Cysteine/Penicillamine Ligation Independent of Terminal Steric Demands for Chemical Protein Synthesis

Abstract

The chemical ligation of two unprotected peptides to generate a natural peptidic linkage specifically at the C‐ and N‐termini is a desirable goal in chemical protein synthesis but is challenging because it demands high reactivity and selectivity (chemo‐, regio‐, and stereoselectivity). We report an operationally simple and highly effective chemical peptide ligation involving the ligation of peptides with C‐terminal salicylaldehyde esters to peptides with N‐terminal cysteine/penicillamine. The notable features of this method include its tolerance of steric hinderance from the side groups on either ligating terminus, thereby allowing flexible disconnection at sites that are otherwise difficult to functionalize. In addition, this method can be expanded to selective desulfurization and one‐pot ligation‐desulfurization reactions. The effectiveness of this method was demonstrated by the synthesis of VISTA (216‐311), PD‐1 (192‐288) and Eglin C.

Enabling chemical protein (semi)synthesis via reducible solubilizing tags (RSTs)

“…Please do not adjust margins Please do not adjust margins The RST strategy allows for the late-stage installing solubilizing tags, making it possible for solubilizing expressed protein segments. Ser/Thr ligation and Cys/Pen ligation conditions do not involve reducing agents [17][18] , therefore we envisaged that the solubilizing tags could be incorporated into the peptide via a disulfide linker which would be stable during the ligation process. After the ligation step, the solubilizing tags could be readily removed upon TCEP treatment or desulfurization [42] , regenerating the free cysteine or alanine (Fig.…”

“…[1][2] The peptide ligation allows for peptide assembly with side chain unprotected peptides specifically at the C-/N-terminus with high chemo-, regio-and stereo-selectivity. [3][4] With the advancement in the ligation toolbox over the past decades, including native chemical ligation (NCL) and its variant [5] , KAHA ligation [6] , Ser/Thr ligation (STL) [7][8][9][10][11][12][13][14][15][16][17] and Cys/Pen ligation (CPL) [18] , there are many options for designing protein retrosynthetic strategies. One of often-met problems is the poor solubility of the peptide segments for ligation.…”

Recent advances in protein modifications techniques for the targeting N‐terminal cysteine

“…[50] Lee and Li recently reported a repurposed chemical strategy based on an O-salicylaldehyde ester. [51,52] This approach has been used previously and is still being used to modify the N-terminal 1,2-aminoalcohol group of serine and threonine in protein semisynthesis (Figure 3A). [53][54][55] To repurpose this strategy, Lee et al…”

Revealing the function of HMGB1 N-terminal acetylation by a protein semi-synthesis approach

“…Thr and Pro). [28] STL and CPL have been widely used for synthesis of cyclic peptides and proteins. [29][30][31][32][33][34][35][36][37][38][39][40][41][42] As a further advance, STL/CPL-based protein semi-synthesis will be of great importance for large protein synthesis.…”

“…Accessing proteins with desired natural or unnatural modifications is important yet challenging to correlate structure to function in the investigation of protein post-translational modifications (PTMs). [1,2] Due to the non-template biogenesis of PTMs, biologically controlled methods cannot be used to produce homogeneous proteins bearing site-specific PTMs with precision and flexibility. Alternatively, advances in chemoselective peptide ligation chemistry enable peptide chemical synthesis to readily reach the protein domains comprising of 100-300 amino acid residues in general.…”