"The Development of Novel Solid Phase JMethodologies for the Synthesis of Atypical Peptides and Non-peptide Entities,»'> by Barrie Kellam, B.Pharm. Hons. Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy, May 1996. Acknowledgements I would like to thank my supervisors, Professor B. W. Bycroft and Dr. S. R. Chhabra for their suggestions, advice and excellent training. Also Dr W. C. Chan, Dr. J. C. Clark and Dr. R. T. Wheelhouse for invaluable contributions. My thanks must also go to the Pharmaceutical Chemistry research technicians, for both their expertise and good humour, and to all my fellow research workers within the Department; conversations of both a chenucal and non- chemical nature will be greatly missed. The people out there that have played a valuable part within my last three years would make a list as long as this thesis, but they know who they are, and thanks, especially to Louise, whose patience over the last few weeks of writing has been a great stiength. Finally, I would like to thank again Dr. S. R. Chhabra for proof reading this thesis, but mainly for his never lacking enthusiasm, friendship and unquestionable wisdom which made this work possible. This thesis is dedicated to my parents - Thank you. "You see things and you say, 'Why ?' But I dream things that never were; and I say 'Why not ?' Thomas Edison Contents Chapter 1. Introduction Page 1 1.1 The biosynthetic origins of peptides and proteins 2 1.1.1 The formation of the peptide bond 6 1.2 Solid phase peptide synthesis 8 1.3 The solid support 11 1.3.1 Base resins commorUy exploited in SPPS 12 1.4 The peptide-resin linker 15 1.4.1 Continuous flow SPPS 16 1.5 A^a-Amino protection 16 1.5.1 Acid-labUe urethane protection 19 1.5.2 B ase-labile urethane protection 21 1.5.3 The A^-1 -(4,4-dimethyl-2,6-dioxocyclohex-1 -ylidene) ethyl (Dde) protecting group 23 1.5.4 The A'-allyloxycarbonyl (Alloc) protecting group 24 1.6 Amide bond protection in SPPS 26 1.7 Carboxy protection 27 1.7.1 AUyl ester protection 1.7.2 The 4-{A^-[I-(4,4-dimethyl-2,6-dioxocyclohexylidene)- 3-methylbutyl]amino}benzyl ester (ODmab) protecting group 29 1.8 Side chain protecting groups 30 1.8.1 Side chain protection employed in Boc SPPS 32 1.8.2 Side chain protection employed in Fmoc SPPS 34 1.9 Synthetic methods of peptide bond formation 36 1.9.1 Via anhydrides 38 1.9.2 Via carbodiunide reagents 41 1.9.3 Via phosphonium and uronium reagents 45 1.9.4 Via active esters 46 1.9.5 Via A^-carboxyanhydrides 48 1.9.6 Via acyl halides 48 1.9.6.1 Amino acid chlorides 49 1.9.6.2 Amino acid fluorides 51 1.10 Racemisation 51 1.10.1 Direct enolisation 52 1.10.2 Oxazolone formation 54 1.11 Common side reactions observed in SPPS 54 1.11.1 DUcetopiperazine formation 55 1.11.2 Aspargine and glutamine 56 1.11.3 Aspartic and glutamic acid 57 1.11.4 Arginine 58 1.11.5 Histidine 58 1.11.6 side reactions during peptide resin cleavage and deprotection Chapter 2. Development of a Primary Amine Protecting Group Orthogonal to the FmocABoc Strategy for SPPS 61 2.1 Introduction 61 2.1.1 Vinylogous amides as amine protection 62 2.1.2 