.9, 29.8, 18.5, 18.0. HRMS (ESI): m/z calcd for C35H35N3O4Na [M+Na]+ 584.2525, found 584.2531 3.5.2.3 Aziridine 67  Trifluoroacetic acid (3.1 mL, 39.2 mmol) was added dropwise to a solution of N-trityl aziridine 66 (4.0 g, 3.1 mmol) in a mixture of chloroform (10 mL) and methanol (5 mL) at 0 °C. The resulting solution was stirred at  0°C for 3 h and then concentrated by rotary evaporation. The residue was partitioned between ethyl acetate (50 mL) and water (30 mL). The organic layer was re-extracted with water (3 x 20 mL). The combined aqueous fractions were neutralized with saturated sodium bicarbonate solution and extracted with DCM (5x50 mL). The organic layer was 88   dried over sodium sulfate, filtered and concentrated under reduced pressure to afford the aziridine 67 as a white solid (1.48 g, 65%). 1H NMR (400 MHz, MeOD) δ 7.42 – 7.27 (m, 5H), 5.16 (dd, J = 12.2, 8.4 Hz, 2H), 4.52 – 4.34 (m, 2H), 2.58 (s, 1H), 1.96 – 1.83 (m, 1H), 1.81 – 1.68 (m, 1H), 1.41 (d, J = 7.3 Hz, 3H), 1.33 (d, J = 7.1 Hz, 3H). 13C NMR (101 MHz, MeOD) δ 173.4, 172.5, 136.0, 128.3, 128.3, 128.3, 128.3, 128.1, 128.0, 66.7, 29.1, 24.8, 17.0, 16.0. HRMS (ESI): m/z calcd for C16H21N3O4Na [M+Na]+ 342.1430, found 342.1429  3.5.2.4 H-D-Glu(t-Bu)-OBn 69  A solution of N-Boc-D-Glu-1-OBn-5-OH (4.0 g, 11.9 mmol) in DCM (20 mL) at 0 °C under nitrogen was treated with BF3·OEt2 (0.30 mL, 2.37 mmol), followed dropwise by tert-butyl 2,2,2-trichloroacetimidate (4.25 mL, 23.7 mmol) in cyclohexane (20 mL) . The ice bath was removed, and the reaction mixture warmed to room temperature while stirring for 18h. The reaction mixture was treated with solid NaHCO3 (1.0 g) and the reaction was filtered. The filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography (30% ethyl acetate in petroleum ether) to give Boc-D-Glu(t-Bu)-OBn as a white solid. The spectroscopic data were identical to reported literature.113 To 20 mL of 4 M HCl in dioxane at 0°C, Boc-D-Glu(t-Bu)-OBn (1.0 g, 2.54 mmol) was added, stirred for 2 h and evaporated under reduced pressure to give oily liquid, which crystallized from petroleum ether overnight to give H-D-Glu(t-Bu)-OBn 69 as a white solid (0.9 g, quantitative yield). 1H NMR (400 MHz, MeOD) δ 7.47 – 7.38 (m, 5H), 5.37 – 5.26 (m, 2H), 4.18 (dd, J = 6.6, 4.6 Hz, 1H), 2.58 – 2.34 (m, 2H), 2.25 – 2.11 (m, 2H), 1.45 89   (s, 9H). 13C NMR (101 MHz, MeOD) δ 171.4, 168.7, 135.0, 128.5, 128.4, 128.4, 80.9, 67.9, 52.0, 30.0, 26.9, 25.4. HRMS (ESI): m/z calcd for C16H24NO4 [M+H]+ 294.1705, found 294.1704 3.5.2.5 Carboxylic acid 70  H-D-Glu(t-Bu)-OBn 69 (2.5 g, 8.52 mmol) and N-Fmoc-L-Ala-NHS ester (68, 3.34 g, 8.52 mmol) were dissolved in a mixture of MeCN: H2O (1:1, 100 mL) , cooled to 0°C, and treated with sodium bicarbonate (2.86 g, 34.1 mmol). The ice bath was removed. The reaction mixture warmed to room temperature while stirring for 24 h. The volatiles were removed under reduced pressure. The residue was dissolved in H2O (100 mL) and the pH was adjusted to 1 using 1 M HCl. The aqueous layer was extracted with DCM (5x50 mL). The organic layer was combined, dried over sodium sulfate, filtered, and evaporated under reduced pressure to give a white foam, which was carried on further without purification,  dissolved in DCM (20 mL) , cooled to 0°C, treated dropwise with TFA (20 mL) and stirred for 2h. The volatiles were co-evaporated with diethyl ether (4x20 mL) under reduced pressure to give a white precipitate of carboxylic acid 70 as a white solid (3.6 g, 80 %). 1H NMR (400 MHz, MeOD) δ 7.80 (d, J = 7.5 Hz, 2H), 7.67 (t, J = 7.4 Hz, 2H), 7.43 – 7.27 (m, 9H), 5.20 – 5.10 (m, 2H), 4.50 (dd, J = 9.3, 5.0 Hz, 1H), 4.42 – 4.31 (m, 2H), 4.26 – 4.12 (m, 2H), 2.36 (t, J = 7.5 Hz, 2H), 2.24 – 2.11 (m, 1H), 2.03 – 1.89 (m, 1H), 1.33 (d, J = 7.2 Hz, 3H). 13C NMR (101 MHz, MeOD) δ 174.9, 174.6, 171.5, 144.1, 144.0, 141.3, 135.9, 128.3, 90   128.0, 128.0, 127.5, 126.9, 125.0, 124.9, 119.6, 66.8, 52.0, 50.8, 29.6, 26.3, 17.1. HRMS (ESI): m/z calcd for C30H31N2O7 [M+H]+ 531.2131, found 531.2134 3.5.2.6 Isopeptide 71  To a solution of aziridine 67 (1.48 g, 4.63 mmol) and glutamate 70 (2.70 g, 5.10 mmol) in 100 mL of DCM, PyBOP (2.65 g, 5.10 mmol) was added.. Then DIPEA (2.43 mL, 13.9 mmol) was added to solution dropwise at rt. The reaction mixture was stirred for 18 h, diluted with DCM (100 mL). , washed successively with 10% aq. citric acid (100 mL), aq. sodium bicarbonate (100 mL) and brine (100 mL), dried over sodium sulfate, filtered, and reduced under pressure to give a residue which was purified by silica gel column chromatography (70 % ethyl acetate in petroleum ether) to give azirdine pentapeptide 71 as a white solid (1.93 g, 50 %). 1H NMR (400 MHz, MeOD) δ 7.79 (d, J = 7.5 Hz, 2H), 7.66 (t, J = 8.1 Hz, 2H), 7.44 – 7.12 (m, 15H), 5.19 – 5.01 (m, 4H), 4.54 – 4.43 (m, 2H), 4.41 – 4.28 (m, 3H), 4.26 – 4.14 (m, 2H), 3.22 (dd, J = 5.3, 3.1 Hz, 1H), 2.52 – 2.34 (m, 4H), 2.29 – 2.14 (m, 1H), 1.97 – 1.84 (m, 1H), 1.37 – 1.30 (m, 9H). 13C NMR (101 MHz, MeOD) δ 183.8, 174.6, 173.1, 172.6, 171.6, 167.9, 156.9, 144.2, 144.0, 141.3, 141.3, 136.0, 135.9, 128.3, 128.3, 128.0, 128.0, 128.0, 127.5, 126.9, 125.0, 125.0, 119.7, 66.7, 66.7, 52.0, 50.9, 49.2, 48.3, 35.7, 32.4, 29.5, 25.7, 17.3, 16.8, 16.0.HRMS (ESI): m/z calcd for C46H49N5O10Na  [M+Na]+ 854.3377, found 854.3383 91   3.5.2.7 oxa-pimelate 72  In a flame dried round bottom flask containing oven dried 3Å molecular sieves under an argon atmosphere, aziridine 71 (500 mg, 0.60 mmol) and a solution of N-Pht-D-Ser(OH)-OBn (391 mg, 1.20 mmol) in DCM (10 mL) were added sequentially. The mixture was cooled to -78 °C, stirred for 30 min, treated dropwise with BF3·OEt2 (190 µl, 1.50 mmol) over 20 min and stirred for 3 h. The cold bath was removed. The reaction mixture warmed to rt and was filtered. The volatiles were evaporated under reduced pressure. The residue was purified by silica gel column chromatography (100% ethyl acetate) to give oxa-Dap 72 as a white solid (63 mg, 9 % yield). 1H NMR (400 MHz, CDCl3) δ 7.88 – 7.80 (m, 2H), 7.76 (d, J = 7.5 Hz, 2H), 7.72 – 7.65 (m, 2H), 7.59 (d, J = 7.5 Hz, 2H), 7.39 (t, J = 7.5 Hz, 3H), 7.36 – 7.27 (m, 14H), 7.26 – 7.23 (m, 2H), 5.74 (d, J = 7.2 Hz, 1H), 5.23 – 4.89 (m, 8H), 4.62 (d, J = 8.8 Hz, 1H), 4.53 – 4.45 (m, 3H), 4.40 – 4.34 (m, 2H), 4.29 (t, J = 6.9 Hz, 1H), 4.23 – 4.13 (m, 2H), 4.09 (dd, J = 11.0, 5.1 Hz, 1H), 3.97 (d, J = 8.4 Hz, 1H), 3.61 (dd, J = 9.3, 4.9 Hz, 1H), 2.49 – 2.37 (m, 1H), 2.35 – 2.17 (m, 2H), 1.94 – 1.83 (m, 1H), 1.82 – 1.70 (m, 1H), 1.34 – 1.25 (m, 9H). 13C NMR (101 MHz, CDCl3) δ 172.7, 172.4, 172.3, 171.8, 171.6, 169.8, 167.6, 167.3, 156.1, 143.7, 141.3, 135.4, 135.2, 134.8, 134.4, 131.7, 128.6, 128.6, 128.6, 128.5, 128.5, 128.5, 128.3, 128.2, 128.1, 127.8, 127.1, 127.1, 125.1, 123.7, 92   120.0, 68.4, 67.8, 67.3, 67.2, 67.0, 53.1, 51.5, 50.6, 49.1, 48.2, 47.1, 31.5, 29.7, 28.0, 18.8, 17.9, 17.4. HRMS (ESI): m/z calcd for C64H64N6O15Na [M+Na]+ 1179.4327, found 1179.4308 3.5.2.8 Oxazoline 73   Second to elute was oxazoline 73 ( elution with 2% MeOH in ethyl acetate) as a white solid (0.15 g, 30% yield) 1H NMR (400 MHz, Chloroform-d) δ 7.76 (d, J = 7.6 Hz, 2H), 7.61 (m, 2H), 7.40 (t, J = 7.5 Hz, 3H), 7.38 – 7.25 (m, 12H), 6.78 (d, J = 7.1 Hz, 1H), 6.05 (d, J = 7.1 Hz, 1H), 5.23 – 5.02 (m, 4H), 4.87 (d, J = 4.6 Hz, 1H), 4.67 – 4.49 (m, 3H), 4.50 – 4.27 (m, 5H), 4.27 – 4.16 (m, 2H), 2.51 – 2.41 (m, 1H), 2.40 – 2.29 (m, 1H), 2.20 – 2.03 (m, 2H), 1.52 – 1.37(m, 6H), 1.35 (d, J = 7.1 Hz, 3H) 13C NMR (101 MHz, CDCl3) δ 172.8, 172.6, 172.0, 171.7, 171.6, 169.9, 156.2, 144.1, 143.9, 141.4, 141.4, 135.4, 128.8, 128.7, 128.7, 128.6, 128.3, 127.9, 127.2, 125.3, 120.1, 70.3, 68.6, 67.3, 51.2, 51.0, 48.7, 48.4, 47.3, 29.8, 27.3, 26.3, 23.7, 18.7, 18.2, 18.0. HRMS (ESI): m/z calcd for C46H50N5O10 [M+H]+ 832.3554, found 832.3552   93   3.5.3 Synthesis of Pentapeptide 61 3.5.3.1 Dipeptide 74  N, N-Diisopropylethylamine (6.3 mL, 36.2 mmol) was added dropwise to a solution of triethylammonium trityl serinate (8.9 g, 19.9 mmol), D-Ala-Oallyl-NH2·HCl (3.0 g, 18.1 mmol), and PyBOP (10.4 g, 19.9 mmol) in DCM (50 mL) at rt. The resulting solution was stirred for 18 h , diluted with DCM (100 mL), washed subsequently with 10% aq. citric acid (2x200 mL), aq. sodium bicarbonate (2X200 mL), and brine (200 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (20% ethyl acetate in petroleum ether) to afford Alcohol 74 as a white solid (5.98 g, 72%). 1H NMR (400 MHz, CDCl3) δ 7.70 (d, J = 7.8 Hz, 1H), 7.48 – 7.38 (m, 5H), 7.34 – 7.20 (m, 10H), 5.99 – 5.83 (m, 1H), 5.35 (dd, J = 17.2, 1.5 Hz, 1H), 5.28 (dd, J = 10.4, 1.6 Hz, 1H), 4.74 – 4.56 (m, 2H), 4.45 (m, 1H), 3.68 (dd, J = 11.2, 2.6 Hz, 1H), 3.34 (dd, J = 4.4, 2.6 Hz, 1H), 2.74 (dd, J = 11.2, 4.4 Hz, 1H), 2.63 ( broad s, 1H), 1.40 (d, J = 7.3 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 174.1, 173.2, 145.8, 131.6, 128.8, 128.3, 127.0, 119.1, 71.8, 66.4, 64.5, 59.5, 48.3, 17.6. HRMS (ESI): m/z calcd for C28H30N2O4Na [M+Na]+ 481.2103, found 481.2105   94   3.5.3.2 N-trityl aziridine 75  Triethylamine (2.64 mL, 18.9 mmol) was added dropwise to a solution of alcohol 74 (5.8 g, 12.7 mmol) and methane sulfonyl chloride (1.2 mL, 15.2 mmol) in DCM (50 mL) at 0 °C. The resulting solution was stirred at this temperature for 1 h, diluted with DCM (50 mL), subsequently with 10% aq. citric acid (100 mL), water (100 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was dissolved in THF (18 mL), treated dropwise with triethylamine (3.5 mL, 25.3 mmol) heated to reflux (bath temperature of 70 ° ) for 2 h, cooled to rt, diluted with DCM (100mL), washed subsequently with 10% aqueous citric acid (200 mL), saturated aqueous sodium bicarbonate (200 mL), and water (200 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (10% ethyl acetate in petroleum ether) to afford N-trityl aziridine 75 as a white solid (5.0 g, 91%). 1H NMR (400 MHz, CDCl3) δ 7.47 – 7.42 (m, 5H), 7.35 – 7.23 (m, 10H), 6.02 – 5.88 (m, 1H), 5.38 (dd, J = 17.2, 1.4 Hz, 1H), 5.30 (dd, J = 10.7, 1.3 Hz, 1H), 4.80 – 4.63 (m, 3H), 2.11 (d, J = 2.2 Hz, 1H), 2.04 (dd, J = 6.6, 2.7 Hz, 1H), 1.58 (d, J = 7.3 Hz, 3H), 1.51 (d, J = 6.8 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 172.7, 170.5, 143.4, 131.7, 129.5, 127.9, 127.3, 118.9, 74.7, 66.1, 47.6, 34.1, 29.9, 18.9. HRMS (ESI): m/z calcd for C28H29N2O3 [M+H]+ 440.2160, found 440.2177 95   3.5.3.3 Aziridine 76  Trifluoroacetic acid (5.3 mL, 68.1 mmol) was added dropwise to a solution of aziridine dipeptide 76 (5.0 g, 11.4 mmol) in a mixture of DCM (15 mL) and methanol (10 mL) at 0 °C. The resulting solution was stirred at this temperature for 3 h then concentrated by rotary evaporation. The residue was partitioned between ethyl acetate (50 mL) and water (30 mL). The organic layer was extracted with water (2x30 mL). The combined aqueous fractions were neutralized with sodium bicarbonate (aq) and extracted with DCM (5x50 mL). The organic layer was combined, dried over sodium sulfate, filtered and concentrated under reduced pressure to afford the free aziridine 76 as a colorless oil (1.57g, 70 %). 1H NMR (400 MHz, MeOD) δ 6.02 – 5.91 (m, 1H), 5.36 (dd, J = 17.3, 1.5 Hz, 1H), 5.25 (dd, J = 10.3, 1.6 Hz, 1H), 4.67 – 4.62 (m, 2H), 4.50 (q, J = 7.3 Hz, 1H), 2.60 (dd, J = 5.7, 3.2 Hz, 1H), 1.90 – 1.82 (m, 2H), 1.44 (d, J = 7.4 Hz, 3H)  13C NMR (101 MHz, MeOD) δ 172.2, 171.2, 132.0, 117.3, 65.4, 48.3, 29.0, 24.8, 16.3. HRMS (ESI): m/z calcd for C9H15N2O3 [M+H]+ 199.1083, found 199.1083   96   3.5.3.4 Tripeptidyl aziridine 77  N, N-Diisopropylethylamine (578 µL, 3.31 mmol) was added dropwise to a solution of N-Fmoc-D-Glu-α-OBn (0.95 g, 2.07 mmol), aziridine 76 (0.33 g, 1.66 mmol) and PyBOP (1.08 g, 2.07 mmol) in DCM (20 mL) at rt, stirred for 18 h, diluted with DCM (50 mL), washed sequentially with 10% aq. citric acid (100 mL), aq. sodium bicarbonate (100 mL), and brine (100 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (70% ethyl acetate in petroleum ether) to afford the aziridine tripeptide 77 as a white solid (0.66 g, 62%). 1H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 7.5 Hz, 2H), 7.64 – 7.57 (m, 2H), 7.43 – 7.29 (m, 9H), 6.76 (d, J = 7.7 Hz, 1H), 5.92 – 5.78 (m, 1H), 5.56 (d, J = 8.2 Hz, 1H), 5.33 – 5.15 (m, 4H), 4.64 – 4.52 (m, 3H), 4.50 – 4.31 (m, 3H), 4.21 (t, J = 7.0 Hz, 1H), 3.05 (dd, J = 6.5, 3.1 Hz, 1H), 2.58 – 2.41 (m, 3H), 2.38 – 2.23 (m, 2H), 2.11 – 1.99 (m, 1H), 1.44 (d, J = 7.2 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 183.2, 172.3, 171.8, 167.3, 156.3, 143.8, 141.4, 135.2, 131.5, 128.8, 128.8, 128.5, 127.9, 127.2, 125.2, 120.1, 119.0, 67.6, 67.4, 66.2, 53.6, 48.0, 47.2, 36.1, 32.7, 31.2, 27.6, 18.0. HRMS (ESI): m/z calcd for C36H37N3O8Na [M+Na]+ 662.2478, found 662.2475   97   3.5.3.5 Tripeptide 78  The tripeptide 78 was prepared using procedurial steps mentioned in section 3.6.2.7., staring from tripeptide  aziridine 77 (1 g, 1.56 mmol). The crude residue was purified by silica gel column chromatography (90% ethyl acetate in petroleum ether) to give protected tripeptide 78 as a white solid (0.49 g, 33% yield). 