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TITLE
DEDICATION
CERTIFICATE
DECLARATION
ACKNOWLEDGEMENT
CONTENTS
ABBREVIATIONS
I. INTRODUCTION AND OBJECTIVES
OBJECTIVES OF THE PRESENT WORK
1. Correlation of the macromolecular structural parameters with the reactivity of the functional groups relevant for peptide synthesis.
2. Probing the optimum conditions for effective synthetic converaions
3. Delineation of the structural paremeters for efficient supports and illustration of the application of suitable resins for peptide synthesis.
SUMMARY OF RESULTS
i. Synthesis of acrylamide-based polymeric supports
ii. Polymer reactivity
iii. Water-binding studies and hydrophilicity scale of the polymer supports
iv. Polysterene-based supports
v. Peptide synthesis
OROANISATION OF THE THESIS
II. SOLID PHASE PEPTIDE SYNTHESIS: AN OVERVIEW
1. Chemical synthesis of peptides
2. Solid phase peptide synthesis
3. Problems involved in solid phase peptide synthesis
4. Recent trends in solid phase peptide synthesis
5. Solvation within the solid phase
Fig. 11. 2. Hypothetical models of polystyrene-bound peptides
6. Incomplete coupling and deprotection reactions
7. Impact of secondary structure formation during synthesis
8. Non-equivalence of reaction sites
9. Optimization of the polymer support
10.Effect of macromolecular matrix
11. Molecular character and extent of cross linking
III. MACROMOLECULAR SYSTEMS SELECTED AND METHODOLOGY: SYNTHETIC AND PHYSICOCHEMICAL STUDIES
1. Polymer synthesis
a. Poly (acrylamide) s cross linked with N, Nmethylene bisacrylamide
b. Poly (acrylamide) s crosslinked with tetraethyleneglycol diacrylate
c. Poly (acrylamide) s cross linked with triethyleneglycol dimethacrylate
d. Poly (acrylamide) s cross linked with divinylbenzene
e. Terpolymers of acrylamide, N, N--dimethylacrylamide and N, N-metbylene bisacrylamide
f. Terpolymers of acrylamide, N, N—dimethylacrylamide and divinylbenzene
g. Poly (styrene) s crosslinked with triethyleneglycol dimethacrylate
2. Functionalisation of the cross linked polymers
a. Transamidation of crosslinked poly (acrylamide) s to poly (N-2-aminoethylacrylamide) s
b. Transamidation of poly (acrylamide) s to poly (N-2-aminohexylacrylamide) s
c. Functionalisation of crosslinked poly (styrene) s by chloromethylation
3. Functional group analysis
a. Amino capacity
b. Extent of chloromethylation
4. Reactivity studies
a. Effect of nature of monomers and resulting polymeric system on reactivity
b. Effect of nature of crosslinking agent
c. Effect of degree of crosslinking
d. Effect of solvent
e. Effect of spacer on reactivity
5. Swelling and solvation
a. Polyacrylamide-based resins
b. Polystyrene-TEGDMA resins
6. Water binding studies
a. Equilibrium water content
i. Effect of nature of monomers and cross linking agent on EWC
ii. Effect of degree of crosslinking
iii. Effect of method of polymerisation
iv. Effect of temperature on water sorption
v. Time-course of hydration: effect of degree. of crosslinks
b. Desorption kinetics
Effect of crosslink density on desorption kinetics
c. Water structuring in poly (acrylamide) gels
d. Weight percent oxygen content
e. Water-swollen poly (acrylamide) s: physical nature and mechanical characteristics
7. Morphological studies
a. Effect of crosslinking agent on morphology
Fig. III. 3 5. (a,b) Scanning Electron Micrographs of 10% NNMBA- crosslinked poly (acrylamide) s
Fig. III. 36 (a,b) Scaning Electron Micrographs of 10% TTEGDA- crosslinked poly (acrylamide) s
Fig. III. 3 7. (a,b) Scanning Electron Micrographs of 10% TEGDMA- crosslinked poly (acrylamide) s
Fig. III. 38. (a,b) Scanning Electron Micrographs of 10% DVB- crosslinked poly (acrylamide) s
b. Effect of extent of crosslinking on morphology
Fig. III. 39 Scanning Electron Micrographs of (a) 5% and (b) 10% TTEGDA- crosslinked poly (acrylamide) s
c. Linear Vs. crosslinked poly (acryl amides)
Fig. III. 40 Scanning Electron Micrographs of (a) 15% and (b) 20% TTEGDA- crosslinked poly (acrylamide) s
