Pyrimidodiazepine, a ring-strained cofactor for phenylalanine hydroxylase

Abstract

Homologues of 6-methyl-7,8-dihydropterin (6-Me-7,8-PH2) and 6-methyl-5,6,7,8-tetrahydropterin (6-Me-PH4), expanded in the pyrazine ring, were synthesized to determine the effect of increased strain on the chemical and enzymatic properties of the pyrimidodiazepine series. 2-Amino-4-keto-6-methyl-7,8-dihydro-3H,9H-pyrimido[4,5-b] [1,4]diazepine (6-Me-7,8-PDH2) was found to be more unstable in neutral solution than 6-Me-7,8-PH2. Its decomposition appears to proceed by hydrolytic ring opening of the 5,6-imine bond, followed by autooxidation. 6-Me-7,8-PDH2 can be reduced, either chemically or by dihydrofolate reductase (Km = 0.16 mM), to the 5,6,7,8-tetrahydro form (6-Me-PDH4). This can be oxidized with halogen to quinoid dihydropyrimidodiazepine (quinoid 6-Me-PDH2), which is a substrate for dihydropteridine reductase (Km = 33 microM). Whereas quinoid 6-methyldihydropterin was found to tautomerize to 6-Me-7,8-PH2 in 95% yield in 0.1 M tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 7.4, quinoid 6-Me-PDH2 gives only 53% 6-Me-7,8-PDH2, the remainder decomposing via an initial opening of the diazepine ring. Additional evidence for the extra strain in the pyrimidodiazepine system is the cyclization of quinoid 6-N-(2'-aminopropyl)divicine to quinoid 6-Me-PH2 in 57% yield in 0.1 M Tris-HCl, pH 7.4. By comparison, no quinoid 6-Me-PDH2 is formed from the homologue quinoid 6-N-(3'-aminobutyl)divicine. A small (2%) yield of 6-Me-PDH4 is found if the unstable C4a-carbinolamine intermediate is trapped by enzymatic dehydration and reduction. Although phenylalanine hydroxylase utilizes 6-Me-PDH4 (Km = 0.15 mM), the maximum velocity of tyrosine production is 20 times slower than that with 6-Me-PH4, indicating that a ring opening reaction is not a rate-limiting step in the hydroxylase pathway. Further, the maximum velocities of 2,5,6-triamino-4(3H)-pyrimidinone, 2,6-diamino-5-(methylamino)-4(3H)-pyrimidinone, and 2,6-diamino-5-(benzylamino)-4(3H)-pyrimidinone span a 35-fold range. These cofactors would theoretically form the same oxide of quinoid divicine if oxygen activation involves a carbonyl oxide intermediate. Thus, the limiting step is also not transfer of oxygen from this hypothetical intermediate to the phenylalanine substrate.

Pterin-Dependent Amino Acid Hydroxylases

“…Another pterin analogue that is a functional cofactor is 6-methylpyrimidodiazepine (6MPDH 4 ), which has an additional methylene inserted into the pyrazine ring (Figure ). It has about 5% of the 6MPH 4 activity when tyrosine formation is assayed …”

“…Were it to occur, observation of the ring-opened oxidized intermediate (the first point mentioned above) would probably be difficult because pathway would have to support a recyclization rate in excess of 10 s -1 . In order to slow down any recyclization occurring with the enzyme, 6MPDH 4 was synthesized and found to be a tightly coupled cofactor for PAH ( q -6MPDH 2 and 7,8-6MPDH 2 were efficiently reduced by DHPR and DHFR) . The K m values for cofactor and phenylalanine were comparable to those with 6MPH 4 (intriguingly, the K m for O 2 was >10 times higher) but the rate of reaction was quite slow, about 5% of the 6MPH 4 V max .…”

“…Unlike the relatively stable 7,8-dihydropterin, the ring-strained 7,8-6MPDH 2 degrades further over the course of a day. Instead of forming an analogue of 6-methylpterin, it is converted into far-UV absorbing species proposed to arise from oxidation of the 7,8-hydrate (Figure B) 32 Opening of the diazepine cofactor during turnover. …”

Synthesis of a naturally occurring dihydrohomopteridine insect metabolite, 6-acetyl-2-amino-7,8-dihydro-9H-pyrimido[4,5-b][1,4]diazepin-4(3H)-one

“…These results are consistent with observations made earlier by Nair and cow o r k e r ~, ~~ and have been explained by a possible interaction between the 5-nitro group and the carbonyl group on the side chain. The initial 5-aminopyrimidine reduction product (16) was unstable in air and was not isolated, although its formation could be monitored by TLC. It was converted into (1) by treatment with a solution of barium chloride, the pH being maintained at 3 for 20 min at room temperature, and then neutralisation to pH 7.…”

Synthesis of Spiropyrimidodiazepines and Spirodiazepinopurines by Tandem Nitroso‐ene/Diels–Alder Reactions

“…As a rule, the products possessing different substituents at C(6) and C (8) are obtained as mixtures of regioisomers, a disadvantage that is avoided by condensing 4-chloro-5-nitropyrimidines with b-amino ketones (Polonovski -Boon-type reactions) [9] [10]. Only a few examples of spirobenzodiazepines are known [4] [11] [12] and, to the best of our knowledge, no synthesis of spiropyrimido [4,5-b] [1,4]diazepines has been reported.…”