Oxidation and Phenolysis of Peptide/Protein C-Terminal Hydrazides Afford Salicylaldehyde Ester Surrogates for Chemical Protein Synthesis

With the growing popularity of serine/threonineligation(STL)and cysteine/penicillamine ligation (CPL) in chemical protein synthesis,facile and general approaches for the preparation of peptide salicylaldehyde(SAL) esters are urgently needed, especially those viable for obtainingexpressed protein SAL esters. Herein, we report the access of SALester surrogates from peptide hydrazides (obtained either syntheticallyor recombinantly) via nitrite oxidation and phenolysis by 3-(1,3-dithian-2-yl)-4-hydroxybenzoicacid (SAL(-COOH)(PDT)). The resulting peptide SAL(-COOH)(PDT) esters can be activated to afford the reactive peptideSAL(-COOH) esters for subsequent STL/CPL. While being operationallysimple for both synthetic peptides and expressed proteins, the currentstrategy facilitates convergent protein synthesis and combined applicationof STL with NCL. The generality of the strategy is showcased by theN-terminal ubiquitination of the growth arrest and DNA damage-inducibleprotein (Gadd45a), the efficient synthesis of ubiquitin-like protein5 (UBL-5) via a combined N-to-C NCL-STL strategy, and the C-to-N semisynthesisof a myoglobin (Mb) variant.

Automated summary

In order to meet the demand for peptide salicylaldehyde esters, we developed a new method for the preparation of these esters from peptide hydrazides through nitrite oxidation and phenolysis. This method is simple and suitable for both synthetic peptides and expressed proteins, facilitating peptide synthesis and the combined application of NCL and STL.