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

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.

Automated summary

Thiazine formation during the conjugation of N-terminal cysteine peptides to maleimides is an underreported side reaction. Different amino acid substitutions near the N-terminal cysteine and variations in the N-substitution of the maleimide affect the rate of thiazine formation. Performing the conjugation under acidic conditions or avoiding the use of N-terminal cysteine with a free amino group can prevent the formation of the thiazine impurity.