Fig. III. 41. Scanning Electron Micrographs of (a) linear poly (acrylamide) and (b) 5% TTEGDA crossliked poly (acrylamide)
d. Effect of addition of a third component: terpolymerisation and subsequent morphology
e. Effect of method of polymerisation on morphology
Fig. III. 42. Scanning Electron Micrographs of (a) 10% DVB- crosslinked acrylamide-dimethylacrylamide terpolymer and (b) acrylamide copolymer
Fig. III. 43 Scanning Electron Micrographs of 10% NNMBA crosslinked acrylamide prepared (a) in bulk and (b) in film form
f. Effect of functionalisation on morphology
Fig. III. 44. (a,b) Scanning Electron Micrographs of 10% TTEGDA- crosslinked poly (acrylamide) in film form
Fig. III. 45. Scanning Electron Micrographs of 10% TTEGDA- crosslinked poly (acrylamide) after functionalisation with ethylenediamine
g. Morphology of poly (styrene) -TEGDMA resins
Fig. III. 46. (a,b) Scanning Electron micrographs of 2% TEGDMA- crosslinked poly (styrene) beads
8. Thermal stability of the polymeric supports
a. Effect of nature of crosslinking
b. Effect of polymerisation conditions
c. Effect of addition of a third component
IV. SYNTHESIS OF MODEL PEPTIDES AND BIOACTIVE PEPTIDES
1. Preparation of 4-bromomethyl benzoyl amino ethylpoly (acrylamide)
2. Synthesis of Gly-Ala
3. Synthesis of Ala-Ala-Gly-Gly
4. Synthesis of Phe-Leu-Leu
Synthesis of biologically relevant sequences
5. Synthesis of Crabrolin: Phe-Leu-Pro-Leu-Ile Leu-Arg-Lys-Ile-Val-Thr-Ala-Leu -a peptide toxin
6. Problems encountered in the synthesis
7. Synthetic attempts with other acrylamide based supports
8. Gisins method Vs. triethylammonium salt method for C-terminal amino acid attachment
9. Attempt to attach the first amino acid as its acid chloride
10. Inherent problems with the acrylamide-based supports
11. Cross linked terpolymers of acrylamide and N, N-dimethylacrylamide as supports
12. Synthesis of a drug targeting tetrapeptide: Ala-Leu-Ala-Leu
a. The polymer support for peptide synthesis
b. Deprotection and cleavage
c. purification by FPLC
d. Amino acid analysis
13. Synthesis of human growth hormone releasing factor (hGRF) segment (12-15): Lys-Leu-Val-Gly
a. Peptide Synthesis
b. Assembly of Lys-Val-Leu-Gly (K V L G)
c. Deprotection and cleavage
d. Purification of the crude peptide
Fig. IV. 6. Scanning Electron Micrographs of chloromethylcopoly (PS-2%-TEGDMA) resin (a) before synthesis (b) after synthesis.
e. Amino acid analysis
14. Synthesis of a contraceptive tetrapeptide: Thr-Pro-Arg-Lys
a. Attachment of Boc (C12) - Lysine to chloromethyl resin
b. Syntheels of Thr-Pro-Arg-Lys (T P R K)
d. Cleavage and purification
e. Amino acid analysis
15. Synthesis of a delicious octapeptide: Lys-Gly Asp--Glu--Glu-Ser-Leu-Ala
a. Assembly of the peptide Lys-Gly-Asp-Glu-Glu-Ser-Leu-Ala (K G D E E S L A)
b. Cleavage of the peptide, purification and amino acidanalysis
16. Synthesis of seminalplasmin segment (14-26): Ser--Leu-Ser-Arg-Tyr-Ala-Lys-Leu-Ala-Asn-Arg Leu-Ala
a. Peptide Synthesis
b. Purification of the peptide
c. Analysis of the peaks
d. Manual microsequencing of seminalplasmin segment
e. Circular dichroism spectra of Ser-Leu-Ser-Arg-Tyr-Ala-Lys-Leu-Ala-Asn-Arg-Leu-Ala
17. Synthesis of seminalplasmin segment (28-40): Pro-Lys-Leu-Leu-Lys-Thr-Phe-Leu-SerLys-Trp Ile-Gly
a. Synthesis of Pro-Lys-Leu-Leu-Lys-Thr-Phe-Leu-Ser-Lys-Trp-Ile-Gly
b. Cleavage, purification and amino acid analysis
c. Sequencing of the peptide
d. CD studies
e. Biological activity
f. Stability of the PS-TEGDMA support
V. EXPERIMENTAL
Part A. Preparation of polymers and functionalisation Materials and methods
1. Source of chemicals
2. Polymer synthesis
a. NNMBA- crosslinked poly (acrylamide) e: general procedure
b. TTEODA- crosslinked poly (ecrylamide) s: general procedure
c. TEGDMA- crosslinked poly (acrylamide) s: general procedure
d. DVB- crosslinked poly (acrylamide) s: genera1 procedure
