published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

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The thesis discusses the design of potential inhibitors of Angiotensin Converting Enzyme (ACE). The synthesis of pep tide inhibitors containing arginine and histidine-type residues is described. Successful incorporation of these residues during peptide synthesis requires the use of protecting groups on the side-chains* and new developments in this area are described. Ch. 1 reviews the currently available protecting groups for histidine. A methodology for regiospecific introduction of protecting groups of type ROCH₂-, via their corresponding chloromethyl ethers, is described. A convenient synthesis of these reagents (specifically t-Butoxymethylchloride, Dum-Cl and 2,4,6-TrimethyIbenzyloxymethyl- chloride, Tom-Cl) is given. Ch. 2 demonstrates that a knowledge of the location of histidine protecting groups has become mandatory, both in peptide synthesis and elsewhere. Two methods) a), nuclear Overhauser enhancement measurements and b), a procedure involving methylation, deprotection and amino-acid analysis are presented, which have allowed the differentiation of ? and ? derivatised histidines. Ch. 3 reviews the currently available protecting groups for arginine. Using 2-phenylethyIguanidine as a model for arginine, a number of haloacylguanidines and 5,5-disubstituted pyrimidinones wBe synthesised, and this chapter describes their structures, and the potential use of the corresponding reagents in protecting arginine during peptide synthesis. Ch. 4 describes the synthesis of his tidyIphenylalanylarginine and several variants on this structure. Biological data showing the level of inhibition both of A.C-E- and of Renal ^ndopeptidase by these compounds is presented. The syntheses also provide a further demonstration of the efficacy of the recently introduced benzyloxymethyI, (Bom)protecting group.

The role of the amino-acid histidine in biologically important molecules and the problems encountered in its incorporation whether in protected or unprotected form into peptides, and recent work¹ establishing the importance of the location of the im-protecting group -N(π) being desirable are reviewed in the introduction. In Chapter 1 the electrolytic deprotection of π-phenacyl-thyroliberin is described: it was found to produce a complex mixture. This and otherdifficulties encountered by co-workers led to the conclusion that π-phenacyl was unsuitable for general use as a histidine protecting group. In Chapter 2, the investigation of the 1,1,l-trichlorobut-2-enyl group for histidine protection is described. A model compound- NI(1,1,l-trichlorobut-2-enyl)imidazole-was prepared and subjected to screening tests. A number of undesirable side-reactions including cis-trans isomerisation of the double bond were observed indicating that this is not a practical blocking group. A novel strategy of histidine protection involving reducing the basicity of the imidazole ring by temporary substitution with electronwithdrawing groups is outlined in Chapter 3. A new synthesis of N(α) -protected diiodohistidine derivatives and tests demonstrating their stability to routine conditions encountered in peptide synthesis and deprotection by hydrogenolysis are described. A synthesis of thyroliberin indicated the potential of this strategy. In Chapter 4 preparations of the corresponding 2,5-dibromoderivatives are described, and a solid phase synthesis of glycyl- L-histidyl-L-phenylalanine and a classical synthesis of thyroliberin are outlined. It was found however that although diiodination inhibited racemisation and dibromination was even more effective that direct blockade of the N(π) was indispensable for its complete prohibition. A new optimised preparation for N(α) -t-butoxycarbonyl,N(π) - benzyloxymethyl-L-histidine² is described in Chapter 5, this being the first simple three-step synthesis of a N(π)-protected histidine derivative. In Chapter 6 the use of N(α)-t-butoxycarbonyl ,N(π)-benzyloxymethyl- L-histidine as a protected intermediate in a number of demanding exercises, including the solid phase synthesis of angiotensin II and a solution synthesis of a histidyl-tryptophan dipeptide are demonstrated. No problems due to the histidine derivatives were encountered. Methods of evaluating the degree of racemisation occurring in activated histidine derivatives are discussed in Chapter 7.

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The preparation of 11 new oxytocin analogs is described. The synthesis of the protected peptides were performed using solid phase peptides synthetic methodology. The protected peptides were deprotected and cyclized using sodium in liquid ammonia followed by aqueous potassium ferricyanide treatment. The purification of each peptide was accomplished using partition chromatography followed by gel filtration. Final purity was checked using high-performance liquid chromatography. Several amino-acid derivatives were prepared and incorporated as racemates into the synthetic peptides. The synthetic diastereomeric peptides were separated and purified by high pressure liquid chromatography using aqueous trifluoroacetic acid:acetonitrile mixtures. The oxytocin analogs prepared in this dissertation were divided into 2 classes: oxytocin agonists and oxytocin antagonists. The oxytocin agonist analogs prepared are [2-cycloleucine]oxytocin and [8-cycloleucine]oxytocin. The oxytocin antagonists described in this dissertation are [Pen¹,Cle²]oxytocin, [Pen¹,Cle⁸]oxytocin, [Pen¹,L-TyrMe²,Thr⁴,Orn⁸]oxytocin, [Pen¹,L-TyrEt²,Thr⁴,Orn⁸]oxytocin, [Pen¹,D-TyrEt²,Thr⁴,Orn⁸]oxytocin, [Pen¹,L-PheMe²,Thr⁴,Orn⁸]oxytocin, [Pen¹,L-PheEt²,Thr⁴,Orn⁸]oxytocin, [Pen¹,D-PheMe²,Thr⁴,Orn⁸]oxytocin and [Pen¹,D-PheEt²,Thr⁴,Orn⁸]oxytocin. A conformational study of the synthetic peptides was also undertaken in order to determine possible solution conformations for the various peptides. Two biophysical methods were used in the conformational study of these peptides; they include nuclear magnetic resonance spectroscopy (H-1 and C-13) and circular dichroism spectroscopy. Two somewhat different solution conformations were discovered for peptides containing all L-amino acids and for peptides containing a D-amino acid residue in position 2. A possible correlation between biological potency and observed solution conformation is suggested; the proposed models may aid in the design of more potent peptide inhibitor analogs.

