Copper assisted sequence-specific chemical protein conjugation at a single backbone amide

Abstract

Direct, site-specific methods of protein functionalization are highly desirable for biotechnology. However, such methods are challenging due to the difficulty of chemically differentiating a single site within a large protein. Herein, we propose “metal binding targeting” strategy and develop a Copper Assisted Sequence-specific conjugation Tag (CAST) method to achieve rapid (second order rate 8.1 M⁻¹ s⁻¹), site-specific protein backbone chemical modification with pinpoint accuracy. We demonstrate the versatility of CAST conjugation by preparing various on-demand modified recombinant proteins, including a homogeneous antibody-drug conjugate with high plasma stability and potent efficacy in vitro and in vivo. Thus, CAST provides an efficient and quantitative method to site-specifically attach payloads on large, native proteins.

Direct, site-specific methods of protein functionalization are of interest, but challenging due to difficulty in chemically differentiating a single site within a large protein. Here, the authors develop a Copper Assisted Sequence-specific conjugation Tag (CAST) method to achieve rapid, site-specific protein backbone chemical modification with pinpoint accuracy, and prepare various on-demand modified recombinant proteins using CAST.