Liu Weibing, Khan Aziz, Demyanenko Yana, Mohammed Shabaz, Davis Benjamin G
The Rosalind Franklin Institute, Harwell Oxfordshire, UK.
Department of Pharmacology, University of Oxford, Oxford, UK.
Nat Commun. 2025 Jul 1;16(1):6028. doi: 10.1038/s41467-025-60527-5.
N-Hydroxysuccinimide (NHS)-ester derivatives are widely used reagents in biological chemistry and chemical biology. Their efficacy relies critically on the exclusive chemoselectivity of activated acyl over that of the imidic acyl moieties in the succinimide. Here, through systematic structural variation that modulates acyl reactivity, coupled with a statistically controlled ultra-rapid screen for unknown modifications in tandem mass spectra as well as lysine profiling across complex lysine environments, including those within proteomes containing many thousands of proteins, we reveal that ring-opening to afford N-succinamide derivatives is a present, sometimes dominant, side-reaction. The extent of side-reaction is shown to be site-dependent, with side-reaction and desired reaction occurring within the same protein substrate. The resulting formation of bioconjugates with unintended, unstable linkages and modifications suggests the re-evaluation of: (i) known commercial reagents; and (ii) functional conclusions previously drawn using NHS esters in areas as diverse as antibody-drug biotherapy, vaccination and cross-link-enabled structural analyses.
N-羟基琥珀酰亚胺（NHS）酯衍生物是生物化学和化学生物学中广泛使用的试剂。它们的功效关键取决于活化酰基相对于琥珀酰亚胺中亚胺酰基部分的独特化学选择性。在此，通过调节酰基反应性的系统结构变化，结合用于串联质谱中未知修饰的统计控制超快速筛选以及跨复杂赖氨酸环境（包括含有数千种蛋白质的蛋白质组中的赖氨酸环境）的赖氨酸分析，我们发现开环生成N-琥珀酰胺衍生物是一种存在的、有时占主导的副反应。结果表明，副反应的程度取决于位点，副反应和期望反应会在同一蛋白质底物中发生。生成具有意外、不稳定连接和修饰的生物共轭物表明需要重新评估：（i）已知的商业试剂；以及（ii）先前在抗体-药物生物疗法、疫苗接种和交联结构分析等不同领域使用NHS酯得出的功能结论。
