Bioconjugation Using Mutant Glycosyltransferases for the Site-Specific Labeling of Biomolecules with Sugars Carrying Chemical Handles

Abstract

This chapter presents a technique that employs mutant glycosyltransferase enzymes for the site-specific bioconjugation of biomolecules via a glycan moiety to facilitate the development of a targeted drug delivery system. The target specificity of this methodology is based on unique sugar residues that are present on glycoproteins or engineered glycopeptides. The glycosyltransferases used in this approach have been manipulated in a way that confers the ability to transfer a modified sugar residue with a chemical handle to a sugar moiety of the glycoprotein or to a polypeptide tag of an engineered nonglycoprotein. The availability of the modified sugar moiety thus makes it possible to link cargo molecules at specific sites. The cargo may be comprised of, for example, biotin or fluorescent tags for detection, imaging agents for magnetic resonance imaging (MRI), or cytotoxic drugs for cancer therapy.

Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry

“…They first homogenize mAb glycoforms via galactosidase digestion and subsequently integrate keto-or azide-modified galactose residues using mutant galactosyltransferases (40,41). Similarly, a recent report by Senter and co-workers exploits the promiscuity of native fucosyltransferases to incorporate thiol-functional fucose analogues for thiolmaleimide conjugation to MMAE (42).…”

Glycans of Antibodies as a Specific Site for Drug Conjugation Using Glycosyltransferases

“…32,33 The addition of the modified galactose at a specific glycan residue of a mAb permits the coupling of a biomolecule that carries an orthogonal reactive group (Figure 4 A,B). The transferred C2-keto-Gal is coupled to any molecule carrying an aminooxy group, 29 e.g.…”

“…The transferred C2-keto-Gal is coupled to any molecule carrying an aminooxy group, 29 e.g. aminooxy-biotin, aminooxyfluoroprobes, [31][32][33][34] or aminooxy-drug 35, 36 ( Figure 4A). The GalNAz that was transferred from UDP-GalNAz to the GlcNAc residues on the mAb glycan with the mutant enzyme β4Gal-T1-Y289L is then conjugated with alkyne residue using Cu-catalyzed azidealkyne cycloaddition click chemistry 23 ( Figure 4B) or Cu-free strain-promoted azidealkyne Huisgen cycloaddition reaction.…”

“…24,37 The linking of alkyne derivatives carrying a fluorescent dye selectively occurs with the modified galactose at the heavy chain of these mAbs, without altering their antigen-binding activities, as shown by indirect enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell sorting (FACS) methods. 33,34 These studies demonstrated that linking cargo molecules to mAbs via glycans could prove to be an invaluable tool for potential drug targeting by immunotherapeutic methods. 25,34 This technique of labeling mAbs using the mutant enzyme β4Gal-T1-Y289L has recently been used by several researchers.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2011–2012

“…Details have also beem published for bioconjugation reactions using mutant glycosyltransferases for site-specific labeling with sugars carrying chemical handles (Ramakrishnan et al, 2011). Liu et al (2011a) have evaluated a method for extracting drugs from pharmaceutical preparations with maintenance of quality and structural integrity, particularly of the N-glycans.…”