Studies on the synthesis of CDP-diacylglycerol: Stimulation by GTP and inhibition by ATP and fluoride

The rat liver microsomal enzyme CTP: phosphatidate cytidylyltransferase (EC 2.7.7.41) which catalyzes the formation of CDP-diacylglycerol has been found to be markedly stimulated by GTP. The requirement for GTP is absolute, the novel GTP analogues such as guanosine 5′-[β,γ-methylene]-triphosphate, guanosine 5′-[α,β-methylene]-triphosphate, guanosine 5′-[β,γ-imido]-triphosphate and guanosine 3′-diphosphate 5′-diphosphate are without significant effect. Maximal stimulation occurs at 1 mM GTP. ATP at a concentration of 5 mM totally inhibits the formation of CDP-diacylglycerol even in the presence of optimal GTP concentration. Analogues of ATP such as adenosine 5′-[α,β-methylene]-triphosphate, adenosine 5′-[β,γ-methylene]-triphosphate and adenosine 5′-[β,γ-imido]-triphosphate are without effect on the reaction. The addition of fluoride (8 mM) likewise abolishes the stimulatory effect of GTP.     

Enzymatic synthesis of 2′-ara and 2′-deoxy analogues of c-di-GMP

The substrate specificity of recombinant full-length diguanylate cyclase (DGC) of Thermotoga maritima with mutant allosteric site was investigated. It has been originally shown that the enzyme could use GTP closest analogues – 2′-deoxyguanosine-5′-triphosphate (dGTP) and 9-β-D-arabinofuranosyl-guanine-5′-triphosphate (araGTP) as the substrates. The first demonstrations of an enzymatic synthesis of bis-(3′-5′)-cyclic dimeric deoxyguanosine monophosphate (c-di-dGMP) and the previously unknown bis-(3′-5′)-cyclic dimeric araguanosine monophosphate (c-di-araGMP) using DGC of T. maritima in the form of inclusion bodies have been provided.     

The Synthesis of Cyclonucleotides with Fixed Glycosidic Bond Linkages as Putative Agonists for P2-Purinergic Receptors

Abstract

Cyclonucleotides with fixed glycosidic bond linkages were investigated as possible ligands for purinoceptors in PC12 cells. P₂Y₂-purinoceptors were not activated by the ATP analogue, 8,2′-thioanhydroadenosine-5′-triphosphate (4) and only weakly by the UTP analogue, 2,2′-anhydrouridine-5′-triphosphate (6). However, both analogues were agonists for P2X₂-purinoceptors although the potencies were approximately 30-fold less than that of the parent nucleotides.     

NMR study of dideoxynucleotides with anti-human immunodeficiency virus (HIV) activity

The molecular structures of 3′-azido-2′,3′-dideoxyribosylthymine 5′-triphosphate (AZTTP), 2′,3′-dideoxyribosylinosine 5′-triphosphate (ddITP), 3′-azido-2′,3′-dideoxyribosylthymine 5′-monophosphate (AZTMP) and 2′,3′-dideoxyribosyladenine 5′-monophosphate (ddAMP) have been studied by NMR to understand their anti-HIV activity. For ddAMP and ddITP, conformations are almost identical with their nucleoside analogues with sugar ring pucker equilibriating between C3′-endo (∼75%) and C2′-endo (∼25%). AZTMP and AZTTP on the other hand show significant variations in the conformational behaviour compared with 3′-azido-2′,3′-dideoxyribo-sylthymine (AZT). The sugar rings for these nucleotides have a much larger population of C2′-endo (∼75%) conformers, like those observed for natural 2′-deoxynucleosides and nucleotides. The major conformers around C5′-O5′, C4′-C5′ and the glycosidic bonds are the β^t, γ⁺ and anti, respectively.     

Studies on uridine diphosphate-galactose pyrophosphatase and uridine diphosphate-galactose: glycoprotein galactosyltransferase activities in microsomal membranes.

Rat liver microsomes showed very active uridine diphosphate-galactose pyrophosphatase activity leading to the hydrolysis of uridine diphosphate-galactose into galactose1-phosphate and finally into galactose. The activity was observed in presence of buffers with wide ranges of pH. Different concentrations of divalent cations, such as Mn²⁺, Mg²⁺, and Ca²⁺ had no significant effect on the enzyme activity. A number of nucleotides and their derivatives inhibited the pyrophosphatase activity. Of these, different concentrations of uridine monophosphate, cytidine 5′-phosphate and cytidine 5′-diphosphate have slight or no effect; cytidine 5′-triphosphate, adenosine 5′-triphosphate, guanosine 5′-triphosphate, cytidine 5′-diphosphate-glucose and guanosine 5′-diphosphate-glucose showed strong inhibitory effect whereas cytidine 5′-diphosphate-choline showed a moderate effect on the pyrophosphatase. All these nucleotides also showed variable stimulatory effects on uridine diphosphate-galactose:glycoprotein galactosyltransferase activity in the microsomes which could be partly related to their inhibitory effects on uridine diphosphate-galactose pyrophosphatase. Among them uridine monophosphate, cytidine 5′-phosphate, and cytidine 5′-diphosphate stimulated galactosyltransferase activity without showing appreciable inhibition of pyrophosphatase, cytidine 5′-diphosphate-choline, although did not inhibit pyrophosphatase as effectively as cytidine 5′-triphosphate, guanosine 5′-triphosphate, adenosine 5′-triphosphate, cytidine 5′-diphosphate-glucose, and guanosine 5′-diphosphate-glucose but stimulated galactosyltransferase activity as well as those. The fact that cytidine 5′-diphosphate-choline stimulated galactosyltransferase more effectively than cytidine 5′-phosphate, cytidine 5′-diphosphate, and cytidine 5′-triphosphate suggested an additional role of the choline moiety in the system. It has been also shown that cytidine 5′-diphosphate-choline can affect the saturation of galactosyltransferase enzyme at a much lower concentration of uridine diphosphate-galactose. Most of the pyrophosphatase and galactosyltransferase activities were solubilized by deoxycholate and the membrane pellets remaining after solubilization still retained some galactosyltransferase activity which was stimulated by cytidine 5′-diphosphate-choline. In different membrane fractions a concerted effect of both uridine diphosphate-galactose pyrophosphatase and glycoprotein:galactosyltransferase enzymes on the substrate uridine diphosphate-galactose is indicated and their eventual controlling effects on the glycopolymer synthesis in vitro or in vivo need careful evaluation.     

