The Chemistry of Peptide Synthesis

INTRODUCTION

Peptides and proteins exhibit the largest structural and functional variation of all classes of biologically-active macromolecules. Biological functions as diverse as sexual maturation and reproduction, enzyme inhibition, blood pressure regulation, glucose metabolism, thermal control, analgesia and learning and memory are now thought to be regulated by peptides.

Synthetic peptides are valuable tools in analysis of naturally occurring peptides or proteins. Since Emil Fischer's pioneering work in the early 1900's (Fischer, 1906), the methods for the preparation of synthetic peptides have improved continually. These advances include the development of selectively-removable protecting groups (Bergmann & Zervas, 1935), the complete synthesis of biologically-active peptides (du Vigneaud et al., 1953), more efficient and useful coupling reagents (Sheehan and Hess, 1955), means to deprotect the side chain-protecting groups on the fully assembled linear peptide chain readily (Sakikabara et al., 1976), and the use of insoluble resin supports to simplify and speed up the synthesis process greatly (Merrifield, 1963). Numerous critical, but less fundamental, changes in the chemistry and assembly methods since 1960 have further improved the yield and purity of synthetically prepared peptides (Barany & Merrifield, 1979). As a result of all these advancements, examples of rapid synthesis of large polypeptides of up to 140 amino acids are beginning to appear in literature. In spite of the many improvements, peptide chemistry has remained a difficult, costly and exacting science. The subject of peptide synthesis has been reviewed extensively and therefore, in this introduction only some of the major aspects relevant to the synthetic work described in this manual will be accounted briefly.

The "classical" methods for synthesis in solution have been summarized in numerous technical monographs and reviews (Wünsch, E. et al., 1974; Bodanszky, M. et al., 1984; Bodanszky, M.& Bodanszky, A., 1984; Bodanszky, M. 1985). Unfortunately these methods are labour, time, and skill intensive largely due to the unpredictable solubility characteristics of intermediates. Solid-phase peptide synthesis (SPPS), as developed by R. B. Merrifield; has proven to be the method of choice for producing peptides and small proteins of specific sequences.

The concept of the solid-phase approach involves covalent attachment (anchoring) of the growing peptide chain to an insoluble polymeric support (resin carrier), so that unreacted soluble reagents can be removed by simple filtration and washing without manipulative losses. Subsequently, the insoluble peptide-resin is extended by a series of additional cycles, which are required to proceed with exquisitely high yields and fidelities (Figure 1). Excess soluble reagents are used to drive reactions to completion. Because of the speed and simplicity of the repeated steps, the major portion of the solid-phase procedure is amenable to automation. Once chain elaboration has been accomplished, it is necessary to release (cleave) the crude peptide from the support under conditions that are minimally destructive towards sensitive residues in the sequence. Finally, prudent purification and scrupulous characterisation of the synthetic peptide product must follow to ensure and verify that the desired structure is indeed the one obtained.

SOLID PHASE PEPTIDE SYNTHESIS

Solid-phase peptide synthesis (SPPS) consists of three distinct sets of operations: 1) chain assembly on a resin; 2) simultaneous or sequential cleavage and deprotection of the resin-bound, fully protected chain; and 3) purification and characterisation of the target peptide. Various chemical strategies exist for the chain assembly and cleavage / deprotection operations, but purification and characterisation methods are more or less invariant to the methods used to generate the crude peptide product.

The two currently most popular solid-phase synthesis strategies utilize either an acid labile a-amino protecting group (t-Boc) or a base labile (Fmoc) protecting group (Figure 1), each method involving fundamentally different amino acid side-chain protection and consequent cleavage/deprotection methods. The t-Boc methodology has undergone extensive refinement over the last 25 years and has enabled synthesis of many convincingly characterised, exquisitely complex and long peptides, IGF-I & IGF-II being excellent examples. The application of Carpino's development of Fmoc protection has been more recent and its excellent potential is suggested by the steadily growing list of successfully synthesized shorter peptides.

The principles of stepwise solid-phase synthesis were first enuciated by Merrifield: an Na-tert.-butyloxycarbonyl (Boc) amino acid corresponding to the C-terminal of the target peptide is covalently attached to an insoluble polymeric support (the resin). The Boc group is removed by TFA, and the free amino terminus is neutralized by TEA. The next amino acid, with a protected a-amino group, is activated and reacted with the resin-bound amino acid to yield an amino-protected dipeptide on the resin. Dichloromethane (DCM) or dimethylformamide (DMF) is the primary solvent for deprotection, coupling, and washing. Excess reactants and coproducts are removed by simple filtration and washing. The amino-protecting group is removed and chain extension is continued with the third and subsequent protected amino acids. After the target-protected peptide chain has been built up in this stepwise-fashion, all side-chain protecting groups are removed and the anchoring bond between the peptide and the resin is cleaved by suitable chemical means (HF or TFMSA), releasing the crude peptide product into solution. The desired peptide is then purified and characterised.

A summary of an optimized chemistry for the stepwise solid-phase synthesis of peptides is given in Table 3. Despite the use of an optimized chemistry, not all peptides can be made with equal ease by solid-phase peptide synthesis. It has been observed that some amino acid sequences are more difficult to assemble on the resin support than others.In general, areas of difficulty involve four consecutive low yield (85-98%) coupling in DCM and are observed between 5 and 20 residues into a synthesis, the problems are sequence-dependent rather than specific to individual amino acid residues, they get worse if larger amounts of peptides are grown per gram of resin, and they show a strong dependency on the solvent used for the coupling reaction.

OPTIMIZED SOLID-PHASE PEPTIDE SYNTHESIS

若您有任何意见或建议，请及时E-mail通知我们，谢谢！