Sequence sensitivity and pH dependence of maleimide conjugated N-terminal cysteine peptides to thiazine rearrangement.

Abstract

Thiazine formation during the conjugation of N‐terminal cysteine peptides to maleimides is an underreported side reaction in the peptide literature. When the conjugation was performed at neutral and basic pH, we observed the thiazine isomer as a significant by‐product. Nuclear magnetic resonance (NMR) spectroscopy confirmed the structure of the six‐membered thiazine and ultra‐high performance liquid chromatography (UHPLC) combined with tandem mass spectrometry (MS/MS) allowed for facile, unambiguous detection due to a unique thiazine mass fragment. Furthermore, substitution of various amino acids adjacent to the N‐terminal cysteine in a tripeptide model system resulted in different rates of thiazine formation, albeit within the same order of magnitude. We also determined that varying the N‐substitution of the maleimide affects the thiazine conversion rate. Altogether, our findings suggest that thiazine rearrangement for N‐terminal cysteine‐maleimide adducts is a general side reaction that is applicable to other peptide or protein systems. Performing the conjugation reaction under acidic conditions or avoiding the use of an N‐terminal cysteine with a free amino group prevents the formation of the thiazine impurity.