Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02139, USA.
Chembiochem. 2022 Jul 19;23(14):e202200258. doi: 10.1002/cbic.202200258. Epub 2022 Jun 1.
The S-alkylation of Cys residues with a maleimide and the N -acylation of Lys residues with an N-hydroxysuccinimide (NHS) ester are common methods for bioconjugation. Using Cys and Lys derivatives as proxies, we assessed differences in reactivity depending on the position of Cys or Lys in a protein sequence. We find that Cys position is exploitable to improve site-selectivity in maleimide-based modifications. Reactivity decreases substantially in the order N-terminal>in-chain>C-terminal Cys due to modulation of sulfhydryl pK by the α-ammonium and carboxylate groups at the termini. A lower pK value yields a larger fraction thiolate, which promotes selectivity while somewhat decreasing thiolate nucleophilicity in accord with =0.41. Lowering pH and salt concentration enhances selectivity still further. In contrast, differences in the reactivity of Lys towards an NHS ester were modest due to an appreciable decrease in amino group nucleophilicity with a lower pK of its conjugate acid. Hence, site-selective Lys modification protocols will require electrophiles other than NHS esters.
半胱氨酸残基的 S-烷基化与赖氨酸残基的 N-酰化反应是常见的生物偶联方法。我们使用半胱氨酸和赖氨酸衍生物作为替代物，根据蛋白质序列中半胱氨酸或赖氨酸的位置评估反应性的差异。我们发现，半胱氨酸的位置可用于提高基于马来酰亚胺的修饰的位点选择性。由于末端的α-铵和羧酸盐基团对半胱氨酸巯基 pK 的调节，N 端＞链中＞C 端半胱氨酸的反应性按顺序显著降低。较低的 pK 值产生更大比例的硫醇盐，这促进了选择性，同时根据 =0.41 略微降低了硫醇盐的亲核性。降低 pH 值和盐浓度可进一步提高选择性。相比之下，由于其共轭酸的较低 pK 值导致氨基亲核性明显降低，赖氨酸对 NHS 酯的反应性差异不大。因此，除 NHS 酯外，还需要其他亲电试剂来进行选择性赖氨酸修饰。
