Synthesis of 5-ethynyl-2'-deoxyuridine-5'-boranomono phosphate as a potential thymidylate synthase inhibitor

The 5-ethynyl-2'-deoxyuridine nucleoside and the 5'-boranomonophosphate nucleotide were synthesized as analogs of 5-fluoro-2'-deoxyuridine monophosphate (5-FdUMP), a widely used mechanism-based inhibitor of thymidylate synthase. Synthesis was carried out from protected 5-iodo-2'-deoxyuridine and trimethylsilylacetylene by Sonogashira palladium-catalyzed cross coupling reaction followed by selective phosphorylation and finally boronation.     

Purification of thymidine phosphorylase from Escherichia coli and its photoinactivation in the presence of thymine, thymidine, and some halogenated analogs.

Isoelectric focusing was used as the final step in the isolation of thymidine phosphorylase which was found to have an isoelectric point of 4.1. Analytical acrylamide gel electrophoresis showed the purified enzyme preparation contained one major protein band which stained for thymidine phosphorylase activity and usually a minor, faster migrating band devoid of activity. Inactivation of thymidine phosphorylase alone or in the presence of sensitizers by ultraviolet light, primarily at 253.7 nm, followed first order inactivation kinetics. The rate of inactivation of the enzyme was the same at pH 5 and 7.4 and the addition of various pyrimidine bases and nucleosides enhanced the inactivation rate at both pH values, but to a greater extent at pH 5. Linear plots of inactivation rates versus concentrations of thymidine or thymine were the same. At 7.8 mM thymidine or thymine, 11- and 4.4-fold increases in photoinactivation of thymidine phosphorylase were observed at pH 5 AND 7.4 RESPECTIVELY. Parabolic curves were obtained with increasing concentrations of either 5-iodo-2'-deoxyuridine or 5-iodouracil. 5-Iodouracil at 5.2 mM caused 212- (pH 5) and 100- (pH 7.4) FOLD INCREASES IN THE RATES OF PHOTOINACTIVATION OF THYMIDINE PHOSPHORYLASE. However, 5-iodo-2'-deoxyuridine at 5.0mM only enhanced the photoinactivation of enzyme by factors of 83 (pH 5) and 21 (pH 7.4). Neither 5-bromo-2'-deoxyuridine or 5-bromo-uracil was as potent in sensitizing the enzyme as the iodo analogs. Combinations of 5-iodouracil or 5-iodo-2'-deoxyuridine with thymine resulted in higher inactivation rates than the additive inactivation rates of individual compounds, whereas combinations of either iodo analog with thymidine resulted in lower inactivation rates. Increasing concentrations of phosphate or NaCl lessened the photoinactivation rate of thymidine phosphorylase alone and protected the enzyme from the sensitization caused by the different bases and nucleosides. No quantitative changes in the number of primary amino groups in thymidine phosphorylase was evident as a result of irradiation in the presence or absence of 5-iodouracil or 5-iodo-2'-deoxyuridine. Examination of the irradiated enzyme on Sephadex G-150 indicated that a larger protein species is formed and that 5-iodouracil promotes this process.     

Mechanism-based inactivation of thymidylate synthase by 5-(3-fluoropropyn-1-yl)-2'-deoxyuridine 5'-phosphate

5-Fluoropropynyl-2'-deoxyuridine 5'-phosphate (3) was designed as a mechanism-based inactivator of thymidylate synthase (TS). The inhibitor was synthesized from 5-iodo-2'-deoxyuridine and propargyl alcohol by palladium-catalyzed coupling, followed by fluorination and selective phosphorylation. Incubation of TS with 3, in the presence or absence of the CH2H4folate cofactor, caused rapid, irreversible inactivation of the enzyme.     

