Oxidation Mechanisms of Tetrahydrofolic Acid, Tetrahydrobiopterin and Related Compounds

Abstract

The autoxidation of tetrahydrofolic acid and tetrahydrobiopterin, 6-substituted derivatives of 2-amino-4-hydroxy-5,6,7,8-tetrahydropteridine (tetrahydropterin), has been investigated by product analysis and kinetic methods using gas manometric and spectrophotometric techniques. The overall reaction is first order in tetrahydropterin and shows a combination of zero- and first order dependence on oxygen, the relative contributions of which are affected by the exclusion of visible light. There is a linear dependence of rate on percentage ionisation of the 3,4-amide group and the reaction is retarded by protonation of N(5), suggesting that oxygen attack occurs at C(4a). Use of appropriately deuteriated compounds leads to no kinetic isotope effects, showing that 6- and 7- CH bond cleavages are not rate-determining. Kinetic data suggest a free radical chain reaction in which the chain carrier is the hydroperoxyl radical (HO2.) and that intermediate hydroperoxides are not involved. Quinonoid dihydropterins are obligatory intermediates, their rearrangement leading to the observed products. Oxygen-18 labelling experiments show that the autoxidative ring contraction of 1,3,6,7,8-pentamethyl-5,6,7,8-tetrahydropteri-2,4-dione does not provide evidence for the intermediacy of 8a-hydroperoxides as suggested in the literature. The mechanism is rationalised on the basis of simple Huckel molecular orbital calculations, Solutions of autoxidising tetrahydrobiopterin in the presence of ferrous ion are shown to induce non-specific hydroxylation of phenylalanine, and a radiochemical product analysis method is described. Effects of catalase indicate the partial involvement of hydrogen peroxide, generated during tetrahydrobiopterin autoxidation. Use of superoxide dismutase indicates that the hydroperoxyl radical is not active in the system, but N.I.H. shift determinations suggest a free radical species. The complexity of the reaction is recognised, and the involvement of iron-oxygen complexes and hydroxyl radicals (OH.) is considered.