Protein Assembly Using the Staudinger Ligation

Introduction New methods are facilitating the total chemical synthesis of proteins. In particular, the chemical ligation of synthetic peptides provides a convergent route to proteins. Currently, the most common ligation method is “native chemical ligation” [1]. In native chemical ligation, the thiolate of an N-terminal cysteine residue of one peptide attacks the C-terminal thioester of a second peptide. An amide linkage forms after S N acyl transfer. “Expressed protein ligation” is an extension of native chemical ligation in which the C-terminal thioester is produced by recombinant DNA (rDNA) technology rather than chemical synthesis [2]. A limitation of native chemical ligation is its intrinsic reliance on having a cysteine residue at each ligation juncture. Cysteine is uncommon, comprising only 1.7% of all residues in proteins. Modern peptide synthesis is typically limited to peptides of 40 residues [3]. Hence, most proteins cannot be prepared by any method that requires peptides to be coupled only at cysteine residues. The removal of the cysteine limitation by the development of a more general ligation reaction would greatly expand the scope and utility of total protein synthesis. We have developed such a reaction. Specifically, we have used the Staudinger reaction to unite two peptides, one with a C-terminal phosphinothioester and the other with an N-terminal azide [4–6]. A putative mechanism for this version of the ‘Staudinger ligation’ is shown in Figure 1. The reaction of a phosphinothioester with a peptide azide leads to the formation of the reactive iminophosphorane. Attack of the iminophosphorane nitrogen on the thioester leads to an amidophosphonium salt. Hydrolysis of the amidophosphonium salt produces the desired amide bond and a phosphine oxide. Significantly, no residual atoms remain in the amide product.