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TITLE
CERTIFICATE
DECLARATION
ACKNOWLEDGEMENT
CONTENTS
ABBREVIATIONS
1. Introduction and Objectives
Fig.1.1 Amino acid sequence of E.Coli thioredoxin.
1.1 Objectives of the thesis
1.2 Organisation of the thesis
2. Recent Advances in Solid phase Peptide Synthesis
2.1 Chemical synthesis of peptides
2.2 Solid phase peptide synthesis (SPPS)
Fig.2.1. Typical outline of the solid phase peptide synthesis.
2.3 Improvements in the original solid phase peptide synthesis
2.4 Synthesis of hydrophobic peptides
Table 2.1. Relative hydrophilicities and hydrophobicities of amino acid sidechains
3. Experimental
3.1 Preparation of polymer supports and functionalisation
3.1.1 Materials and methods
3.1.2 Source of chemicals
3.1.3 Polymer synthesis
3.1.4 Functionalisation of PS-HDODA support with chloromethyl groups: general procedure.
(a) Preparation of 1 Manh. ZnClz in THF
(b) Preparation of chlorornethyl methylether
(c) Estimation of chlorine capacity by pyridine fusion219 method general procedure
3.2 Peptide synthesis
3.2.1 Source of chemicals
3.2.2 Physical measurements
3.2.3 Purification of reagents and solvents
3.2.4 Detection
3.2.5 Identification of the peptides on TLC
3.2.6 Visualisation
3.2.7 Preparation of amino acid derivatives
(a) Reparation of Boc-azide from t-butyl carbazate
(b Synthesis of Boc amino acids: Schnabels method: 222 general procedure
(c) Preparation of Boc-glycine
(d) Boc-ON method: general procedure
(e) Purity of Boc-amino acids
3.2.8 Preparation of 1-Hydroxybenzotriazole (HOBt)
3.2.9 General procedure for solid phase peptide synthesis
3.2.10 Attachment of first amino acid to the resin
(a) Merrifields method (TEA method): general procedure
(b) Gisins cesium salt method: general procedure
3.2.11 Estimation of amino groups by picric acid method
3.2.12 Deprotection procedure
(a) Removal of t-butyloxy carbonyl group225
3.2.13 Methods of activation and coupling
3.2.14 Cleavage of the peptide from the resin: TFA / thioanisol method
3.2.15 Purification
(a) Thin layer chromatography (TLC)
(b) High performance liquid chromatography
3.2.16 Amino acid analysis
3.2.17 Synthesis of partial sequences of thioredoxin
a) Synthesis of Asp-Lys-Ile-Ile-His-Leu- Thr (T 2-81
Table 3.2. Protocol used for the synthesis of Asp-Lys-lle-lle-His-Leu-Thr
(b) Synthesis of Ser-Phe-Asp-Thr-Asp-Leu-Val-Lys (Tll-18)
(c) Synthesis of Ala-lle-Leu- Val-Asp-Phe- Trp--AIa (T22-29)
(d) Synthesis of Met-lle-Ala-Pro-lle-Leu-Asp-Glu-lle-AIa-AspGIu-Tyr-Gln-Gly-Lys (T37-52)
(e) Synthesis of Leu-Thr- Val-Ala-Lys-Leu (T53-58)
(f) Synthesis of Asn-Ile-Asp-Gln-Asn-Pro-Gly-Thr-Ala (T59-67)
(g) Synthesis of Pro-Lys- Tyr-Ile-Gly (T68-72)
(h) Synthesis of lle-Gly-Arg-Gly-lle-Pro-Thr-Leu-Leu-Leu-Phe (T71-81)
(i) Synthesis of Thr-Leu-Leu-Leu-Phe (T77-81)
(j) Synthesis of Ala-Thr-Lys-Val (T88-91)
(k) Synthesis of Gly-Ala-Leu-Ser-Lys-Gly-Gln-Leu-Lys-Glu-Phe-Leu-Asp-Ala-Asn-Leu (T92-107)
(l) Synthesis of Ser-Lys-Gly-Gln-Leu-Lys-Glu-Phe-Leu-Asp-Ala-Asn-Leu (T95-107)
3.2.18 Synthesis of hairpin peptides
(a) Synthesis of Glu -Val -Lys- Val-Dpro-Gly- Val-Glu- Val-Lys
(b) Synthesis of Ala-Cys- Val-Leu- Val-Dpro-Gly- Val-Leu- Val-Cys-Ala
4. Results and Discussion
4.1.Preparation of polymer supports and functionalisation
4.1.1 Preparation of polystyrene--cross linked hexanediol diacrylate supports for solid phase peptide synthesis
4.1.2 Polymer synthesis
Table 4.1. Preparation of HDODA-crosslinked polystyrene
Fig.4.3. SEM of PS-HDODA resin beads.
4.1.3 Functionalisation of HDO DA-cross linked polystyrene by chloromethylation
4.1.4 Functional group analysis
Table 4.2. Chlorine Capacities of 2% HDODA-crosslinked poly (styrene) s.
4.2.Peptide synthesis
4.2.1 Synthesis of thioredoxin partial sequences using HDODA-crosslinked polystyrene support
4.2.2 Synthesis of thioredoxin partial sequences
(a) AspLys-lle-Ile-His-Leu-Thr (T2-8)
(b) Synthesis of Ser-Phe-Asp -Thr-Asp-Leu- Val-Lys (Tll-18)
(c) Synthesis of Ala-lle-Leu-Val-Asp-Phe-Trp-Ala (T22-29)
(d) Synthesis of Met-lle-Ala -Pro-lle-Leu-Asp-Glu-lle-Ala-Asp-GIu-Tyr-Gln-Gly-Lys (T37-52)
Fig.4.13. The scanning electron rnicrographs of (a) the chloromethylated resin and (b) the peptide bearing resin.
(e) Synthesis of Leu-Thr- Val-Ala-Lys-Leu (T53-58)
(f) Synthesis of Asn-lle-Asp-Gln-Asn-Pro-Gly- Thr-Ala (T59-67)
(g) Synthesis of Pro-Lys-Tyr-lle-Gly (T68-72)
(h) Synthesis of lle-Gly-Arg-Gly-lle-Pro-Thr-Leu-Leu--Leu-Phe (T71-81)
(i) Synthesis of Thr-Leu-Leu-Leu-Phe (T77-81)
(j) Synthesis of Ala-Thr-Lys-Val (T88-91)
(k) Synthesis of Gly-Ala-Leu-Ser-Lys-Gly-Gln-Leu-Lys-Glu-Phe-Leu-Asp-Ala-Asn-Leu (T92-107)
(l) Synthesis of Ser-Lys-Gly-Gln-Leu-Lys-Glu-Phe-Leu-Asp-Ala-Asn-Leu (T95-107)
4.2.3 Synthesis of designed R-hairpin peptides
(a) Synthesis of Glu-Val-Lys-Val-Dpro-Gly-Val-Glu-Val-Lys
(b) Synthesis of Ala-Cys-Val-Leu-Val-Dpro-Gly-Val-Leu-Val-Cys-Ala
5. Summary and Conclusion
References