Chemical Biology Department, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Rössle-Strasse 10, 13125, Berlin, Germany.
Department of Chemistry, Humboldt Universität zu Berlin, Brook-Taylor-Strasse 2, 12489, Berlin, Germany.
Chembiochem. 2020 Jan 15;21(1-2):113-119. doi: 10.1002/cbic.201900587. Epub 2020 Jan 7.
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.
本文报道了 N-羟基琥珀酰亚胺修饰的膦酰胺酯砌块在生理 pH 条件下，将半胱氨酸选择性乙炔基膦酰胺酯引入蛋白质赖氨酸残基中的应用，随后与小分子和蛋白质进行硫醇加成。结果表明，与常用的琥珀酰亚胺 4-（N-马来酰亚胺甲基）环己烷-1-羧酸酯（SMCC）相比，砌块显著降低了不期望的同型交叉连接副产物的形成。此外，膦酰胺酯部分的稳定性还解决了马来酰亚胺过早水解的问题，这可能会阻碍后续硫醇加成的效率。此外，还开发了一种分离膦酰胺酯对映体的方法，以便能够以确定的构型合成蛋白质缀合物。最后，该砌块被用于构建功能性抗体-药物偶联物，类似于 FDA 批准的、与 SMCC 相连的 Kadcyla，以及功能性抗体-蛋白质偶联物的合成。
