A straightforward method for automated Fmoc-based synthesis of bio-inspired peptide crypto-thioesters

Abstract

Despite recent advances, the direct Fmoc-based solid phase synthesis of peptide α-thioesters for the convergent synthesis of proteins via native chemical ligation (NCL) remains a challenge in the field. We herein report a simple and general methodology, enabling access to peptide thioester surrogates. A novel C-terminal N-(2-hydroxybenzyl)cysteine thioesterification device based on an amide-to-thioester rearrangement was developed, and the resulting peptide crypto-thioesters can be directly used in NCL reactions with fast N → S shift kinetics at neutral pH. These fast kinetics arise from our bio-inspired design, via intein-like intramolecular catalysis. Due to a well-positioned phenol moiety, an impressive >50 fold increase in the kinetic rate is observed compared to an O-methylated derivative. Importantly, the synthesis of this new device can be fully automated using inexpensive commercially available materials and does not require any post-synthetic steps prior to NCL. We successfully applied this new method to the synthesis of two long naturally-occurring cysteine-rich peptide sequences.

Fig. 1. N → S shift-based in situ synthesis of peptide thioesters for NCL. (A) Mechanism. (B) Crypto-thioesters bearing thioesterification devices operating under NCL conditions.

Fig. 2. Rational design of N-(2-hydroxybenzyl)cysteine thioesterification devices. (A) Mechanism of the intein self-catalyzed N → S shift in water. (B) Bio-inspired putative mechanism for the self-catalysis of the N → S shift in N-acyl-N-(2-hydroxybenzyl)cysteine peptides in water. (C) Mechanism of the intramolecular O → N shift in organic solvents shared by N-Hmb peptides and the new N-(2-hydroxybenzyl)cysteine thioesterification devices. PG: protecting group.

Fig. 3. NCL of the model crypto-thioester peptides 7–11 bearing an N-Hnb-cysteine device, and an analog (12) with the hydroxyl group masked as a methyl ether. ^a 300 mM MPAA, pH 6.6, 50 °C. ^b Hnb methyl ether (12).

Fig. 4. Application of the N-(2-hydroxy-5-nitrobenzyl)cysteine thioesterification device to the NCL-based syntheses of two long and demanding peptide sequences.