An Enzyme Cascade for Selective Modification of Tyrosine Residues in Structurally Diverse Peptides and Proteins.

Bioorthogonal chemistry enables a specific moiety in a complex biomolecule to be selectively modified in the presence of many reactive functional groups and other cellular entities. Such selectivity has become indispensable in biology, enabling biomolecules to be derivatized, conjugated, labelled or immobilized for imaging, biochemical assays or therapeutic applications. Methyltransferase enzymes (MTase) that accept analogs of the cofactor S adenosyl methionine have been widely deployed for al-kyl-diversification and bioorthogonal labelling. However, MTases typically possess tight substrate specificity. Here we introduce a more flexible methodology for selective derivatization of phenolic moieties in complex biomolecules. Our approach relies on the tandem enzymatic reaction of a fungal tyrosinase and the mammalian catechol-O-methyltransferase (COMT), which can effect the sequential hydroxylation of the phenolic group to give an intermediate catechol moiety that is subsequently O-alkylated. When used in this combination, the alkoxylation is highly selective for tyrosine residues in peptides and proteins, yet remarkably tolerant to changes in the peptide sequence. Tyrosinase-COMT are shown to provide highly versatile and regioselective modification of a di-verse range of substrates including peptide antitumor agents, hormones, cyclic peptide antibiotics and model proteins.