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DEDICATION
CERTIFICATE
DECLARATION
ACKNOWLEDGEMENT
ABBREVIATIONS AND ACRONYMS
CONTENTS
1. Introduction and Objectives
1.1 Objectives of the thesis
1.2 Organization of the thesis
2. Solid Phase Peptide Synthesis
2.1 Principle and strategy of solid phase peptide synthesis
2.2 Polymer supports and resin linkages
2.2.a Styrene based polymers
Fig.11.1. Hypothetical models of polystyrene-bound peptides in (A) non polar solvent, (B) polar solvent, (C) inter molecularassociation, and (D) ideal situation.
Fig.11.2 Dimethyl acrylamide (2) Ethylene bis acrylamide (3) Acryloylsacrosine methyl ester (4) Polyacrylamide support
Fig.11.3 General scheme of oligoethyleneglycol diacrylate crosslinked polystyrenes.
Fig.11.4 HDODA - crosslinked polystyrene
2.2.b The resin linkages
2.2.c Photolytically detachable anchoring groups
2.3 Effects of solvents in solid phase peptide synthesis
2.4 Novel protecting groups
2.5 Coupling reagents
Fig.II.5. 1, Hydroxy -7 - azobenzotriazole (HOAt)
Fig.II.6. Benzotriazyloxy - bis (pyrrolidine) carbonium hexaflourophosphate (BBC)
Fig.II.7. 1- (β-napthalenes sulfonyloxy) -benzotriazole (NSBT)
Fig.II.8.2- (1-HB, enzotriazol-1-yl) 1, 1, 3, 3 -tetramethyl uronium hexaflourophosphate (HBTU)
Fig.II.9.2- (1-H Benzotriazol-1-yl) 1, 1, 3, 3-tetramethyl uronium tetraflouroborate (TBTU)
Fig.II.10. (1-H, Benzotriazo1) -1-yloxy tris (dimethylamino) phosphonium hexafluorophosphate (BOP)
Fig.II.11. 1H- (1, 2, 3-benzotriazol-1-gloxy) -tris (pyrrolidino) phosphonium hexafluorophosphate (PyBOP)
Fig.II.12. Bromo-tris- (pyrrolidino-) phosphoniumhexafluorophosphate (PyBrop)
2.6 Final cleavage
2.7 Purification and characterization of peptides
2.8 Problems associated with solid phase peptide synthesis
3. Results and Discussion
3.1 Preparation of polymer supports and functionalization
3.1.a Preparation of 2% 1, 6-hexanediol diacrylate (HDODA) -crosslinked polystyrene
3.1.b Functionalization of the polymer
3.1.c Functional group assay
3.2 Synthesis of peptides
3.2.a Synthesis of model peptides
i. Ala-Pro-Ala
ii. Ala-Ala-Pro-Ala
iii. Ala-Ala-Ala-Ala-Ala
iv. Ala-Ala-Pro
v. Ala-Ala-Ala-Pro-Ala
vi. AIa-Pro-Gly-Pro-Arg
3.2.b Synthesis of short fragments of rubber elongation factor (REF) protein
i. Gln-GIn-Gly-Gln-Gly (7-11)
ii. Val- Gln-Asp-Ala-Ala-Thr-Tyr-Ala (20- 27)
iii. Pro-Leu-Gln-Pro-Gly-Val-Asp-lle-Ile Glu-Gly-Pro (44-55)
iv Val-Lys-Asn-Val-Ala-Val-Pro (56-62)
v. Tyr- Ile-Pro-Asn-Gly-Ala- Leu-Lys-Phe-Val Asp-Ser-Thr-Val-Val-Ala (69-84)
vi Gln-Thr-Lys- Ile- Leu-Al a-Lys-Val-Phe Tyr-Gly (125-135)
4. Experimental
4.1 Preparation and functionalization of hexanediol diacrylate-crosslinked polystyrene
4.1.a Materials and methods
4.1.b Source of Chemicals
4.1.c Preparation of 2% HDODA-crosslinked polystyrene by suspension polymerization
4.1.d Chloromethylation of HDODA-crosslinked polystyrene: General procedure
i. Preparation of chloromethyl methyl ether
ii. Preparation of 1M anhydrous ZnCl2 in THF
iii Estimation of chlorine capacity by pyridine fusion method: General procedue
4.2 Solid phase peptide synthesis
4.2.a Source of chemicals
4.2.b Purification of solvents and reagents
4.2.c Preparation of reagents and amino acid derivtives
i. Preparation of Boc-azide from t-butyl carbazate
ii. Synthesis of Boc-amino acids - Schanabels method General procedure
iii Boc-ON method: General procedure
4.2.d Purity of Boc-amino acids
4.2.e Preparation of 1-Hydroxybenzotriazole (HOBt)
4.2.f Solid phase synthesis: General procedure
4.2.g Attachment of first amino acid to the resin
i. Cesium salt method: General procedure
ii. Estimation of first amino acid substitution: General procedure
4.2.h Deprotection of t-Boc group: General procedure
4.2.i Methods for activation and coupling
i. Anhydride method
ii. HOBt-active ester method
4.2.j Cleavage of peptide from the resin by TFA / Thioanisole method
4.2.k Purification and analysis
i. Purification by TLC
ii. High performance liquid chromatography
iii. Amino acid analysis
iv Mass spectroscopy
4.2.l Synthesis of Model Peptides
i. Synthesis of Ala - Pro - Ala
ii Synthesis of Ala-Ala-Pro-Ala
iii Synthesis of AIa - Ala - Ala - Ala -Ala
iv. Synthesis of Ala-Ala -Pro
v. Synthesis of Ala-Ala-Ala-Pro-Ala
vi Ala-Pro-Gly-Pro-Arg
4.2.m Synthesis of short fragments of rubber elongation factor (REF) protein
i Synthesis of Gln - Gln-Gly -Gln-Gly (7-11)
ii. Synthesis of Val-Gln-Asp-Ala-Ala-Thr-Tyr-Ala (20-27)
iii Synthesis of Pro-Leu-Gln-Pro-Gly-Val-Asp-lle-lle-Glu-Gly-Pro (44-55)
iv.Synthesis of Val-Lys-Asn-Val-Ala-Val-Pro (56-62)
v. Synthesis of Tyr-lle-Pro-Asn-Gly-Ala-Leu-Lys-Phe-Val-Asp-Ser-Thr-Val- Val-Ala (69-84)
vi Synthesis of Gln-Thr-Lys-lle-Leu-Ala-Lys-Val-Phe-Tyr (125-135)
5. Summary and Outlook
References