Enzymatic Incorporation of a Coumarin–Guanine Base Pair

Previous expansions beyond nature's preferred base‐pairing interactions have utilized either nonpolar shape‐fitting interactions or classical hydrogen bonding. Reported here is a hybrid of these systems. By replacing a single N−H with C−H at a Watson–Crick interface, the design space for new drug candidates and fluorescent nucleobase analogues is dramatically expanded, as demonstrated here by the new, highly fluorescent deoxycytidine mimic 3‐glycosyl‐5‐fluoro‐7‐methoxy‐coumarin‐2′‐deoxyribose (d C C ). dGTP is selectively incorporated across from a template d C C during enzymatic DNA synthesis. Likewise, d C C is selectively incorporated across from a template guanine when d C C is provided as the triphosphate d C C TP . DNA polymerase I (Klenow fragment) exhibited about a 10‐fold higher affinity for d C C TP than dCTP, allowing selective incorporation of d C C in direct competition experiments. These results demonstrate that a single C−H can replace N−H at a Watson–Crick‐type interface with preservation of functional selectivity and enhanced activity.