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Synthesis of 4a-hydroxytetrahydropterins and the mechanism of their nonenzymatic dehydration to quinoid dihydropterins

4a-Hydroxytetrahydropterins 2 (R = Me, Pr, and 1'(R),2'(S)-dihydroxypropyl) were synthesized by intramolecular Schiff base condensation of 2'-substituted 2-amino-6-[(aminoethyl)amino]-4,5-pyrimidinediones 6. The rate vs pH of cyclization of 6 (which is predominately monohydrated) follows a bell-shaped curve with maxima at pH 9.8 in H2O (consistent with pK values of 8.8 and 10.8) and pH 9.1 in MeOH. Although almost insignificant in organic solvents, substantial 7-substituted dihydropterin was generated in water, particularly with 6 (R = dihydroxypropyl). The minimum rate of dehydration of carbinolamine 2 (R = Me or Pr) in 'zero' buffer (0.0035 s-1 at 17°C) occurs at pH 8.25, and is catalyzed by proton (1.3 x 105 M-1 s-1) and general acids. Between pH 8.4 and pH 7.4 ΔH* decreases from 15 to 12 kcal/mol, while ΔS* decreases from -18 to -26 eu for 2 (R = Me or Pr), consistent with concerted proton transfer in the dehydration transition state. Surprisingly, the rate also increases in more alkaline conditions up to 12-fold (in zero buffer), coincident with formation of a pteridine anion (pK = 9.8). Below pH 11.5 catalysis by buffer base was also observed. A solvent kinetic isotope effect (k(H2O)/k(D2O)) of 2.6, 2.2, and 3.5 was found in dilute buffers at pH 7.4, 8.2, and 10.9, respectively. The overall rate of disappearance of 2 (R = dihydroxypropyl) is similar to that of the alkyl analogs, but a second pathway competes with dehydration to produce a compound tentatively identified as a side-chain 4a-cyclic adduct, which subsequently decays to quinoid 6(R)-dihydrobiopterin. These synthetic substrates have permitted the first kinetic characterization of 4a-hydroxytetrahydropterin dehydratase (Rebrin, I.; et al. Biochemistry 1995, 34, 5801-5810), the enzyme involved in cofactor regeneration during aromatic amino acid hydroxylation.