Four-Segment Tandem Ligation of Proteins with Unusual Branched Architecture

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Chemoselective and orthogonal (NT-amino acid-specific) peptide ligation for coupling free peptides, proteins, and biopolymers afford non-amide and amide bonds, respectively. These peptide ligation methods are attractive because they are not burdened by the use of a protecting group scheme or a coupling reagent. Thus, they are applicable to both chemistry and biology. Our laboratory has recently reported several three-segment tandem ligation strategies [1,2] that utilize two orthogonal ligation approaches. To further exploit tandem ligation methods, we have combined chemoselective and orthogonal peptide ligation to produce a four-segment tandem peptide ligation strategy. This tandem ligation scheme, suitable for synthetic vaccines and combinatorial protein synthesis, couples consecutively four different segments S1-S4 to produce two amide bonds and a thioether bond at three ligation sites (Figure 1). To demonstrate the versatility of this new strategy, we describe the synthesis of branched proteins of 56–70 residues with the S1-S4 segments derived from Bac7, NK-lysin, melittin, and gpl20 sequences.