Carbon-13 nuclear magnetic resonance investigations into the interactions of bisulfite with pyrimidine nucleosides and nucleotides

Abstract

Carbon-13 NMR is utilized to demonstrate the attack of bisulfite anion on uridine, 5-fluorouridine, and uridine 5'-monophosphate. The attack produces a pair of diastereomeric adducts similar in structure to those seen in the uracil series. Intensity data from the equilibrium system give an estimate for the individual equilibrium constants. Thymidine and thymidine 5'-monophosphate show no evidence of nucleophilic attack by bisulfite. This evidence indicates that bisulfite addition to nucleosides and nucleotides models the enzymatic methylation of uridine by the enzyme thymidylate synthetase better than the uracil bisulfite system.

Indiscriminate Binding by Orotidine 5‘-Phosphate Decarboxylase of Uridine 5‘-Phosphate Derivatives with Bulky Anionic C6 Substituents

“…In the case of UMP-SO 3 , the signals arising from the vinyl protons at C 5 and C 6 of the pyrimidine ring are replaced by those of the aliphatic protons of the 5,6-dihydro species. Because chiral centers are present in both substituent ribose and C 6 of the saturated pyrimidine ring, diastereomeric products would be expected to result from sulfite addition (9,17,19) and were observed in the present experiments. Here, we refer to these diastereomers as I and II, the resonances of I appearing downfield from those of II.…”

“…The time course of formation of the two diastereomeric adducts of UMP and OMP was followed by 1 H NMR. Integrated intensities of the resonances arising from the C 6 , C 5 , and C 1′ protons of UMP, and the C 5 , and C 1′ protons of OMP, for each diastereomer and unreacted nucleotide, were substituted into Equation 1 (17) to calculate the concentrations of each diastereomer and unreacted nucleotide at each time point. For example, to calculate the concentration of diastereomer I,…”

“…At this stage, no correction was made for the state of ionization of sulfite at pH 7. From the above concentrations, apparent equilibrium constants for the formation of each adduct, at each time point, were calculated using Equation 2 (17).…”

“…Apparent equilibrium constants for sulfite addition to UMP were 26 and 27 M −1 for diastereomers I and II respectively. Earlier, Triplett, et al (17) used 13 C NMR resonance intensities to estimate equilibrium constants of 6.9 and 7.2 M −1 for addition of sulfite (1 M) to UMP (1 M) after 24 hrs at room temperature. The conditions employed by those investigators were different from those in the present experiments, in which the concentration of nucleotide was very much lower.…”

Determination of pK values for the bisulfite adducts of cytidine 5'-Monophosphate by carbon-13 nuclear magnetic resonance

“…The system under study using 13C NMR spectroscopy initially consists of an aqueous solution containing only CMP (4) and its bisulfite adducts (5A and 5B). However, after 24 h it was found to contain five discrete chemical species (Scheme II): CMP, its two diastereomeric bisulfite adducts (5A and 5B), and two diastereomeric bisulfite adducts of uridine 5'monophosphate (UMP) (6A and 6B).…”