Altered thymidylate kinase substrate specificity in mammalian cells selected for resistance to iododeoxyuridine

A clone of Syrian hamster melanoma cells was selected for resistance to high levels of the thymidine (dT) analog 5-iododeoxyuridine (IdU). Unlike cell lines previously isolated as IdU resistant (IdU^r), these IdU^r lines had normal levels of thymidine kinase (EC 2.7.1.21) activity, grew in HAT medium, and readily incorporated exogenous dT and 5-bromodeoxyuridine (BrdU) into DNA. However, these IdU^r cells were found to preferentially exclude IdU from their DNA. Analyses of the soluble nucleotide pools of the IdU^r cells showed that they were able to take up and phosphorylate exogenous dT as well as the wild-type cells, and both mutant and wild-type cells accumulated dTTP as the major phosphorylated component. In contrast, while the wild-type cells in the presence of exogenous IdU accumulated significant levels of IdUTP (as well as IdUMP), the IdU^r cells accumulated only IdUMP. Thus, the mutant cells appear to have a markedly decreased ability to phosphorylate IdU beyond the monophosphate level. Assays of thymidylate kinase (EC 2.7.4.9) activity in extracts of the IdU^r cells indicated a marked preference for dTMP as substrate over IdUMP (in comparison to the wild-type enzyme activity). The cell lines described in this study represent a new phenotype arising from selection for resistance to a halogenated dT analog. The resistance appears to involve a change in the substrate specificity of thymidylate kinase, such that the enzyme in the IdU^r cells has an enhanced ability to discriminate between very closely related compounds.     

Inactivation of S-adenosyl-L-homocysteine hydrolase with fluorinated analogs of 2'- and 3'-deoxy-5'-methylthioadenosine

Fluorinated analogs of 2'- and 3'-deoxy-5'-methylthioadenosine 1-4 caused irreversible inactivation of AdoHcy hydrolase. Based on the ESI-Mass spectra analysis of the inactivated enzyme with the fluorinated analog 1 a mechanism of inactivation is proposed.     

Substrate specificity of vaccinia virus thymidylate kinase

Anti-poxvirus therapies are currently limited to cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine], but drug-resistant strains have already been characterized. In the aim of finding a new target, the thymidylate (TMP) kinase from vaccinia virus, the prototype of Orthopoxvirus, has been overexpressed in Escherichia coli after cloning the gene (A48R). Specific inhibitors and alternative substrates of pox TMP kinase should contribute to virus replication inhibition. Biochemical characterization of the enzyme revealed distinct catalytic features when compared to its human counterpart. Sharing 42% identity with human TMP kinase, the vaccinia virus enzyme was assumed to adopt the common fold of nucleoside monophosphate kinases. The enzyme was purified to homogeneity and behaves as a homodimer, like all known TMP kinases. Initial velocity studies showed that the Km for ATP-Mg2+ and dTMP were 0.15 mm and 20 microM, respectively. Vaccinia virus TMP kinase was found to phosphorylate dTMP, dUMP and also dGMP from any purine and pyrimidine nucleoside triphosphate. 5-Halogenated dUMP such as 5-iodo-2'-deoxyuridine 5'-monophosphate (5I-dUMP) and 5-bromo-2'-deoxyuridine 5'-monophosphate (5Br-dUMP) were also efficient alternative substrates. Using thymidine-5'-(4-N'-methylanthraniloyl-aminobutyl)phosphoramidate as a fluorescent probe of the dTMP binding site, we detected an ADP-induced conformational change enhancing the binding affinity of dTMP and analogues. Several thymidine and dTMP derivatives were found to bind the enzyme with micromolar affinities. The present study provides the basis for the design of specific inhibitors or substrates for poxvirus TMP kinase.     

Metabolism of 4'-azidothymidine. A compound with potent and selective activity against the human immunodeficiency virus.

