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Full Screen- TITLE
- CERTIFICATE
- DECLARATION
- ACKNOWLEDGEMENT
- CONTENTS
- ABBREVIATIONS
- I. INTRODUCTION AND OBJECTIVES
- INTRODUCTION AND OBJECTIVES
- Organisation of the thesis
- II. MACROMOLECULAR SUPPORTS IN SOLID PHASE PEPTIDE SYNTHESIS
- II.1 Polymers as aids in organic synthesis
- II.2 Solid phase peptide synthesis
- II.3 Problems involved in solid phase peptide synthesis
- a) Incomparibility of polystyrene supports
- Fig. II.1. Hypothetical models of polystyrene-bound peptides
- b) Non-equivalence of reaction sites
- II.4 Polyacrylamides as polymeric supports
- II.5 Hydrophilic tentacle polymers
- FignreII.2. Conformations of the graft copolymers PS and PEG.
- II.6 Recent developments in solid phase peptide synthesis
- II.7 Modified polyocrylamide supports
- II.8 Modified cross linked polystyrene supports
- III. HEXANEDIOL DIACRYLATE-CROSSLINKED POLYSTYRENE SUPPORTS FOR SOLID PHASE PEPTIDE SYNTHESIS
- III.1 Polymer synthesis
- Fig. III.1. IR spectrum of HDODA-PS resin (2%)
- III.2. Functionalisation
- III.2.1 Functionalisation of HDODA-cross linked polystyrene by chloromethylation
- III.2.2 Functional group analysis
- III.3. Introduction of amino methyl groups to the HDODA-PS resins
- III.4. Swelling and salvation
- III.5. Reactivity studies
- III.6. Stability of the support used for solid phase peptide synthesis
- Fig. III.2. IR spectra of MDODA-PS resin
- IV. SYNTHESIS OF MODEL PEPTIDES AND BIOACTIVE PEPTIDES USING HDODA-CROSSLINKED POLYSTYRENE SUPPORT
- IV. 1 Synthesis of model peptides
- IV. 1.1 Synthesis of Leu-Phe
- IV. 1.2 Synthesis of Thr-Val-Leu
- IV. 1.3 Synthesis of Val-Pro-Leu
- IV. 1.4 Synthesis of Gly-Pro-Gly-Gly
- IV. 1.5 Synthesis of Phe-Leu-Phe-Leu-Phe
- IV. 1.6 Synthesis of Lys-Val-Ile-Leu-Phe
- IV. 2 Synthesis of biologically important sequences
- IV.2.1 Synthesis of signal sequences
- IV.2.1a Synthesis of Lys-Gln-Ser-Thr-Ile-Alr -Leu-A a-Leu Leu-Pro-Leu-Leu-Phe-Thr-Pro-VaI Thr-Lys-Ala
- Fig. IV. 1. PPLC profile of crude signal- Lys-Gln-Ser-Thr-IIc-Ala-Lcu-Ala-Leu-Leu-Pro-Leu-Leu-Phe-Thr-Pro-Val-Thr-Lys-Als
- IV.2.1 b Synthesis of Lys-Gln-Ser-Thr-Ile-Alo-LewAkrLeu Leu-Pro-Leu-Arg-Phe-Thr-Pro-VaI-Thr-Lys-AIa
- Fig. lV.2. PPLC profile of crude signal Lys-Gln-Ser-Thr-IIc-Ala-Lea-Lcu-Pro-Leu-Arg-Phe-Thr-Pro-Val-Thr-Lys-Ala
- IV.2.2 Synthesis of Magainin-2
- Fig. IV.3. PPLC profile of crude Magainin.2.
