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Abstract
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, labeled, or immobilized for imaging, biochemical assays, or therapeutic applications. Methyltransferase enzymes (MTase) that accept analogues of the cofactor S-adenosyl methionine have been widely deployed for alkyl-diversification and bioorthogonal labeling. 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 diverse range of substrates including peptide antitumor agents, hormones, cyclic peptide antibiotics, and model proteins.
Highlights
IntroductionThe introduction of bioorthogonal functional groups into biomolecules has found widespread application in biomolecular conjugation for imaging, therapeutics, and pull-down assays.[1−5] For example, biomolecules that have been derivatized with terminal alkynyl groups can undergo highly efficient and selective Cu(I)-catalyzed cycloaddition reactions, with azidefunctionalized coupling partners in a reaction that is entirely orthogonal to any other biological entities.[1−4] The exquisite selectivity of enzymes, coupled with the capability to reengineer their properties, offers a powerful means to introduce bioorthogonally reactive functionality into biomolecules of interest.[6−14] To this end, a number of enzymatic reactions have been exploited to selectively label biomolecules including peptides, proteins, nucleic acids, and glycans.[6−17]One class of enzymes that has proven to be versatile for functionalization of small metabolites through to biopolymers are the S-adenosyl methionine (AdoMet)-dependent methyltransferases (MTases).[15]
As far as we are aware, there are no reports of COMT-catalyzed methylation of LDOPA residues that were incorporated into peptides or proteins
This suggests that the sequence of the peptide has influence on the methylation reactions, with the more flexible and accessible terminal L-DOPA residue preferred by COMT
Summary
The introduction of bioorthogonal functional groups into biomolecules has found widespread application in biomolecular conjugation for imaging, therapeutics, and pull-down assays.[1−5] For example, biomolecules that have been derivatized with terminal alkynyl groups can undergo highly efficient and selective Cu(I)-catalyzed cycloaddition reactions, with azidefunctionalized coupling partners in a reaction that is entirely orthogonal to any other biological entities.[1−4] The exquisite selectivity of enzymes, coupled with the capability to reengineer their properties, offers a powerful means to introduce bioorthogonally reactive functionality into biomolecules of interest.[6−14] To this end, a number of enzymatic reactions have been exploited to selectively label biomolecules including peptides, proteins, nucleic acids, and glycans.[6−17]One class of enzymes that has proven to be versatile for functionalization of small metabolites through to biopolymers are the S-adenosyl methionine (AdoMet)-dependent methyltransferases (MTases).[15]. We set out to develop an alternative and more generic MTase based bioalkylation system This system would recognize and modify a specific and unique functional group within a range of proteinogenic and nonproteinogenic peptide scaffolds. To this end, we identified catechol-O-methyl-transferase (COMT), an MTase with more relaxed substrate specificity, as a potential bioalkylation tool. COMT accepts a range of catecholamines, including L-3,4dihydroxyphenylalanine (L-DOPA),[30] a nonproteinogenic
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