e. NNMBA- crosslinked acrylamide- N, N-dimethylacrylamide terpolymer by inverse suspension polymerisation
f. DVB- crosslinked terpolymers of acrylamide and N, N-dimethylacrylamide
g. TEGDMA- crosslinked poly (styrene) s by suspension polymerisation
h. Prepration of NNMBA- crosslinked polyIacrylamide) s in film form
i. Preparation of TEGDMA- crosslinked poly (acrylamide) in film form
3. Amino functionalisation
4. Preparation of anhydrous zinc chloride in THF
5. Preparation of chloromethyl methylether
6. Estimation of capacity
7. Preparation of N-benzoylglycine 4-nitrophenyl ester
8. Aminolysis of polymeric amines by the active ester
9. Estimation of functional group reactivity towards peptide coupling
10. Swelling and solvation
11. Determination of equilibrium water content of crosslinked poly (acrylamide) s
12. Estimation of freezing and non-freezing water content
13. Water sorption / desorption experiments
14. Polymer morphological studies
15. Thermal stability of the supports
Part B. Peptide synthesis
16. Source of chemicals
17. Physical measurements
18. Purification of reagents and solvents
19. Detection
i. Thin layer chromatography
ii. Identification of the peotide on TLC
20. Visualisation
21. Amino acid analysis
22. Preparation of derivatives
a. Preparation of Boc azide from t-butyl carbazate
b. Synthesis of Boc amino acids by Schnabels method
c. Boc-ON method: general Procedure
d. Purity of Boc amino acids
23. Methods of coupling
a. Dicyclohexylcarbodiimide method
b. Active ester method
24. General method for solid phase peptide synthesis
25. Deprotection procedure
26. Purification
a. Column chromatography
b. Fast protein liquid chromatography
27. Amino acid analysis
28. Peptide sequencing
29. Circular dichroism measurement
30. Preparation of 4-bromomethyl benzoic acid from 4-methyl benzoic acid
31. Preparation of 4-methyl benzoyl chloride from 4-bromomethyl benzoic acid
32. Preparation of 4-bromomethyl benzoyl aminoethyl DVB-crosslinked poly (acrylamide)
33. Estimation of bromine content in the bromo resin
34. Capping the residual amino groups by acetylation
35: Attachment of the first amino acid to the bromo resin via esterification
36. Attachment of first Boc amino acid by Gisins cesium salt method
37. Estimation of first amino acid substitution by picric acid method
38. Preparation of 4N HCI / dioxane
39. Synthesis of Ala-Leu-Ala-Leu
a. Preparation of cesium salt of Boc-Leu
b. Attachment of Boc-Leu to chloromethyl resin
c. Synthesis of Ala-Leu-Ala-Leu (A L A L)
d. Cleavage, purification and amino acid analysis
e. Amino acid analysis
40. Synthesis of Lys-Val-Leu-Gly
a. Attachment of the first amino acid to the functionalised resin
b. Stepwise synthesis of hQRF segement (12-15)
c. Cleavage of the peptide
d. Purification and analysis
41. Synthesis of Thr-Pro-Arg-Lys
a. Attachment of first amino acid to chloromethyl resin and estimation of the level of substitution
b. Synthesis of Thr-Pro-Arg-Lys (TPRK)
c. Deprotection and cleavage
d. Purification and amino acid analysis
42. Synthesis of Lys-Gly-Asp-Glu-Glu-Ser-Leu-Ala
a. Attachment of the first amino acid to the chloromethyl resin
b. Stepwise addition of Boc-Amino acids
c. Cleavage of the finished peptide from petidyl resin
d. Purification of crude peptide
e. Amino acid analysis
43. Synthesis of Ser-Leu-Ser-Arg-Tyr-Ala--Lys-Leu Ala-Asn-Arg-Leu-Ala
a. Attachment of the C-terminal aminoacid to the functionalised resin
b. Stepwise synthesis of the peptide
c. Cleavage of the finished peptide from the resin
d. Purification of the peptide
e. Manual microsequencing of seminalplasmin (14-28)
f. CD studies
44. Synthesis of Pro--Lys-Leu-Leu-Lys-Thr-Phe-Leu Ser-Lys-Trp-Ile-Gly
a. Attachment of the first amino acid and synthesis of SPF (28-40)
b. Cleavage of the peptide from the resin support
c. Purification of the crude peptide
d. Amino acid analysis
e. Amino acid Sequencing
f. CD Spectrum
VI. SUMMARY AND OUTLOOK
VII. REFERENCES