BIOPTERIN .5. DENOVO SYNTHESIS OF DIHYDROBIOPTERIN - EVIDENCE FOR ITS QUINONOID STRUCTURE AND LACK OF DEPENDENCE OF ITS REDUCTION TO TETRAHYDROBIOPTERIN ON DIHYDROFOLATE-REDUCTASE

Samples of quinonoid‐l‐erythrodihydrobiopterin (q‐BH2) and quinonoid‐6‐methyl‐dihydro‐pterin (q‐6‐MPH2) were prepared by oxidation of l‐erythro‐5,6,7,8‐tetrahydrobiopterin (BH4) and 5,6,7,8‐tetrahydro‐6‐methylpterin (6‐MPH4) and separated from D‐erythro‐7,8‐dihydrobiopterin (7,8‐BH2) and 6‐methyl‐7,8‐dihydropterin (7,8‐6‐MPH2) as well as from the tetrahydropterins on phosphocellulose column by high‐pressure liquid chromatography. The quinonoid dihydropterins were identified and quantitated by scan of their ultraviolet absorption and fluorescence emission spectra through their rearrangement to their 7,8‐tautomer and also by gas chromatography of their rapidly synthesized trimethylsilyl derivative. Identification was also achieved by the enzymatic reduction of [3H]q‐BH2to [3H]BH4 by dihydrofolate reductase (DHFR). Direct proof for the enzymatic synthesis of the q‐BH2 from GTP or from 2‐amino‐6‐(5′‐triphosphoribosyl)‐amino‐5‐ or ‐6‐formamido‐6‐hydroxypyrimi‐dine (FPyd‐P3) was obtained by isolation of the compound which was identical in all respects to the q‐BH2 obtained by chemical synthesis from BH4. The reduction of enzymatically synthesized q‐BH2 by dihydropteridine reductase (DHPR) to BH4 was not inhibited by methotrexate (MTX). When the enzymatically synthesized q‐BH2 was converted to 7,8‐BH2, it was reduced only by DHFR. This reduction, however, was inhibited by MTX. On the biosynthetic pathway from GTP to dihydrobiopterin, the enzyme responsible for the appearance of the quinonoid structure is the d‐erythro‐dihydroneopterin triphosphate synthetase, the product of which (quinonoid d‐erythro‐dihydroneopterin triphosphate) is converted to quinonoid dihydrobiopterin by l‐erythro‐dihydrobiopterin synthetase. Experiments in vivo established that DHFR does not participate in the reduction of dihydrobiopterin to tetra‐hydrobiopterin when the former is synthesized from GTP de novo. MTX at 5 × 10−6M exerted no inhibition on the reduction of the biosynthetic dihydrobiopterin to tetrahydrobiopterin in vivo, yet completely inhibited the reduction of intraventricularly injected tritiated dihydrofolate ([3H]FH2) to tritiated tetrahydrofolate ([3H]FH4). Copyright © 1979, Wiley Blackwell. All rights reserved