High-performance liquid chromatography (HPLC) is an essential analytical and purification tool in peptide chemistry. Although the demand for peptide hydrazides, which are key intermediates for native chemical ligation (NCL), is increasing, peptide hydrazides often exhibit significant peak tailing during HPLC purification, especially when using degraded columns, leading to reduced resolution and purification efficiency. Therefore, developing a method to improve the HPLC peak shapes of peptide hydrazides is crucial.
To address this issue, we developed a removable dialkoxybenzyl (Dab) linker. The Dab linker was synthesized in a four-step reaction without requiring column chromatography. Investigation of global deprotection conditions revealed its unique reactivity: treatment with TFA–triisopropylsilane–H₂O at 37 °C completely removed the Dab moiety to yield free hydrazides, whereas using a TMSBr-containing cocktail retained the Dab linker intact while removing all other protecting groups.
Peptide hydrazides alkylated with the Dab linker exhibited sharp and highly symmetrical peaks in HPLC analysis. Utilizing this method for ubiquitin synthesis, we compared the isolated yields of intermediate peptide fragments bearing either the Dab group or as free hydrazides, and found that the yields of the Dab-alkylated fragments were two to three times higher than those of the free hydrazide counterparts.