3-Mercaptopropionic acid-mediated synthesis of peptide and protein thioestersw

Native chemical ligation (NCL) is an extremely useful method for the production of synthetic and semi-synthetic proteins. Since it is widely accepted that approximately fifty amino acid residues represents the limit for efficient automated solid phase peptide synthesis (SPPS) the use of recombinant methods for the production of the required thioester and cysteine-containing components has been widely exploited. However, while the number of available synthetic methods for the production of the thioester component has risen dramatically, microorganism-derived thioesters are most frequently generated by the commercially available intein-fusion expression system. Recently, several variations on a central theme of thioester production via an N to S acyl shift, that are mechanistically more similar to the intein system, have been reported (Scheme 1). Following the work of Kawakami and Aimoto on the use of cysteinylprolyl esters (CPEs) for the transient production of thioesters which can participate in NCL reactions, we had attempted to utilise CPEs in our studies geared towards the semi-synthesis of erythropoietin (EPO) and had managed to apply them with limited success (Scheme 2(a)). Consequently, we attempted to ‘‘rescue’’ CPE 1 employing 3-mercaptopropionic acid (MPA) to produce thioesters via a N S acyl shift in aqueous MPA as reported by Hojo and co-workers. In doing so we observed formation of an undesired glycine thioester 3 at the expense of our desired alanine thioester. Interestingly the ‘‘isolated’’ cysteine residue appeared to facilitate conversion to a glycine-MPA thioester in the absence of any additional stimulus such as a CPE, a mercaptomethyl proline residue, or cysteine N-alkylation and led us to predict that some selectivity may be achievable in peptide and protein fragmentation leading to thioesters in a single step, using MPA as the sole thioesterification reagent. The desired alanine thioester was observed in trace quantities only and although it was possible that any Ala thioester could be further processed to the Gly-thioester, it was not yet clear if this was the case. To validate that the glycine thioester 3 had indeed formed it was isolated by semi-preparative HPLC and successfully ligated to model peptide 2 (Scheme 2(b)). To determine whether or not MPA could effect this transformation more widely we investigated the MPA mediated fragmentation of a recombinant 21 kDa protein sample, His10-WT human erythropoietin (EPO). This 166 residue protein contains four cysteine residues in the arrangement IC, GC, HC and AC and was treated with 20% MPA over time periods of 1–48 h and at temperatures ranging from 40 to 80 1C. The protein appeared to fragment in 20% v/v MPA at temperatures as low as 40 1C (Fig. 1(a)) and surprisingly there appeared to be an accumulation of the His10-EPO(1-28)Gly-MPA thioester and His10-EPO(1-32)His-MPA thioester Scheme 1 (a) NCL, and (b) acid mediatedNS thioester formation compared. R is usually an acyl-transfer facilitating group such as alkyl, benzyl, or d-mercaptomethyl prolyl.