4'-Azidothymidine (ADRT) is a novel nucleoside analog, that selectively inhibits human immunodeficiency virus replication in human lymphocytes. Unlike the dideoxyribonucleoside analogs and 3'-azido-2',3'-dideoxythymidine (AZT), ADRT retains the 3'-hydroxy group. The pathways of ADRT metabolism were elucidated by determining: (i) the kinetics of the interactions of ADRT and its metabolites with enzymes of thymidine metabolic pathways, (ii) the pool sizes of phosphorylated metabolites, and (iii) the nature of ADRT incorporation into human DNA. ADRT is not a substrate for thymidine phosphorylase, but is metabolized by kinases. Thymidine kinase phosphorylates ADRT to ADRT monophosphate (ADRT-MP). For this enzyme, ADRT has a Ki value of 5.2 microM, in comparison to a Km value of 0.7 microM for thymidine. The Km value of ADRT toward thymidine kinase is 8.3 microM and the rate of ADRT phosphorylation is 1.4% that of thymidine phosphorylation. ADRT-MP has a low affinity toward thymidylate kinase (a Ki value of 28.9 microM versus a Km value of 0.56 microM for thymidylate), and toward thymidylate synthase (a Ki value of 180 microM versus a Km value of 8 microM for deoxyuridylate). The results suggest that ADRT can be activated effectively by cellular kinases without significant interference of normal thymidine metabolism. In cultured human lymphocytes (A3.01, H9, and U937 cells), ADRT was phosphorylated efficiently to ADRT 5'-triphosphate (ADRT-TP), which is the major metabolite of ADRT. The intracellular concentrations of ADRT-TP ranged from 1 to 3.3 microM after 24 h of incubation with 2 microM of ADRT and the half-life of ADRT-TP varied from 3 to 6 h. Although ADRT-TP is a poor competitive inhibitor against dTTP toward DNA polymerases alpha and beta with Ki values of 62.5 and 150 microM, respectively. ADRT-MP was found to be internally incorporated into cellular DNA. The extent of ADRT-MP substitution for dTMP in DNA was 1 in 6979 for A3.01 cells incubated with 2.9 microM ADRT for 24 h. Internal incorporation of ADRT-MP contrasts with the mechanism of other 2',3'-dideoxynucleoside analogs (i.e. AZT, ddC, ddI, d4T...), which are DNA chain terminators. This finding indicates that a 3'-deoxy structure in a nucleoside analog is not a prerequisite for anti-human immunodeficiency virus activity.     

Assay of intracellular thymidylate synthetase activity and inhibition by 5-fluoro-2'-deoxyuridine in lymphocytes.

Thymidylate synthetase catalyses the formation of thymidine monophosphate from deoxyuridine monophosphate. Purified thymidylate synthetase can be assayed radiochemically using labelled deoxyuridine monophosphate as substrate, but cells are impervious to deoxyuridine monophosphate and so intracellular thymidylate synthetase activity cannot be assayed in this way. In this paper we describe the assay of intracellular thymidylate synthetase activity in intact cells using labelled 2'-deoxyuridine. The assay showed linear kinetics with respect to time, concentration of 2'-deoxyuridine, and cell concentration. 5-fluoro-2'-deoxyuridine inhibited intracellular thymidylate synthetase activity measured with this assay by 50% at 5 nM. Cell growth was inhibited by 50% at 6 nM 5-fluoro-2'-deoxyuridine. The assay was specific for thymidylate synthetase and enabled measurement of thymidylate synthetase activity in situ in intact cells.     

Thymidylate synthetase. Catalysis of dehalogenation of 5-bromo- and 5-iodo-2'-deoxyuridylate.

