2-deoxyribose-1-phosphate--(alpha-d-erythro)-isomer and ribose-1-phosphate

A simple and efficient method for the preparation of α-D-ribose 1-phosphate and 2-deoxy-α-D-ribose 1-phosphate, key intermediates in nucleoside metabolism and important starting compounds for the enzymatic synthesis of various modified nucleosides, has been proposed. It consists in near-irreversible enzymatic phosphorolysis of readily prepared hydroiodide salts of 7-methylguanosine and 7-methyl-2'-deoxyguanosine, respectively, in the presence of purine nucleoside phosphorylase. α-D-Ribose 1-phosphate and 2-deoxy-α-D-ribose 1-phosphate are obtained in near quantitative yields (by HPLC analysis) and 74%-94% yields after their isolation and purification. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of α-D-ribose 1-phosphate barium salt (4a) Alternate Protocol 1: Preparation of 2-deoxy-α-D-ribose 1-phosphate barium salt (4b) Basic Protocol 2: Preparation of α-D-ribose 1-phosphate bis(cyclohexylammonium) salt (5a) Alternate Protocol 2: Preparation of 2-deoxy-α-D-ribose 1-phosphate bis(cyclohexylammonium) salt (5b).

Topics: Deoxyguanosine; Guanosine; Ribosemonophosphates

The reversible phosphorolysis of uridine to generate uracil and ribose 1-phosphate is catalyzed by uridine phosphorylase and is involved in the pyrimidine salvage pathway. We define the reaction mechanism of uridine phosphorylase from Trypanosoma cruzi by steady-state and pre-steady-state kinetics, pH-rate profiles, kinetic isotope effects from uridine, and solvent deuterium isotope effects. Initial rate and product inhibition patterns suggest a steady-state random kinetic mechanism. Pre-steady-state kinetics indicated no rate-limiting step after formation of the enzyme-products ternary complex, as no burst in product formation is observed. The limiting single-turnover rate constant equals the steady-state turnover number; thus, chemistry is partially or fully rate limiting. Kinetic isotope effects with [1'-(3)H]-, [1'-(14)C]-, and [5'-(14)C,1,3-(15)N(2)]uridine gave experimental values of (α-T)(V/K)(uridine) = 1.063, (14)(V/K)(uridine) = 1.069, and (15,β-15)(V/K)(uridine) = 1.018, in agreement with an A(N)D(N) (S(N)2) mechanism where chemistry contributes significantly to the overall rate-limiting step of the reaction. Density functional theory modeling of the reaction in gas phase supports an A(N)D(N) mechanism. Solvent deuterium kinetic isotope effects were unity, indicating that no kinetically significant proton transfer step is involved at the transition state. In this N-ribosyl transferase, proton transfer to neutralize the leaving group is not part of transition state formation, consistent with an enzyme-stabilized anionic uracil as the leaving group. Kinetic analysis as a function of pH indicates one protonated group essential for catalysis and for substrate binding.

Topics: Animals; Catalysis; Deuterium Exchange Measurement; Hydrogen-Ion Concentration; Phosphorylation; Ribosemonophosphates; Substrate Specificity; Trypanosoma cruzi; Uracil; Uridine; Uridine Phosphorylase

The metabolism of 5-fluorouracil (5-FU) was studied in biopsy specimens of primary colorectal cancer and healthy colonic mucosa obtained from previously untreated patients immediately after surgical removal. The conversion of 5-FU to anabolites was measured under saturating substrate (5-FU) and cosubstrate concentrations. For all enzymes, the activity was about threefold higher in tumor tissue compared with healthy mucosa of the same patient. The activity of pyrimidine nucleoside phosphorylase with deoxyribose-1-phosphate (dRib-1-P) was about tenfold higher (about 130 and 1200 nmol/hr/mg protein in tumors) than with ribose-1-phosphate (Rib-1-P), both in tumor and mucosa. Synthesis of the active nucleotides (5-fluoro-uridine-5'-monophosphate [FUMP] and 5-fluoro-2'-deoxyuridine-5'-monophosphate [FdUMP]) was studied by adding physiologic concentrations of adenosine triphosphate (ATP) to the reaction mixture; the rate of FdUMP synthesis was 50% of that of FUMP (about 4 and 7 nmol/hr/mg protein in tumors). Direct synthesis of FUMP from 5-FU in the presence of 5-phosphoribosyl-1-pyrophosphate (PRPP) was about 2 nmol/hr/mg protein. With the natural substrate for this reaction, orotic acid, the activity was about 14-fold higher. To obtain insight into the recruitment of precursors for these cosubstrates, the authors also tested the enzyme activity of pyrimidine nucleoside phosphorylase with inosine and ribose-5-phosphate (Rib-5-P, as precursors for Rib-1-P) and deoxyinosine (as a precursor for dRib-1-P); enzyme activities were approximately 7%, 7%, and 3%, respectively, of that with the normal substrates, both in tumors and mucosa. However, when ATP and Rib-5-P were combined, the synthesis of FUMP was about 70% of that with PRPP, but only in tumors. In normal tissues no activity was detectable. These data suggest a preference of colon tumor over colon mucosa for the conversion of 5-FU to active nucleotides by a direct pathway; a selective antitumor effect of 5-FU may be related to this difference.

