¹⁹ F Nuclear Magnetic Resonance Analysis of 5-Fluorouracil Metabolism in Four Differently Pigmented Strains of Nectria haematococca

Abstract

9F nuclear magnetic resonance spectroscopy was used to study the metabolism of 5-fluorouracil in four strains of Nectria haematococca which displayed similar sensitivities to growth inhibition by this compound but differed in their pigmentation. The major metabolites, 5-fluorouridine and a-fluoro- ,-alanine, were excreted into the medium by all four strains. The classical ribofluoronucleotides (5-fluorouridine-5'-monophosphate,-diphosphate, and-triphosphate) and a-fluoro-,l-alanine were identified in the acid-soluble fraction of perchloric acid extracts of mycelia. Two hydrolysis products of 5-fluorouracil incorporated into RNA were found in the acid-insoluble pool. They were unambiguously assigned to 5-fluorouridine-2'-monophosphate and 3'-monophosphate with specific hydrolysis reactions on isolated RNA. The lack of fluorodeoxyribonucleotides and the fact that the four strains incorporated similar amounts of fluororibonucleotides into their RNAs strongly suggest an RNA-directed mechanism of cytotoxicity for 5-fluorouracil. The heavily pigmented wild type differed from the three low-pigmented strains in its low uptake of 5-fluorouracil and, consequently, in its reduced biosynthesis of 5-fluorouridine and a-fluoro- ,-alanine. At present, it is not clear whether this change in 5-fluorouracil metabolism is a side effect of pigment production or results from another event.

Assignment and pH dependence of the19F-NMR resonances from the fluorouracil anabolites involved in fluoropyrimidine chemotherapy

“…FUra at pH 5.3 and 25°C. 33 From our data we calculate for pH 5.30 at 4°C the following relative shifts: FUMP = 4.174, FUDP = 4.213, FUTP = 4.239, FUDPG = 4.313. In agreement with our results Parisot et al assigned the first two signals to FUMP and FUDP by spiking with commercial compounds.…”

“…Parisot et al have used 19 F-NMR spectroscopy at 7 T for the analysis of FUra metabolism in the fungus Nectria haematococca. 26,33 In acid-soluble extracts they observed four F-Nuctd signals at 4.18, 4.28, 4.31 and 4.45 ppm rel. FUra at pH 5.3 and 25°C.…”

“…This metabolite was converted to FUrd by alkaline phosphatase, ruling out an FUDP-Hex compound. 33 Parisot et al also examined acid-insoluble material which was solubilized at 70°C in 10% PCA to hydrolyze nucleic acids. Two species were observed with chemical shifts of À89.60 and À89.85 ppm, on our shift scale, at pH 5.35 and 25°C.…”

“…[24][25][26][27][28][29] Further resolution of F-Nucsd and F-Nuctd signals into individual components can be achieved only at very high field strengths, to some extent in intact tissue samples, [30][31][32] but most readily in extracts. 23,[33][34][35][36][37] Biofluids such as plasma and urine yield high-resolution spectra but generally contain only fluoropyrimidine catabolites and their by-products and do not provide information about the cytotoxic anabolites which are of interest in chemotherapy. 38 The antitumor activity of fluoropyrimidines can be due to (i) the incorporation of FUTP into RNA and/or (ii) the inhibition of DNA biosynthesis via the binding of FdUMP to thymidylate synthase, 1-3 depending on cell phenotype 21,[26][27][28] and phase of the cell cycle.…”

“…Over the past decade, several groups have carried out 19 F-NMR experiments on cells, extracts or reference compounds and have assigned many of the F-Nuc (F-Nuc = F-Nucsd F-Nuctd) signals observed. [23][24][25][26][27]29,[33][34][35][36][37]39,40 This has usually been accomplished by spiking NMR samples at a given pH with authentic reference compounds or by observing signal intensity changes caused by specific enzymes. 33,34,37 However, a considerable variety of samples in terms of composition, presence or absence of chelator, and pH (between 5.0 and 8.3) have been examined, making comparisons difficult.…”