One‐Step Maleimide‐Based Dual Functionalization of Protein N‐Termini

Abstract Maleimide derivatives are privileged reagents for chemically modifying proteins through the Michael addition reaction with cysteine due to their selectivity, operational simplicity, and commercial availability. However, since accessible free cysteine is rarely found in natural proteins, it is highly desirable to find alternative targets to enable direct bioconjugation of proteins with maleimides. In this study, we have developed an operationally simple and straightforward method for the N‐terminal modification of proteins without the need for mutagenesis via a copper(II)‐mediated [3+2] cycloaddition reaction with maleimides and 2‐pyridinecarboxaldehyde (2‐PCA) derivatives under non‐denaturing conditions at pH 6 and 37 °C in aqueous media. Our method utilizes commercially available maleimides to attach diverse functionalities to various N‐terminal amino acids. We demonstrate the preparation of a ternary protein complex cross‐linked at the N‐termini and dually modified trastuzumab equipped with monomethyl auristatin E (MMAE), a cytotoxic agent, and a Cy5 fluorophore (MMAE‐Cy5‐trastuzumab). MMAE‐Cy5‐trastuzumab retained human epidermal growth factor receptor 2 (HER2) recognition activity and exerted cytotoxicity against HER2‐positive cells. Furthermore, MMAE‐Cy5‐trastuzumab allowed successful visualization of HER2‐positive cancer cells in mouse tumors. This straightforward method will expand the accessibility of protein conjugates with well‐defined structures in a wide range of research fields.