Removable dialkoxybenzyl linker for enhanced HPLC purification of peptide hydrazides

High-performance liquid chromatography (HPLC) plays a crucial role in purifying peptides and proteins and monitoring their reactions. Peptide hydrazides are widely employed intermediates in modern peptide/protein chemistry. However, they often exhibit peak tailing during HPLC purification and analysis. Herein, we describe a dialkoxybenzyl-linker approach to improve HPLC performance in the purification and analysis of peptide hydrazides. A dialkoxybenzyl linker is designed for the solid-phase synthesis of peptide hydrazides, enabling selective cleavage under controlled global deprotection conditions to afford alkylated or free hydrazides. The peptides with this linker exhibit superior HPLC peak shapes, even on degraded columns, compared with those of free hydrazides. In addition, the dialkoxybenzyl linker enhances the overall yield in solid-phase peptide synthesis compared with that achieved using a conventional trityl-based linker. The utility of this linker is validated by the successful synthesis of ubiquitin from three peptide segments using native chemical ligation, achieving higher isolated yields of the peptide segments because of the improved purification efficiency. Thus, this approach is a powerful tool for the synthesis and purification of peptide hydrazides. In conclusion, the dialkoxybenzyl linker enhances HPLC peak symmetry, separation, and solid-phase peptide synthesis yields for peptide hydrazides, providing a practical solution to key purification challenges in peptide chemistry.