Development of the A^-l-(4,4-dimethyl-2,6- dioxocyclohexylidenemethylene) (Dmc) amino protecting group 65 2.1.3 Development of the N-l-(4,4-dimethyl-2,6- dioxocyclohex-l-ylidene)ethyl (Dde) amino protecting group 65 2.2 Development of the N-1-(4-nitro-1,3-dioxoindan-2-ylidene)ethyl (Nde) amino protecting group 70 2.2.1 Synthesis of N«-Nde-L-aniino acids 72 2.2.2 Compatibility of A^o^Nde-amino acids in SPPS 75 2.2.3 Racemisation of A^«-Nde-amino acids during synthesis 77 2.2.4 Racemisation of iVa-Nde-amino acids during coupling 78 2.3 SPPS synthesis of Neuromendin N amide using A^a-Nde amino acids 80 2.4 SPPS synthesis of the angiotensin II receptor binding protein fragment as its corresponding amide using A^o-Nde-amino acids 82 2.5 SPPS synthesis of Leu-enkephalinamide using A^«-Nde amino acids 82 2.6 StabiUty of Nde to TFA 83 2.7 Stability of Nde to piperidine 83 2.8 Solid phase synthesis of trypanothione disulphide utilising Nde as a primary amine protecting group 91 2.9 Conclusions Chapter 3. The Development of Reversible Chromatographic Probes for the Purification of Synthetic Peptides 92 3.1 Introduction 94 3.1.1 Previously reported reversible affinity or hydrophobic chromatographic probes 98 3.2 2-Acetyl-4-nitroindane,l,3-dione (71) as a potential precursor for a chromatographic probe 100 3.2.1 Acylation of a resin-bound amine using 2-acetyl-4- nitroindane-1,3-dione 100 3.2.2 Reduction and concomitant acetylation of A^o^Nde-Ala via catalytic hydrogenation in the presence of acetic anhydride 102 3.3 A new synthesis of 2-acetyldimedone 103 3.4 Development of a hydrophobic chromatographic probe 104 3.4.1 Synthesis of 2-hexanoyldimedone 105 3.4.2 On resin A^-acylation of the angiotensin receptor binding fragment using 2-hexanoyldimedone 106 3.4.3 Deprotection and recovery of the target peptide 107 3.5 Development of an affinity chromatographic probe 110 3.5.1 Synthesis of 2-biotinyldimedone 111 3.5.2 On resin A^-acylation of the angiotensin receptor binding protein fragment using 2-biotinyldimedone (94) 111 3.5.3 Immobilisation and deprotection of the biotinylated angiotensin receptor binding protein fragment on an immobiUsed avidin column 113 3.6 Future work Chapter 4. Development of a IMultiple Antigenic Vector, Expressing a Non-Peptidic Hapten, Via Solid Phase IVIethodology 116 4.1 Introduction 117 4.2 OHHL; stracture and function 117 4.2.1 Autoinduction of biolununescence in Vibrio fisheri 118 4.2.2 Other bacteria possessing autoinducer molecules 122 4.3 Synthesis of a A^-p-ketoacyl-L-homoserine lactone containing hapten 125 4.4 Initial attempt at the MAP synthesis 127 4.4.1 Attempted coupling of hapten (103) to L-valine methyl ester 129 4.4.2 Attempted coupling of the ketal protected free acid (107) to L-valine methyl ester 130 4.5 Second attempt at the MAP synthesis 130 4.5.1 Synthesis of a 4-branch MAP core 131 4.6 Attempted coupUng of the MAP type vector to a carrier protein 133 4.7 Exploitation of a solubilisable resin suitable for injection into experimental animals 133 4.7.1 Synthesis of the MAP vector on NovaSyn® KDIOO resin 134 4.7.2 Preparation and injection of the homogenised resin into experimental animals 134 4.7.3 Analysis of plasma samples for anti-A^-p-ketoacyl-L- homoserine lactone antibodies 135 4.8 Conclusions and future work Chapter 5. Preliminary Solution Phase Synthesis of Methyl (3S,5/?)