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

In this paper we report the syntheses of the 39-peptide C(Acm)LNELDADEQADL-CESLHDHADEunderbar-LYRSCLARFGDDGENL, 1, and of the 25-peptide C(Acm)LNELDADEQADL-CLARFGDDGENLunberbar, 2, by means of thiol capture ligations using precursor peptides bearing blocking 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 O,N-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 t1/2 = 5 min was seen for the leucine-cysteine amide bond-forming reaction. A model leucine-cysteine O,N-acyl transfer as well as leucine-cysteine O,N-acyl transfers between protected peptide fragments, however, showed the expected t1/2 = 4h. 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 a possible intramolecular general base catalyst for the proton-transfer step during the O,N-acyl transfer.