1H NMR (400 MHz, MeOD) δ 7.88 – 7.73 (m, 6H), 7.66 (t, J = 7.2 Hz, 2H), 7.44 – 7.20 (m, 14H), 5.92 – 5.75 (m, 1H), 5.29 – 5.08 (m, 7H), 4.53 (t, J = 4.8 Hz, 1H), 4.48 – 4.35 (m, 3H), 4.33 – 4.08 (m, 6H), 3.77 – 3.67 (m, 2H), 2.37 – 2.16 (m, 3H), 1.94 – 1.80 (m, 1H), 1.25 (d, J = 7.3 Hz, 3H). 13C NMR (101 MHz, MeOD) δ 173.3, 172.0, 171.9, 170.1, 167.6, 167.4, 157.3, 143.9, 143.8, 141.2, 141.2, 135.8, 135.3, 134.3, 131.9, 131.6, 128.2, 128.2, 128.0, 127.9, 127.8, 127.4, 126.8, 125.0, 124.9, 123.1, 119.5, 117.0, 70.2, 67.4, 67.2, 66.8, 66.6, 65.2, 53.5, 53.0, 51.3, 31.3, 27.0, 15.9. HRMS (ESI): m/z calcd for C54H52N4O13Na [M+Na]+ 987.3429, found 987.3427 3.5.3.6 Oxazoline 79   98   The Heine rearrangement product 79 was obtained in the same protocol for 3.6.3.5 as described earlier (elution with 2% MeOH in ethyl acetate) as a white solid (0.74 g, 37% yield) 1H NMR (400 MHz, MeOD) δ 7.78 (d, J = 7.6 Hz, 2H), 7.64 (m, 2H), 7.39 – 7.34 (m, 3H), 7.32 – 7.24 (m, 6H), 5.98 – 5.73 (m, 1H), 5.32 – 5.08 (m, 4H), 4.65 (dd, J = 10.8, 7.8 Hz, 1H), 4.58 – 4.52 (m, 2H), 4.52 – 4.42 (m, 3H), 4.40 – 4.25 (m, 3H), 4.17 (t, J = 7.1 Hz, 1H), 2.42 (t, J = 7.3 Hz, 2H), 2.32 – 2.21 (m, 1H), 2.06 – 1.93 (m, 1H), 1.40 (d, J = 7.3 Hz, 3H). 13C NMR (101 MHz, MeOD) δ 172.5, 172.1, 172.1, 171.0, 157.4, 144.1, 143.9, 141.3, 135.9, 132.0, 128.3, 128.1, 128.0, 127.6, 126.9, 126.9, 125.0, 125.0, 119.7, 117.3, 70.3, 68.2, 66.8, 65.5, 53.3, 27.1, 23.8, 16.1.HRMS (ESI): m/z calcd for C36H38N3O8  [M+H]+ 640.2659, found 640.2657 3.5.3.7 Tetrapeptide 80  To solution of tripeptide 78 (100 mg, 0.10 mmol) in 5 mL DCM, morpholine (12 μL, 0.14 mmol) followed by Pd(PPh3)4 (2.4 mg, 2 μmol) were added. The reaction was covered with aluminum foil and stirred for 1 h. The volatiles were removed under reduced pressure. Water (10 mL) was added to the crude yellow solid and the pH was adjusted to 2-3 using 1M HCl. The aqueous layer was extracted with DCM (3x10 mL). The organic layer was combined, dried over sodium sulfate, filtered, and evaporated under reduced pressure to give a yellow solid which was used without further purification. To a solution of the crude acid in DCM (5 mL) was added PyBOP 99   (60 mg, 0.11 mmol) followed by D-Ala-OBn·tosylate salt (40 mg, 0.11 mmol). Then DIPEA (54 µL, 0.31 mmol) was added dropwise to the reaction mixture, which was stirred for 1 h at rt. The reaction mixture was diluted with DCM (10 mL), washed with water (3x10 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure to give a residue which was purified using silica gel column chromatography (80% ethyl acetate in petroleum ether) to give tetrapeptide 80 as a white solid (55 mg, 49% yield). 1H NMR (400 MHz, MeOD) δ 7.87 – 7.82 (m, 2H), 7.80 – 7.75 (m, 4H), 7.64 (d, J = 7.6 Hz, 2H), 7.40 – 7.23 (m, 19H), 5.23 (dd, J = 9.5, 5.1 Hz, 1H), 5.19 – 5.11 (m, 4H), 5.10 – 5.05 (m, 2H), 4.43 (t, J = 5.3 Hz, 1H), 4.40 – 4.30 (m, 3H), 4.30 – 4.16 (m, 5H), 4.16 – 4.07 (m, 2H), 3.77 – 3.68 (m, 2H), 2.29 (t, J = 7.2 Hz, 2H), 2.23 – 2.15 (m, 1H), 1.95 – 1.84 (m, 1H), 1.32 (d, J = 7.3 Hz, 3H), 1.20 (d, J = 7.2 Hz, 3H). 13C NMR (101 MHz, MeOD) δ 173.6, 173.2, 172.4, 172.1, 170.4, 167.7, 167.5, 157.4, 144.0, 143.9, 141.3, 136.0, 135.9, 135.4, 134.5, 131.7, 128.3, 128.3, 128.2, 128.0, 128.0, 128.0, 127.9, 127.5, 127.5, 126.9, 126.9, 125.1, 125.0, 123.3, 119.7, 70.0, 67.6, 67.3, 66.9, 66.7, 66.6, 53.7, 51.3, 31.3, 27.0, 16.6, 16.0. HRMS (ESI): m/z calcd for C61H59N5O14Na [M+Na]+ 1108.3951, found 1108.3962   100   3.5.4 Synthesis of pentapeptide 62 3.5.4.1 Azide 82  Modification of the protocol for 3.6.2.7 in which the differences in amounts and details are specified. The azide 82 was obtained as a white solid (0.16 g, 24 % yield) (80% ethyl acetate in petroleum ether ) from aziridine tripeptide 77 (0.5 g, 0.78 mmol). 1H NMR (400 MHz, MeOD) δ 7.83 – 7.75 (m, 2H), 7.67 (t, J = 6.9 Hz, 2H), 7.57 – 7.12 (m, 14H), 5.93 – 5.79 (m, 1H), 5.33 – 5.08 (m, 6H), 4.59 (t, J = 5.4 Hz, 1H), 4.52 – 4.49 (m, 2H), 4.47 – 4.36 (m, 2H), 4.34 – 4.24 (m, 2H), 4.23 – 4.15 (m, 2H), 3.91 – 3.80 (m, 2H), 3.78 – 3.66 (m, 2H), 2.42 – 2.24 (m, 3H), 1.97 – 1.84 (m, 1H), 1.36 (d, J = 7.4 Hz, 3H). 13C NMR (101 MHz, MeOD) δ 173.4, 172.1, 171.9, 170.1, 168.5, 157.3, 143.9, 143.8, 141.2, 135.8, 135.4, 131.9, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.4, 126.8, 126.8, 125.0, 124.9, 119.5, 117.1, 70.9, 70.5, 67.2, 66.8, 66.6, 65.3, 61.5, 53.4, 53.0, 31.3, 27.0, 16.0. HRMS (ESI): m/z calcd for C46H48N6O11Na [M+Na]+ 883.3279, found 883.3273  101   3.5.5 Synthesis of pentapeptide 63 3.5.5.1 Carbamate 84  Tripeptide 78 (400 mg, 0.42 mmol) was dissolved in THF (5mL) , cooled to 0°C, treated with hydrazine (50 % in water) (30 µl, 0.47 mmol) was added at 0 °C and stirred for 2 h, treated with AcOH (1 mL), heated to 50°C and stirred for 10 min. The volatiles were removed under reduced pressure to give the white solid. The solid was digested with 5 mL ethyl acetate and filtered. The filtrate was evaporated to give an oily residue which was dissolved in mixture of water/ THF (1:1, 10 mL) , cooled to 0°C, and treated with sodium carbonate (220 mg, 2.1 mmol) followed by Cbz-Cl (354 mg, 2.11 mmol). The reaction mixture was stirred for 1 h. and volatiles were removed under reduced pressure. The reduced volume was partitioned between water (30 mL) and DCM (30 mL). The organic layer was separated. The aqueous layer was washed with DCM (2 x10 mL). The combined organic layer was collected, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified with silica gel column chromatography (80% ethyl acetate in petroleum ether) to give carbamate 84 as a white solid (360 mg, 90% yield). 1H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 7.5 Hz, 2H), 7.59 (m, 2H), 7.43 – 7.27 (m, 19H), 6.51 (d, J = 7.4 Hz, 1H), 6.09 (d, J = 8.4 Hz, 1H), 5.87 – 5.74 (m, 1H), 5.66 (d, J = 8.2 Hz, 1H), 5.33 – 4.98 (m, 8H), 4.64 – 4.32 (m, 8H), 4.13 (t, J = 7.0 Hz, 1H), 3.98 – 3.85 (m, 2H), 102   3.77 (dd, J = 9.9, 3.0 Hz, 1H), 3.54 (dd, J = 9.3, 5.7 Hz, 1H), 2.39 – 2.27 (m, 1H), 2.27 – 2.18 (m, 1H), 2.18 – 2.07 (m, 1H), 1.87 – 1.75 (m, 1H), 1.35 (d, J = 7.3 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 172.6, 172.1, 172.0, 170.0, 169.4, 156.4, 156.3, 144.0, 141.4, 136.3, 135.3, 131.8, 128.8, 128.8, 128.7, 128.6, 128.5, 128.5, 128.3, 128.2, 127.9, 127.9, 127.2, 127.1, 125.3, 125.2, 120.1, 118.6, 71.4, 70.5, 67.7, 67.6, 67.2, 66.0, 54.7, 53.2, 52.7, 48.4, 47.4, 31.8, 28.6, 17.8.HRMS (ESI): m/z calcd for C54H56N4O13Na  [M+Na]+ 991.3742, found 991.3743 3.5.5.2 Tetrapeptide 85  A solution of 5% piperidine in DMF (2 mL) was mixed with tripeptide 84 (0.36g , 0.37 mmol) and stirred for 5 min at rt. The volatiles were removed under reduced pressure. The white solid obtained was washed with hexane (3x5 mL) to give crude amine which was used further without purification. N-Boc-Ala (84 mg, 0.45 mmol) was dissolved in 2 mL DCM under an inert atmosphere. Then EDC·HCl (85 mg, 0.45 mmol) and HOBt (68 mg, 0.45 mmol) were added sequentially at 0 ◦C. The reaction mixture was stirred for 30 min at rt. Then a solution of crude amine and DIPEA (0.130 mL, 0.74 mmol) was added dropwise at 0 °C. The reaction mixture was stirred for 3 h at rt. The reaction mixture was then partitioned between DCM (20 mL) and water (20 mL). The organic layer was separated, washed with water (3x 20 mL), and dried over sodium sulfate and concentrated under reduced pressure to give residue which was purified by silica gel 103   column chromatography (80 % ethyl acetate in petroleum ether) to give tetrapeptide 85 as a white foamy solid (212 mg, 62 % yield). 1H NMR (400 MHz, MeOD) δ 7.51 – 7.12 (m, 15H), 5.98 – 5.79 (m, 1H), 5.33 – 5.00 (m, 8H), 4.63 – 4.36 (m, 6H), 4.15 – 4.00 (m, 1H), 3.90 (dd, J = 9.7, 4.5 Hz, 1H), 3.76 (m, 2H), 3.64 (dd, J = 9.8, 4.5 Hz, 1H), 2.42 – 2.22 (m, 3H), 1.92 – 1.83 (m, 1H), 1.43 (s, 9H), 1.36 (d, J = 7.3 Hz, 3H), 1.26 (d, J = 7.2 Hz, 3H). 13C NMR (101 MHz, MeOD) δ 174.9, 173.7, 172.3, 171.4, 170.7, 170.3, 157.4, 156.7, 136.8, 135.9, 135.9, 132.1, 128.4, 128.3, 128.3, 128.2, 128.1, 128.0, 128.0, 127.8, 127.8, 127.7, 117.3, 79.7, 70.5, 66.9, 66.8, 66.6, 65.5, 54.7, 53.7, 53.5, 51.1, 51.0, 31.1, 27.5, 27.1, 16.8, 16.1. HRMS (ESI): m/z calcd for C47H59N5O14 [M+H]+ 918.4137, found 918.4134 3.5.5.3 Pentapeptide 63  To solution of tetrapeptide 85 (212 mg, 0.23 mmol) in 5 mL DCM was added morpholine (26 µL, 0.30 mmol). Then Pd(PPh3)4  (5.3 mg, µmol) was added. The reaction was covered with Al foil and stirred for 1 h. The volatiles were removed under reduced pressure. Water (10 mL) was added to the crude yellow solid and the pH was adjusted to 2-3 using 1M HCl. The aqueous layer was extracted with DCM (3x10 mL). The organic layer was collected, dried over sodium sulfate and evaporated under reduced pressure to give a yellow solid which was used without further purification. To a solution of the crude acid in DCM (5 mL) was added EDC·HCl (66 mg, 0.35 104   mmol) followed by HOBt (53 mg, 0.35 mmol) at 0 °C. After 30 min, a solution of D-Ala-OBn·tosylate salt (122 mg, 0.35 mmol) and DIPEA (0.121 mL, 0.69 mmol) in DCM (5 mL) was added. The reaction mixture was stirred for 1 h at rt. The reaction mixture was diluted with DCM (10 mL) and then washed with water (3x10 mL). The organic layer was collected, dried over sodium sulfate and concentrated under reduced pressure to give residue which was purified using silica gel column chromatography (90% ethyl acetate in petroleum ether) to give pentapeptide 63 as a white solid (150 mg, 62% yield). 1H NMR (400 MHz, MeOD) δ 7.41 – 7.22 (m, 20H), 5.24 – 4.97 (m, 8H), 4.52 – 4.33 (m, 5H), 4.13 – 4.02 (m, 1H), 3.90 (dd, J = 9.7, 4.4 Hz, 1H), 3.76 (m, 2H), 3.65 (dd, J = 9.7, 4.6 Hz, 1H), 2.39 – 2.22 (m, 3H), 2.01 – 1.80 (m, 1H), 1.44 (s, 9H), 1.38 (d, J = 7.3 Hz, 3H), 1.32 (d, J = 7.2 Hz, 3H), 1.23 (d, J = 7.2 Hz, 3H). 13C NMR (101 MHz, MeOD) δ 174.9, 173.7, 173.1, 172.4, 171.3, 170.6, 170.2, 157.2, 156.6, 136.7, 135.9, 135.8, 128.2, 128.2, 128.2, 128.1, 128.0, 128.0, 127.9, 127.9, 127.9, 127.8, 127.8, 127.7, 127.6, 79.5, 70.5, 70.2, 66.8, 66.6, 66.5, 66.4, 54.6, 54.0, 51.0, 50.8, 49.1, 30.9, 27.4, 26.7, 16.7, 16.6, 15.9.HRMS (ESI): m/z calcd for C54H66N6O15Na [M+Na]+ 1061.4484, found 1061.4488         105   Chapter 4: Progress towards the synthesis and testing of phosphonamidate and phosphonate inhibitors of the Csd4 enzyme As discussed in Chapter 1, H. pylori is a pathogenic bacterium present in half the world's population. H. pylori colonize the stomach epithelial cells and cause gastric ulcers, which, if not treated in time, can lead to gastric cancer.63, 64 It is thought that the bacteria’s helical shape is necessary for its pathogenicity. Recently, a series of genes were identified in H. pylori that encoded for proteins that were required for maintaining the helical shape of the bacteria. These proteins were called cell shape-determining proteins (Csds).54-57 Homologs of Csds are also found in C. jejuni and were called peptidoglycan peptidases (Pgps).59, 60 One of the H. pylori proteins responsible for cell-straightening is Csd4, which hydrolyzes the bond between the meso-Dap and iso-D-Glu of an uncrosslinked PG tripeptide ( a D, L-carboxypeptidase) to give a PG dipeptide. The homologue in C. jejuni is called Pgp1. Deletion of csd4 or pgp1 genes leads to bacteria with a straight-rod phenotype and a reduced ability for colonization.54, 56, 60 It is postulated that the inability of the PG dipeptide to form cross-links with other neighbouring peptide chains leads to a less localized crosslinking and a looser mesh in areas where the Csds are present. If the localization of the Csds is controlled, the differential extent of cross-linking could promote curvature of the overall sacculus structure. Structural and functional studies, along with homology comparisons, clearly indicate that the Csd4/Pgp1 peptidase are members of the zinc-dependent metalloprotease family of enzymes.54, 56, 60 This chapter details our progress towards the design and synthesis of phosphonamidate (P-N bond) and phosphonate (P-O bond) based inhibitors for the Csd4 enzyme. Potent Csd4 inhibitors 106   could result in cell straitening and reduced pathogenicity of H. pylori and serve as leads in the design of antibacterial agents.58  We will first briefly summarize the previous work on the synthesis and testing of a phosphinic acid (P-C bond) based inhibitor. 