Tymidylate synthetase catalyzes the facile dehalogenation of 5-bromo-2'-deoxyuridylate (BrdUMP) and 5-iodo-2'-deoxyuridylate )IdUMP) to give 2'-deoxyuridylate (dUMP), the natural substrate of the enzyme. The reaction does not require folate cofactors and stoichiometrically consumes 2 equiv of thiol. In addition to dUMP, a minor product is formed during the debromination of BrdUMP which has been identified as a 5-alkylthio derivative formed by displacement of bromide ion by thiolate. The reaction has been found to proceed with a substantial alpha-secondary inverse tritium isotope effect (kT/kH = 1.212--1.258) with [2-14C,6-3H]-BrdUMP as the substrate. Similarly, an inverse tritiumisotope effect of 1.18 was observed in the nonenzymatic chemical counterpart of this reaction, the cysteine-promoted dehalogenation of [2-14C,6-3H]-5-bromo-2'-deoxyuridine. Previous evidence for the mechanism of action of this enzyme has rested largely on chemical model studies and on information obtained from its stoichiometric interaction with the inhibitor 5-fluoro-2'-deoxyuridylate. The magnitude of the secondary isotope effect during the enzymatic dehalogenation described here provides direct proof for nucleophilic catalysis and formation of 5,6-dihydroprimidine intermediates in a reaction catalyzed by thymidylate synthetase.     

An optimized thymidylate kinase assay, based on enzymatically synthesized 5-[125I]iododeoxyuridine monophosphate and its application to an immunological study of herpes simplex virus thymidine-thymidylate kinases

The biological synthesis and purification of 5-[125I]iododeoxyuridine monophosphate (IdUMP) are described. The specificity of IdUMP as substrate in the thymidylate monophosphate kinase (TMPK) assay is demonstrated, and a 100-fold gain in sensitivity as compared to the conventional TMPK assay is shown. TMPK measurements of isozymes derived from herpes simplex virus (HSV)-infected cells, uninfected cells, and tumor biopsies were performed. The results showed a significant difference in dependence of phosphate donor concentration present for TMPK activity from HSV-infected cells compared to the corresponding activity from uninfected cells, while only a minor difference in pH optima was observed for these enzyme activities. The increased sensitivity made it possible to detect and quantify HSV TMPK-blocking antibodies (ab) present in human sera. Sera from HSV ab-positive individuals were found to block the two HSV TMPKs to varying degrees and with different specificities. The immunological relationship between the TMPK and thymidine kinase (TK) induced by HSV-1 and HSV-2, respectively, was studied by comparing the capacities of different sera to block the two enzymatic activities. The results showed that the capacity to block HSV-1 TK and TMPK was proportional for all of the sera studied, while sera that preferentially blocked only the HSV-2 TMPK or HSV-2 TK were found. It was concluded that the HSV-2 TMPK and TK activities are less related than the corresponding activities for HSV-1 and that the HSV-2 enzyme activities are mediated by different catalytic sites.     

Rapid quantitation of plasma 2'-deoxyuridine by high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry and its application to pharmacodynamic studies in cancer patients

A novel method employing high-performance liquid chromatograph-mass spectrometry (LC-MS) has been developed and validated for the quantitation of plasma 2'-deoxyuridine (UdR). It involves a plasma clean-up step with strong anion-exchange solid-phase extraction (SAX-SPE) followed by HPLC separation and atmospheric pressure chemical ionization mass spectrometry detection (APCI-MS) in a selected-ion monitoring (SIM) mode. The ionization conditions were optimised in negative ion mode to give the best intensity of the dominant formate adduct [M+HCOO]- at m/z 273. Retention times were 7.5 and 12.5 min for 2'-deoxyuridine and 5-iodo-2'-deoxyuridine, an iodinated analogue internal standard (IS), respectively. Peak area ratios of 2'-deoxyuridine to IS were used for regression analysis of the calibration curve. The latter was linear from 5 to 400 nmol/l using 1 ml sample volume of plasma. The average recovery was 81.5% and 78.6% for 2'-deoxyuridine and 5-iodo-deoxyuridine, respectively. The method provides sufficient sensitivity, precision, accuracy and selectivity for routine analysis of human plasma 2'-deoxyuridine concentration with the lowest limit of quantitation (LLOQ) of 5 nmol/l. Clinical studies in cancer patients treated with the new fluoropyrimidine analogue capecitabine (N4-pentoxycarbonyl-5'-5-fluorocytidine) have shown that plasma 2'-deoxyuridine was significantly elevated after 1 week of treatment, consistent with inhibition of thymidylate synthase (TS). These findings suggest that the mechanism of antiproliferative toxicity of capecitabine is at least partly due to TS inhibitory activity of its active metabolite 5-fluoro-2'-deoxyuridine monophosphate (FdUMP). Monitoring of plasma UdR concentrations have the potential to help clinicians to guide scheduling of capecitabine or other TS inhibitors in clinical trials. Marked differences of plasma 2'-deoxyuridine between human and rodents have also been confirmed. In conclusion, the LC-MS method developed is simple, highly selective and sensitive and permits pharmacodynamic studies of TS inhibitors in several species.     

