Site-selective chemical modifications of proteins have emerged as a potent technology in chemical biology, materials science, and medicine, facilitating precise manipulation of proteins with tailored functionalities for basic biology research and developing innovative therapeutics. Compared to traditional recombinant expression methods, one of the prominent advantages of chemical protein modification lies in its capacity to decorate proteins with a wide range of functional moieties, including non-genetically encoded ones, enabling the generation of novel protein conjugates with enhanced or previously unexplored properties. Among these, approaches for dual or multiple modifications of proteins are increasingly garnering attention, as it has been found that single modification of proteins is inadequate to meet current demands. Therefore, in light of the rapid developments in this field, this review provides a timely and comprehensive overview of the latest advancements in chemical and biological approaches for dual functionalization of proteins. It further discusses their advantages, limitations, and potential future directions in this relatively nascent area.
Funding
This work was supported by the grant from Zhejiang Provincial Natural Science Foundation of China under Grant No. LY24B020008 and Zhejiang University of Technology High-Level Talents Startup Funds (2021414800229, X. B. Bi). X. Hemu is the recipient of National Natural Science Foundation of China (No. 32371324), National Key Research and Development Program of China (No. 2023YFA0916000) and the High-level Talent Startup Fund by China Pharmaceutical University. W.L. is the recipient of NHMRC Investigator Grant EL1 (APP2018256) and Ramaciotti Health Investment Grant.