Site-Selective Protein Chemical Modification of Exposed Tyrosine Residues Using Tyrosine Click Reaction

Abstract

Targeting less abundant amino acid residues on the protein surface may realize site-selective protein modification of natural proteins. The relative hydrophobicity of tyrosine combined with the π–π stacking tendency of the aromatic rings results in generally low accessibility. In this study, site-selective protein modification was achieved by targeting surface-exposed tyrosine residues without using a genetic encoding system. Tyrosine residues were modified with N-methylated luminol derivative under single-electron transfer (SET) reaction conditions. Horseradish peroxidase (HRP)-catalyzed SET and electrochemically activated SET modified surface-exposed tyrosine residues selectively. N-Methylated luminol derivative modified tyrosine residues more efficiently than 4-arylurazole under tyrosine click conditions using HRP and electrochemistry. Tyrosine residues that are evolutionarily exposed only in the complementarity-determining region (CDR) of an antibody were selectively modified by tyrosine click reactions. CDR-modified antibodies were applied to in vivo imaging and antibody–drug conjugated (ADC).

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.…”

Tyrosine bioconjugation – an emergent alternative

“…Sato et al not only demonstrated that the luminol derivatives are more efficient for tyrosine modification than the PTADs under these conditions, but also are better substrates for the e-Y-click. 31 In addition, they succeeded in site-selective modification of several proteins owing to the unique high solvent accessibility of one or more tyrosine residues, which further reinforces the robustness of tyrosine modification in particular cases. 31 For example, streptavidin was selectively modified on Y83, trastuzumab on Y57 and rituximab on all four of its solvent accessible tyrosine residues.…”

“…This pioneering work also inspired further research in the field of electrochemically promoted tyrosine-selective protein labeling. 30,31 Another way to avoid side reactions was to use luminol derivatives 12 that do not decompose to isocyanates. 32 N-Methyl luminol derivatives activated in situ with hydrogen peroxide and hemin as a catalyst were conjugated to angiotensin II with 95% conversion (Scheme 3c).…”

“…This pioneering work also inspired further research in the field of electrochemically promoted tyrosine-selective protein labeling. 30 , 31 …”

Site-selective modification strategies in antibody–drug conjugates

“…20G). 327 In this protocol, horseradish peroxidase (HRP) catalysed single electron transfer modification of tyrosine phenol with N-methylated luminol derivatives containing a pendant azide. Modification was found to occur on tyrosines in the complementarity determining region (HC-Y53).…”