Stereospecific Synthesis of 2-Desamino-Tetrahydropterins as Probes of Hydroxylase Cofactor Recognition

Abstract

6(S-Alkyl-tetrahydropteridin-4(3H)ones have been synthesized with specific C6-chirality as probes of the properties of hydroxylase cofactor binding domain. We have previously observed that tetrahydropterin cofactor analogs with moderately large hydrophobic 6-substituents have much lower Km values for phenylalanine and tyrosine hydroxylases than 6-methyl-tetrahydropterin (1,2). For example, the Km’s for 6(S)-propyl-tetrahydropterin are 2 µM and 0.3 µM compared to 57 µM and 40 µM for 6(S)-methyl-tetrahydropterin with phenylalanine hydroxylase and phosphorylated tyrosine hydroxylase, respectively (3). The current goal was to determine the effect of removing the 2-amino group on binding. The 6(S)-methyl and 6(S)-propyl-2-desamino-tetrahydropterins (i.e. having the natural C6 configuration) were synthesized from L-alanine and L-norvaline by the stereospecific oxidative cyclization method developed in our laboratory (4,5). The optimum pH of cyclization was shifted from that of the original procedure, related to a change in the pK of the quinoid 6-alkylamino-pyrimidin-4,5-dione intermediate (due to a lack of electron donation by the 2-amino group). In contrast to the tetrahydropterin analogs, the affinity of 6(S)-propyl-2-desamino-tetrahydropterin was not much greater than with the methyl analog, indicating that the 2-amino group is required to achieve full recognition of the 6-substituent at the opposite end of the cofactor.