Topics: Adenosine Triphosphate; Aged; Aged, 80 and over; Colon; Colonic Neoplasms; Fluorodeoxyuridylate; Fluorouracil; Humans; Intestinal Mucosa; Middle Aged; Orotate Phosphoribosyltransferase; Pentosyltransferases; Phosphoribosyl Pyrophosphate; Pyrimidine Phosphorylases; Rectal Neoplasms; Ribosemonophosphates; Uracil Nucleotides; Uridine Triphosphate

An adenylate-specific purine nucleoside phosphorylase (purine nucleoside:orthophosphate ribosyltransferase, EC12.4.2.1) (PNP) was isolated from a cytoplasmic fraction of Acholeplasma laidlawii B-PG9 and partially purified (820-fold). This partially purified PNP could only ribosylate adenine and deribosylate adenosine and deoxyadenosine. The A. laidlawii partially purified PNP could not use hypoxanthine, guanine, uracil, guanosine, deoxyguanosine, or inosine as substrates, but could use ribose-1-phosphate, deoxyribose-1-phosphate, or xylose-1-phosphate as the pentose donor. Mg2+ and a pH of 7.6 were required for maximum activity for each of the pentoses. The partially purified enzyme in sucrose density gradient experiments had an approximate molecular weight of 108,000 and a sedimentation coefficient of 6.9, and in gel filtration experiments it had an approximate molecular weight of 102,000 and a Stoke's radius of 4.1 nm. Nondenaturing polyacrylamide tube gels of the enzyme preparation produced one major and one minor band. The major band (Rf, 0.57) corresponded to all enzyme activity. The Kms for the partially purified PNP with ribose-1-phosphate, deoxyribose-1-phosphate, and xylose-1-phosphate were 0.80, 0.82, and 0.81 mM, respectively. The corresponding Vmaxs were 12.5, 14.3, and 12.0 microM min-1, respectively. The Hill or interaction coefficients (n) for all three pentose phosphates were close to unity. The characterization data suggest the possibility of one active site on the enzyme which is equally reactive toward each of the three pentoses. This is the first report of an apparently adenine-specific PNP activity.

Topics: Acholeplasma laidlawii; Adenine; Centrifugation, Density Gradient; Chromatography, Gel; Hexosephosphates; Hydrogen-Ion Concentration; Pentosephosphates; Pentosyltransferases; Purine-Nucleoside Phosphorylase; Ribosemonophosphates; Substrate Specificity

The pools of free ribose 1-phosphate and deoxyribose 1-phosphate have been measured in Bacillus cereus. It is shown that crude extracts of the same organism can actively utilize the sugar phosphates to convert adenine to ATP and deoxyATP, via a 'salvage' pathway, involving adenine ribosylation (or deoxyribosylation), followed by multiple phosphorylation steps. The biosynthetic pathway operates even in the presence of excess P(i') thus showing that purine nucleoside phosphorylases may function in vivo, contrary to what is generally assumed, as anabolic rather than catabolic enzymes.

Topics: Adenosine Triphosphate; Bacillus cereus; Deoxyadenine Nucleotides; Ribosemonophosphates

A method has been developed to measure deoxyribose 1-phosphate in the presence of ribose 1-phosphate and other sugar phosphates. The specificity of the method is based on the observation that only deoxyribose 1-phosphate is hydrolyzed by heating at pH 7.4, while both deoxyribose 1-phosphate and ribose 1-phosphate remain unchanged when heated at pH 10. A tissue extract is heated at pH 10. The amount of deoxyribose 1-phosphate plus ribose 1-phosphate is determined from that of deoxyinosine plus inosine formed in a coupled enzymatic reaction, based on the following two-stage transformation: deoxyribose 1-phosphate (ribose 1-phosphate) + adenine in equilibrium deoxyadenosine (adenosine) + inorganic phosphate, catalyzed by adenosine phosphorylase; deoxyadenosine (adenosine) + H2O----deoxyinosine (inosine), catalyzed by adenosine deaminase. By taking advantage of its unique heat lability, deoxyribose 1-phosphate is eliminated by heating the tissue extract at pH 7.4, and ribose 1-phosphate is determined as above. The amount of deoxyribose 1-phosphate stems from the difference between the amount of deoxyinosine plus inosine measured in the tissue extract heated at pH 10 and that of inosine measured in the tissue extract heated at pH 7.4. Free deoxyribose 1-phosphate has been found in rat tissues, as well as in Bacillus cereus during stationary phase of growth.

