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Synthesis and Conformational Studies of the Lipid II-Binding Rings of Nisin and Mutacin I

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Synthesis and Conformational Studies of the Lipid II-Binding Rings of Nisin and Mutacin I University College London UCL Synthesis and Conformational Studies of the Lipid II-Binding Rings of Nisin and Mutacin I Rachael Dickman Submitted as partial fulfilment for the degree of Doctor of Philosophy Declaration I, Rachael Dickman, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Signed: Dated: 29-06-2018 Abstract Antibiotic resistance is a huge global health threat, and there is urgent need for new classes of antimicrobials to combat the spread of resistant organisms. In recent years, a class of antimicrobial peptides called the lantibiotics has emerged as a promising potential source of new antibiotics. The first discovered lantibiotic, nisin, has become a model compound for the class. It displays extremely potent antibacterial activity, but its poor pharmacokinetic properties mean that it is currently unable to be used therapeutically. This thesis describes the synthesis and structural analysis of the target-binding region of nisin and its close structural relative mutacin I. The lantibiotics are characterised by their complex cyclic structures formed by the unusual bis-amino acids lanthionine and methyllanthionine, and frequently contain the dehydrated amino acids dehydroalanine and dehydrobutyrine. To synthesise these peptides, methods to introduce each of these unusual residues are required. Therefore, the first aim of this research was to synthesise two orthogonally protected (methyl)lanthionines, as well as various precursors to the dehydrated residues. With these in hand, several novel truncated analogues of nisin and mutacin I were synthesised. Simpler analogues with fewer unusual amino acids were prepared, as well as peptides with the wild-type sequence. Finally, the solution state structure of each of the peptides was calculated from NMR data. From these studies, key conformational differences were observed between the synthesised truncated analogues in comparison to both full length and truncated wild-type nisin. In addition, significant differences were seen between the wild-type peptides and the simplified analogues. Taken together, these results indicate which of the unusual amino acids it may be possible to substitute whilst still maintaining native solution structure. It is hoped that this will guide future analogue design, and aid in the development of new semisynthetic antibiotics based on the structure of nisin. Table of Contents Declaration ...………………………………………………………………………….... 3 Abstract ………………………………………………………………………………… 5 Table of Contents ………………………………………………………………………. 7 List of Tables, Figures and Schemes ……………………………………………...….. 11 Acknowledgements ……………………………..……………………………………. 17 Abbreviations …………………………………………………………………………. 19 1. INTRODUCTION....................................................................................................... 23 1.1. The Need for New Antimicrobials ...................................................................... 23 1.2. Antimicrobial Peptides ........................................................................................ 23 1.2.1. The Lantibiotics........................................................................................... 25 1.3. Nisin .................................................................................................................... 29 1.3.1. Biosynthesis of Nisin .................................................................................. 30 1.3.2. Nisin Mode of Action.................................................................................. 32 1.3.3. Lantibiotics with Nisin-Like AB Ring Bridging Patterns ........................... 36 1.3.3.1. Mutacins ......................................................................................... 37 1.4. Synthesis of Lantibiotic Peptides ........................................................................ 38 1.4.1. Biosynthesis of Lantibiotics ........................................................................ 38 1.4.1.1. In vitro Engineering ........................................................................ 39 1.4.1.2. In vivo Engineering ......................................................................... 40 1.4.2. Chemical Synthesis of Lantibiotics............................................................. 42 1.4.2.1. Lanthionine Formation by Desulfurisation .................................... 43 1.4.2.2. Biomimetic Lanthionine Formation ............................................... 43 1.4.2.3. Incorporation of Orthogonally Protected Lanthionines into SPPS 44 1.4.2.4. Use of Lanthionine Mimics in SPPS.............................................. 46 1.5. Syntheses of Orthogonally Protected Lanthionine and Methyllanthionine ........ 47 1.5.1. Synthesis of (Methyl)Lanthionines via β-Haloalanines .............................. 48 1.5.2. Synthesis of (Methyl)Lanthionines via Ring-Opening ............................... 50 1.6. Incorporation of Dehydro Residues in SPPS ...................................................... 51 1.6.1. Dehydroalanine (Dha) Incorporation .......................................................... 52 1.6.2. Dehydrobutyrine (Dhb) Incorporation ........................................................ 54 1.7. Structural Studies of Nisin by NMR ................................................................... 56 1.7.1. Solution State Conformation of Nisin ......................................................... 56 1.7.2. Conformation of Nisin in Lipidic Environments ........................................ 57 1.8. Project Aims........................................................................................................ 60 1.8.1. Analogues Chosen for Synthesis in this Work ........................................... 62 2. SYNTHESIS OF LANTHIONINES & DEHYDRO PRECURSORS ....................... 65 2.1. Introduction.......................................................................................................... 65 2.2. Lanthionine Synthesis .......................................................................................... 65 2.2.1. Synthesis of Fmoc-Cys-OTce (104) ............................................................. 66 2.2.2. Synthesis of Trt-β-Iodo-Ala-OTMSE (109) ................................................. 67 2.2.3. Synthesis of (Teoc, TMSE/Fmoc) Lanthionine (38) .................................... 68 2.3. Methyllanthionine Synthesis................................................................................ 69 2.3.1. Synthesis of N-DNs/Allyl Aziridine (71) ..................................................... 69 2.3.2. Synthesis of (Alloc, Allyl/Fmoc) Methyllanthionine (70) ........................... 70 2.4. Dehydroalanine Precursor Synthesis ................................................................... 71 2.4.1. Synthesis of Se-phenylselenocysteine (123) ................................................ 71 2.4.2. Synthesis of Methyl 2,5-dibromopentanoate (80) ........................................ 72 2.5. Dehydrobutyrine Precursor Synthesis ................................................................. 72 2.5.1. Synthesis of Ile-Dhb and Ile-Thr Dipeptides ................................................ 73 2.5.2. Synthesis and Use of Fmoc-β-methyl-cysteine (136)................................... 73 2.6. Summary .............................................................................................................. 75 3. SYNTHESIS OF SINGLE RINGS............................................................................. 77 3.1. Introduction ......................................................................................................... 77 3.1.1. General Strategy for Single Lantibiotic Ring Synthesis ............................. 77 3.2. Synthesis of Ring B Peptides .............................................................................. 79 3.2.1. Mutacin I Ring B WT (96).......................................................................... 79 3.2.2. Nisin Ring B Lan Analogue (98) ................................................................ 80 3.2.3. Nisin Ring B WT (97)................................................................................. 81 3.3. Synthesis of Ring A Peptides.............................................................................. 81 3.3.1. Mutacin I Ring A Ser/Ala Analogue (101) ................................................. 82 3.3.2. Mutacin I Ring A Ser Analogue (102)........................................................ 84 3.3.3. Mutacin I Ring A WT (99) ......................................................................... 86 3.3.3.1. Synthesis Using Sec(Ph) as a Dha Precursor ................................. 86 3.3.3.2. Synthesis Using Cys as a Dha Precursor ....................................... 91 3.3.4. Nisin Ring A WT (100) .............................................................................. 93 3.3.4.1. Synthesis Using Ile-Dhb and Ile-Thr Dipeptides ........................... 93 3.3.4.2. Synthesis Using β-Me-Cys as a Dhb Precursor ............................. 96 3.4. Summary ........................................................................................................... 100 4. SYNTHESIS OF DOUBLE RINGS ......................................................................... 103 4.1. Introduction ....................................................................................................... 103 4.1.1. General Strategy
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