Chemoselective and Site‐Selective Lysine‐Directed Lysine Modification Enables Single‐Site Labeling of Native Proteins

Abstract

The necessity for precision labeling of proteins emerged during the efforts to understand and regulate their structure and function. It demands selective attachment of tags such as affinity probes, fluorophores, and potent cytotoxins. Here, we report a method that enables single‐site labeling of a high‐frequency Lys residue in the native proteins. At first, the enabling reagent forms stabilized imines with multiple solvent‐accessible Lys residues chemoselectively. These linchpins create the opportunity to regulate the position of a second Lys‐selective electrophile connected by a spacer. Consequently, it enables the irreversible single‐site labeling of a Lys residue independent of its place in the reactivity order. The user‐friendly protocol involves a series of steps to deconvolute and address chemoselectivity, site‐selectivity, and modularity. Also, it delivers ordered immobilization and analytically pure probe‐tagged proteins. Besides, the methodology provides access to antibody‐drug conjugate (ADC), which exhibits highly selective anti‐proliferative activity towards HER‐2 expressing SKBR‐3 breast cancer cells.

Investigating Ugi/Passerini Multicomponent Reactions for the Site‐Selective Conjugation of Native Trastuzumab**

“…[11][12][13] In parallel, site-selective chemical strategies for the conjugation of native and natural proteins have also flourishedo ver the past few years, giving rise to methods targeting varioust ypes of amino acids (for example, lysine, cysteine, tryptophan, tyrosine) that proved to be effective on proteins of all sizes, including antibodies. [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] With the aim of pursuing the efforts in this field, we could not help but notice that the vast majority of previously reported strategies for the site-selective conjugation of native proteins were focusedo nt he modificationo faunique residue. We hypothesized that targeting two different amino acid side chainss imultaneously would lower the enormouss ubset of possibilities given by single-residue bioconjugation techniques, thus increasing our chances of developing as ite-selective method by minimising the number of potentially reactive sites;apath that has also been successfully explored by others in the meantime.…”

“…Regioselective (i.e., site‐specific) methods were thus developed and are currently dominated by the use of recombinant proteins, incorporating exogenous amino acid residues (natural or unnatural) or peptide sequences that can be specifically targeted by a tailored reagent or strategy [11–13] . In parallel, site‐selective chemical strategies for the conjugation of native and natural proteins have also flourished over the past few years, giving rise to methods targeting various types of amino acids (for example, lysine, cysteine, tryptophan, tyrosine) that proved to be effective on proteins of all sizes, including antibodies [14–28] …”

A Reagent for Amine‐Directed Conjugation to IgG1 Antibodies

“…Analysis of crystal structures of Fab domains of Rituximab, Cetuximab and Belimumab showed that these labelling sites indeed have a nearby lysine residue in the 3D structure (Supporting information Figure S19–S21). We know from earlier studies, using the same salicylaldehyde guiding moiety for protein labeling, that labeling of the LC was also observed [26] . We were however, pleased to see that for the LDLR 1 a reagent most sites were located on the HC (Data shown in Supporting information pages S14–16).…”

“…In other studies, mentioned above, where the same guiding moiety is used, much harsher conditions were required with up to 25 equiv. of reagent and in some cases 20 % DMSO [25, 26] . Furthermore, the labeling from the LDLR 1 a occurred predominantly once on the heavy chain.…”

Site-selective modification strategies in antibody–drug conjugates

“…14A). 224 2.6.3 Selective arginine modification. The lower abundance of arginine residues in native antibodies compared to lysine residues makes arginine-selective modification attractive for the preparation of homogeneous conjugates.…”