Serine‐Protease‐Assisted Synthesis of Peptide Substrates for α‐Chymotrypsin

Abstract

Summary6-Chymotrypsin catalyzes peptide-bond formation between acylated ,amino-acid and peptide esters as the carboxyl component and amino-acid and peptide amides as the amino component. The conditions under which enzyme-catalyzed coupling can be used for fragment condensation in peptide synthesis is investigated. To illustrate the method the synthesis of tetra-, penta-and hexapeptides of thewith L,,= Tyr, designed as substrates for a-chymotrypsin, is described. Introduction'). -Kinetic investigations of the interactions occurring between serine proteases and peptide substrates as carried out in our laboratory for several years [l] require a large number of peptide substrates with systematically varied structure. Often the desired structural variations involve substitution of amino-acid residues at a single position or at a limited number of adjacent positions, the rest of the peptide chain remaining unchanged. Similar synthetic problems are also met in other fields, e.g. in the study of structure-activity relationships with peptide hormones. In all these cases assembly of preformed building blocks by fragment condensation represents the most appropriate synthetic pathway. Conventional fragment condensation involves considerable hazards of azlactone-induced racemization at the C-terminal residue of the carboxyl component, which is activated and not urethan-protected. Thus for studies, like those mentioned, where high optical purity is essential, safe application of conventional methods is restricted to post-glycine and post-proline condensations. In contrast, enzyme-catalyzed fragment condensation using a variety of tissue and bacterial proteases with different specificities is entirely free of racemization and likely to meet the requirement for structural flexibility. Application of proteases for catalyzing peptide-bond formation was first reported in the late thirties

Enzyme‐Catalyzed Peptide‐Bond Formation: Elastase‐ and δ‐Chymotrypsin‐Assisted Synthesis of Oligopeptides

“…Methods: Procedures I-IV, Isolation of Peptides, HPLC. Standard procedures for coupling with N-hydroxysuccinimide esters of N-(t-butoxycarbony1)amino acids (Procedure I), removal of the amino-protecting t-butoxycarbonyl group (Procedure II), acylation with N-acetoxysuccinimide (Procedure III) have been described previously [2].…”

“…The fractions were collected at 15 min intervals. HPLC and TLC analysis were performed as described previously [2]. The solv.…”

“…for C13H24N205 (288.34): C 54.15, H 8.39, N 9.72; found: C 53.98, H 8.24, N 9.66. (2). Compound 1 (15.0 g, 52 mmol) was deprotected with HCI-sat.…”

“…The former fragments were prepared by conventional stepwise synthesis (Scheme 2), whereas the synthesis of the latter involved d-chymotrypsin-catalyzed coupling of Z-Tyr-OH or Z-Phe-OH with the required amino-acid or dipeptide amides (Scheme 3 ) . Scheme which alkyl esters X-OR of the carbonxyl components serve as acyl-enzyme precursors according to a mechanism considered in detail previously [2]. Concurrent nucleophilic attack of the acyl-enzyme X-E by the amino component H -Y , water, and alcohol (if present as cosolvent) lead to the formation of peptide product X-Y, carboxylic acid X-OH as a by-product, and regeneration of the starting ester X-OR, respectively.…”

Chymotrypsin catalysed synthesis of fluorescent amino acid amide and peptide derivatives

“…After the discovery that a-chymotrypsin can be employed to accomplish both hydrolysis and synthesis of a peptide bond (I), the enzymecatalysed synthesis of peptides has undergone intensive development (2)(3)(4)(5)(6)(7) and is considered at present a viable alternative t o the purely chemical synthesis.…”