5, 10-Methylene-5, 6, 7, 8-tetrahydrofolate. Conformation of the Tetrahydropyrazine and Imidazoline Rings†

It is suggested from analysis of its 1H NMR spectrum and from model building that methylenetetrahydro-folate in solution at neutral and alkaline pH has a rigid conformation for its tetrahydropyrazine and imidazolidine rings. The proposed conformation in solution has three noteworthy features: (1) the tetrahydropyrazine ring is in a half-chair conformation with C6-H axial; (2) the imidazolidine ring is on the same side of the pyrimidine ring as C7 and is nearly planar, and the plane is at an angle of 120° to the pyrimidine ring plane; (3) the aromatic ring of the p-aminobenzoyl-l-glutamate moiety extends away from the pteridine rings, but this possibly may not be true for methylenetetrahydrofolate at neutral pH. The chemical shifts and spin-spin coupling constants of the 15 carbon-bound hydrogens of a diastereo-meric mixture of the two C6 stereoisomers and of the natural C6 stereoisomer of methylenetetrahydrofolate were measured at 25 °C and 300 MHz in 0.1 M NaOD and in 0.1 M sodium phosphate, pH 6.79. In 0.1 M NaOD and at pH∗ 6.79, these compounds had the same coupling constants between their C6, C7, C9, and C11 hydrogens. Also examined was a methylenetetrahydrofolate specifically deuterated at C7. The resonances corresponding to the two hydrogens on C7 had a geminal coupling to one another of 12 Hz and spin-spin couplings to C6-H of 10.0 and 3.0 Hz for the hydrogens trans and cis to C6-H, respectively. The two C9 resonances had geminal coupling of 9.5 Hz and spin-spin couplings to C6-H of 6.5 and 1.5 Hz for the hydrogens cis and trans to C6-H, respectively. The two resonances for the hydrogens on the carbon between N5 and N10, C11, had a chemical shift difference greater than 1 ppm and a geminal coupling of 5.0 Hz.