4.1 Design and synthesis of phosphinate (P-C bond) based inhibitor Chan et al., in their structural studies of Csd4 with a truncated version of natural PG tripeptide ( N-Ac-L-Ala-iso-D-Glu-meso-Dap), revealed that the iso-D-Glu-meso-Dap moiety is essential for the binding as it was found to be located in the center of the active site, adjacent to the zinc-binding pocket and catalytic water (Fig. 4.1).54  A common strategy for inhibiting metalloprotease is to use tetrahedral phosphorus as a mimic of the tetrahedral intermediate formed when water is delivered to the carbonyl of the scissile bond. As the enzyme is expected to bind tightly to the tetrahedral intermediate, with the oxyanion coordinating to the active site metal, such inhibitors can prove to be very potent.80, 81 In order to make a non-hydrolyzable inhibitor of Csd4, a previous group member, Yanjie Liu, decided to make the dipeptidyl phosphinate inhibitor 4.58 107    Figure 4.1: Comparison between tripeptide substrate (left), tetrahedral intermediate (middle) and dipeptidyl phosphinate inhibitor 4 (right). The scissile peptide bond and the tetrahedral centers are coloured in red The phosphinate-based inhibitors are easier to prepare than phosphonate or phosphonamidate-based inhibitors as the P-C bonds are not susceptible to hydrolysis. The commercially available D-glutamic acid derivative 89 was converted to alkene 90 using copper acetate and lead acetate (Fig. 4.2). The alkene 90 was then treated with ammonium hypophosphite and triethyl borane to give hypophosphite 91. The conjugate addition of compound 91 with benzyl acrylate derivative 92 in the presence of hexamethyldisilazane gave phosphinic acid 93, which was then immediately protected with adamantyl bromide to give an ester 94 as a diastereomeric mixture.  Both the benzyl and Cbz group were removed by hydrogenolysis, and treatment with acetic anhydride gave acetamide 95. The acetamide 95 was then treated with TFA to give phosphinate inhibitor 4 as a diastereomeric mixture.58 108    Figure 4.2: Synthesis of the phosphinate inhibitor 4 4.1.1 Kinetic studies of inhibitor 4  To measure the activity of Csd4 with the tripeptide substrate (N-Ac-L-Ala-iso-D-Glu-meso-Dap),   Chan et al. employed a continuous coupled assay in which diaminopimelate dehydrogenase (DAPDH) acts as a coupling enzyme (Fig. 4.3).54 The formation of NADPH was measured using UV spectroscopy. The kinetic constants for the tripeptide substrate were found to be kcat = 0.015 ± 0.001 s-1 and KM = 112 ± 5 µM.54 Using the same continuous coupled assay, Liu et al. determined the KI value of inhibitor 4 to be 1.4 ± 0.2 µM.58  As expected for an intermediate analog, the value of KI  was considerably lower than the KM value of the tripeptide substrate.  109    Figure 4.3: The continuous coupled assay used in the Csd4 kinetic analysis 4.1.2 Structural studies of Csd4 with the phosphinate inhibitor 4 A crystal structure of the Csd4-inhibitor 4 complex clearly showed the phosphinate inhibitor bound in the active site (Fig. 4.4A). Csd4 selectively bound the isomer with the (S)- stereocenter adjacent to the phosphinic acid when incubated with a diastereomeric mixture of inhibitor 4 (1:1), consistent with the stereochemistry of the normal substrate. Both oxygens of the phosphonic acid were found to be coordinated with the zinc atom, and the zinc-bound water was displaced. This is expected for an inhibitor that mimics the tetrahedral intermediate formed in a metalloprotease active site.58 A detailed mechanism of catalytic activity is presented in Chapter 1, section 1.4.3.2. The Csd4-inhibitor 4 complex's overall structure was very similar to that of the Csd4-tripeptide substrate complex (Fig4.4 B). The main difference is that the phosphinate inhibitor has slightly twisted to coordinate with the Zn+2 ion in the active site.58     110      Figure 4.4: A) Electron density map of inhibitor 4 in the Csd4-Inhibitor complex. The (S) configuration of the inhibitor stereocenter that is close to the phosphinate is shown (left) and Csd4-inhibitor complex (right). B) Structural alignment of the active site of the Csd4-Inhibitor and the Csd4-Tripeptide complex. (images are taken from Liu et al., 2015) 4.1.3 Studies of inhibitor 4 with H. pylori and C. jejuni   After demonstrating that compound 4 was an effective inhibitor, Liu et al. performed morphological studies to see if inhibitor 4 can cause cell straightening, as was observed upon deletion of the Csd4 gene in H. pylori. First, they tested inhibitor 4 with H. pylori strain KBH19, 111   a strain with a pronounced helical shape. After ten hours of treatment with 2.1 mM inhibitor 4, a loss in curvature was observed (Fig. 4.5). A histogram of the cell population vs side curvature also summarizes these results. This observation indicates that inhibitor 4 induces cell straightening in H. pylori. The high concentration required (relative to the 1.4 µM value of KI) is likely due to the inability of the highly charged inhibitor to penetrate the outer membrane of the bacterial cell wall.58   Figure 4.5: Phase microscopy images of H. pylori with inhibitor 4 and its histogram ((images are taken from Liu et al., 2015) A similar study was also done with the C. jejuni strains, wild type (81-176) and acapsular mutant ΔkpsM.  Cell straightening was observed after 24 h at an inhibitor concentration of 2.3 mM (Fig. 4.6). Cell straightening was more pronounced with the acapsular strain indicating that the polysaccharide capsule hinders the ability of the inhibitor to cross the outer membrane. These results indicated that inhibitor 4 also targets Pgp1 and that the polysaccharide capsules hinder the ability of the molecule to enter the cell. Overall, these studies show that the phosphinate-based inhibitor can directly target cell shape-determining enzymes in helical bacteria.58  112    Figure 4.6: Phase microscopy images of C. jejuni cells (81-176 and ΔkpsM ) with inhibitor 4 and their histograms (images are taken from Liu et al., 2015) 4.2 Design and synthesis of phosphonamidate and phosphonate inhibitors While the results obtained with inhibitor 4 were encouraging, the high concentrations required to observe cell straightening were problematic. One way to circumvent this issue is to increase the potency of the inhibitors. In many cases, the replacement of a phosphinate for a phosphonate or a phosphonamidate can lead to inhibitors that bind 2-3 orders of magnitude more tightly.82, 114, 115  This could be due to the reduced sterics, more H-bonding possibilities, and an altered electron density around the phosphorus atom. We, therefore, decided to target the second 113   generation of inhibitors, compounds 5 and 6, bearing either phosphonamidate or phosphonate functionalities, respectively (Fig. 4.7).  We chose to incorporate meso-oxa-Dap because we can easily access this compound via our aziridine ring-opening approach. As observed in the Csd4-inhibitor 4 complex, the enzyme preferentially bound to the isomer of inhibitor 4 bearing an (S)-stereocenter β to the phosphorus atom. Therefore, we decided to attach iso-D-Glu-acid to the (S)-stereocenter of the meso-oxa-Dap.   Figure 4.7: Structure comparison of phosphinate inhibitor 4 with phosphonamidate inhibitor 5 and phosphonate inhibitor 6. These inhibitors can be obtained from the coupling of phosphochloridates with either appropriately protected meso-oxa-Dap or the corresponding hydroxy-meso-oxa-Dap (Fig. 4.8). Our retrosynthetic analysis indicated that the desired dimethyl phosphonate could be obtained via the Michaelis-Arbuzov reaction between trimethyl phosphite and suitably protected alkyl bromide derived from D-glutamic acid.116 The meso-oxa-Dap and its analogous hydroxy-meso-oxa-Dap can be obtained by the aziridine ring-opening using appropriately protected serine 95, or 2-deamino-2-114   hydroxy-serine could be prepared from the free amine of meso-oxa-Dap via the Sandmeyer reaction.117   Figure 4.8: Retrosynthetic analysis for the preparation of inhibitors 5 and 6 4.3 Attempted synthesis of the phosphonamidate inhibitor 6 4.3.1 Synthesis of the meso-oxa-Dap fragment In order to prepare the meso-oxa-Dap fragment of inhibitor 6, the pNZ protected aziridine 98  was first synthesized from D-serine benzyl ester using a four-step literature known procedure.118, 119 The D-serine benzyl ester was protected by using trityl chloride to give compound 96. This compound was treated with mesyl chloride and then refluxed in the presence of a base to give aziridine 97. The trityl group was removed under acidic conditions, and the free amine obtained was treated with pNZ-Cl to give aziridine 98. Ring-opening of aziridine 98 was done 115   using N-Pht-Ser-OBn as a nucleophile in the presence of BF3.OEt2 to give protected meso-oxa-Dap 99 in 50% yield.95 Deprotection of phthalimide group using hydrazine and the treatment with 4M HCl in dioxane gave the desired meso-oxa-Dap 100 in good yield.  Figure 4.9: Synthesis of the HCl salt of meso-oxa-Dap 100 4.3.2 Synthesis of the phosphonic acid fragment The easiest way to synthesize a dialkyl phosphonate is by heating the corresponding alkyl bromide with trimethyl phosphite in an Arbuzov reaction.116 Therefore, we decided to synthesize phosphonate derivative 103 from an alkyl halide (Fig. 4.10).  Protected glutamic acid 101 was first converted into bromide 102 via a Barton decarboxylation using 2-mercaptopyridine oxide and bromotrichloromethane.120 The bromide derivative 102 was then refluxed with trimethyl phosphite, but the desired product 103 was not obtained, likely because of a competing cyclization involving the acetamido group. After multiple attempts to synthesize phosphonate 103, we decided to prepare Cbz-protected bromide 105 that could be converted into phosphonate 106. The bromide 105 was prepared from Cbz-protected glutamic acid 104 via a Barton decarboxylation in a 116   moderate yield.120 Treatment of this bromide 105 with trimethyl phosphite gave the phosphonate 106  with two side products, 107 and 108.  Figure 4.10: Synthesis of alkyl phosphonates 103 and 106 An ESI-MS and 31P NMR analysis of the phosphonate 106 obtained after column chromatography showed that the side product dimethyl benzyl phosphonate 107 co-eluted with it. All further attempts to purify the phosphonate 106 were unsuccessful. The cyclized by-product 108 is formed by an intramolecular attack of the carbonyl oxygen of the Cbz group onto the alkyl bromide to form intermediate 109 under refluxing conditions (Fig. 4.11). The dimethyl benzyl phosphonate 107 could be formed by an attack of trimethyl phosphite at the benzylic position of the intermediate 109. The cyclized by-product 108 could be isolated but in a lower yield. A similar reaction was also reported by Malachowski et al. during their synthesis of a phosphorus-based inhibitor of glutamyl-γ-glutamate synthetase.116 117    Figure 4.11: A postulated mechanism for the formation of compounds 107 and 108  After our failure with the synthesis of phosphonate 106, we decided to synthesize the more sterically hindered Boc-protected derivative, which could then be reacted with trimethyl phosphite via an Arbuzov reaction to prepare an N-Boc-protected phosphonate. The Boc group be removed under acidic conditions and then replaced with either a Cbz or an acetyl group to give phosphonates 103 and 106, respectively. 4.3.2.1 Synthesis of Boc-protected phosphonate fragment The Boc-protected bromide 111 was prepared by treatment of glutamic acid derivative 110 with 2-mercaptopyridine N-oxide and CBrCl3 in the presence of light (Fig 4.12).120 Compound 111 was then refluxed in trimethyl phosphite to give phosphonate 112, albeit in a lower yield. The major product obtained was cyclic carbamate 108, which is formed by an intramolecular attack on the carbonyl carbon of the Boc group, as shown in Figure 4.11.116 It should be noted that dimethyl t-butyl phosphonate was not detected in this reaction. 118    Figure 4.12: Synthesis of Boc-protected phosphonate 112 The Boc group of phosphonate 112 was removed under acidic conditions to give a free amine, which was further reacted with either Cbz-Cl or acetic anhydride to give the corresponding phosphonates, 103 and 106 (Fig 4.13). A mono-demethylation was then performed on both compounds using sodium iodide. All attempts to isolate 113 were unsuccessful; however, the phosphonic acid 114 could be isolated as a yellow solid. The next step was the coupling of phosphonic acid 114 with meso-oxa-Dap derivative 100 to form a protected phosphonamidate inhibitor. 