5-Formyl- and 10-formyl-5,6,7,8-tetrahydrofolate. Conformation of the tetrahydropyrazine ring and formyl group in solution

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Abstract
The chemical shifts and spin-spin coupling constants for the 300 MHz PMR spectra of (6RS)-10-formyl- and (6RS)-5-formyl-5,6,7,8-tetrahydro-L-folate at 25.degree. 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 resonaces 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 10-formyl- and 5-formyl-H4folate are in a half-chair conformation with C(6)-H equatorial. The 5-formyl group and C(9) of 5-formyl-H4 folate are probably trans. In the PMR spectrum of 5-CHO-H4folate, there was a minor species which was present at a concentration roughly 1/6 of 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. 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-C bond. During the preparation of 10-formyl-H4folate from 5,10-methenyl-H4folate in D20, .apprx. 60% of the formyl H was exchanged for D. 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) .times. 106 M-1 in D2O at 25.degree. C. [The formic acid adducts of 5,6,7,8-tetrahydrofolate are of interest because they are enzymatic cofactors essential in purine biosynthesis. The effectiveness of antifolates like methotrexate in cancer chemotherapy may be due to purineless death accompanying deprivation of vertebrate neoplastic cells of these formyltetrahydrofolates.