Investigating a Boronate‐Affinity‐Guided Acylation Reaction for Labelling Native Antibodies

Abstract

The excellent molecular recognition capabilities of monoclonal antibodies (mAbs) have opened up exciting opportunities for biotherapeutic discovery. Taking advantage of the full potential of this tool necessitates affinity ligands capable of conjugating directly with small molecules to a defined degree of biorthogonality, especially when modifying natural Abs. Herein, a bioorthogonal boronate‐affinity‐based Ab ligand featuring a 4‐(dimethylamino)pyridine and an S‐aryl thioester to label full‐length Abs is reported. The photoactivatable linker in the acyl donor facilitated purification of azide‐labelled Ab (N3‐Ab) was quantitatively cleaved upon brief exposure to UV light while retaining the original Ab activity. Click reactions enabled the precise addition of biotin, a fluorophore, and a pharmacological agent to the purified N3‐Abs. The resulting immunoconjugate showed selectivity against targeted cells. Bioorthogonal traceless design and reagentless purification allow this strategy to be a powerful tool to engineer native antibodies amenable to therapeutic intervention.

Chemical technology principles for selective bioconjugation of proteins and antibodies

“…Another similar method involved reversible boronic acid complexation with glycans to direct a nucleophilic catalyst. 224 The linchpin-guided platform inspired the development of 10 H -phenoxazine-3,7-dicarboxaldehyde for photoredox catalysed Tyr modification. 225 It would be fascinating to find out if this chemo- and site-selective approach can add modularity by incorporating spacers or linkers in these reagents.…”

“…393 These linchpins could also direct an acylium ion for selective generation of amide with Lys residues. 224…”

“…Another set of bifunctional reagents is equipped with boronic acid and DMAP moiety at two ends. 224 The reaction with a native antibody enables glycan-directed DMAP installation. The latter activates an external acylating reagent to generate an electrophilic center for labeling the antibody.…”

Disintegrate (DIN) Theory Enabling Precision Engineering of Proteins

“…In a principally similar approach, the reversible complexation of boronic acids (BA) with an antibody's F C -N-glycan directs the 4-(dimethylamino)pyridine (DMAP). 105 The latter forms N-acylpyridinium intermediate with thioester-based acyl donors to yield the acylation of a proximal Lys residue. Encouraged by the linchpin-guided protein modification platform, 10H-phenoxazine-3,7-dicarboxaldehyde was designed for Tyr modification via photoredox catalysis.…”

“…It required disintegrating the acylating reagent into two components, the catalyst ( 12f ) and the proelectrophile ( 12g ), both of which were equipped with a linchpin handle for proximity control (LDC). In a principally similar approach, the reversible complexation of boronic acids (BA) with an antibody’s F C - N -glycan directs the 4-(dimethylamino)­pyridine (DMAP) . The latter forms N -acylpyridinium intermediate with thioester-based acyl donors to yield the acylation of a proximal Lys residue.…”