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

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Sequence sensitivity and pH dependence of maleimide conjugated N-terminal cysteine peptides to thiazine rearrangement
Journal of Peptide Science ( IF 1.8 ) Pub Date : 2021-03-30 , DOI: 10.1002/psc.3323
Isaiah N Gober ₁ , Alexander J Riemen ₁ , Matteo Villain ₂

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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.

中文翻译：

马来酰亚胺偶联的 N 端半胱氨酸肽对噻嗪重排的序列敏感性和 pH 依赖性

在 N 端半胱氨酸肽与马来酰亚胺缀合期间形成噻嗪是肽文献中报道不足的副反应。当结合在中性和碱性 pH 值下进行时，我们观察到噻嗪异构体是一种重要的副产物。核磁共振 (NMR) 光谱证实了六元噻嗪的结构，超高效液相色谱 (UHPLC) 与串联质谱 (MS/MS) 相结合，由于具有独特的噻嗪质量碎片，因此可以轻松、明确地进行检测。此外，在三肽模型系统中替换与 N 端半胱氨酸相邻的各种氨基酸会导致不同的噻嗪形成率，尽管在同一数量级内。我们还确定改变马来酰亚胺的 N 取代会影响噻嗪转化率。总之，我们的研究结果表明 N 端半胱氨酸 - 马来酰亚胺加合物的噻嗪重排是适用于其他肽或蛋白质系统的一般副反应。在酸性条件下进行缀合反应或避免使用带有游离氨基的 N 端半胱氨酸可防止形成噻嗪杂质。

更新日期：2021-06-04

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