Synthesis of carbocyclic nucleotides as potential substrates for thymidylate kinase

Enantiomerically pure carbocyclic 2'-deoxy-3'-azidothymidine monophosphate (AZTMP) and carba-2'deoxy-3'-thiocyanatothymidine monophosphate were synthesized to study their behavior toward their phosphorylation by thymidylate kinase. The nucleotides were synthesized starting from the parent nucleosides by an alkaline hydrolysis of the corresponding cycloSal-phosphate triesters.     

5-propynylpyrimidine nucleoside derivatives: rationally designed mechanism-based inactivators of thymidylate synthase

A novel series of 5-propynyl-dUMP derivatives, with a variety of leaving groups on the side-chain, was designed as potential mechanism-based inhibitors of thymidylate synthase (TS), and synthesized from 5-iodo-2'-deoxyuridine by Pd(0)-catalyzed coupling, followed by direct phosphorylation with POCl3. All members of the series inhibited TS competitively with Ki-values of 0.015-18 microM. Analogs with fluorine or imidazole-based leaving groups caused rapid, irreversible inactivation of TS.     

Induction and inhibition of Friend erythroleukemia cell differentiation by pyrimidine analogs: analysis of the requirement for intracellular accumulation and incorporation into DNA.

Alkyldeoxyuridines which differ from thymidine by a C5 substitution of straight or branched alkyl chains of two to six carbon atoms have been tested for their ability to be taken up, phosphorylated, and incorporated into DNA. Analysis of the uptake of 5-ethyl-2'-deoxyuridine and 5-propyl-2'-deoxyuridine (n-PrdU)--similar to both thymidine and 5-bromo-2'-deoxyuridine--indicates that transport is dependent upon a functional cellular thymidine kinase. All of the aforementioned pyrimidines with the exception of n-PrdU are phosphorylated to the triphosphate and incorporated into DNA. The homologs 5-iso-propyl-2'-deoxyuridine (iso-PrdU) and 5-hexyl-2'-deoxyuridine are neither transported into the cell, phosphorylated, nor incorporated into DNA. These analogs were tested (i) for their ability to induce in the absence of dimethyl sulfoxide and (ii) to determine whether they enhance or inhibit dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells. Inhibition of erythroid differentiation appears to require the incorporation of thymidine analogs into DNA, and thus only 5-ethyl-2'-deoxyuridine and 5-bromo-2'-deoxyuridine were effective in inhibiting dimethyl sulfoxide-induced differentiation. The observation that iso-PrdU, and to a lesser extent n-PrdU and 5-propyldeoxyuridine monophosphate, induce differentiation under conditions in which they are not detectable intracellularly is strong evidence that this class of inducer acts at the cell membrane.     

Carbocyclic thymidine derivatives efficiently inhibit Plasmodium falciparum thymidylate kinase (PfTMK)

During the course of our research into new anti-malaria drugs, Plasmodium falciparum thymidylate kinase (PfTMK) has emerged as an important drug target because of its unique substrate specificity. Compared with human thymidylate kinase (HsTMK), PfTMK shows broader substrate specificity, which includes both purine and pyrimidine nucleotides. PfTMK accepts both 2'-deoxyguanosine monophosphate (dGMP) and thymidine monosphosphate (TMP) as substrates. We have evaluated the inhibitory activity of seven carbocyclic thymidine analogs and report the first structure-activity relationship for these inhibitors against PfTMK. The 2',3' dideoxycarbocyclic derivative of thymidine showed the most potent inhibition of the enzyme. The K(i)(dTMP) and K(i)(dGMP) values were 20 and 7 μM respectively. Thus, further modifications of carbocyclic thymidine analogs represent a good strategy for developing more powerful thymidylate kinase inhibitors.     