- V. EXPERIMENTAL
- PART A: HEXANEDIOL DIACRYLATE-CROSSLINKED POLYSTYRENE RESIN
- Materials and methods
- V. l Source of chemicals
- V.2 Preparation of HDODA.cross linked polystyrene
- V.3 Functionalisation
- V.3.1 Functionalisation of HDODA-cross linked poly (styrene) s with chloromethyl groups: General procedure
- a) Preparation of chloromethyl methylether
- b) Preparation of IM anhydrous ZnCl2 in THF
- V.3.2 Estimation of chlorine capacity by pyridine fusion meisod: General procedure
- V.4 Swelling studies
- V.4.1 Swelling capacity
- V.4.2 Swelling ratio
- V.5 Preparation of amino methyl polystyrene from chloromethyl polystyrene: Gabriels phthalimide process: General procedure
- V.5.1 Estimation of functional groups
- a) Estimation of amino capacity
- V.6 Preparation of N-benzoyl glycine
- V.7 Preparation of N-benzoyl glycine-4-nitrophanylester
- V.8 Aminolysis of N-benzoyl glycine-4- nitrophenylester by polymeric amines
- V.8.1 Preparation of standard curve
- V.8.2 Aminolysis of 4-nitrophenyl ester by polymeric amines
- PART B: PEPTIDE SYNTHESIS
- V.9 Source of chemicals
- V.10 Physical measurements
- V.11 Purification of reagents and solvents
- V. 12 Preparation of reagents and amino acid derivatives
- V.12.1 Preparation of Boc-azide from t-butyl corbazate
- V.12.2 Synthesis of Boc amino acids-Schnabels method: General procedure
- V.12.3 Boc-ON method: General procedure
- V.12.4 Purity of Boc amino acids
- V.12.5 Preparation of 1-hydroxybenzotriazole (HOW)
- V.13 Solid phase synthesis: General procedure
- V.14 Attachment of first amino acid to the resin
- V.1 4.1 Merrifields method: General procedure
- V.1 4.2 Gisins cesium salt method: General procedure
- V.15 Estimation of first amino acid substitution: Picric acid test: General procedure
- V.16 Deprotection procedure
- V.16.1 Removal of t-butyloxy carbonyl group
- V.17 Methods of activation and coupling
- V.18 Cleavage of the peptide from the resin: TFA / thioanisole method
- V.19 Purification
- V.19.1 Assessment of peptide purity: TLC of peptides
- V.20 Identification sprays
- a) Ninhydrin spray
- b) Chlorine gaslstarch spray (Rydons reagent)
- c) Iodine
- d) Sakaguchi spray
- V.21 Fast protein liquid chromatography (FPLC)
- V.22 Amino acid analysis
- V.23 Synthesis of model peptides using the new support
- V.23.1 Synthesis of Leu-Phe
- a) Attachment of Boc-Phe to the chloromethyl resin
- b) Synthesis of Leu-Phe
- c) Cleavage and purification
- V.23.2 Synthesis of Thr-Val-Leu
- a) Preparation of cesium salt of Boc-Leu
- b) Attachment of cesium salt of Eoc-Leu to chloromethyl resin
- c) Synthesis of Thr- Val-Leu
- V.23.3 Synthesis of Vat-Pro-Leu
- V.23.4 Synthesis of Gly-Pro-Gly-Gly
- a) Attachment of Boc-Gly to the functionalised resin
- b) Stepwise synthesis of the peptide
- c) Cleavage of the peptide from the support
- V.23.5 Synthesis of Phe-Leu-Phe-Leu-Phe
- a) Attachment of the first amino acid to the chloromethyl resin
- b) Solid phase synthesis of Phe-Leu-Phe-Leu-Phe
- c) Cleavage of the synthesised peptide from the support
- d) Amino acid anaIysis
- V.23.6 Synthesis of Lys VaI-Ile-Leu-Phe
- a) Cleavage of the peptide
- b) Amino acid analysis
- V.24 Synthesis of signal sequences
- a) Attachment of Boc-Ala to the functionalised resin
- V.24.1 Synthesis of Lys, -Gln-Thr-IIe-Ala-Leu-Alo- Leu-Leu-Pto Leu-Leu-Phe-Thr-Pro-Val-Thr-Lys-Ala
- a) Cleavage and purification
- b) Amino acid analysis
- V.24.2 Synthesis of signal sequence: Lys-Gln-Ser-Thr- IIe-Ala.eu AIa.Leu-Leu-Pro-Leu-Arg-Phe-ThrPro. Val-Thr-Lys-Ala
- V.25 Synthesis of Magainin-2
- a) First amino acid attachment to the chloromerhylated resin
- b) Synthesis of magainin-2
- VI. SUMMARY AND OUTLOOK
- REFERENCES