本論文中我們將介紹一種新穎且便利的利用微波的固相多肽合成方法，以及在此方法協助下對一組混合肽之間相互作用的研究。與傳統的多肽合成相比，微波輻射幫助提高耦合過程的效率，從而大大縮短了合成時間，以及提高了反應效率。家庭用和實驗室用微波爐的比較表明，家用微波爐在實驗室條件下足以勝任微波輔助固相多肽合成。在第二部分，我們利用微波固相多肽合成合成了一組多肽用以探究其之間相互作用。一對在之前文獻報導的，通過庫篩選得到的結合肽被用來與另一組捲曲肽組合構成混合肽，期望得到強而且具特異性的相互作用以用作蛋白的標記。然而文獻報導的這對多肽並未達到預期的相互作用，因此我們需要在後續實驗中重新設計混合肽。 / This thesis summarizes an investigation into a novel solid-phase peptide synthesis protocol assisted by microwave irradiation, as well as an attempt to design hybrid peptides for enhanced binding properties. Compared with conventional peptide synthesis, a brief microwave irradiation during coupling and deprotection was shown to significantly reduce the time for peptide synthesis, at the same time yielding satisfactory product purity. A comparison of a domestic and a laboratory microwave oven indicated that the former could be easily adapted for conducting microwave-assisted solid phase peptide synthesis in a research laboratory, a facile and budget-efficient solution for enhanced efficiency in solid phase synthesis. In the second part of the research, we utilized the developed protocol to synthesize a set of peptides to investigate peptide-peptide interaction. A pair of binding peptides previously identified through library screening and genetic selection reported in literature was fused with designed coiled coil peptides to afford hybrid peptides, which was expected to result in exceptionally strong interaction and enhanced specificity. These hybrid peptide pairs could find applications in protein labeling, immunoblotting, and purification. However, the peptide pair did not demonstrate the claimed binding affinity in the literature. Therefore, the current design is flawed, and we shall re-design hybrid peptides based on other binding pairs in the following research. / Detailed summary in vernacular field only. / Zhang, Han. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references. / Abstracts also in Chinese. / 摘要 --- p.I / ABSTRACT --- p.II / TABLE OF CONTENTS --- p.III / ACKNOWLEDGEMENT --- p.V / ABBREVIATION --- p.VI / Chapter Chapter 1 --- Microwave -assisted synthesis of a defensin peptide: a comparison of domestic and laboratory microwaves --- p.1 / Chapter 1. --- INTRODUCTION --- p.1 / Chapter 1.1. --- Defensins --- p.1 / Chapter 1.2. --- Solid-phase peptide synthesis (SPPS) --- p.5 / Chapter 1.3. --- Microwave (MW) heating in organic synthesis --- p.6 / Chapter 1.4. --- Microwave (MW) heating assisted solid phase peptide synthesis --- p.7 / Chapter 2. --- METHODS --- p.8 / Chapter 2.1. --- General synthetic approach of SPPS --- p.8 / Chapter 2.2. --- Microwave-assisted SPPS --- p.9 / Chapter 2.3. --- Analysis and characterization of the product --- p.12 / Chapter 3. --- RESULT AND DISCUSSION --- p.12 / Chapter 3.1. --- Exploring the coupling step under microwaving --- p.12 / Chapter 3.2. --- Nαdeprotection under microwave heating --- p.14 / Chapter 3.3. --- Synthesis of the target peptide --- p.16 / Chapter 3.4. --- SPPS in laboratory MW reactor --- p.18 / Chapter 3.5. --- SPPS by standard conventional method --- p.20 / Chapter 3.6. --- Comparison of the three methods --- p.21 / Chapter 3.6.1. --- Product p Product p urity --- p.21 / Chapter 3.6.2. --- Time and cost --- p.24 / Chapter 3.6.3 --- Sc alabiltiy of SPPS --- p.24 / Chapter 4. --- Exploration of the mechanism of microwave-assisted SPPS: thermal or nonthermal effect? --- p.25 / Chapter 5. --- CONCLUSION --- p.29 / REFERENCE --- p.30 / Chapter Chapter 2 --- Hybrid peptides with enhanced affinity and specificity for protein labeling --- p.33 / Chapter 1. --- INTRODUCTION --- p.33 / Chapter 1.1. --- Protein labeling --- p.33 / Chapter 1.2. --- Hybrid peptides sequences --- p.35 / Chapter 2. --- EXPERIMENTAL SECTION --- p.36 / Chapter 2.1. --- HPLC result --- p.37 / REFERENCE --- p.48