74. NHS-modified ethynylphosphonamidates enable the synthesis of configurationally defined protein conjugates
M.-A. Kasper, M. Gerlach, A. Schneider, C. Groneberg, S. Boldt, P. Ochtrop, D. Schumacher, J. Helma, H. Leonhardt, M. Christmann, C. P. R. Hackenberger
Herein, we describe the application of N‐hydroxy‐succinimide‐(NHS‐)‐modified phosphonamidate building blocks for the incorporation of Cysteine‐selective ethynylphosphonamidates to lysine residues of proteins followed by thiol addition with small molecules and proteins. We demonstrate that our 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 the subsequent thiol addition. Furthermore, we developed a method to separate the phosphonamidate enantiomers to be able to synthesize protein‐conjugates in a defined configuration. Finally, we apply our building blocks to the construction of functional Antibody Drug Conjugates (ADCs), analogously to the FDA‐approved, SMCC‐linked Kadcyla and to the synthesis of a functional antibody‐protein conjugate.