Synthesis and evaluation of 6-aza-2'-deoxyuridine monophosphate analogs as inhibitors of thymidylate synthases, and as substrates or inhibitors of thymidine monophosphate kinase in Mycobacterium tuberculosis

A series of 5-substituted analogs of 6-aza-2'-deoxyuridine 5'-monophosphate, 6-aza-dUMP, has been synthesized and evaluated as potential inhibitors of the two mycobacterial thymidylate synthases (i.e., a flavin-dependent thymidylate synthase, ThyX, and a classical thymidylate synthase, ThyA). Replacement of C(6) of the natural substrate dUMP by a N-atom in 6-aza-dUMP 1a led to a derivative with weak ThyX inhibitory activity (33% inhibition at 50?μM). Introduction of alkyl and aryl groups at C(5) of 1a resulted in complete loss of inhibitory activity, whereas the attachment of a 3-(octanamido)prop-1-ynyl side chain in derivative 3 retained the weak level of mycobacterial ThyX inhibition (40% inhibition at 50?μM). None of the synthesized derivatives displayed any significant inhibitory activity against mycobacterial ThyA. The compounds have also been evaluated as potential inhibitors of mycobacterial thymidine monophosphate kinase (TMPKmt). None of the derivatives showed any significant TMPKmt inhibition. However, replacement of C(6) of the natural substrate (dTMP) by a N-atom furnished 6-aza-dTMP (1b), which still was recognized as a substrate by TMPKmt.     

Photoaffinity labeling and partial purification of the beta cell sulfonylurea receptor using a novel, biologically active glyburide analog

An iodinated analog of the sulfonylurea, glyburide, has been synthesized which can be labeled to high specific activity and used to photolabel the sulfonylurea receptor. 5-Iodo-2-hydroxy-"glyburide", has an iodo group replacing the chlorine at position 5 and a methoxy residue replacing the hydroxy group at position 2 on the benzamido ring. This analog retains biologic activity stimulating insulin secretion from a hamster beta cell line (HIT cells) at the same ED50 (0.4 nM) as glyburide. Scatchard analysis demonstrated high and low affinity binding sites on HIT cell membranes (Kd values of 0.36 nM and 277 nM and Bmax values of 1.6 and 100 pmol/mg of membrane protein, respectively). Competitive binding assays with unlabeled glyburide or 5-iodo-2-hydroxyglyburide yield Ki values of 0.5 and 1.0 nM, respectively. The analog can be covalently linked by ultraviolet irradiation to a membrane protein of Mr = 140,000. The photolabeling is completely blocked by unlabeled glyburide or the analog. Two other species of Mr = 65,000 and 43,000 are also photolabeled; these may be the low affinity sites. After photolabeling, the receptor has been purified partially by chromatographic procedures and is suitable for obtaining peptide sequence. The 140,000 molecular weight protein is identified as the sulfonylurea receptor since its binding constant, 0.36 nM, is closely correlated with its ability to stimulate insulin secretion (ED50 congruent to 0.4 nM).     