Topics: Animals; Bacillus cereus; Female; Pentosephosphates; Radiochemistry; Rats; Rats, Inbred Strains; Ribosemonophosphates; Spectrophotometry, Ultraviolet; Tissue Distribution

We studied the uptake of radioactive 5-fluorouracil (FUra) by the intact cells of Ehrlich ascites tumor in the presence of various coreactants with FUra such as uridine (Urd), deoxyuridine (dUrd), ribose 1-phosphate (Rib1P), and deoxyribose 1-phosphate (dRib1P). Radioactivity uptake by the cells was increased when FUra-6-14C was incubated with Rib1P or dRib1P, while the uptake was not stimulated with Urd or dUrd. The increased formation of antineoplastic ribo- and deoxyribo-nucleotides of FUra in the acid-soluble fraction was also observed in the same incubation with Rib1P or dRib1P. Also, some detergents, ethylenediaminetetraacetic acid (EDTA), adenosine 5'-triphosphate (ATP), and polyamines were examined. EDTA stimulated the uptake of radioactivity from the FUra by the cells. However, the other compounds exhibited no effect on the uptake of FUra alone or FUra plus dRib1P, except of ATP showing somewhat increase of radioactivity uptake. The above results suggest that the coadministration of FUra together with Rib1P or/and dRib1P, which are stimulants for the formation of FUra-deoxynucleotides from FUra, may be able to potentiate the chemotherapeutic effect of FUra.

Topics: Adenosine Triphosphate; Animals; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Deoxyuridine; Detergents; Edetic Acid; Fluorouracil; Male; Mice; Mice, Inbred Strains; Polyamines; Ribosemonophosphates; RNA, Neoplasm; Uridine

In order to acquire a biochemical fundamental knowledge on the enhancement of antitumor effect with 5-fluorouracil or its masked derivatives, this investigation was undertaken to determine the amounts of antineoplastic metabolic precursors formed from 5-fluorouracil-6-14C in the presence of various substrates by crude enzyme extracts from Ehrlich mouse ascites tumor cells and normal mouse liver. Combinations of uridine plus ribose 1-phosphate and deoxyuridine plus deoxyribose 1-phosphate were effective for the enhancement of 5-fluorouridine and 5-fluorodeoxyuridine formation, respectively. 5-Fluorouridine and 5-fluorodeoxyuridine are respectively utilizable for the syntheses of the antineoplastic compounds, 5-fluorouridine 5'-triphosphate and 5-fluoro-2'-deoxyuridine 5'-monophosphate. These results suggest that the coadministration of the above substrates together with 5-fluorouracil would be advantageous in the enhancement of the antitumor effect of 5-fluorouracil or its masked derivatives.

Topics: Animals; Ascites; Carcinoma, Ehrlich Tumor; Deoxyuridine; Fluorouracil; In Vitro Techniques; Liver; Male; Mice; Ribosemonophosphates; Uridine

The metabolism of 5-fluorouracil (5-FU) in tumors and normal tissues of humans was investigated in vitro. Phosphorylation of 5-FU was faster in tumor tissues than in normal tissues. Phosphorylating activity with 2-deoxy-alpha-D-ribose 1-phosphate (dRiblP) and ATP as cofactors was more active than that with alpha D-ribose 1-phosphate (RiblP) and ATP, or 5-phospho-alpha-D-ribosyldiphosphate (PPRibP) as cofactors. Phosphorylating activity in squamous cell carcinoma of the lung was similar to that in adenocarcinomas. Degradation of 5-FU was much faster in the liver than in other tissues including tumor tissues.

Topics: Adenocarcinoma; Adenosine Triphosphate; Carcinoma, Squamous Cell; Digestive System; Fluorouracil; Gastrointestinal Neoplasms; Humans; Liver; Lung; Lung Neoplasms; Neoplasms; Phosphoribosyl Pyrophosphate; Phosphorylation; Ribosemonophosphates