Spectroscopic Characterization of the 5,10-Methenyltetrahydrofolate Cofactor and Its Degradation Products

5,10-methenyltetrahydrofolate (MTHF) is a folic acid-derived metabolic intermediate produced by the Wood-Ljungdahl metabolic pathway. The molecule is a cofactor in DNA repairing photolyases and in cryptochromes involved in signal transduction. It is suspected to have a photoprotective role within these proteins. MTHF has three degradation products: 10-formyltetrahydrofolate (10-HCO-H4folate, first intermediate), 10-formyldihydrofolate (10-HCO-H2folate, second intermediate), and 10-formylfolate (10-HCO-folate, final product). The goal of this project is to find conditions to capture each intermediate and to spectroscopically characterize these molecules. To accomplish this, in-house synthesized MTHF is analyzed by means of absorbance spectroscopy in phosphate buffers of varying concentrations at specific pH values, as well as by fluorescence and NMR spectroscopy to provide a comprehensive molecular profile of the cofactor. From MTHF, 10-HCO-H4folate is formed in the absence of oxygen, and 10-HCO-H2folate is formed in the presence of oxygen with 10-HCO-H4folate as an undetectable intermediate. Thus far, we have successfully characterized both MTHF and the two degradation products by absorption and fluorescence spectroscopy. In addition, we have successfully characterized both MTHF and 10-HCO-H2folate by NMR spectroscopy for structural elucidation. Our results demonstrate that the fluorescence spectra of 10-HCO-H4folate and 10-HCO-H2folate have been incorrectly assigned in the literature, most likely due to lack of NMR spectroscopic data that could properly identify the molecular structure of each intermediate.