Excited State Electronic Structures of 5,10-Methenyltetrahydrofolate and 5,10-Methylenetetrahydrofolate Determined by Stark Spectroscopy

Folates are ubiquitous cofactors that participate in a wide variety of critical biological processes. 5,10-Methenyltetrahydrofolate and its photodegradation product 5,10-methylenetetrahydrofolate are both associated with the light-driven DNA repair protein DNA photolyase and its homologues (e.g., cryptochromes). The excited state electronic properties of these folate molecules have been studied here using Stark spectroscopy and complementary quantum calculations. The tetrahydrofolates have relatively large difference dipole moments (ca. 6–8 Debye) and difference polarizabilities (ca. 100 Å3). This extensive excited state charge redistribution appears to be due largely to the pendant p-aminobenzoic acid group, which helps shuttle charge over the entirety of the molecule. Simple calculations based on the experimental difference dipole moments suggest that tetrahydrofolates should have large two photon cross sections sufficient to enable two photon microscopy to selectively detect and follow folate-containing proteins both in vitro and in vivo.