-Carbapenam-3- carboxylate for Potential Transposition onto Solid Phase 137 5.1 Introduction 137 5.2 The development of p-lactam antibiotics 138 5.2.1 The carbapenems 140 5.2.2 Biosynthetic studies 143 5.3 Solution phase synthesis of ci5'-(35,5i?)-carbapenem-3- carboxylic acid biosynthetic precursors with potential for the generation of a carbapenem Ubrary 144 5.3.1 Attempted synthesis of methyl (35,5/?)-carbapenam-3- carboxylate 147 5.3.1.1 Synthesis of tert-butyl {2R,5S)-5- methoxycarbonyl-2-pyrrolidineacetate via chiraUy induced catalytic hydrogenation 151 5.3.2 Synthesis of a Co-enzyme A analogue of the cis- pyrroUdine intermediate 153 Experimental 155 Chapter 2 181 Chapter 3 188 Chapter 4 200 Chapter 5 207 References 229 Appendix Abbreviations Abbreviations for amino acids and peptides follow the lUPAC-IUB nomenclature where applicable {Eur. J. Biochem., 1984, 9-37). Ac Acetyl (Ac)20 Acetic anhydride AcOH Acetic acid Acm Acetamidomethyl Alloc Allyloxycarbonyl BASEC 2-[(A^-Biotinyl)-aminoethylsulphonyl]ethyl/7-nitrophenyl carbonate Boc rerr-Butoxycarbonyl BOP Benzotriazol-1 -yloxy-tris-pyrroUdinophosphonium hexafluorophosphate BSA Bovine semm albumin 'Bu fe/t-Butyl n-BuLi n-Butyl Uthium Bzl Benzyl CD Circular dichroism CDCI3 Deuteriochloroform CE Capillary electrophoresis 2-ClZ 2-Chlorobenzyloxycarbonyl DBU l,8-Diazabicyclo[5.4.0]undec-7-ene 2,6-DCB 2,6-Dichlorobenzene DCC A'.A^'-Dicyclohexylcarbodiimide DCM Dichloromethane DCU A/,A^'-Dicyclohexylurea Dde (4,4-Dimethyl-2,6-dioxocyclohex-1 -ylidene)ethyl DIEA DUsopropylamine DIPCDI A/^,A^'-DUsopropylcarbodiinude DKP Diketopiperazine DMAP 4-Dimethylaminopyridine Dmc 4,4-Dimethyl-2,6-dioxocyclohexylidenemethylene DMMD 5,5 -dime thyl-2 -(die thy la minom ethyl ene)cy clohe xane-1,3 dione DMF Dimethylformamide DNP Dinitrophenyl DSC A'.A^'-Disuccirumidyl carbonate DSS 3-(Trimethylsilyl)-l-propanesulphoruc acid sodium salt DVB Divinylbenzene EDC l-(3-Dunethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDT 1,2-Ethanedithiol EI Electron impact (MS) EMS Ethyl methyl sulphide Eq. Molar equivalent ES-MS Electrospray mass spectroscopy Eton Ethanol FAB-MS Fast atomic bombardment mass spectroscopy Fmoc 9-Fluorenylmethoxycarbonyl Fmoc-Cl 9-Fluorenylmethylchloroformate For Formyl HATU 0-(7-Azabenzotiiazol-1 -yl)-1,1,3,3-tetramethyluronium hexafluorophosphate. HBTU 0-(Benzotriazol-1 -yl)-1,1,3,3-tetramethyluronium hexafluorophosphate. HF Hydrogen fluoride HMB 2-Hydroxy-4-methoxybenzyl HMPA 4-Hydroxymethylphenoxyaceticacid HOAt 1 -Hydroxy-7-azabenzotriazole HOBt I -Hydroxybenzotiiazole HPLC High performance liquid chromatography KLH Keyhole Umpet haemocyanin LDA Lithium dUsopropylamide MALDI-TOF MS Matrix assisted laser desorption time of flight mass spectroscopy MAP Multiple antigenic peptide 4-MeBzl 4-Methylbenzyl MeOH Methanol m.p.