Page 44 - Tyrosine-Based Bioconjugations - Jorick Bruins
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Chapter 2
2.1. Introduction
Functional modification of proteins can be achieved in a wide variety of ways. Early generation approaches, entailing the reaction with amino or thiol groups of amino acid side chains,1-3 are effective and facile but in most cases lack selectivity due to relatively high natural abundance of lysines and cysteines in proteins.3 Other methods involving conjugation to tyrosine, histidine, or tryptophan side chains or to the N-terminus of proteins have also been developed. As an alternative to native proteins, full control of regioselectivity can be achieved by introduction of a non-natural amino acid containing a functional handle such as an azide, a ketone or an ortho- aminophenol but often at the expense of protein expression yields.4-6 Site-specific conjugation can also be achieved through enzymatic means, as, for example, with sortase-mediated conjugation, formyl-generating enzyme (FGE), tubulin tyrosine ligase, or activation of dihydrotetrazines by oxidation for tetrazine-TCO ligation via horseradish peroxidase or a photocatalytic agent.7-10
Tyrosine residues show promise as selective conjugation sites, as the relative hydrophobicity of tyrosine combined with the tendency of π–π stacking of the aromatic rings results in the limited exposure of tyrosine residues on the periphery of proteins, resulting in generally low accessibility.11, 12 As a consequence, reactive small molecules may conjugate to tyrosines for less selective conjugation,13-15 while more exposed tyrosine residues can allow for a wide variety of enzymatic reactions.12, 16-20 For example, exposed tyrosine residues can be oxidized by mushroom tyrosinase to generate a 1,2-quinone, which can undergo nucleophilic attack by amines or thiols from the side chains of lysine, histidine, or cysteine.21-23
We recently showed that a 1,2-quinone undergoes fast strain-promoted oxidation-controlled quinone–alkyne cycloaddition (SPOCQ) with bicyclo[6.1.0]nonyne (BCN).24-26 Here, we report that SPOCQ finds useful application in protein labelling via in situ generation of a quinone by oxidation under the action of mushroom tyrosinase (mTyr). We demonstrate that fast and complete C-terminal labelling of proteins, including an enzyme and a monoclonal antibody, can be readily achieved with fluorescent BCN-lissamine 1. The potential usefulness of the SPOCQ labelling approach is exemplified by fully controlled, site-specific generation of an antibody-drug conjugate based on anti-influenza AT1002 and BCN bearing the highly potent tubulin binder monomethyl auristatin F (BCN-MMAF) 2.
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