Herein, the application of N-hydroxysuccinimide-modified phosphonamidate building blocks for the incorporation of cysteine-selective ethynylphosphonamidates into lysine residues of proteins, followed by thiol addition with small molecules and proteins, is reported. It is demonstrated that the building blocks significantly lower undesired homo-crosslinking side products that can occur with commonly applied succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) under physiological pH. The previously demonstrated stability of the phosphonamidate moiety additionally solves the problem of premature maleimide hydrolysis, which can hamper the efficiency of subsequent thiol addition. Furthermore, a method to separate the phosphonamidate enantiomers to be able to synthesize protein conjugates in a defined configuration has been developed. Finally, the building blocks are applied to the construction of functional antibody-drug conjugates, analogously to FDA-approved, SMCC-linked Kadcyla, and to the synthesis of a functional antibody-protein conjugate.

中文翻译：

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N-羟基琥珀酰亚胺修饰的乙炔基膦酰胺能够合成构型确定的蛋白质缀合物。

在此，报道了应用 N-羟基琥珀酰亚胺修饰的膦酰胺结构单元将半胱氨酸选择性乙炔基膦酰胺掺入蛋白质的赖氨酸残基中，然后用小分子和蛋白质添加硫醇。结果表明，这些结构单元可显着降低生理 pH 下常用的琥珀酰亚胺基 4-(N-马来酰亚胺甲基)环己烷-1-羧酸酯 (SMCC) 可能出现的不良同源交联副产物。先前证明的膦酰胺部分的稳定性还解决了马来酰亚胺过早水解的问题，该水解可能会妨碍随后硫醇添加的效率。此外，还开发了一种分离膦酰胺对映体的方法，以便能够合成确定构型的蛋白质缀合物。最后，这些构建模块被应用于构建功能性抗体-药物缀合物（类似于 FDA 批准的、SMCC 连接的 Kadcyla），并用于合成功能性抗体-蛋白质缀合物。