5-Methyl-5,6,7,8-tetrahydrofolic acid. Conformation of the tetrahydropyrazine ring.

It is suggested from analysis of proton spin-spin coupling constants that the tetrahydropyrazine ring of 5methyltetrahydrofolate in solution is in a half-chair conformation, with the methyl group at N-5 and the methylene group C-9 truns, and with C-6-H in an equatorial position. The chemical shifts and spin-spin coupling constants for the carbon-bound hydrogens of (+)-L-, (-)-L, (+)-L-~-‘H, 7-‘H (50% 2H), and (+-)-L-7-“H (50% 2H)-5-methyl-5,6,7,8-tetrahydrofolate have been measured at 25°C and neutral pH at 300 MHz. The resonances corresponding to the two C-7 hydrogens have a spin-spin coupling of 12.8 Hz to one another and a chemical shift difference of 0.24 ppm and have spinspin couplings of 2.2 and -1 Hz to C-6-H for the lowand high-field C-7-H resonances, respectively. The C-7 hydrogens are proposed to be a geminally coupled axial-equatorial pair that are coupled to an equatorial C6 hydrogen. In (+)-L-~-‘H, 7-‘H (50% 2H)-5-methyl5,6,7,8&etrahydrofolate, the two C-9 hydrogens constitute an AB quartet with JAB of 14 Hz and chemical shift difference of 0.103 ppm. The nonequivalence of these two protons is attributed to the adjacent chiral center at C-6. The proposed conformation for B-methyltetrahydrofolate allows a great deal of conformational freedom for the p-aminobenzoyl-L-glutamate moiety, which is the portion of the molecule which is recognized by cellular transport systems; the conformational freedom explains the ability of cellular transport systems to carry both C-6 stereoisomers of Smethyltetrahydrofolate. The proposed conformation has a fixed position for the B-methyl group on the same face of the tetrahydropyrazine ring as the C-6 hydrogen. Thus, the two C-6 stereoisomers of 5-methyltetrahydrofolate have the 5methyl group on opposite faces of the tetrahydropyrazine ring, which may explain why the vitamin B12-5methyltetrahydrofolate-dependent cysteine methyltransferase enzyme can use only one of the C-6 stereoisomers.