Ascorbate Peroxidase (APEX2) Activates Dihydrotetrazine Oxidation for Rapid Bioorthogonal Chemistry in Living Cells

It is shown that genetically expressible APEX2 efficiently catalyzes the oxidation of dihydrotetrazine (DHTz) to give tetrazine products that engage in rapid bioorthogonal reactions with trans-cyclooctene dienophiles. Through in vitro studies, it is shown that the APEX2-catalyzed DHTz oxidation is accelerated by superoxide dismutase— an enzyme found in all mammalian cells that regulates oxidative stress by disproportioning superoxide into O2 and H2O2. While the addition of H2O2 is not required for APEX2-catalyzed DHTz oxidation, the catalytic efficiency is increased substantially to kcat/KM 4.90 × 103 M–1s–1 by the addition of a small amount of H2O2 (10 μM). These ‘minimal peroxide’ conditions are mild compared to the higher H2O2 concentrations that are typically employed in phenolic oxidation reactions promoted by APEX2. The ability of APEX2 to promote DHTz oxidation for subsequent Diels-Alder chemistry was demonstrated in live HeLa cells that, through a dual-transfection protocol, contained cytosolic APEX2 and a HaloTag-DHTz conjugate. Upon oxidation, the resulting tetrazine was conjugated in situ to a fluorophore-tagged TCO, and conjugation efficiency was assayed through in-gel fluorescence, western blot analysis, and confocal microscopy. In live PC3 cells, it was further shown that APEX2 catalyzed the oxidation of a DHTz that had been selectively conjugated to endogenous monoacylglycerol lipase (MAGL) via a selective covalent warhead.