Traceless cysteine-linchpin enables precision engineering of lysine in native proteins

Abstract

The maintenance of machinery requires its operational understanding and a toolbox for repair. The methods for the precision engineering of native proteins meet a similar requirement in biosystems. Its success hinges on the principles regulating chemical reactions with a protein. Here, we report a technology that delivers high-level control over reactivity, chemoselectivity, site-selectivity, modularity, dual-probe installation, and protein-selectivity. It utilizes cysteine-based chemoselective Linchpin-Directed site-selective Modification of lysine residue in a protein (LDMC-K). The efficiency of the end-user-friendly protocol is evident in quantitative conversions within an hour. A chemically orthogonal C-S bond-formation and bond-dissociation are essential among multiple regulatory attributes. The method offers protein selectivity by targeting a single lysine residue of a single protein in a complex biomolecular mixture. The protocol renders analytically pure single-site probe-engineered protein bioconjugate. Also, it provides access to homogeneous antibody conjugates (AFC and ADC). The LDMC-K-ADC exhibits highly selective anti-proliferative activity towards breast cancer cells.

Disintegrate (DIN) Theory Enabling Precision Engineering of Proteins

“…The LDM platform also extends the site-selective modification of His or Asp using an alkylating reagent such as sulfonate esters ( 12c , F X ) . Additionally, we demonstrated that nitroolefins could shift the linchpin sites from a high-frequency residue (Lys) to Cys with low occurrence . This considerably reduces the number of competitors and creates the opportunity to translate the method for protein selectivity in a complex biological milieu.…”

“…100 Additionally, we demonstrated that nitroolefins could shift the linchpin sites from a high-frequency residue (Lys) to Cys with low occurrence. 101 This considerably reduces the number of competitors and creates the opportunity to translate the method for protein selectivity in Here, the chemoselective thio-Michael addition (13b) enables the site-selective Lys acylation (15b). The linchpin's release from the thio-Michael adduct is coupled with a retro-Henry reaction to render an aldehyde under mild conditions for downstream utility.…”

Human Behavior-Inspired Linchpin-Directed Catalysis for Traceless Precision Labeling of Lysine in Native Proteins

“…We initiated the search for building blocks by selecting ortho-hydroxyaldehyde as LF. In our earlier investigations, − we established its capability for chemoselective imine (linchpin) formation with all of the solvent-accessible Lys residues to regulate the proximal placement of an electrophile. For pIP, an electrophilic system must display negligible reactivity with proteinogenic residues in both intermolecular and intramolecular processes.…”

“…In our previous work on LDM, − we demonstrate how a chemical platform can use a reaction sequence to control one or more reactive species’ localization factors (LFs, Figure ). Here, we decoupled the interacting partner (IP) into a pro-interacting partner (pIP) and its activator (pIPa).…”

“…While the N-terminus imine is captured irreversibly by a proximal thiol in the first case, the latter involves the nucleophilic intermediate generation that subsequently renders the N-Gly adduct. On the other hand, chemoselectively placed imine-based linchpins could enable site-selective modification of His, Lys, and Asp. − However, this LDM platform is not traceless. Consequently, we cannot translate the method for post-translational modifications or regulate the linkers in the antibody conjugates.…”