Polymerase recognition and stability of fluoro-substituted pyridone nucleobase analogues

Recently much effort has been focused on designing unnatural base pairs that are stable and replicated by DNA polymerases with high efficiency and fidelity. This work has helped to identify a variety of nucleobase properties that are capable of mediating the required interbase interactions in the absence of Watson-Crick hydrogen-bonding complementarity. These properties include shape complementarity, the presence of a suitably positioned hydrogen-bond donor in the developing minor groove, and fluorine substitution. In order to help characterize how each factor contributes to base pairing stability and replication, we synthesized and characterized three fluoro-substituted pyridone nucleoside analogues, 3FP, 4FP, and 5FP. Generally, we found that the specific fluorine substitution pattern of the analogues had little impact on unnatural pair or mispair stability, with the exception of mispairs with dG, which were also the most stable. The mispair between dG and 3FP was less stable than that with 4FP or 5FP, which likely resulted from specific interbase interactions. While fluorine substitution had little impact on the synthesis of the unnatural base pairs, it significantly enhanced mispairing with dG. Remarkably, the mispair between dG and 3FP was the most efficiently synthesized, due to a favorable entropy of activation, which possibly resulted from the displacement of water molecules from dG in the phosphoryl transfer transition state. The more efficient synthesis of the 3FP-dG mispair, despite its being the least stable of the three, suggests that the determinants of synthesis and stability are distinct. Finally, we found that fluorine substitution significantly increased the rate at which the pyridone-based unnatural base pairs were extended; this suggests that both minor groove hydrogen-bond acceptors and fluorine substituents could be used to simultaneously optimize unnatural base pairs.