119    Figure 4.13: Synthesis of phosphonic acids 4.3.3 Coupling of phosphonic acid 114 with meso-oxa-Dap 100 Several papers report the coupling of a phosphonic acid with a free amine using coupling reagents like PyBOP or BOP to make phosphonamidate inhibitors with good yields.121-123 Therefore, we decided to use PyBOP as a coupling reagent for the preparation of the protected phosphonamidate inhibitor 115 (Fig 4.14). However, in our case, no formation of the product was observed after monitoring the reaction for several days. There was also a report of the use of COMU and Oxyma for the coupling reactions, but this method also did not give the coupled product.124 Therefore, we decided to carry out the coupling via a phosphochloridate intermediate.  Figure 4.14: Attempted synthesis of phosphonamidate 115 using PyBOP as a coupling reagent 120   Many chlorinating agents such as SOCl2, PCl5 and oxalyl chloride have been used for preparing phosphochloridates.116, 125, 126 We decided to use oxalyl chloride as a chlorinating agent to prepare phosphochloridate 116 (Fig 4.15). This chloridate was then reacted with the meso-oxa-Dap derivative 100 to prepare phosphonamidate 115. Unfortunately, we were never able to isolate the final product. It is well documented that the P-N bond is very acid-labile,127, 128 and we thought that this compound might decompose on the silica gel column (Silicycle, F460) during purification. During the synthesis of this compound, we also found that phosphochloridate 116 decomposed when we used a rotary evaporator connected to a water aspirator during concentration.  Figure 4.15: Synthesis of phosphonamidate 115 via phosphochloridate 116 To ameliorate these problems, we decided to use high-grade silica gel (Davisil grade 643, Sigma-Aldrich) for future purification as well as use a rotary evaporator connected to a vacuum pump for the concentration of phosphochloridates.  Since the coupling was proving too problematic, and the phosphonic acid was not readily available due to the low yield of the Arbuzov reaction, we decided to look for alternative ways to synthesize the phosphonic acid. The Montchamp group has reported a radical addition of hypophosphites to olefins at room temperature in the presence of triethyl borane to give H-121   phosphinic acid (Fig 4.16).129 This H-phosphinic acid could be protected with an appropriate protecting group and then oxidized to give phosphonic acid derivative.130, 131 We decided to utilize the same strategy to synthesize a mono-benzyl-protected phosphonic acid derivative starting from a vinyl glycine derivative.  Figure 4.16: Synthesis of an H-phosphinic acid developed by the Montchamp group 4.3.3.1 Synthesis of vinyl glycine derivative 118 The vinyl glycine derivative 118 was prepared in two steps starting from bromide 105 (Fig 4.17). Bromide 105 was treated with phenyl selenide to give compound 117.132 An oxidative elimination using hydrogen peroxide afforded the vinyl glycine 118.133 This vinyl glycine was then converted to H-phosphinic acid 119 using ammonium hypophosphite and triethyl borane. 129, 134 Compound 120 was then prepared from H-phosphinic acid 119 via the Hewitt reaction, using Cbz-Cl and pyridine.131 In this reaction,  the chloroformate reacts with the tautomer of H-phosphinic acid 119 to form a mixed anhydride 112 (Fig 4.18). The nucleophilic phosphorus reacts with the benzylic oxygen atom to form compound 120 with the release of CO2.131 Oxidation using sodium 122   periodate gave the mono-benzyl-phosphonic acid 121,130 which was then used for the coupling with the meso-oxa-Dap derivative 100.  Figure 4.17: Synthesis of mono-benzyl phosphonic acid 121  Figure 4.18: Possible mechanism of the Hewitt reaction 123   The phosphonic acid 121 was converted to phosphochloridate 123 using oxalyl chloride, and the product was immediately reacted with compound 100 to give the protected phosphonamidate 124 in a moderate yield (Fig 4.19). It should be noted that yields were not reproducible because of the unstable nature of the P-N bond. This compound was then subjected to catalytic hydrogenation to prepare the final inhibitor 5. However, we could not isolate the final product. We thought that compound 5 might have decomposed after catalytic hydrogenation as the overall pH of the solution would become acidic due to the presence of the free phosphonic acid. Therefore, a base was added to later hydrogenation attempts to maintain a basic pH. Unfortunately, inhibitor 5 could still not be isolated. After closer analysis of the ESI-MS data, we found that the P-N bond breaks to give free phosphonic acid 125 and free meso-oxa-Dap 126, as well as a cyclized phosphonic acid derivative 127 formed via an intramolecular attack of a free amine at the phosphorus atom.  Figure 4.19: Attempted synthesis of inhibitor 5 After the failure in preparing inhibitor 5, we decided to use truncated phosphonic acid in which the γ-Glu mimic was replaced by a methyl group. This would simplify its preparation as 124   well as avoid any intramolecular cyclization during catalytic hydrogenation. As Csd4 is known to bind to meso-Dap, and the phosphonamidate would still retain the tetrahedral center that binds to the metal ion, such a truncated inhibitor may still be potent despite lacking the γ-Glu portion of the original design. 4.3.3.2 Coupling of mono-benzyl methyl phosphonic acid with meso-oxa-Dap derivative 100 The mono-benzyl methyl phosphonic acid was prepared in two steps using a literature known procedure (Fig 4.20). The commercially available dibenzyl phosphite 128 is converted to methyl phosphonate 129 using DBU and methyl iodide.135 The mono debenzylation was done using sodium iodide to furnish phosphonic acid 130. The phosphonic acid 130 was converted to phosphochloridate 131, which was added to the solution of meso-oxa-Dap 100 and TEA to give protected phophonamidate 132.  The catalytic hydrogenation of 132 did not yield the inhibitor 133. Several attempts were made to get the final product; however, it was ultimately unsuccessful.  Figure 4.20: Attempted synthesis of inhibitor 133 125   There have been reports of the successful removal of benzyl groups from phosphonamidates under mild catalytic conditions 127, 136, 137, but in our case, cleavage of the P-N bond was observed. Precedence for this can be found in reports by Gobec et al. and Chen et al., who also observed P-N bond cleavage during hydrogenolysis in their synthesis of phosphonamidate inhibitors.123, 138 Thus, it is not entirely clear that this deprotection method is generalizable to all P-N-containing compounds. Due to our problems preparing phosphonamidate inhibitors, we decided to synthesize the phosphonate (P-O bond) based inhibitors instead. These compounds are more stable in acidic conditions as compared to phosphonamidate (P-NH) compounds and can be easily purified by column chromatography. 4.4 Synthesis of phosphonate inhibitors Phosphonate inhibitors can be synthesized in a similar manner as phosphonamidate inhibitors, using appropriately protected phosphonic acid and an alcohol, in this case, hydroxy-meso-oxa-Dap, via a formation of phosphochloridate (Fig 4.21).  Figure 4.21: Schematic for the preparation of  a protected phosphonate inhibitor  4.4.1 Synthesis of hydroxy-meso-oxa-Dap  We considered two ways to synthesize hydroxy-meso-oxa-Dap. The first is the conversion of the free amine of meso-oxa-Dap into a hydroxyl group via a diazotization method using sodium nitrite117, and the second method is the ring-opening of an aziridine using a protected glyceric acid 126   (Fig 4.22).95 The diazotization method has been used for converting the free amine of amino acids into a hydroxyl group with retention of stereochemistry.117 However, when using sodium nitrite and HCl, we were never able to isolate the hydroxy-meso-oxa-Dap. Therefore, we decided to use the aziridine ring-opening approach to prepare the hydroxy-meso-oxa-Dap.  Figure 4.22: Different methods to prepare hydroxy-meso-oxa-Dap 4.4.1.1 Synthesis of glyceric acid 138 The transesterification of commercially available 134 into benzyl ester 135 was done using dibutyltin oxide and benzyl alcohol (Fig 4.23).139 The acetonide protecting group of compound 135 was removed, and the primary hydroxyl was protected using trityl chloride to give compound 136.139 The protection of the secondary hydroxyl group of 136 using allyl bromide in the presence of silver oxide gave compound 137.140 Deprotection of trityl group under acidic conditions furnished the desired glyceric acid 138.  127    Figure 4.23: Synthesis of hydroxy-serine 138 4.4.1.2 Ring-opening of protected aziridine using glyceric acid The ring-opening of aziridine 98 was performed using glyceric acid 138 as a nucleophile in the presence of the Lewis acid, BF3.OEt2 (Fig. 4.24).95 The protected hydroxy-Dap 139 was isolated in good yield, and it should also be noted that the Heine rearrangement product was only formed in trace amount. This might be due to the enhanced nucleophilicity of the glyceric acid hydroxyl group, as it is unable to act as a hydrogen bond acceptor with the secondary hydroxyl group.65 Instead, it likely acts as a hydrogen bond donor, and this may increase its reactivity. In any event, the formation of the ring-opening product was faster than the intramolecular rearrangement.   Figure 4.24: Synthesis of protected hydroxy-meso-oxa-Dap 128   4.4.1.3 Deprotection of allyl group using polymethylhydrosiloxane (PMHS) Several methods were initially explored to remove the allyl moiety from hydroxy-meso-oxa-Dap 139, such as morpholine/ Pd(PPh3)4, barbituric acid/ Pd(PPh3)4 as well as via isomerization of the allyl ether to an enol ether and then subsequent hydrogenation using an iridium catalyst. However, the free hydroxyl was not be obtained in any of these attempts.  A report from the Chandrasekhar group showed that the allyl moiety could be selectively removed from allyl ethers, amines, and esters using PMHS in the presence of Pd(PPh3)4 and zinc chloride (Fig 4.25).141  Figure 4.25: Cleavage of the allyl moiety employed by the Chandrasekhar group We decided to use this method for the cleavage of the allyl group from compound 139, which gave the hydroxy-meso-oxa-Dap 140 in a good yield (Fig 4.26).  Figure 4.26: Synthesis of hydroxy-meso-oxa-Dap 140 A possible mechanism for the cleavage of the allyl group starts with the formation of the π-allyl complex of 139 with the Pd(0) catalyst (Fig 4.27). Then the zinc chloride activates the 129   PMHS to promote a hydride transfer to the π-complex, giving propene and the deallylated product 140 after workup.141  Figure 4.27: Schematic representation for allyl deprotection mechanism  4.4.1.4 Coupling of mono-benzyl methyl phosphonic acid 130 with hydroxy-meso-oxa-Dap With hydroxy-meso-oxa-Dap 140 in hand, we first tried coupling it to mono-benzyl methyl phosphonic acid 130 and prepare compound 141 (Table 4.1). After deprotection, this would give the truncated phosphonate inhibitor that lacks the γ-Glu portion of the original design (analogous to the phosphonamidate inhibitor 133 in section 4.3.3.2). Initially, we employed similar conditions to those used for the preparation of protected phosphonamidates, 124 and 132, i.e., one equivalent of phosphonic acid and two equivalents of amine (Table 4.1, entry 1). However, in this case, no product was observed after monitoring the reaction for several days (entry 2-3).  The coupling of phosphonic acid 130 and alcohol 140  using PyBOP was also unsuccessful (entry 4).123 We also 130   tried a Mitsonubu reaction in the synthesis of the protected phosphonate 141, which would give the desired phosphonate with an (R)-configuration at the carbon attached to the hydroxyl group (entry 5).116 After several failed attempts, we decided to change the equivalency of phosphonic acid 130 and alcohol 140 (entry 6-10). A 2:1 ratio of 130:140 gave product, but in a low yield of  10 %. Increasing the ratio to 5:1 resulted in a better yield of 30 % (entry 7).  Table 4. 