Synthesis of a 39-peptide and a 25-peptide by thiol capture ligations: observation of a 40-fold rate acceleration of the intramolecular O,N-acyl-transfer reaction between peptide fragments bearing only cysteine protective groups

Abstract

In this paper we report the syntheses of the 39-peptide C(Acm)LNELDADEQADL-CESLHDHADE-LYRSCLARFGDDGENL, 1, and of the 25-peptide C(Acm)LNELDADEQADL-CLARFGDDGENL, 2, by means of thiol capture ligations using precursor peptides bearing b l o c b g groups only on cysteine residues. The ligations were made in each case at the underlined cysteine, cleanly and in high yield. For each of the above syntheses, an acidolytically deblocked 13-peptide dibenzofuranyl ester, 6-[C(Acm)LNELDADEQADLeucinyloxy]-4-mercaptodibenzofuran, 3, was prepared in pure form in 52% overall yield through three stages: (1) stepwise synthesis on a solid-phase resin loaded with the dibenzofuran template, (2) acidolytic removal of the tert-butyl esters of the resin-bound peptide, and (3) preparative cleavage of the deblocked peptidyloxydibenzofuran ester from the resin. In the case of both the 39-peptide and the 25-peptide, significant rate enhancements were seen for the 08-acyl-transfer step of the thiol capture sequence when both the N-terminal and C-terminal fragments had been previously side-chain deblocked, in comparison with the cases when only the C-terminal fragment had been side-chain deblocked. In the 13-peptide + 12-peptide ligation to form the 25-peptide 2, a t1p = 5 min was seen for the leucine-cysteine amide bond-forming reaction. A model leucine-cysteine 08-acyl transfer as well as leucinecysteine OJV-acyl transfers between protected peptide fragments, however, showed the expected t1p = 4 h. Rationalization of this observed 40-fold rate enhancement is offered that identifies the aspartic acid side chain carboxylate, 12 residues in sequence from the N-terminus and penultimate to the amide ligation site, as apossible intramolecular general base catalyst for the proton-transfer step during the OJV-acyl transfer.

Synthesis of Native Proteins by Chemical Ligation

“…A number of attempts were made to take advantage of the ability to make, characterize, and handle unprotected peptides (41)(42)(43)(44). Noteworthy is the development of enzymatic ligation methods for the preparation of large polypeptides from synthetic peptide segments, with ligase enzymes specifically engineered for this purpose by the methods of molecular biology (45).…”

“…It was evident (41)(42)(43)(44)48) that a truly useful approach to chemical protein synthesis would be based on the ability to routinely make unprotected peptides ≤50 amino acid residues in length and would consist of a practical way to stitch such synthetic peptides together to give polypeptides of any desired length, and hence the corresponding folded protein molecules.…”

Diels–Alder Ligation of Peptides and Proteins

“…The desire to assemble proteins with native backbone structures by chemoselective ligation reaction inspired Kent and co-workers to develop a novel thiol capture ligation approach [31] that generates amide bonds at the ligation site. The chemoselective step involves the reversible transthioesterifcation of a thioester modified C-terminus peptide with the thiol group of an N-terminal cysteine residue ( Figure 6B).…”

Protein chemical synthesis by serine and threonine ligation

“…In general, protein synthesis consists of two key phases: (i) solid-phase peptide synthesis (SPPS) allows for the generation of moderately sized peptide segments (up to ∼30 amino acids) (12); and (ii) chemical ligation serves to chemoselectively join these synthetic peptide fragments (13-16). In the 1970s, Kemp and coworkers conceptually devised a promising peptide ligation strategy, involving prior capture followed by acyl transfer, which has laid the foundation for the development of chemical ligation in the convergent peptide synthesis (17)(18)(19)(20)(21)(22). A milestone advance in the field was the discovery, by Kent and coworkers, of native chemical ligation (NCL) (13), in which a C-terminal peptide thioester and an N-terminal cysteine-containing peptide--both in side-chain unprotected forms--are selectively coupled to generate a natural peptidic linkage (Xaa-Cys) at the site of ligation.…”