ABSTRACT Aryl fluorosulfate warheads, possessing sulfur(VI) fluoride exchange (SuFEx) reaction, hold significant promise for the development of covalent protein drugs. However, their SuFEx reactivity remains limited within the complex microenvironment of protein interactions. To address this challenge, we sought to enhance their reactivity by adjusting the electronic and steric properties of warheads. Herein, we synthesized various maleimide‐functionalized aryl fluorosulfate (MFS) bearing different substituents (e.g., o‐F, o‐CF 3 , o‐NO 2 , o‐CH 3 , o‐OCH 3 , o‐Cl, o‐Br, o‐I, 2,6‐diF, and m‐F), which were then chemically conjugated to Adnectin (an EGFR‐targeting protein). The SuFEx reactivity of the resulting xMFS‐modified Adnectin was systematically investigated by comparing their covalent cross‐linking efficiency to EGFR. Notably, the meta‐Fluoro‐substituted MFS warhead, featuring moderate electrophilicity and minimal steric hindrance, exhibited the highest reactivity, achieving a 3.5‐fold increase in cross‐linking efficiency compared to unsubstituted control. The m‐F MFS‐modified Adnectin was further attached to the surface of albumin‐bound DXd. Leveraging its enhanced SuFEx reactivity, the resulting covalent albumin‐bound drug exhibited 6.4‐fold higher intracellular accumulation, 3.0‐fold greater tumor retention, and 4.0‐fold higher antitumor efficiency compared to unsubstituted control. Overall, fine‐adjusting the electronic and steric properties of warheads significantly enhances their SuFEx reactivity, enabling the rational design of SuFEx‐based warheads and facilitating the application in covalent protein drugs.
Gao et al. (Fri,) studied this question.