1 Optimization of the coupling reaction for the  formation of phosphonate 141  Entry Phosphonic acid (Eq.) Hydroxyl   (Eq.) Reagents Temperature (°C) Solvent (time) Yield (%) 1 1 2 (COCl)2/ TEA 0 to rt CH2Cl2  (24 h) - 2 1 2 (COCl)2/ TEA 0 to rt CH2Cl2  (48 h) - 3 1 2 (COCl)2/ TEA 0 to rt CH2Cl2  (72 h) - 4 1 2 PyBOP rt CH2Cl2  (24 h) - 5 1 2 DIAD/PPh3 0 to rt THF  (24 h) - 6 2 1 (COCl)2/ TEA 0 to rt CH2Cl2 (24h) 10 7 5 1 (COCl)2/ TEA 0 to rt CH2Cl2 (24h) 30a 8 5 1 (COCl)2/ TEA 0 to rt CH2Cl2  (1h) 80b 9 5 1 (COCl)2/ TEA rt to100 Toluene  (1h) 75b 10 5 1 (COCl)2/ TEA rt to100 Toluene (1h) 70a 131   Method a- phosphochloridate was added to the solution of alcohol and TEA at the mentioned temperature and then stirred for the given time. Method b- alcohol was added to the solution of formed phosphochloridate and TEA at the mentioned temperature and then stirred for the given time.  Hirschmann et al. reported that the treatment of phosphonochloridates with a tertiary amine prior to the addition of the nucleophile results in the formation of more reactive species than the phosphonochloridate,  which they called a phosphonyltriethylammonium salt.125 Therefore, we changed the order of addition and first added triethylamine to the phosphochloridate, followed by the alcohol. This resulted in an excellent yield of 80 % (entry 8). Similar yields were obtained when the reaction was done at an elevated temperature using toluene as a solvent (entry 9, 10). Thus, the protected phosphonate inhibitor 141 could be prepared in good yields, and all that remained was to remove the protecting groups (Fig. 4.28). The removal of all three benzyl groups and the pNZ were achieved using hydrogenation conditions, and purification with a C18 column gave the phosphonate inhibitor 142. This inhibitor will be tested for binding to the Csd4 enzyme. Our collaborator Dr. Anson Chan in the Murphy lab (UBC Microbiology), will use isothermal calorimetry (ITC) to measure the affinity. This method is superior to the kinetic assay used previously as it is not necessary to synthesize the tripeptide substrate, and problems with having an excess of enzyme over inhibitor are avoided. If inhibitor 142 shows a high affinity to Csd4, a structure of the Csd4-inhibitor complex will be obtained, and the morphological studies will be done by our collaborator Dr. Erin Gaynor (UBC Microbiology, C. jejuni) and Dr. Nina Salama (Fred Hutchison Cancer Research Center, H. pylori). 132    Figure 4.28: Synthesis of phosphonate inhibitor 142 4.5 Conclusion and summary  In this chapter, we have detailed the design and synthesis of the phosphonate inhibitor 142, which mimics the tetrahedral intermediate formed in the Csd4-catalyzed reaction. Initially, we planned to synthesize phosphonamidate (P-N bond) inhibitors, which have been reported to be potent inhibitors of metalloprotease enzymes due to the presence of hydrogen bonding with NH functionality. 82, 114, 115 However, several challenges were faced during this work. Firstly, the synthesis of monoalkyl phosphonates using trimethyl phosphite gave two side products, benzyl dimethyl phosphonate 107 and cyclized carbamate product 108 (section 4.3.2). To resolve this issue, we used an alternate method that was developed by the Montchamp group. 129 The radical addition of ammonium hypophosphite to the alkene gave the phosphinic acid 119 that was benzylated and oxidized to give the monoalkylated phosphonic acid 121 (section 4.3.3.1). The most common method employed for the synthesis of phosphonamidate inhibitors is the conversion of the phosphonic acid into phosphochloridate and then coupling to an amine. 116, 125, 126 We used the same strategy to synthesize the protected phosphonamidate 124; however, the yield was low and irreproducible (section 4.3.3.1). It is well-known that the P-N bond is acid-labile, and 133   decomposition can occur during the column chromatography. 127, 128 The catalytic hydrogenation of the protected phosphonate resulted only in the formation of by-products, some of which were formed from an intramolecular cyclization. Therefore, we decided to use a truncated, mono-benzyl methyl phosphonic acid 130 for coupling with the free amine of the meso-oxa-Dap derivative 100 (section 4.3.3.2). Unfortunately, the attempted removal of the protecting group's hydrogenation did not give the desired phosphonamidate inhibitor.  Following our difficulties in preparing a phosphonamidate inhibitor, we moved towards the synthesis of phosphonate inhibitor 142. Phosphonates have also been shown to acts as extremely potent inhibitors of metalloenzymes such as carboxypeptidase A.115 The hydroxy-serine 138 was prepared in five overall steps starting from commercially available dioxolane (section 4.4.1.1). This was used in the ring-opening of protected aziridine 98 to give the protected hydroxy-meso-oxa-Dap (section 4.4.1.2). Deprotection of allyl group using PHMS/ZnCl2/Pd(PPh3)4 gave the alcohol 140 that was used for coupling with mono-benzyl methyl phosphonic acid 130. This coupling was optimized by using an excess of the phosphonic acid and by adding the triethylamine prior to the alcohol. The protecting groups were removed without incident to give the final phosphonate inhibitor 142 in good overall yield. Inhibition studies with Csd4 are currently underway in the Murphy laboratory.       134   4.6 Experimental procedures 4.6.1 General information All reactions were performed using flame-dried glassware unless otherwise stated. All reagents were purchased from Sigma-Aldrich, AK Scientific or Combi-blocks. DCM and triethylamine were distilled under Argon from calcium hydride. Anhydrous toluene was purchased from Sigma-Aldrich. All other solvents and reagents were used without further purification. NMR spectra were recorded on a Bruker AV400 spectrometer or a Bruker AV300 spectrometer at a field strength of 400 MHz or 300 MHz for 1H NMR , 101 MHz or 75 MHz for 13C NMR and 122MHz for 31P NMR. Column chromatography was performed using SiliaFlash silica gel F60, 40 - 63 µm purchased from Silicycle. High purity Davisil grade 635 (Sigma-Aldrich) silica gel was used for the purification of phosphonamidate and phosphonate precursors. Thin-layer chromatography was performed using silica gel 60 F254 purchased from Merck and visualized under short wave UV or KMnO4 stain. High-resolution mass spectra were recorded on a Waters/Micro mass LCT TOF spectrometer equipped with electrospray (ESI) ionization. 4.6.2 Synthesis of meso-oxa-Dap 100 4.6.2.1 Aziridine 98  Trifluoroacetic acid (10 mL) was added dropwise over 10 min to a solution of 97 (5.57 g, 13.3 mmol) in DCM (10 mL) and methanol (10 mL) at 0 °C. The solution was stirred for 1 h at 0 °C. Volatiles were removed by azeotroping with Et2O (3 x10 mL). The residue was partitioned 135   between Et2O (50 mL) and H2O (50 mL), and the ether layer was extracted with water (3 x20 mL). The combined aqueous layers were basified to pH 9 with NaHCO3 at 0 °C. Ethyl acetate (100 mL) was added to the aqueous solution followed by 4-nitrobenzyl chloroformate (2.86 g, 13.3 mmol) at 0 °C. The resulting immiscible layers were warmed to room temperature and stirred vigorously for 24 h. After completion of the reaction, the two layers were separated, and the aqueous layer was extracted with EtOAc (3 x 30 mL). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated in vacuo. The residue was further purified by silica gel chromatography (50% ethyl acetate in petroleum ether) to give aziridine 98 as a colourless liquid (3.3 g, 70%).1H NMR (400 MHz, CDCl3) δ 8.20 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 8.7 Hz, 2H), 7.42 – 7.30 (m, 5H), 5.26 – 5.10 (m, 4H), 3.20 (dd, J = 5.2, 3.2 Hz, 1H), 2.67 (dd, J = 3.2, 1.3 Hz, 1H), 2.53 (dd, J = 5.2, 1.3 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 168.1, 160.3, 147.9, 142.7, 134.9, 128.9, 128.8, 128.6, 128.6, 123.9, 67.8, 67.0, 35.2, 31.7. HRMS (ESI): m/z calcd for C18H16N2O6Na  [M+Na]+ 379.0906, found 379.0900. 4.6.2.2 Protected meso-oxa-Dap 99  BF3.OEt2 ( 0.18 mL, 1.40 mmol) was added dropwise to a stirred solution of compound 98  (1.00 g, 2.81 mmol) and N-Pht-Ser-OBn (1.83 g, 5.61 mmol) in toluene (15 mL) at rt. The resulting reaction mixture was then refluxed at 110 ˚C for 3 h. After completion of the reaction, the solvent was removed under reduced pressure and the residue was purified by silica gel chromatography (5% ethyl acetate in dichloromethane) to give oxa-Dap 99 as a white solid ( 0.96 g, 50%). 1H NMR (400 MHz, CDCl3) δ 8.19 (d, J = 8.6 Hz, 2H), 7.85 – 7.80 (m, 2H), 7.75 – 7.68 (m, 2H), 7.49 (d, 136   J = 8.4 Hz, 2H), 7.35 – 7.26 (m, 9H), 7.24 – 7.19 (m, 2H), 5.75 (d, J = 8.8 Hz, 1H), 5.26 – 5.11 (m, 6H), 4.90 (q, J = 12.3 Hz, 2H), 4.51 – 4.43 (m, 1H), 4.23 – 4.08 (m, 2H), 3.92 (dd, J = 9.4, 3.0 Hz, 1H), 3.81 (dd, J = 9.4, 3.0 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 169.6, 167.5, 167.1, 155.7, 147.7, 144.0, 135.2, 135.0, 134.4, 131.9, 128.8, 128.7, 128.7, 128.6, 128.4, 128.2, 128.0, 123.9, 123.7, 71.0, 68.1, 67.9, 67.3, 65.5, 54.6, 51.5. HRMS (ESI): m/z calcd for C36H32N3O11  [M+H]+ 682.2037, found 682.2039. 4.6.2.3 Amine 100  Compound 99 (440 mg, 0.65 mmol) was dissolved in THF (5 mL) and hydrazine (50 % in water) (50 µl, 0.78 mmol) was added at 0 °C. After 2 h, 1 mL AcOH was added and the solution was stirred for 10 min at 50 °C. Volatiles were removed under reduced pressure to give a white solid. The solid was triturated with 5 mL ethyl acetate and filtered. The filtrate was evaporated to give an oily crude which was dissolved in diethyl ether (10mL) and 4 M HCl in dioxane (20 mL) was added to it. The solution was stirred for 15 min at rt and the volatiles were removed azeotropically with diethyl ether ( 3x20 mL) under reduced pressure to give amine 100 as a white solid ( 0.32 g, 90 %). 1H NMR (400 MHz, MeOD) δ 8.19 (d, J = 8.4 Hz, 2H), 7.58 (d, J = 8.3 Hz, 2H), 7.45 – 7.28 (m, 10H), 5.32 – 5.13 (m, 6H), 4.54 (t, J = 4.4 Hz, 1H), 4.36 (t, J = 3.4 Hz, 1H), 4.03 (dd, J = 10.7, 3.8 Hz, 1H), 3.95 – 3.81 (m, 3H). 13C NMR (101 MHz, MeOD) δ 170.0, 167.0, 157.0, 147.7, 144.4, 135.7, 134.9, 128.5, 128.5, 128.4, 128.3, 128.2, 128.0, 127.9, 123.3, 70.9, 137   68.2, 68.1, 67.1, 65.2, 54.5, 53.2. HRMS (ESI): m/z calcd for C28H30N3O9  [M+H]+ 552.1982, found 552.1982. 4.6.3 Synthesis of mono-protected phosphonate derivative 121 4.6.3.1 General procedure for preparation of bromo derivatives 102,105, and 111 The preparation of bromo derivatives of glutamic acid was done using the procedure developed by Takahashi et al., 2001.120 The protected glutamic acid (1.0 eq) was dissolved in THF (15 mL). Then 2-mercaptopyridine N-oxide (1.1 eq) , DCC (1.1 eq) and DMAP (0.1 eq) were added sequentially at 0 °C. The reaction mixture was covered with Al foil for 3 h. After 1 h, CBrCl3 (25 eq.) was added, and the reaction mixture kept stirring at 0 °C. After 3 h, the Al foil was removed, and the reaction mixture was irradiated with a 100 W lamp for 2 h at rt. The reaction mixture was filtered and concentrated under reduced pressure to give the residue, which was purified by silica gel column chromatography. Bromo 102:   The general procedure was used to prepare compound 102 from N-Ac-D-Glu-1-OBn (3.0 g, 10.74 mmol) to gave a white solid (1.00 g, 29 %) (silica-gel column chromatography: 15% ethyl acetate in petroleum ether). 1H NMR (400 MHz, CDCl3) δ 7.48 – 7.31 (m, 5H), 6.19 (d, J = 7.8 Hz, 1H), 5.22 (s, 2H), 4.88 – 4.64 (m, 1H), 3.50 – 3.30 (m, 2H), 2.57 – 2.42 (m, 1H), 2.39 – 2.13 (m, 1H), 2.06 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 171.4, 170.0, 134.9, 128.7, 128.7, 128.4, 67.7, 51.5, 35.5, 27.9, 23.2. HRMS (ESI): m/z calcd for C13H16BrNO3Na [M+Na]+ 336.0211, found 336.0216. 138   Bromo 105 :   The general procedure was used to prepare compound 105 from N-Cbz-D-Glu-1-OBn (3.0 g, 8.08 mmol)  and gave a white solid (1.64 g, 50 %) (silica-gel column chromatography: 10% ethyl acetate in petroleum ether). 1H NMR (300 MHz, CDCl3) δ 7.49 – 7.26 (m, 10H), 5.39 (d, J = 7.9 Hz, 1H), 5.23 – 5.10 (m, 4H), 4.63 – 4.51 (m, 1H), 3.47 – 3.33 (m, 2H), 2.54 – 2.37 (m, 1H), 2.37 – 2.18 (m, 1H).The spectral data match with literature values.142  Bromo 111:   The general procedure was used to prepare compound 111 from N-Boc-D-Glu-1-OBn (3.2 g, 9.49 mmol)  and gave a white solid (2.7 g, 76 %) (silica-gel column chromatography: 15% ethyl acetate in petroleum ether). 