Development of New Thioester Equivalents for Protein Chemical Synthesis

Abstract

The chemical synthesis of proteins provides synthetic chemists with an interesting challenge and supports biological research through the generation of proteins that are not produced naturally. Although it offers advantages, studies of solid phase peptide synthesis have established limits for this technique: researchers can only prepare peptides up to 50 amino acids in length in sufficient yields and purity. Therefore, researchers have developed techniques to condense peptide segments to build longer polypeptide chains. The method of choice for chemical synthesis of these longer polypeptides is convergent condensation of unprotected protein fragments by the native chemical ligation reaction in aqueous buffer. As researchers apply this strategy to increasingly difficult protein targets, they have needed to overcome diverse problems such as the requirement for a thiol-containing amino acid residue at the ligation site, the difficulty in synthesizing thioester intermediates under mild conditions, and the challenge of condensing multiple peptide segments with higher efficiency. In this Account, we describe our research toward the development of new thioester equivalents for protein chemical synthesis. We have focused on a simple idea of finding new chemistry to selectively convert a relatively "low-energy" acyl group such as an ester or amide to a thioester under mild conditions. We have learned that this seemingly unfavorable acyl substitution process can occur by the coupling of the ester or amide with another energetically favorable reaction, such as the irreversible hydrolysis of an enamine or condensation of a hydrazide with nitrous acid. Using this strategy, we have developed several new thioester equivalents that we can use for the condensation of protein segments. These new thioester equivalents not only improve the efficiency for the preparation of the intermediates needed for protein chemical synthesis but also allow for the design of new convergent routes for the condensation of multiple protein fragments.

From protein total synthesis to peptide transamidation and metathesis: playing with the reversibility of N,S-acyl or N,Se-acyl migration reactions

“…One important step toward the design of mild transamidation and amide metathesis reactions that operate with peptide substrates in water was inspired by recent advances in the design of N,S-acyl shift systems ( Figure 3) [13][14][15][16][17][18][19][20]21 ]. Indeed, some N-(2-sulfanylethyl) amides are able to rearrange into transient thioesters by spontaneous acyl migration from nitrogen to sulfur.…”

New Methods for Chemical Protein Synthesis

“…These methods were problematic for several reasons, including the favorable Curtius rearrangement which acyl azides readily undergo. In an attempt to overcome these limitations, Liu and co-workers have investigated extensively peptide hydrazines and their conversion to acyl azides [134]. They found that the conversion of hydrazide 96 to acyl azide 97 could be completed nearly C. Subsequent addition of thiols, such as MPAA, to the reaction mixture, followed by an increase in pH to 7.0, reliably generated the required thioester 98 in a convenient one-pot procedure.…”

Chemical and Genetic Approaches to Study Histone Modifications

“…The two peptide fragments may be obtained by solid-phase peptide synthesis using well-established fluorenylmethoxycarbonyl (Fmoc-) or tertbutyloxycarbonyl (Boc-) amine protecting group strategies. Several chemical linkers and derivatization strategies are available for the synthesis of peptide α-thioesters by both chemistries [49,50]. Since most histone marks are found in the N-terminal tails, the C-terminal fragment may be larger and obtained by heterologous expression in Escherichia coli (Figure 8.2).…”