Qitao Yu

Orthogonal ligation strategies for peptide and protein

This review focuses on the concept, criteria, and methods of an orthogonal amide ligating strategy suitable for syntheses of peptides, peptide mimetics, and proteins. Utilizing unprotected peptides or proteins derived from chemical or biosynthetic sources, this ligation strategy has been shown to be general and exceptionally mild. Its orthogonality in ligating two unprotected segments with free N-terminal (NT)-amines at a specific NT-amine is achieved through a chemoselective capture step and then an intramolecular acyl transfer reaction. Both coupling reagents for enthalpic activation and protection schemes therefore become unnecessary. More than a dozen orthogonal ligation methods based on either imine or thioester captures have been developed to afford native and unusual amino acids at ligation sites of linear, branched, or cyclic peptides. Because unprotected peptides and proteins of different sizes and forms can be obtained from either chemical or recombinant sources, orthogonal ligation removes the size limitation imposed on the chemical synthesis of a protein with a native or non-native structure. Furthermore, by using building blocks from biosynthetic sources, orthogonal ligation provides a unifying operational concept for both total and semisynthesis of peptides and proteins.

Methionine ligation strategy in the biomimetic synthesis of parathyroid hormones.

In biological systems, both proteolysis and aminolysis of amide bonds produce activated intermediates through acyl transfer reactions either inter- or intramolecularly. Protein splicing is an illustrative example that proceeds through a series of catalyzed acyl transfer reactions and culminates at an O- or S-acyl intermediate. This intermediate leads to an uncatalyzed acyl migration to form an amide bond in the spliced product. A ligation method mimicking the uncatalyzed final steps in protein splicing has been developed utilizing the acyl transfer amide-bond feature for the blockwise coupling of unprotected, free peptide segments at methionine (Met). The latent thiol moiety of Met can be exploited using homocysteine at the alpha-amino terminal position of a free peptide for transthioesterification with another free peptide containing an alpha-thioester to give an S-acyl intermediate. A subsequent, proximity-driven S- to N-acyl migration of this acyl intermediate spontaneously rearranges to form a homocysteinyl amide bond. S-methylation with excess p-nitrobenezensulfonate yields Met at the ligation site. The methionine ligation is selective and orthogonal, and is usually completed within 4 h when performed at slightly basis pH and under strongly reductive conditions. No side reactions due to acylation were observed with any other alpha-amines of both peptide segments as seen in the synthesis of parathyroid hormone peptides. Furthermore, cyclic peptide can also be obtained through the same strategy by placing both homocysteine at the amino terminus and the thioester at the carboxyl terminus in an unprotected peptide precursor. These biomimetic ligation strategies hold promise for engineering novel peptides and proteins.

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.

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).

Design of Cyclic α-Defensin Dimers as Channel Forming BuildingBlocks

Defensins exhibit antimicrobial activity toward bacteria, yeast, fungi and enveloped viruses. Studies indicate that the microbe killing activity is related to their pore forming and membrane permeabilizing properties [1]. Also, the microbicidal activities of defensins are salt sensitive. This leads to the hypothesis that salt-sensitivity of defensins may exacerbate certain pathological conditions such as those found in cystic fibrosis in which the NaCl concentration on lung mucosal surface is increased to a level that inactivates endogenous defensins.