1H NMR (400 MHz, CDCl3) δ 7.42 – 7.31 (m, 5H), 5.25 – 5.14 (m, 2H), 5.14 – 5.04 (m, 1H), 4.52 – 4.41 (m, 1H), 3.41 (m, 2H), 2.48 – 2.35 (m, 1H), 2.29 – 2.14 (m, 1H), 1.45 (s, 9H). The spectral data match with literature values.142  4.6.3.2 N-boc phosphonate 112  In a round bottom flask contaning compound 111 (2.6 g, 6.98 mmol) was added trimethyl phosphite (16.6 mL, 140 mmol). The reaction mixture was refluxed at 120 °C for 24 h, and then 139   concentrated under reduced pressure. Silica gel chromatography (50% ethyl acetate in petroleum ether) gave phosphonate 112 as a colorless liquid (0.48 g, 17 %) 1H NMR (400 MHz, CDCl3) δ 7.40 – 7.29 (m, 5H), 5.24 – 5.11 (m, 3H), 4.43 – 4.32 (m, 1H), 3.71 (d, J = 2.9 Hz, 3H), 3.68 (d, J = 2.7 Hz, 3H), 2.20 – 2.09 (m, 1H), 1.95 – 1.86 (m, 1H), 1.85 – 1.66 (m, 2H), 1.43 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 171.8, 155.5, 135.3, 128.8, 128.7, 128.5, 80.3, 67.5, 53.7 (d, J = 17.9 Hz), 52.6 (d, J = 6.6 Hz), 28.4, 25.9 (d, J = 3.9 Hz), 20.9 (d, J = 143.4 Hz).31P NMR (162 MHz, CDCl3) δ 34.0. HRMS (ESI): m/z calcd for C18H28NO7NaP [M+Na]+ 424.1501, found 424.1502. Oxazinone 108:   The cyclized product 108 was isolated during the purification of compound 112 by silica gel column chromatography (45% ethyl acetate in petroleum ether) as a white solid (0.66 g, 40 %) 1H NMR (400 MHz, CDCl3) δ 7.45 – 7.32 (m, 5H), 5.95 (s, 1H), 5.29 – 5.12 (m, 2H), 4.34 – 4.24 (m, 2H), 4.23 – 4.16 (m, 1H), 2.40 – 2.26 (m, 1H), 2.22 – 2.03 (m, 1H). 13C NMR (101 MHz, CDCl3) δ 170.1, 152.7, 134.9, 129.0, 128.9, 128.6, 68.0, 65.1, 52.5, 24.2. HRMS (ESI): m/z calcd for C12H14NO4  [M+H]+ 236.0923, found 236.0921. 4.6.3.3 N-acetyl phosphonate 103  140   In a round bottom flask containing compound 111 (0.500 g, 1.25 mmol) was added 4M HCl in dioxane (10 mL) at 0 °C . After stirring for 1 h, volatiles were removed under reduced pressure and co-evaporated with diethyl ether (3 x10 mL) to give a white solid that was used without further purification. The crude free amine salt was dissolved in H2O (5 mL), and the pH was adjusted to 8 using saturated NaHCO3 solution. Acetic anhydride ( 1.12 mL, 12.5 mmol) was added to it and the mixture was stirred for 18 h at rt. The reaction mixture was partitioned between H2O (10 mL) and ethyl acetate (10 mL). The organic layer was separated and the aqueous layer was extracted with ethyl acetate (3X10 mL). The combined organic layer was dried over Na2SO4, filtered, and evaporated under reduced  pressure to give the residue. Silica-gel column chromatography (70 % ethyl acetate in petroleum ether ) gave phosphonate 103 as a white solid (0.25 g, 57 %). 1H NMR (400 MHz, CDCl3) δ 7.41 – 7.29 (m, 5H), 6.59 (d, J = 7.8 Hz, 1H), 5.24 – 5.12 (m, 2H), 4.73 – 4.61 (m, 1H), 3.71 (d, J = 4.7 Hz, 3H), 3.68 (d, J = 4.7 Hz, 3H), 2.23 – 2.09 (m, 1H), 2.03 (s, 3H), 2.02 – 1.88 (m, 1H), 1.83 – 1.63 (m, 2H).  13C NMR (101 MHz, CDCl3) δ 171.6, 170.3, 135.2, 128.8, 128.7, 128.5, 67.5, 52.6 (dd, J = 6.6, 1.9 Hz), 52.4 (d, J = 16.3 Hz), 25.5 (d, J = 4.0 Hz), 23.2, 20.9 (d, J = 143.0 Hz). 31P NMR (162 MHz, CDCl3) δ 34.15. HRMS (ESI): m/z calcd for C15H23NO6P  [M+H]+ 344.1263, found 344.1261. 4.6.3.4 N-Cbz phosphonate 106  In a round bottom flask containing compound 111 ( 0.355 g, 0.88 mmol) was added 4M HCl in dioxane (5 mL) at 0 °C. After stirring for 1 h, volatiles was removed under reduced pressure 141   and co-evaporated with diethyl ether (3 x10 mL) to give a white solid that was used without further  further purification. The crude free amine salt was dissolved in H2O (5 mL) and the pH was adjusted to 8 using saturated NaHCO3 solution. Cbz-Cl (0.19 mL, 1.33 mmol) was added dropwise at 0 °C and the mixture was stirred for 18 h at rt. The reaction mixture was partitioned between H2O (10 mL) and ethyl acetate (10 mL). The organic layer was separated and the aqueous layer was extracted with ethyl acetate (3X10 mL). The combined organic layers were dried over Na2SO4, filtered and evaporated under reduced pressure to give residue. Silica gel column chromatography (50 % ethyl acetate in petroleum ether) gave phosphonate 106 as a colourless liquid (0.35 g, 91%). 1H NMR (300 MHz, CDCl3) δ 7.41 – 7.35 (m, 10H), 5.51 (d, J = 8.2 Hz, 1H), 5.23 (s, 2H), 5.14 (s, 2H), 4.65 – 4.45 (m, 1H), 3.74  (s, 3H), 3.78 (s, 3H), 2.32 – 2.13 (m, 1H), 2.15 – 1.93 (m, 1H), 1.82 – 1.73 (m, 2H). The spectral data matches with the literature values (Watanabe et al., 2017). 4.6.3.5 General procedure for mono-dealkylation of protected phosphonates 106 and 129 Sodium iodide (3 eq) was added to the solution of protected phosphonate (1 eq) in acetone. The reaction mixture was refluxed at 60 °C for 24 h. The mixture was concentrated under reduced pressure and partitioned between H2O and ethyl acetate. The aqueous layer was washed with ethyl acetate three times. The aqueous layer was acidifed to pH 2-3 using dilute HCl solution and then extracted with ethyl acetate. The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to give residue which was used without further purification. Phosphonic acid 114:   142   The general procedure was used to prepare compound 114 from compound 106 (400 mg, 0.92 mmol) to give a colorless liquid (311 mg, 80%). 1H NMR (400 MHz, CDCl3) δ 7.42 – 7.29 (m, 10H), 5.19 (s, 2H), 5.12 (s, 2H), 4.52 – 4.41 (m, 1H), 3.66 (d, J = 11.2 Hz, 3H), 2.27 – 2.12 (m, 1H), 2.04 – 1.93 (m, 1H), 1.89 – 1.69 (m, 2H). The spectral data matches with the literature values.134  Phosphonic acid 130:   The general procedure was used to prepare compound 130 from compound 129 (0.85 g, 3.10 mmol) to give a colourless liquid (0.46 g, 80%). 1H NMR (400 MHz, CDCl3) δ 7.44 – 7.31 (m, 5H), 5.71 (s, 2H), 5.06 (d, J = 8.1 Hz, 2H), 1.54 (d, J = 18.0 Hz, 3H). The spectral data matches with the literature values.143  4.6.3.6 Selenide 117  The preparation of selenium derivative of N-Cbz protected glutamic acid was done using the procedure developed by Xiao et al., 2008.132 NaBH4 (0.13 g, 3.51 mmol) was added slowly to a solution of diphenyl diselenide (0.48 g, 1.40 mmol) in EtOH (20 mL), and the mixture was stirred under N2 atmosphere at rt until the yellow solution became colourless. A solution of 105 (0.57 g, 1.40 mmol) in EtOH (10 mL) was then added dropwise and the mixture stirred at rt for 1 h. After EtOH was removed under reduced pressure, and residue was dissolved in water (20 mL). The 143   desired product 117 was extracted with diethyl ether (4 × 20 mL). The combined organic phases were washed with saturated aqueous NaHCO3 solution (2 × 25 mL), water (2 × 25 mL), and brine (25 mL). The resulting solution was dried over Na2SO4, filtered, and then concentrated under reduced pressure. Purification by silica gel chromatography (30 % ethyl acetate in petroleum ether) gave the Selenide 117 ( 0.57 g, 84%) as a light yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.52 – 7.46 (m, 2H), 7.43 – 7.32 (m, 10H), 7.30 – 7.22 (m, 3H), 5.53 (d, J = 8.4 Hz, 1H), 5.26 – 5.04 (m, 4H), 4.65 – 4.57 (m, 1H), 2.89 (t, J = 7.7 Hz, 2H), 2.34 – 2.18 (m, 1H), 2.15 – 2.00 (m, 1H). 13C NMR (101 MHz, CDCl3) δ 171.8, 156.0, 135.2, 133.1, 129.2, 128.7, 128.6, 128.6, 128.3, 128.3, 128.2, 127.2, 67.4, 67.1, 54.1, 33.3, 23.0. HRMS (ESI): m/z calcd for C25H26NO4Se  [M+H]+ 484.1027, found 484.1023. 4.6.3.7 Alkene 118  Oxidative elimination of compound 117 was done using procedure developed by Bartley et al. 2005.133 To a solution of compound 117 (2.40 g, 4.2 mmol) in DCM (25 mL) were added 30% H2O2 (10 mL) and pyridine (10 mL) and the solution was refluxed at 45 °C for 3 h. The reaction was cooled to rt and diluted with DCM (30mL). The mixture was washed with H2O, saturated aq. CuSO4, 1 M HCl, saturated aq. NaHCO3, and brine (50 mL each). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. Purification by silica gel chromatography (15 % ethyl acetate in petroleum ether ) gave the alkene 118 ( 0.81 g, 50%) as a light yellow solid. 1H NMR (300 MHz, CDCl3) δ 7.43 – 7.28 (m, 10H), 5.99 – 5.83 (m, 1H), 5.45 (d, J = 8.1 Hz, 1H), 5.35 (dd, J = 17.1, 1.9 Hz, 1H), 5.27 (dd, J = 10.3, 1.7 Hz, 1H), 5.22 – 144   5.16 (m, 2H), 5.14 – 5.11 (m, 2H), 5.04 – 4.93 (m, 1H). The spectral data matches with the literature values.144  4.6.3.8 H-phosphinate 119  The compound 119 was prepared using procedure developed by Watanabe et al. 2017.140 Triethylborane solution (1 M in THF, 4.92 mL, 4.92 mmol) was added to the solution of compound 118 (0.80 g, 2.5 mmol) and ammonium hypophosphite (0.61 g, 7.4 mmol) in anhydrous methanol (15 mL). The reaction mixture was stirred vigorously for 16 h at rt . The mixture was concentrated under reduced pressure and to the residue was added 10% aqueous solution of potassium hydrogen carbonate (50 mL) and extracted with ethyl acetate (3X50 mL). The combined organic layer was washed with brine (50 mL), over Na2SO4, filtered, and concentrated under reduced pressure to give phosphinate 119 (0.95 g, quant) as a colorless oil. This was used without purification. 1H NMR (300 MHz, CDCl3) 7.40-7.32 (m, 10H), 7.01 (d, J= 554 Hz, 1H), 5.60 (br s, 1H), 5.16 (br s, 2H), 5.08 (s, 2H), 4.49-4.49 (m, 1H), 2.24-2.01(m, 1H), 2.02-1.84(m, 1H), 1.83-1.65(m, 2H). 31P NMR (121 MHz, CDCl3) δP: 36.5. The spectral data matches with the literature values.134  4.6.3.9 Benzyl phosphinate 120  Pyridine (0.18 mL, 2.16 mmol) was carefully added to a vigorously stirred solution of Cbz-Cl (0.31 mL, 2.16 mmol) and compound 119 (0.77 g, 1.97 mmol) in DCM (20 mL) at rt. Once 145   effervescence had stopped, the solution was refluxed for 20 min, then allowed to cool to rt. The solution was poured into 0.1 M hydrochloric acid (30 mL) and the organic layer was separated. The organic layer was washed with H2O (50 mL), dried over Na2SO4, filtered and concentrated in vacuo to give phosphinate 120 as a colourless oil (0.66 g, 70 %). It should be noted that this compound is susceptible to decomposition and should be used immediately. 1H NMR (400 MHz, CDCl3) δ 7.4 – 7.3 (m, 15H), 7.3 (d, J = 544.6 Hz, 1H), 5.7 (t, J = 7.9 Hz, 1H), 5.2 – 5.0 (m, 6H), 4.5 (q, J = 6.9 Hz, 1H), 2.3 – 2.1 (m, 1H), 2.0 – 1.9 (m, 1H), 1.9 – 1.7 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 171.3, 156.1, 136.2, 135.5 (dd, J = 5.7, 1.6 Hz), 135.1, 128.9, 128.8, 128.8, 128.7, 128.6, 128.4, 128.3 (d, J = 1.9 Hz), 128.3, 67.9 (dd, J = 6.5, 2.0 Hz), 67.7, 67.3, 65.4, 54.0 (d, J = 17.7 Hz), 24.9 (d, J = 93.7 Hz), 24.2. 31P NMR (162 MHz, CDCl3) δ 37.41, 37.32. HRMS (ESI): m/z calcd for C26H29NO6P  [M+H]+ 482.1732, found 482.1733 (in acetonitrile) analysis using MeOH as solvent showed transesterification. 4.6.3.10 Phosphonic acid 121  Compound 120 (0.66 g, 1.37 mmol) was dissolved in dioxane (5 mL) and treated with NaIO4 (0.35 g, 1.65 mmol) in water (5 mL). After stirring at rt for 18 h, the mixture was concentrated under reduced pressure and then partitioned between ethyl acetate and 2% KHSO4 (20 mL each). The organic layer was washed successively with water (20 mL), 5% NaHSO3(20 mL) and saturated NaCl solution (20 mL). The solution was dried over Na2SO4 and evaporated under reduced pressure to give phosphonic acid 121 as a colourless oil (0.66 g, quantitative). 1H 146   NMR (300 MHz, CDCl3) δ 8.41 (broad s, 1H), 7.31 (d, J = 2.7 Hz, 15H), 5.48 ( broad s, 1H), 5.19 – 5.02 (m, 4H), 4.97 (d, J = 8.1 Hz, 2H), 4.47 – 4.33 (m, 1H), 2.30 – 2.05 (m, 1H), 2.05 – 1.86 (m, 1H), 1.84 – 1.60 (m, 2H). 13C NMR (75 MHz, CDCl3) δ 171.5, 156.1, 136.2 (d, J = 3.7 Hz), 136.1, 135.2, 128.8, 128.7, 128.7, 128.6, 128.4, 128.4, 128.3, 128.0, 67.6, 67.3, 66.9 (d, J = 6.1 Hz), 54.1 (d, J = 18.7 Hz), 25.7, 22.2 (d, J = 145.3 Hz). 31P NMR (122 MHz, CDCl3) δ 33.29. HRMS (ESI): m/z calcd for C26H30NO7P  [M+H]+ 498.1862, found 498.1860. 4.6.4 Synthesis of protected phosphonamidate inhibitors 124 and 132 4.6.4.1 General procedure for coupling of phosphonic acids 121 and 130 with meso-oxa-Dap 100 Oxalyl chloride ( 2 eq) was added dropwise to a solution of phosphonic acid (1 eq) and DMF (0.05 eq) in anhydrous DCM at 0 °C and the reaction mixture was stirred for 2 h at rt. The volatiles were removed under reduced pressure to give crude phosphochloridate which was used immediately for coupling. A solution of the phosphochloridate in anhydrous DCM (2 mL) was added dropwise to a solution of meso-oxa-Dap 100 (1.5 eq) and triethylamine (3 eq) in DCM (2mL) at 0 °C over 20 min. The mixture was stirred for 24 h, then concentrated under vacuum to give crude phosphonamidate which was by silical gel column chromatography. Phosphonamidate 124:   The general procedure was used to prepare compound 124 from compound 121 (50 mg, 0.10 mmol) to give compound 124 as a colourless liquid  (36 mg, 35%, diastereomeric mixture) ( 147   silical gel chromatography: 70% ethyl acetate in petroleum ether ).  1H NMR (300 MHz, CDCl3) δ 8.18 – 8.06 (m, 2H), 7.44 (t, J = 9.2 Hz, 2H), 7.38 – 7.24 (m, 26H), 7.00 (d, J = 8.3 Hz, 1H), 6.50 (d, J = 8.3 Hz, 1H), 6.04 (d, J = 8.3 Hz, 1H), 5.76 (d, J = 8.1 Hz, 1H), 5.21 – 5.05 (m, 10H), 5.04 – 4.88 (m, 2H), 4.58 – 4.46 (m, 1H), 4.46 – 4.32 (m, 1H), 4.29 – 4.18 (m, 1H), 4.17 – 4.05 (m, 1H), 3.94 – 3.83 (m, 1H), 3.77 – 3.66 (m, 2H), 3.65 – 3.50 (m, 2H), 2.18 (s, 1H), 2.09 – 1.95 (m, 1H), 1.91 – 1.71 (m, 2H). 