5-Formyl- and 10-Formyl-5,6,7,8-tetrahydrofolate. Conformation of the Tetrahydropyrazine Ring and Formyl Group in Solution

The chemical shifts and spin-spin coupling constants for the 300-MHz¹H NMR spectra of (6RS)-10-formyl and (6RS)-5-formyl-5,6,7,8-tetrahydro-L-folate at 25 °C in 0. 1 M NaOD and at neutral pH are reported and analyzed. Assignments of most resonances are based on comparison to closely related compounds; the resonances of C(6)-H, C- (7)-2H, and C(9)-2H are assigned by preparation of specifically deuterated formyltetrahydrofolates. From analysis of the C(7)-2H spin-spin coupling constants, it is proposed that the tetrahydropyrazine rings of both 10-formyl- and 5- formyl-H₄folate are in a half-chair conformation with C(6)-H equatorial. It is further proposed from model building that the 5-formyl group and C(9) of 5-formyl-H⁴folate are trans. In the¹H NMR spectrum of 5-CHO-H₄folate, there was a minor species which was present at a concentration roughly one-sixth that of the major species; this species did not correspond to oxidized 5-CHO-H₄folate and could not be separated from the major species by gel permeation or ion-exchange chromatography. It is proposed that this minor species corresponds to a conformer of 5-CHO-H₄folate in slow exchange with the major conformer; this slow exchange is attributed to slow rotation due to a partial double bond character of the N(5) to 5-formyl-carbon bond. During the preparation of 10-formyl-H₄folate from 5,10-methenyl-H₄folate in D₂O, ~60% of the formyl hydrogen was exchanged for deuterium. The equilibrium constant for the interconversion of 5,10- methenyl-H₄folate and 10-formyl-H₄folate, K' = [5,10- methenyl-H₄folate]/([D⁺][10-formyl-H₄folate]), was measured to be (1.4 ± 0.6) × 10⁶M^-1in D₂O at 25 °C.