Highly efficient one-pot ligation and desulfurization

Abstract

The combination of native chemical ligation and desulfurization is considered a powerful strategy in protein synthesis. Homogeneous desulfurization conditions based on a radical induced reaction have been widely used in the syntheses of various challenging proteins and their analogues. However, the presence of aryl thiols in the reaction mixture as a ligation catalyst hampers one-pot ligation/ desulfurization, hence mandating additional purification/lyophilization steps prior to desulfurization. This significantly reduces the yield and prolongs the ligation process. Here we report that the use of preformed peptide-aryl thioester allows for efficient one-pot ligation and desulfurization. This approach was tested successfully for various model peptides including the synthesis of ubiquitin from two fragments. However, in the case of the synthesis of di-ubiquitin chains, where the ligation is mediated by d-mercaptolysine to form an isopeptide bond, excess aryl thiol was required for efficient ligation, necessitating purification prior to desulfurization. To overcome these obstacles, we found that functionalization of the aryl thiol with a hydrazide moiety enabled, after the ligation step, its capture by resin-aldehyde to permit direct desulfurization. Altogether, these approaches should facilitate protein synthesis with improved efficiency in yields and time.

Chemoselective Peptide Ligation–Desulfurization at Aspartate

“…[7] Further expansion of the native chemical ligation-desulfurization concept has been made possible through synthetic amino acids that bear side-chain thiol groups, which can facilitate ligation reactions in a similar manner to a Cys residue when incorporated at the N-terminus of peptide fragments. These amino acids can be efficiently desulfurized to afford the native residue following the ligation event (Scheme 1).…”

Modern Extensions of Native Chemical Ligation for Chemical Protein Synthesis

“…The prevalence of this byproduct was dramatically reduced when ligation-deselenization reactions were performed as a one-pot protocol in the presence of exogenous MPAA. Under these conditions, the rate of deselenization also dramatically decreased, perhaps because of the ability of aryl thiols such as MPAA to act as competitive radical scavengers [126,127,144]. Nonetheless, native peptide products, including those bearing unprotected, non-ligation site Cys residues, were isolated in good yields following the one-pot protocol for ligation-deselenization at β-selenophenylalanine [149].…”

“…Interestingly, for the construction of the target protein, the authors also reported the use of a novel thiol additive, trifluoroethanethiol (TFET) 124, which facilitates the facile application of a one-pot ligation-desulfurization protocol [124]. Previous attempts to streamline the ligation-desulfurization strategy into a one-pot process have been hampered by the use of aryl thiol ligation additives which, because of their radical quenching ability [125], complicate the radical desulfurization process [126][127][128]. Given the importance of aryl thiol additives in modulating thioester reactivity and promoting rapid ligation reactions [15,16], a number of approaches have aimed to facilitate the post-ligation removal of aryl thiols.…”

“…Given the importance of aryl thiol additives in modulating thioester reactivity and promoting rapid ligation reactions [15,16], a number of approaches have aimed to facilitate the post-ligation removal of aryl thiols. The liquid-liquid extraction of thiophenol [128] and the development of bifunctional aryl thiol catalysts which can be captured using a resin-bound aldehyde following the ligation reaction [126] have recently been employed to facilitate one-pot ligation desulfurization reactions.…”

Postligation-Desulfurization: A General Approach for Chemical Protein Synthesis

“…Subsequent desulfurization with free radical conditions and transformation of Cys to Ala in one pot afforded the desired Rev in 15% overall yield (Scheme 41). The Brik group [99] further demonstrated that a highly activated aryl thioester (MPAA) could proceed with NCL in the absence of additional aryl thiol, which would produce only 1 equiv. of aryl thiol and hardly influence the desulfurization step.…”