31P NMR (121 MHz, CDCl3) δ 34.70, 34.24. . HRMS (ESI): m/z calcd for C54H56N4O15P  [M+H]+ 1031.3480, found 1031.3479. Phosphonamidate 132:   The general procedure was used to prepare compound 132 from compound 130 (50 mg, 0.27 mmol) to give compound 132 as a colourless liquid  (58 mg, 30%, diastereomeric mixture) (silica gel chromatography: 70% ethyl acetate in petroleum ether). 1H NMR (400 MHz, CDCl3) δ 8.21 – 8.14 (m, 2H), 7.53 – 7.46 (m, 2H), 7.38 – 7.31 (m, 15H), 6.60 (d, J = 8.4 Hz, 0H), 6.36 (d, J = 8.3 Hz, 1H), 5.31 – 5.24 (m, 1H), 5.21 – 5.12 (m, 6H), 5.07 – 4.91 (m, 3H), 4.54 – 4.47 (m, 1H), 4.26 – 4.17 (m, 1H), 3.97 – 3.89 (m, 1H), 3.81 – 3.72 (m, 2H), 3.71 – 3.55 (m, 2H), 1.54 (d, J = 16.9 Hz, 3H). 31P NMR (162 MHz, CDCl3) δ 33.78, 33.75. HRMS (ESI): m/z calcd for C36H39N3O11P  [M+H]+ 742.2142, found 742.2143.  148   4.6.5 Synthesis of hydroxy meso-oxa-Dap 140 4.6.5.1 Synthesis of glycerate 137  The protection of the secondary alcohol of hydroxy-serine 136 was done using the procedure developed by Eichelberger et al., 2001.140 Allyl bromide (0.56 mL, 6.48 mmol) was added dropwise to a solution of compound 136 (1.42 g, 3.24 mmol) in dry diethyl ether (30 mL). The mixture was stirred under reflux for 10 min, and silver oxide (2.25 g, 9.71 mmol) was added in three portions over a period of 15 min. The stirring was continued at reflux for 2 h and at 20°C for 48 h (exclusion of light). The reaction mixture was filtered and washed several times with diethyl ether.  The combined organic solutions were concentrated under vacuum to give a residue which was purified by silica gel column chromatography (15% ethyl acetate in petroleum ether) to give glycerate 137 ( 1.34 g, 87 %) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.47 – 7.38 (m, 6H), 7.33 – 7.20 (m, 14H), 5.99 – 5.86 (m, 1H), 5.36 – 5.12 (m, 4H), 4.27 – 4.17 (m, 1H), 4.13 (dd, J = 5.3, 4.2 Hz, 1H), 4.07 – 3.98 (m, 1H), 3.47 (dd, J = 9.5, 4.2 Hz, 1H), 3.39 (dd, J = 9.5, 5.3 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 170.9, 143.8, 135.7, 134.2, 128.8, 128.6, 128.4, 128.4, 127.9, 127.1, 117.9, 86.8, 77.9, 71.8, 66.8, 64.6. HRMS (ESI): m/z calcd for C32H30O4Na [M+Na]+ 501.2042, found 501.2034. 4.6.5.2 O-allyl glycerate 138  149   TFA ( 10 mL) was added dropwise to a solution of compound 137 ( 1.92 g, 4.01 mmol) in DCM (10 mL) at 0 °C. The reaction mixture was stirred for 1 h at 0 °C and then concentrated under reduced pressure to give yellow liquid. The remaining volatiles was removed azeotropically using diethyl ether ( 3x 20 mL) under reduced pressure to give a residue which was purified by silica gel column chromatography ( 50% ethyl acetate in petroleum ether) to give O-allyl glycerate 138 as a colourless liquid. 1H NMR (300 MHz, CDCl3) δ 7.40 – 7.32 (m, 5H), 5.97 – 5.79 (m, 1H), 5.34 – 5.14 (m, 4H), 4.32 – 4.20 (m, 1H), 4.09 (dd, J = 5.8, 3.7 Hz, 1H), 4.05 – 3.97 (m, 1H), 3.94 – 3.80 (m, 2H). 13C NMR (75 MHz, CDCl3) δ 170.6, 135.5, 133.8, 128.8, 128.6, 128.4, 118.6, 78.6, 72.0, 67.0, 63.6. HRMS (ESI): m/z calcd for C13H16O4Na [M+Na]+ 259.0946, found 259.0938. 4.6.5.3 Hydroxy-Dap 139  To a stirred solution of compound 98 (0.80 g, 2.25 mmol) and compound 138 (1.06 g, 4.49 mmol) in toluene (10 mL) was added BF3.OEt2 (0.14 mL, 1.12 mmol) dropwise at rt. The resulting reaction mixture was then refluxed at 110 ˚C for 3 h. The solvent was removed under reduced pressure and the residue was purified by silica gel chromatography (50% ethyl acetate in petroleum ether) to give Dap 139 as a white solid (0.93 g, 70%). 1H NMR (300 MHz, CDCl3) δ 8.17 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 8.5 Hz, 2H), 7.38 – 7.28 (m, 10H), 5.92 (d, J = 8.8 Hz, 1H), 5.89 – 5.76 (m, 1H), 5.31 – 5.01 (m, 9H), 4.53 – 4.42 (m, 1H), 4.23 – 4.11 (m, 1H), 4.11 – 3.99 (m, 2H), 3.97 – 3.87 (m, 1H), 3.87 – 3.68 (m, 3H). 13C NMR (75 MHz, CDCl3) δ 170.3, 170.0, 155.8, 147.7, 144.0, 135.5, 135.4, 133.8, 128.8, 128.7, 128.6, 128.3, 128.3, 128.1, 123.9, 118.2, 78.2, 72.0, 71.9, 150   71.8, 67.5, 67.0, 65.5, 54.9. HRMS (ESI): m/z calcd for C31H33N2O10 [M+H]+ 593.2135, found 593.2133. 4.6.5.4 Hydroxyl 140  Polymethylhydrosiloxane (0.41 mL, 6.83 mmol, 5H eq.), Pd(PPh3)4 (0.16 g, 0.14 mmol), and ZnCl2 (1M in diethyl ether) (0.37 g, 2.73 mmol) were added to a solution of 139 (0.81 g, 1.37 mmol) in dry THF (10 mL) at room temperature under an argon atmosphere, and the mixture was stirred for 18 h. The mixture was diluted with water (20 mL) and extracted with diethyl ether (3x 20 mL). The organic layers were washed with brine ( 50 mL), dried over Na2SO4, and evaporated under reduced pressure to give a residue which was purified by silica gel column chromatography  (70 % ethyl acetate in petroleum ether ) to give the hydroxyl 140  (0.61 g, 80%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 8.17 (d, J = 8.6 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 7.38 – 7.23 (m, 10H), 5.87 (d, J = 8.8 Hz, 1H), 5.18 (dd, J = 12.2, 4.2 Hz, 6H), 4.55 – 4.46 (m, 1H), 4.28 (t, J = 3.3 Hz, 1H), 3.97 (dd, J = 9.8, 3.3 Hz, 1H), 3.84 – 3.67 (m, 3H), 2.60 ( broad s, 1H).  3C NMR (75 MHz, CDCl3) δ 172.3, 169.9, 155.8, 147.7, 143.8, 135.3, 135.1, 128.8, 128.8, 128.6, 128.4, 128.3, 128.1, 123.9, 73.0, 71.8, 71.1, 67.7, 67.6, 65.6, 54.8. HRMS (ESI): m/z calcd for C28H29N2O10 [M+H]+ 553.1822, found 553.1818.  151   4.6.6 Synthesis of phosphonate inhibitor 142 4.6.6.1 Phosphonate 141  Oxalyl chloride ( 48 µL, 0.54 mmol) was added dropwise to a solution of phosphonic acid 130 (50.5 mg, 0.27 mmol) and DMF (2 µL, 0.03 mmol) in anhydrous DCM (1 mL) at 0 °C and the reaction mixture was stirred for 30 min at 0 °C and then at rt for 1.5 h . After 2 h, the volatiles were removed under reduced pressure to give crude phosphochloridate. Triethylamine (83 µL, 0.60 mmol) was added dropwise to a solution of the phosphochloridate in DCM (1 mL) at  0 °C and the mixture was stirred for 15 min. A solution of hydroxy-meso-oxa-Dap 140 (30 mg, 0.05 mmol, limiting reagent) in DCM (1 mL) was added dropwise to the mixture over a period of 15 min at 0 °C. The mixture was stirred for 1 h at rt and then concentrated under vacuum to give the crude phosphonate which was purified by silica gel column chromatography (70% ethyl acetate in hexane ) to give compound 141 (32 mg, 80 %, diastereomeric mixture) as a colourless liquid.  1H NMR (300 MHz, CDCl3, major stereoisomer) δ 8.12 (d, J = 8.3 Hz, 2H), 7.45 (d, J = 8.5 Hz, 2H), 7.39 – 7.26 (m, 15H), 6.53 (d, J = 8.4 Hz, 1H), 5.25 – 4.94 (m, 9H), 4.53 – 4.43 (m, 1H), 3.99 (dd, J = 9.4, 3.1 Hz, 1H), 3.88 – 3.72 (m, 2H), 3.63 – 3.54 (m, 1H), 1.56 (d, J = 18.1 Hz, 3H). 31P NMR (122 MHz, CDCl3) δ 33.12(major), 32.90 (minor). 13C NMR (75 MHz, CDCl3, major stereoisomer) δ 169.8, 168.1, 156.1, 147.6, 144.2, 144.1, 136.3 (d, J = 6.8 Hz), 135.4, 134.9, 128.9, 128.7, 128.6, 128.5, 128.2, 128.0, 127.9, 123.8, 73.8 (d, J = 6.1 Hz), 71.8, 71.5 (d, J = 5.1 Hz), 67.8, 67.4, 66.9 (d, J = 6.5 Hz), 65.4, 54.8, 12.3 (d, J = 145.7 Hz). HRMS (ESI): m/z calcd for C36H38N2O12P [M+H]+ 721.2162, found 721.2153. 152   4.6.6.2 Inhibitor 142  To the solution of compound 141 (32 mg, 0.04 mmol) in ethanol: H2O (2:1) (3 mL) was added 10% Pd/C (approx. 10 mg), and the reaction mixture was stirred under an atmosphere of H2 gas for 18 h at rt. After filtration, the filtrate was concentrated under vacuum to give a clear oily liquid which was dissolved in distilled H2O (1 mL). The pH was adjusted to 7-8 by addition of 0.1 M NaOH. This solution was loaded on a pre-washed Sep-Pak C18 column (20 cc Vac Cartridge, 5 g Sorbent per Cartridge) and then eluted with 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, and 80% CH3CN in distilled water (5 mL each) . The product came out in the initial fractions that were lyophilized to give phosphonate 142 (8.2 mg, 72 %) as a white solid.  1H NMR (300 MHz, D2O) δ 4.61 – 4.45 (m, 1H), 4.03 – 3.82 (m, 4H), 3.76 (dd, J = 11.2, 2.6 Hz, 1H), 1.25 (d, J = 16.4 Hz, 3H).  13C NMR (75 MHz, D2O) δ 177.0, 172.3 (both carbonyl peaks confirmed by HMBC),74.7 (d, J = 4.7 Hz), 73.2 (d, J = 3.9 Hz), 69.4, 54.9, 12.1 (d, J = 136.7 Hz). 31P NMR (122 MHz, D2O) δ 26.31. HRMS (ESI): m/z calcd for C7H15NO8P [M+H]+ 272.0535, found 272.0529.   153   Chapter 5: Conclusions and Future work 5.1 Conclusions for Chapter 2 In this thesis, I focused on two of the cell-shape-determining enzymes found in H. pylori, Csd6 and Csd4, which help in maintaining the helical shape of the bacterium. Csd6 and Csd4 are hydrolases that trim the PG peptides into tripeptides and tetrapeptides, respectively. The helical shape of H. pylori is necessary for colonization, and inhibiting these enzymes could result in cell-straightening and reduced pathogenicity.  Synthetic PG peptides can be very useful in studies on these cell-shape-determining peptidases. However, access to these peptides is hampered by the presence of meso-diaminopimelic acid (meso-Dap) as the (S)-stereocenter of this residue must be linked between the iso-D-Glu and L-Ala residues. For this reason, we decided to prepare peptides containing an analog of meso-Dap, meso-oxa-Dap, where an oxygen atom replaces the central methylene group of meso-Dap.  In the second chapter, we prepared a meso-oxa-Dap containing tripeptide and tetrapeptide and tested them as minimal substrates for Csd6/Pgp2. The initial route for synthesizing these peptides utilized the Vederas group's methodology to prepare orthogonally protected meso-oxa-Dap via the nucleophilic ring-opening of an aziridine. D-Glu and D-Ala, were then sequentially attached to give meso-oxa-Dap containing tripeptide 1. While this synthetic route gave the desired tripeptide 1, the synthesis was lengthy and had many low-yielding steps (aziridine opening and peptide couplings). Therefore, we decided to first prepare a tripeptide with an embedded aziridine and then perform nucleophilic ring-opening at the late stage of synthesis. The overall yield for 154   tripeptide 1 (3.90 %) was better than the previous route (0.63 %), and the new procedure required four less steps as many protections and deprotection steps were avoided. The only low-yielding step was nucleophilic ring-opening of aziridine due to the formation of oxazoline 47 as a side-product. The branched tetrapeptide 2 was also prepared from the protected tripeptide 46. Activity studies showed that both Csd6 and Pgp2 readily cleaved the linear tripeptide 1. In the case of branched tetrapeptide 2, only Pgp2 showed activity. These results were similar to the reported data with intact PG isolated from bacterial sources. This work showed that these enzymes could accept the "minimal" meso-oxa-Dap containing peptide as a substrate.  5.1.1 Future work related to Chapter 2 Future work will focus on kinetic studies of the tripeptide substrate 1 with the Csd6 enzyme. A continuous coupled spectroscopic assay for D-Ala formation is known and could be employed for these studies (Fig. 5.1).145 This assay will utilize the coupling enzymes D-amino acid oxidase and lactate dehydrogenase. Csd6 will hydrolyze the tripeptide substrate to give a dipeptide and free D-alanine. D-Alanine will then be oxidized to pyruvic acid using D-amino acid oxidase, which uses a FAD cofactor. The resulting pyruvic acid will be reduced to L-lactic acid using L-lactate dehydrogenase, which uses NADH as a cofactor. During the reduction step, the NADH will be oxidized to NAD+. Therefore, the reaction kinetics can be monitored by the decrease in UV absorbance at 340 nm using UV spectroscopy. 155    Figure 5.1: Continuous coupled assay for the Csd6 reaction  Other future work will involve the synthesis of inhibitors and testing with the Csd6 enzyme. Our observation that small molecules can act as substrates for the enzyme suggests that small molecule inhibitors may be effective as well. The Csd6 enzyme functions as a cysteine protease and utilizes covalent catalysis.76 Our first approach will be to prepare irreversible covalent inhibitors. Halomethyl ketones are found to be effective inhibitors for the cysteine proteases and result in irreversible inactivation (Fig. 5.2).146  156    Figure 5.2: Potential inactivation mechanisms of cysteine proteases by halomethyl ketones The first target will be to prepare a chloromethyl ketone analogue of dipeptide N-Ac-D-Glu-meso-oxa-Dap, compound 51 (Fig. 5.3). After the synthesis of inhibitor 51, we will examine the in vitro inhibition performance using the continuous coupled assay mentioned above.  