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Title
DECLARATION 1
DECLARATION 2
CONTENTS
ACKNOWLEDGEMENT
ABBREVIATIONS AND ACRONYMS
1. Introduction
Introduction
Objectives of the work
Organization of the Thesis
2. Current trends in solid-phase peptide synthesis
Polymer support
Fig.2.1. TEGDMA-crosslinked polystyrene
Fig.2.1. TTEGDA-crosslinked polystyrene
Protecting groups
Coupling reagents
Table 2.1. Coupling reagents used in peptide synthesis
Purification and characterization of peptide
Problems associated with solid-phase peptide synthesis Polymer effect
Polimer Effect
Fig. 2.3. Possible states of polymer (PS) and peptide chain in solvents of varying polarity
Aggregation of Peptide
3. Results and Discussion
Polymer preparation
Preparation of 2% I, 6-hexanediol diacrylate cross linked polystyrene
Functionalization of the polymer
Fig. 3.1. IR spectrum of 2% hexanediol diacrylate crosslinked polystyrene
Functional group assay
Fig. 3.2. IR spectrum of chloromethylated HDODA-PS
Stability of polymer
Synthesis of model peptides
Synthesis of (Ala) 3
Synthesis of Peptides II to VI
Synthesis of Peptides II to IV
Fig. 3.3.HPLC trace of VAVAAG
Synthesis of peptides V and VI
Fig. 3.4. HPLC trace of Glu-Val-Gly-Gln-Val-Gln-Leu-Gly
Synthesis of acyl carrier protein fragment (65 - 74)
Synthesis of the peptide
Table 3.1. Protocol for the synthesis of ACP 65-74
Amino acid analysis of the peptidyl resin
Table3.2. Coupling difficulty occurred during the synthesis of ACP 65-74 using Merrifield resin and HDODA-PS
Table 3.3. Amino acid analysis of ACP 65-74 bound to HDODA-PS and Merrifield resin
Cleavage of the peptide from the support
Purification of the peptide
Fig.3.5. HPLC trace of Crude ACT 65-74
Synthesis of a 13-residue peptide.corresponding to the fragment of bovine SPLN.
Introduction
Synthsis of peptide
Table 3.4. Protocol for the incorporation of one amino acid in the synthesis of13-residue peptide
Cleavage of the peptide from the support
Purication of the peptide
Table 3.5. Amino acid analysis of 13-residue peptide
Fig. 3.6. FPLC trace of 13-residue peptide
Synthesis of a 31-residue peptide
Introduction
Fig. 3.7.0rientation of stomatitis virus G protein in the Lipid bilayer of cellmembrane of the virus.
Synthesis of the peptide
Table 3.6. Amino acid analysis of decapeptidyl fragment of 31 residue peptide
Fig. 3.8. FPLC trace of the 21-residue peptide
Purification of the peptide
Fig.3.9. FPLC trace of the 31-residue peptide soluble in methanol
Fig. 3.10. FPLC trace of peptide eluted during blank run after methanolic fractions were seperated.
Table 3.7. Amino acid analysis of methanolic fraction of 31-residue crude peptide
Table 3.8. Amino acid analysis of 31-residue peptide soluble in acetonitrile and that of insoluble part
NMR study of 31 -residue peptide
NMR study of biomolecules
Fig. 3.11. Excitation and mixing procedures in multi-dimensional NMR experiments
i) Chemical Shifts
ii) Indirect Spin-Spin Coupling
Fig. 3.12. Perspective drawing of a L-polypeptide chain showing torsional angles
Table 3.9. Different Ф, Ψ and J values of regular structures of protein
iv) Hydrogen bonds
Molecular modelling and minimization methods
Analysis of peptide by NMR
Space correlation method
Fig. 3.13. Schematic representation showing through space connectivities in typical secondary structural elements in peptides that give NOE cross peaks.
Fig. 3.14. 400 MHz 2D NOESY spectrum (at 350 ms mixing time) of the 31 residue peptide. Temperature = 25°C
Fig. 3.15.400 MHz 2D DQF COSY spectrum of the 31 residue peptide
Fig. 3. 16. 400 MHz 2D TOCSY spectrum (at 100 ms mixing time) of the 31 residue peptide
Fig. 3.10. List of chemical shifts for the residues which could be well estimated forthe peptide from the 31 residue peptide in TFA.
Fig.3.17. 3-D structure of 31-residue peptide generated by QUANTA CHARMm program
IR investigation on solid-state conformation of peptides bound to polymer support.
Fig. 3.18. N-methyl acetamide molecule (a), Schematic diagram (b)
Table 3.11. Summary of the characteristic bands associated with the peptide linkage
Table 3.12. Amide I frequencies characteristics of peptide bond
Fig. 3.19. Normal modes of vibration of NMA
Table 3.13. The different peptides and their values used for IR investigation
Table 3.14. The amide I region of peptidyl resin
Fig. 3.20. Amide I regioin of peptidyl resin
Fig. 3.21. Amide I region of VAVAAG bound to resin
Fig. 3.22. Amide I region of peptide bound to resi
Fig. 3.23. Amide I region of ACP fragment bound to resin
Fig. 3.24. IR specra of HDODA-PS corresponding to the ester region
Fig. 3.25. Hypothetical model of peptide on polymer support
4. Experimental section
Materials and methods
Purification of solvents
Preparation of Boc azide
Preparation of Boc amino acids
Schnabels procedure
t- Butyloxycarbonyloximino-2-phenylacetonitrile (Boc ON) Method
Purity of Boc amino acids
Preparation of 2%-HDODA-crosslinked polystyrene
Functionalization of support - chloromethylation
Preparation of chloromethyl methylether
Preparation of I M zinc chloride in THF
Chloromethylation of polymer support
General methods of solid-phase peptide synthesis
Attachment of first amino acid
Estimation of first amino acid attachment
Deprotection procedure
Coupling methods
Anhydride method
HOBt-active ester method
Cleavage of peptide from the support
Checking the homogeneity of the peptide
Thin layer chromatography
Fast protein liquid chromatography
High performance liquid chromatography
Amino acid analysis
IR studies of peptidyl resin
NMR studies of peptide
Solid-phase syntheses of peptides on HDODA-PS
Synthesis of (Ala) 3
Synthesis of Ala-Ala-GIy
Synthesis of Val-Ala-Ala-Gly
Synthesis of Val-Ala-VaI-Ala-AIa-Gly
Synthesis of GIn-Val-Glu-Leu-Gly
Synthesis of Gln-Val-Gly-Gln-Val-Glu-Leu-Gly
Solid-phase syntheses of peptides on Merrifield support Synthesis of Ala-Ala-Gly
Synthesis of Ala-Ala-Gly
Synthesis of Val-Ala-Ala-Gly
Synthesis of Val-Ala-Val-Ala-Ala-Gly
Synthesis of GIn-Val-Glu-Leu-Gly
Synthesis of GIn-Val-GIy-GIn-Val-Glu-Leu-GIy
Synthesis of acyl carrier protein fragment 65-74 on HDODA-PS
Synthesis of acyl carrier protein fragment 65-74 on Merrifield system
Synthesis of a 31 Residue Peptide Corresponding to Vesicular StomatitisVirus G (VSV G) Protein.
Synthesis of a 31-residue peptide
Synthesis of a 13-residue peptide
5. Summary and Outlook
6. References