Orthogonal segment ligation

Orthogonal ligation is a convergent, amide-bond condensation strategy for two unprotected peptide segments regiospecific to a particular N-terminal amino acid. Conceptually, it is similar to other orthogonal strategies such as orthogonal protection, activation, and coupling used in chemistry to distinguish one functional group from another based on chemoselectivity. The ability of orthogonal ligation methods to avoid polymerization reactions may provide a tandem ligation scheme for coupling multiple peptide segments to further enhance the efficiency of convergent synthesis. To achieve the tandem ligation scheme using unprotected peptide segments without any protection or deprotection step, regioselectivity is required to distinguish one Nterminal amino acid from another during the sequential ligation steps. Over the past six years, our laboratory has developed a repertoire of orthogonal ligation methods toward this end [1-4]. These methods are based on two types of capture mechanisms: imine [1] and thioester [2,5]. Thiaproline ligation [1] is the first example demonstrating imine capture and the orthogonal ligation concept (Fig. 1). In aqueous conditions, this ligation employs an acyl segment carrying a glycoaldehyde ester 1 to capture an Nterminal (Nt) Cys segment 2a through an imine 3a, which rapidly tautomerizes to a thiazolidine ester 4a. The O-ester then rearranges to a stable amide bond, thiaproline (SPro) product 5a, at the ligation site. The thiaproline ligation is facile under aqueous conditions at pH 4 to 7. Although similar ligation reactions could occur with five other Nt-amino acids, including Nt-Ser 2b, -Thr 2c, -Trp 2d, -His 2e and -Asn 2f, these N-terminal amino acids do not readily undergo imine capture reaction in aqueous solutions and occur only slowly under non-aqueous conditions. This paper describes a new reaction condition for imine ligation with these six different N-terminal amino acids that leads to a thiaproline bond with Nt-Cys, an oxaproline bond with Nt-Ser or Nt-Thr, as well as other imidic bonds with NtTrp, Nt-His, and Nt-Asn (Fig. 1).