Figure 5.3: Structure of a chloromethyl ketone inhibitor 51 for Csd6   157   5.2  Conclusions for Chapter 3 The next project goal (Chapter 3) was to test whether the methodology of peptidyl-embedded aziridine ring-opening was generalizable and would work with longer peptides. We chose to synthesize a full length meso-oxa-Dap containing PG pentapeptide 63 and attach it to a GlcNAc-AnhMurNAc disaccharide to show its utility. A nucleophilic ring-opening reaction was performed on pentapeptide-embedded aziridine 71. The desired protected PG pentapeptide was isolated but in a lower yield of 9 % and the primary product in this reaction was oxazoline 73 (30%). Therefore, it appears that the peptide embedded aziridine ring-opening is not broadly useful with longer peptides. The presence of multiple Lewis acid coordination sites may result in unreactive conformers. Therefore, we decided to change our strategy and carried out the ring opening on tripeptide-embedded aziridine 84. The resulting tripeptide 78 was then coupled to D-Ala and L-Ala to give the protected PG pentapeptide 63. The pentapeptide 63 was then attached to disaccharide 87 to prepare GlcNAc-AnhMurNAc pentapeptide 3, a substrate for the pore protein AmpG. In these chapters we developed a rapid synthesis of PG peptide isosteres that bear meso-oxa-Dap in place of meso-Dap.  This provides and attractive alternative to the lengthy routes that have been reported for the synthesis of meso-Dap containing PG peptides. 5.2.1 Future work related to Chapter 3 Peptidoglycan fragments also have a very significant biological impact on human health due to their immunostimulatory activities. They can activate the innate immune response system as they are recognized by receptor proteins like NOD1 (Fig 5.4 top).147 Fukase and co-workers, in their stimulatory studies, used synthetic tripeptide (L-Ala-iso-D-Glu-meso-Dap) and tetrapeptide 158   (L-Ala-iso-D-Glu-meso-Dap-D-Ala) adducts of the anhydro disaccharide to show activity with the NOD1 protein (Fig. 5.4 bottom).85 They synthesized the meso-Dap derivatives using a Kocienski-modified Julia olefination, which is covered in detail in Chapter 2. These peptidyl adducts are of interest due to their ability to initiate an immune response upon binding to the NOD1 receptor protein.147 Minor modifications of our synthetic route could also prepare the meso-oxa-Dap analogues of these compounds for use in similar studies.    Figure 5.4: Top: Graphical representation of Nod1 protein interaction with PG fragments (taken from Fukase et al., 2011). Bottom: Structure of meso-Dap containing GlcNAc-anhMurNAc peptides prepared by Fukase et al. for stimulatory studies. 159   5.3 Conclusions for Chapter 4 In previous collaborative work between the Tanner group and the Murphy group (Dept. of Microbiology, UBC) it was shown that a diastereomeric mixture of phosphinic acid 4 inhibits Csd4 by mimicking the tetrahedral intermediate formed during the enzymatic reaction. The work in Chapter 4 aims to prepare the second-generation phosphonamidate- and phosphonate-based inhibitors. We decided to incorporate meso-oxa-Dap instead of meso-Dap as as we had developed a convenient aziridine ring-opening methodology described earlier. The protected phosphonic acid 121 was prepared via radical addition of hypophosphite to olefin 118 in the presence of triethyl borane and oxygen. The phosphonic acid 121 was coupled to meso-oxa-Dap 100 via phosphochloridate formation to give phosphonamidate 124. The removal of the protecting groups to give inhibitor 5 was unsuccessful and gave undesired side-products. To minimize side-product formation, we decided to use a truncated version of glutamic acid, methyl phosphonic acid 130, for coupling with meso-oxa-Dap 100. The obtained phosphonamidate 132 was also resistant to deprotection and only products from the hydrolysis of the P-N bond were observed. Due to the instability of phosphonamidate during deprotection steps, we decided to prepare phosphonate 142. The nucleophilic ring-opening of aziridine 98 using glycerate 138 gave the protected hydroxy-meso-oxa-Dap 139. The removal of the allyl group using PHMS gave hydroxy-meso-oxa-Dap 140. The coupling of methyl phosphonic acid 130 with hydroxy-meso-oxa-Dap 140 gave phosphonate 141. Removal of the protecting groups gave phosphonate inhibitor 142. This inhibitor is currently tested for binding to the Csd4 enzyme. If inhibitor 142 shows a higher affinity than phosphinic acid inhibitor 4, then structural and morphological studies will be done by our collaborators.  160   5.3.1  Future work for Chapter 4 If significant inhibition of Csd4 is observed with phosphonate inhibitor 142, we will try to synthesize inhibitor 6 using a similar synthetic procedure (Fig 5.5). We would expect this inhibitor to bind more tightly as the free carboxylate of the γ-Glu mimic appears to interact with a Lys residue in the active site of Csd4 (Fig. 4.4 A).54 The coupling of phosphonate 121 and hydroxy-meso-oxa-Dap 140 via a phosphochloridate intermediate will give protected phosphonate 143 protecting groups will be removed by hydrogenolysis, and then purification using a Sep-Pak C18 column will give inhibitor 6. One drawback to this method is that it will be difficult to use a large excess of the phosphonic acid, and therefore the yield of the coupling may suffer. The inhibition studies, as well as morphological studies, will be done by our aforementioned collaborators.   Figure 5.5: Proposed synthesis of inhibitor 6 5.3.1.1 Synthesis of hydroxamate inhibitors  Hydroxamic acids have long been known to acts as potent metalloenzyme inhibitors.148, 149 As they have pKa’s of around 8.5-9.5, the anionic form may serve as a potent metal chelator. For example, Holmes et al., in their crystallographic studies, showed that the hydroxamate moiety 161   binds to the zinc metal present in the thermolysin active site in a bidentate fashion (Fig 5.6).150 Therefore, we envisaged that the hydroxamate inhibitor 144 might also show inhibition with the Csd4 enzyme.   Figure 5.6: Cartoon diagram of Zn-hydroxamate binding in thermolysin and structure of hydroxamate inhibitor for Csd4 (box) One way to append a three-carbon unit to an amine is via a conjugate addition to an acrylate.151 We anticipate that the conjugate addition of amine 100 to the hydroxamate-acrylate 145 could result in the formation of product 148 (Fig. 5.7). An alternative method for the preparation of 148 could be the nucleophilic attack of amine 100 onto the protected hydroxamate-alkyl halide, 146 or 147, in the presence of a base.152 Removal of the protecting groups using hydrogenolysis will give hydroxamate inhibitor 144. Once obtained, this inhibitor will be used for inhibition studies as well as morphological studies.  162    Figure 5.7: Synthesis of hydroxamate inhibitor 144  163   Bibliography 1. Costerton, J. W.; Ingram, J. M.; Cheng, K. J., Structure and function of the cell envelope of gram-negative bacteria. Bacteriol. Rev. 1974, 38, 87-110. 2. Golan, D. 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Chem. 2020, 85, 7840-7847.  179   Appendix NMR spectra for selected compounds  180    Figure A.1: 1H NMR (400 MHz, CDCl3 ) for compound 38 Figure A.2: 13C NMR (101 MHz, CDCl3) for compound 38   181    Figure A.3: 1H NMR (400 MHz, CDCl3 ) for compound 39  Figure A.4: 13C NMR (101 MHz, CDCl3) for compound 39   182    Figure A.5: 1H NMR (400 MHz, D2O) for tripeptide 1  Figure A.6: 13C NMR (101 MHz, D2O) for tripeptide 1   183    Figure A.7: 1H NMR (400 MHz, CDCl3) for compound 43  Figure A.8: 13C NMR (101 MHz, CDCl3) for compound 43   184    Figure A.9: 1H NMR (400 MHz, MeOD) for compound 46  Figure A.10: 13C NMR (101 MHz, MeOD) for compound 46   185    Figure A.11: 1H NMR (300 MHz, MeOD) for compound 47  Figure A.12: 13C NMR (75 MHz, MeOD) for compound 47   186    Figure A.13: 1H NMR (400 MHz, CDCl3) for compound 48  Figure A.14: 13C NMR (101 MHz, CDCl3) for compound 48   187    Figure A.15: 1H NMR (400 MHz, D2O) for branched tetrapeptide 2  Figure A.16: 13C NMR (101 MHz, D2O) for branched tetrapeptide 2   188    Figure A.17: 1H NMR (300 MHz, CDCl3) for compound 65  Figure A.18: 13C NMR (75 MHz, CDCl3) for compound 65   189    Figure A.19: 1H NMR (300 MHz, CDCl3) for compound 66  Figure A.20: 13C NMR (75 MHz, CDCl3) for compound 66    190    Figure A.21: 1H NMR (400 MHz, MeOD) for compound 67  Figure A.22: 13C NMR (101 MHz, MeOD) for compound 67   191    Figure A.23: 1H NMR (400 MHz, MeOD) for compound 69  Figure A.24: 13C NMR (101 MHz, MeOD) for compound 69   192    Figure A.25: 1H NMR (400 MHz, MeOD) for compound 70  Figure A.26: 13C NMR (101 MHz, MeOD) for compound 70   193    Figure A.27: 1H NMR (400 MHz, MeOD) for compound 71  Figure A.28: 13C NMR (101 MHz, MeOD) for compound 71   194    Figure A.29: 1H NMR (400 MHz, CDCl3) for compound 72  Figure A.30: 13C NMR (101 MHz, CDCl3) for compound 72   195    Figure A.31: 1H NMR (400 MHz, CDCl3) for compound 73  Figure A.32: 13C NMR (101 MHz, CDCl3) for compound 73     196    Figure A.33: 1H NMR (400 MHz, CDCl3) for compound 74 Figure A.34: 13C NMR (101 MHz, CDCl3) for compound 74   197    Figure A.35: 1H NMR (400 MHz, CDCl3) for compound 75  Figure A.36: 13C NMR (101 MHz, CDCl3) for compound 75   198    Figure A.37: 1H NMR (400 MHz, MeOD) for compound 76  Figure A.38: 13C NMR (101 MHz, MeOD) for compound 76   199    Figure A.39: 1H NMR (400 MHz, CDCl3) for compound 77  Figure A.40: 13C NMR (101 MHz, CDCl3) for compound 77   200    Figure A.41: 1H NMR (400 MHz, MeOD) for compound 78  Figure A.42: 13C NMR (101 MHz, MeOD) for compound 78   201    Figure A.43: 1H NMR (400 MHz, MeOD) for compound 79  Figure A.44: 13C NMR (101 MHz, MeOD) for compound 79   202    Figure A.45: 1H NMR (400 MHz, MeOD) for compound 80  Figure A.46: 13C NMR (101 MHz, MeOD) for compound 80   203    Figure A.47: 1H NMR (400 MHz, MeOD) for compound 82  Figure A.48: 13C NMR (101 MHz, MeOD) for compound 82   204    Figure A.49: 1H NMR (400 MHz, MeOD) for compound 84  Figure A.50: 13C NMR (101 MHz, MeOD) for compound 84   205    Figure A.51: 1H NMR (400 MHz, MeOD) for compound 85  Figure A.52: 13C NMR (101 MHz, MeOD) for compound 85   206    Figure A.53: 1H NMR (400 MHz, MeOD) for compound 63  Figure A.54: 13C NMR (101 MHz, MeOD) for compound 63    207    Figure A.55: 1H NMR (400 MHz, CDCl3) for compound 98  Figure A.56: 13C NMR (101 MHz, CDCl3) for compound 98   208    Figure A.57: 1H NMR (400 MHz, CDCl3) for compound 99  Figure A.58: 13C NMR (101 MHz, CDCl3) for compound 99   209    Figure A.59: 1H NMR (400 MHz, MeOD) for compound 100  Figure A.60: 13C NMR (101 MHz, MeOD) for compound 100   210    Figure A.61: 1H NMR (400 MHz, CDCl3) for compound 102  Figure A.62: 13C NMR (101 MHz, CDCl3) for compound 102     211    Figure A.63: 1H NMR (400 MHz, CDCl3) for compound 103  Figure A.64: 13C NMR (101 MHz, CDCl3) for compound 103   212    Figure A.65: 31P NMR (162 MHz, CDCl3) for compound 103  Figure A.66: 1H NMR (400 MHz, CDCl3) for compound 108   213    Figure A.67: 13C NMR (101 MHz, CDCl3) for compound 108  Figure A.68: 1H NMR (400 MHz, CDCl3) for compound 112   214    Figure A.69: 13C NMR (101 MHz, CDCl3) for compound 112  Figure A.70: 31P NMR (162 MHz, CDCl3) for compound 112   215    Figure A.71: 1H NMR (400 MHz, CDCl3) for compound 117  Figure A.72: 13C NMR (101 MHz, CDCl3) for compound 117   216    Figure A.73: 1H NMR (400 MHz, CDCl3) for compound 120  Figure A.74: 13C NMR (101 MHz, CDCl3) for compound 120   217    Figure A.75: 31P NMR (162 MHz, CDCl3) for compound 120  Figure A.76: 1H NMR (300 MHz, CDCl3) for compound 121   218    Figure A.77: 13C NMR (75 MHz, CDCl3) for compound 121  Figure A.78: 31P NMR (122 MHz, CDCl3) for compound 121   219    Figure A.79: 1H NMR (300 MHz, CDCl3) for compound 124  Figure A.80: 31P NMR (122 MHz, CDCl3) for compound 124   220    Figure A.81: 1H NMR (400 MHz, CDCl3) for compound 132  Figure A.82: 31P NMR (162 MHz, CDCl3) for compound 132   221    Figure A.83: 1H NMR (400 MHz, CDCl3) for compound 137  Figure A.84: 13C NMR (101 MHz, CDCl3) for compound 137   222    Figure A.85: 1H NMR (300 MHz, CDCl3) for compound 138  Figure A.86: 13C NMR (75 MHz, CDCl3) for compound 138   223    Figure A.87: 1H NMR (300 MHz, CDCl3) for compound 139  Figure A.88: 13C NMR (75 MHz, CDCl3) for compound 139   224    Figure A.89: 1H NMR (300 MHz, CDCl3) for compound 140  Figure A.90: 13C NMR (75 MHz, CDCl3) for compound 140   225    Figure A.91: 1H NMR (300 MHz, CDCl3) for compound 141  Figure A.92: 13C NMR (75 MHz, CDCl3) for compound 141   226    Figure A.93: 31P NMR (122 MHz, CDCl3) for compound 141  Figure A.94: 1H NMR (300 MHz, D2O) for compound 142   227    Figure A.95: 13C NMR (75 MHz, D2O) for compound 142  Figure A.96: 31P NMR (122 MHz, D2O) for compound 142   

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