Affiliation: | 1. Chemical Biology Department, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Rössle-Strasse 10, 13125 Berlin, Germany;2. Department of Biology II and, Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universität München, Großhadernerstrasse 2, 82152 Martinsried, Germany
Tubulis GmbH, BioSysM, Butenandtstrasse 1, 81377 Munich, Germany;3. Institut für Chemie und Biochemie, Freie Universität Berlin, Takustrasse. 3, 14195 Berlin, Germany;4. Chemical Biology Department, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Rössle-Strasse 10, 13125 Berlin, Germany
Tubulis GmbH, BioSysM, Butenandtstrasse 1, 81377 Munich, Germany;5. Department of Biology II and, Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universität München, Großhadernerstrasse 2, 82152 Martinsried, Germany |
Abstract: | 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. |