The incorporation of 5-iodo-5'-amino-2',5-dideoxyuridine and 5-iodo-2'-deoxyuridine into herpes simplex virus DNA. Relationship between antiviral activity and effects on DNA structure

Isopycnic centrifugation in CsCl gradients was used to quantify the incorporation of 5-iodo-5'-amino-2',5'-dideoxyuridine and 5-iodo-2'-deoxyuridine into herpes simplex virus type 1 DNA. A parallelism between the degree of incorporation into viral DNA and the inhibition of herpes simplex virus type I replication was found for both thymidine analogs. A concentration of 5-iodo-5'-amino-2',5'-dideoxyuridine approximately 100 times greater than 5-iodo-2'-deoxyuridine was required to achieve similar levels of antiviral activity. However, the inhibitory effects of these compounds are similar when compared with respect to the percent of substitution for thymidine in herpes simplex virus type I DNA. Damage to the viral DNA, as indicated by the presence of single or double-stranded breaks, was assessed by centrifugation in alkaline and neutral sucrose gradients. The incorporation of 5-iodo-5'-amino-2',5'-dideoxyuridine into herpes simplex virus type I DNA produced single and, to a lesser extent, double-stranded breaks in a dose-dependent manner. 5-Iodo-2'-deoxyuridine did not, however, induced DNA breakage. These data indicate that the additional presence of a phosphoramidate bond in the DNA produced the extensive damage detected under these conditions, but that such damage is not required for antiviral activity.     

Mechanism of inactivation of Escherichia coli ribonucleotide reductase by 2'-chloro-2'-deoxyuridine 5'-diphosphate: evidence for generation of a 2'-deoxy-3'-ketonucleotide via a net 1,2 hydrogen shift

Sodium borohydride or ethanethiol protects the Escherichia coli ribonucleoside-diphosphate reductase (RDPR) from inactivation by 2'-chloro-2'-deoxyuridine 5'-diphosphate (ClUDP). Incubation of [3'-3H]ClUDP with RDPR in the presence of NaBH4 allowed trapping of [3H]-2'-deoxy-3'-ketouridine 5'-diphosphate. Degradation of the reduced ketone by a combination of enzymatic and chemical methods indicated that the hydrogen originally present in the 3'-position of ClUDP is transferred to the beta-face of the 2'-position of 2'-deoxy-3'-keto-UDP. RDPR therefore catalyzes a net 1,2 hydrogen shift. Incubation of RDPR with ClUDP in the presence of ethanethiol allowed trapping of 2-methylene-3(2H)-furanone, the species responsible for inactivation of RDPR. Trapped 2-[(ethylthio)methyl]-3(2H)-furanone was identical by 1H NMR spectroscopy with material synthesized chemically. Both subunits of the enzyme are covalently radiolabeled in the reaction of RDPR with [5'-3H]ClUDP. Studies with [3'-3H]ClUDP and prereduced RDPR in the absence of a reductant and with oxidized RDPR indicated that the redox-active thiols of the B1 subunit are not involved in inactivation of the enzyme by ClUDP.     

Properties and inhibition of thymidylate synthetase in Drosophila melanogaster

A quantitative comparison of the incorporation of methyl-³H-thymidine and 6-³H-deoxyuridine into the DNA of Drosophila melanogaster in the presence and in the absence of 5-fluorouracil indicated that 5-fluorouracil inhibits the reaction converting dUMP to dTMP catalysed by thymidylate synthetase (methylenetetrahydrofolate:dUrd-5′-P C-methyltransferase, E.C. 2.1.1.b). The enzyme exhibits maximal activity at pH 7·5 to 8·0 and is protected from heat inactivation by deoxyuridine monophosphate. The addition of thiol compounds to the homogenization buffer results in the enhancement of synthetase activity. The K_m values for deoxyuridine monophosphate and 5,10-methylenetetrahydrofolate are 6·8 × 10^?6 M and 8·3 × 10^?5 M, respectively. Fluorodeoxyuridine monophosphate, trifluoromethyldeoxyuridine monophosphate, and methotrexate are inhibitors of the enzyme. 5-Bromodeoxyuridine and 5-iododeoxyuridine have no inhibitory effect. The results support the contention that, under conditions which induce morphological lesions in Drosophila, fluorinated pyrimidines and methotrexate inhibit the de novo synthesis of thymidylate whereas thymidine analogues function in some other manner.