Formal [4 + 2] Cycloadditions of Maleimides on Duplex DNA

Near-quantitative DNA bioconjugation and detailed mechanistic investigations of reactions involving 5-(vinyl)-2′-deoxyuridine (VdU) and maleimides are reported. According to accelerated reaction rates in solvents with increasing polarity and trends in product stereochemistry, VdU–maleimide reactions proceed via a formal [4 + 2] stepwise cycloaddition. In contrast, 5-(1,3-butadienyl)-2′-deoxyuridine (BDdU) reacts with maleimides in a concerted [4 + 2] Diels–Alder cycloaddition. VdU–maleimide reactions enable high-yielding bioconjugation of duplex DNA in vitro (>90%) as well as metabolic labeling experiments in cells.