Orthogonal ligation of free peptides

Over past eight years, our laboratory has focused on developing new methodologies for ligating free peptide segments to form complex proteins and biopolymers in aqueous or organic solutions. Recently, we and others have developed a novel segment ligation strategy [l-3] in which an amide bond is formed regiospecifically to the desired Nterminal amine between unprotected peptide segments containing more than one free Nterminal amine. This strategy represents a significant methodological advance. We refer it as “orthogonal ligation strategy” in accordance with other orthogonal concepts in chemistry, including orthogonal protection schemes [4], orthogonal activation [5] and coupling [6] in organic chemistry that distinguish two functional sites based on chemoselectivity. The orthogonal ligation is a cascade consisting of two reactions of capture and activation. The capture step utilizes the principle of chemoselective ligation to form a covalent intermediate between two peptide segments. Then, an amide bond is formed via an intramolecular acyl transfer through entropic activation (fig. 1). The intramolecular acylation rate, which is first order and often spontaneous, minimizes side reactions associated with enthalpic activation methods.