Chemical synthesis of proteins with base-labile posttranslational modifications and enhanced serine/threonine ligation with substituted peptide C-terminal salicylaldehyde esters

Abstract

In response to the increasing popularity of serine/threonine (STL) and cysteine/penicillamine (CPL) in protein chemical synthesis, we developed a facile method to access peptide C-terminal salicylaldehyde (SAL) esters, which are the main reaction partner in STL and CPL. Taking advantage of semicarbazone as a temporary masking group for aldehyde and Boc-SPPS, peptide C-terminal SAL esters were facilely obtained after treatment with pyruvic acid in acidic conditions. This method was compatible with all canonical amino acids. In chapter 2, we utilized this strategy to synthesize various peptide SAL esters (6-35 amino acids) with acceptable isolated yields, which were then subjected to corresponding STL or CPL to afford cyclic peptides, linear peptide drugs and LAIR-1 (234-287) peptide. In addition, the property of Boc-SPPS could accommodate base-sensitive species, such as O-acetlyation and S-palmitoylation. Hence, the peptide SAL esters bearing the O-acetylated serine/threonine or S-palmitoylated cysteine were successfully obtained by our strategy. Furthermore, the human histone H3 protein bearing O-acetylated serine/threonine PTMs in the N-terminal was achieved. In addition, taking account of the hydrophobicity of palmitoyl group, we also developed a method to easily install a solubilizing tag (K6-tag) on the benzene ring of peptide SAL ester and could be facilely removed by acidolysis. Finally, combination with the Boc-SPPS and the solubilizing tag, the S-palmitoylated peptide SAL ester was achieved. The S-palmitoylated peptide SAL ester was then subjected to CPL with N-terminal cysteine peptide to afford S-palmitoylated caveolin-1 (131-155) after acidolysis. Importantly, our CPL enabled the S-palmitoylated peptide to ligate with N-terminal cysteine peptide, which was inaccessible with NCL. In chapter 3, we unveiled that the electron-withdrawing group on the benzene ring of C-terminal salicylaldehyde (SAL) ester was able to enhance the reactivity of peptide C-terminal SAL ester, hence expediting the reaction rates of STL. And we found the trifluoromethyl (CF3) group substituted peptide C-terminal SAL ester exhibited the fastest reaction rate in STL compared with other electron-withdrawing groups. The CF3-substituted peptide SAL esters were able to not only expedite the STL reaction rates, but also enable higher conversions, especially for some C-terminal amino acids which exhibited low conversion using non-substituted salicylaldehyde esters. Taking advantage of CF3-substituted peptide SAL esters, we improved the STL conversion in pramlintide and sermorelin synthesis. Importantly, the CF3-substituted peptide SAL esters enabled the access of partial metreleptin (34-101) via peptide Pro-SAL esters. The efficiency of CF3-substituted peptide Pro-SAL esters for STL was fully demonstrated in the protein p53 (1-100) synthesis. The findings documented in this study, including the synthesis of peptide C-terminal SAL esters bearing base-labile O-acetylated Ser/Thr and S-palmitoylated cysteine by Boc-SPPS, and the CF3-substituted C-terminal peptide SAL esters improved the STL reactions, will promote broader applications of STL and CPL in protein chemical synthesis.