6,6-Dimethylpterins: stable quinoid dihydropterin substrate for dihydropteridine reductase and tetrahydropterin cofactor for phenylalanine hydroxylase

Abstract

The tautomeric structure of the cofactor product of aromatic amino acid hydroxylases, quinoid dihydrobiopterin, is still unknown. Characterization of this molecule, which is also the substrate for dihydropteridine reductase (EC 1.6.99.7), has been hindered by the rapid rearrangement of quinoid dihydropterins to 7,8-dihydropterins. This tautomerization can be prevented by disubstitution at the 6-position. A procedure is presented for the synthesis of 6,6-disubstituted pterins from a vicinal diamine and 2-amino-6-chloro-4(3H)-pyrimidinone. The method is illustrated with the specific synthesis of 6,6-dimethyltetrahydropterin (6,6-Me2PH4). 6,6-Me2PH4 is a cofactor for rat liver phenylalanine hydroxylase (EC 1.14.16.1), with enzyme kinetic parameters similar to those of its positional isomer, 6,7-dimethyltetrahydropterin. The resulting quinoid 6,6-dimethyldihydropterin (q-6,6-Me2PH2) is stable; the half-life in 0.1 M Tris-HCl, pH 7.4, at 27 and 37 degrees C is 4 and 1.25 h, respectively. q-6,6-Me2PH2, produced either by phenylalanine hydroxylase or by chemical oxidation of 6,6-Me2PH4, is a substrate for dihydropteridine reductase, with a Km of 0.4 mM and a maximum velocity double that of the natural isomer of quinoid dihydrobiopterin. In concentrations up to 0.4 mM q-6,6-Me2PH2 is not an inhibitor of phenylalanine hydroxylase, in contrast to 6-methyl-7,8-dihydropterin and 7,8-dihydrobiopterin which inhibit competitively, with Ki's of 0.2 mM and 0.05 mM, respectively. The stability of q-6,6-Me2PH2 has facilitated definitive determination of chemical and physical properties of a quinoid dihydropterin.