Search

A new side reaction during Native Chemical Ligation (NCL) at aspartic and glutamic acid/cysteine ligation sites is reported. To exploit NCL at these sites, test peptides having either a C-terminal unprotected aspartyl or glutamyl thioester were ligated with a peptide containing an N-terminal free cysteine generating, in each case, two major compounds having equal masses but different retention times by HPLC analysis. Comparison of these ligation products with reference peptides of identical sequence synthesized by total SPPS (solid phase peptide synthesis) with both natural and unnatural backbones at the cysteinyl residue is in agreement with the hypothesis of migration of the thioester moiety on the side chain carboxyl group of both C-terminal Asp and Glu residues. Enzymatic digestion of the purified ligation products with a V8-protease further confirmed the nature of the side reaction, with a total resistance of the non-natural backbone peptide to the protease. To overcome this problem the use of a protecting group stable to both HF cleavage and ligation conditions, but removable after ligation, became a necessity. A series of side-chain protected derivatives were selected, specifically 9-fluorenylmethyl ester (OFm), (phenylsulfonyl)ethyl ester (OPse), 2,2,2-trichloroethyl ester (OTce), and phenacyl esters (OPac). For the glutamic acid, OPse gave the best results, generating the correct peptide and avoiding the isomerization at the γ-carboxyl group. In the case of the aspartic residue, all the protecting groups were removed under basic conditions, worsening the problem and generating, for example, 70% yield of the β-isomer after OFm deprotection. This prompted us to propose a protecting group derived from the phenacyl ester 1-methyl-2-oxo-2-phenylethyl ester (OMop). This protecting group showed greater stability during the ligation reaction than OPac and OTce, and was easily removed by Zn/acetic acid reduction after the ligation step. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)