Peptide synthesis using unprotected peptides through orthogonal coupling methods.

Abstract

We describe an approach to the synthesis of peptides from segments bearing no protecting groups through an orthogonal coupling method to capture the acyl segment as a thioester that then undergoes an intramolecular acyl transfer to the amine component with formation of a peptide bond. Two orthogonal coupling methods to give the covalent ester intermediate were achieved by either a thiol-thioester exchange mediated by a trialkylphosphine and an alkylthiol or a thioesterification by C"-thiocarboxylic acid reacting with a f8-bromo amino acid. With this approach, unprotected segments ranging from 4 to 37 residues were coupled in aqueous solution to give free peptides up to 54 residues long with high efficiency.Recent advances in the design of artificial proteins with unusual architectures (1) and the ready availability of large peptides and protein domains from both solid-phase synthesis (2, 3) and recombinant DNA have led to the need for the development of convergent strategies in peptide synthesis with these large unprotected peptide segments as building blocks. A requirement of such a strategy is the exceptionally high regiospecificity in the amide bond formation between the a-amine and Ca-acyl moiety of the two segments. Recently, we have proposed (4-7) an orthogonal coupling method to achieve this goal.An orthogonal coupling method to form an amide bond is conceptually different from an orthogonal protecting group strategy (8). It is site-specific and allows only a single specific coupling reaction between the C' moiety of one peptide segment and the Na-amine of another peptide segment, in the presence of other reactive amino moieties. In such a method, a capture step (9) brings the respective N and C termini into close proximity and the amide bond formation is effected by an intramolecular acyl transfer at high effective concentration. So far, all orthogonal couplings have exploited the unique reactivity of the 1,2-aminothiol moiety of an N-terminal cysteine on the amine segment to achieve selectivity to form thiazolidine, thioester, and disulfide with acyl segments bearing glycolaldehyde ester (4, 5), thioester (6, 10), and mixed disulfide (6, 7). In orthogonal couplings involving Ca-thioester and Na-cysteine, the capture step occurs when the covalent thioester is formed between these two segments leading to a spontaneous S-to N-acyl transfer to form a cysteinyl peptide bond (Fig. 1) using fully unprotected peptide segments. The capture is effected by a thiol-thioester exchange in method A and thioesterification in method B. Both will give a covalent thioester leading to a spontaneous intramolecular acyl transfer to give a cysteinyl peptide bond. P-I (shaded oval), peptide-1; P-2, peptide-2 (open square); R, CH2CH2CO2H.N-terminal ,3-bromoalanine (BrAla) and forms a similar covalent thioester that will rearrange rapidly via an intramolecular S-to-N acyl transfer to give the peptide bond (Fig. 1 Table 3), were synthesized by the Fmoc/t-Bu strategy on Wang resin (11) by using the benzotriazol-1-yl-oxy-t...

Bruce Merrifield and solid‐phase peptide synthesis: A historical assessment

“…However, the development of native chemical ligation (NCL) methods using C-terminal peptide-thioesters (obtained from SPPS) that can be coupled without sidechain protection has provided strategies for the synthesis of larger peptides (>100 residues). 53,54 Recently, Torbeev and Kent reported the convergent chemical synthesis of a 203 residue ''covalent dimer'' of HIV-1 protease enzyme using NCL methods. The resulting enzyme molecule showed full catalytic activity and a high resolution crystal structure was reported.…”

Diels–Alder Ligation of Peptides and Proteins

“…[1,35] Two variants of this approach were presented by Tam et al. [36] Both methods are also based on the sequential capture and intramolecular acyl transfer principle where a thioacid is alkylated to form an intermediate thioester that will rearrange to give the cysteine at the ligation site ( Figure 6C). Raines et al have showed that the concept of NCL can also be extended to include selenocysteines.…”

Conformationally driven protease‐catalyzed splicing of peptide segments: V8 protease‐mediated synthesis of fragments derived from thermolysin and ribonuclease A

“…Though DNA-based site-directed mutagenesis is a powerful tool for protein design and engineering, the search for innovative approaches for construction of proteins has continued (Schnolzer & Kent, 1992;Chang et al, 1994;Jackson et al, 1994;Tam et al, 1995). This is in consonance with the protein chemists' desire to expand the range of modification of the covalent structure of proteins, including incorporation of noncoded/nonstandard amino acids or other designer "fixed elements of 3D structure" (Balaram, 1992;Schnolzer & Kent, 1992), since only limited variation is possible through genetic approaches due to inherent problems of the ribosomal protein synthesizing machinery.…”

“…Thus, failure of semisynthesis of S-peptide may be a consequence of high flexibility around the 9-10 peptide bond due to its proximity to the helix stop signal. The results suggest that protease-mediated ligations may be achieved by design and manipulation of the conformational aspects of the product.Keywords: conformation; organic cosolvent; proteosynthesis; reverse proteolysis; semisynthesis Recent years have seen remarkable progress in the area of synthetic protein chemistry toward developing strategies for chemical ligation of polypeptide fragments for construction of natural as well as novel proteins based on de novo design principles (Dawson & Kent, 1993;Dawson et al, 1994;Gaertner et al, 1994;Jackson et al, 1994;Liu & Tam, 1994;Tam et al, 1995;Wallace, 1995). The protease-catalyzed ligation of protein/peptide fragments offers the same flexibility to a selected segment as that available in modular chemical ligation strategy and may act as a bridge between chemical and genetic approaches for construction of pro--Reprint requests to: Rajendra P. Roy, National…”