Inhibition of the activity of DNA polymerase alpha by 2',3'-dideoxythymidine 5'-triphosphate.

2′,3′-Dideoxythymidine 5′-triphosphate was found to strongly inhibit the activity of DNA polymerase α from mouse myeloma in the presence of manganese ion as divalent cation. The extent of inhibition by 2′,3′-dideoxythymidine 5′-triphosphate increased by raising pH of the reaction. The mode of inhibition by 2′,3′-dideoxythymidine 5′-triphosphate was competitive to the substrate, 2′-deoxythymidine 5′-triphosphate. Ki of the DNA polymerase α for 2′,3′-dideoxythymidine 5′-triphosphate (0.035 μM) was much lower than Km for 2′-deoxythymidine 5′-triphosphate (1.8 μM).     

Ability of Adenosine-2′(3′)-deoxy-3′(2′)-triphosphates and Related Analogues to Replace ATP as Phosphate Donor for all Human and Drosphila melanogaster Deoxyribonucleoside Kinases

Abstract

Six non-conventional adenosine-2′- and 3′-triphosphate analogues of ATP were tested as potential phosphate donors for all four human, and D. melanogaster, deoxyribonucleoside kinases. With dCK (only dAdo as acceptor), TK1, TK2 and dNK only 3′-deoxyadenosine-2′-triphosphate was an effective donor (5–60% that for ATP). With dCK (dCyd as acceptor) and dGK (dGuo as acceptor), sharing 45% sequence identity, donor activities ranged from 13 to 119% that for ATP. Products were 5′-phosphates. In some instances, kinetics are dependent on the nature of the acceptor, and donor and acceptors properties are mutually interdependent. Results are highly relevant to studies on the modes of interaction with the enzymes, and to interpretations of reported crystal structures of dCK and dNK with bound ligands.     

Efficient Synthesis of 2′-Amino-2′-deoxypyrimidine 5′-Triphosphates

Abstract

The synthesis of 2′-amino-2′-deoxypyrimidine 5′-triphosphates is described. The 2′-amino-2′-deoxyuridine 5′-triphosphate is obtained from uridine in four steps with 25% overall yield. The 2′-amino-2′-deoxycytidine 5′-triphosphate is obtained from uridine in seven steps with 13% overall yield.     

Automated parallel synthesis of 5′-triphosphate oligonucleotides and preparation of chemically modified 5′-triphosphate small interfering RNA

A fully automated chemical method for the parallel and high-throughput solid-phase synthesis of 5′-triphosphate and 5′-diphosphate oligonucleotides is described. The desired full-length oligonucleotides were first constructed using standard automated DNA/RNA solid-phase synthesis procedures. Then, on the same column and instrument, efficient implementation of an uninterrupted sequential cycle afforded the corresponding unmodified or chemically modified 5′-triphosphates and 5′-diphosphates. The method was readily translated into a scalable and high-throughput synthesis protocol compatible with the current DNA/RNA synthesizers yielding a large variety of unique 5′-polyphosphorylated oligonucleotides. Using this approach, we accomplished the synthesis of chemically modified 5′-triphosphate oligonucleotides that were annealed to form small-interfering RNAs (ppp-siRNAs), a potentially interesting class of novel RNAi therapeutic tools. The attachment of the 5′-triphosphate group to the passenger strand of a siRNA construct did not induce a significant improvement in the in vitro RNAi-mediated gene silencing activity nor a strong specific in vitro RIG-I activation. The reported method will enable the screening of many chemically modified ppp-siRNAs, resulting in a novel bi-functional RNAi therapeutic platform.     

A New Efficient Stereoselective Method for the Synthesis of (E)-5-Aminoallyl-Pyrimidine-5′-Triphosphates Using Palladium-Catalyzed Heck Reaction

An efficient overall two-step strategy for the synthesis of (E)-5-aminoallyl-pyrimidine-5′-triphoshate, starting from commercially available pyrimidine-5′-triphosphate is described. The method involves regioselective iodination of pyrimidine-5′-triphosphate, followed by the palladium-catalyzed Heck coupling with allylamine. The catalytic reaction is highly stereoselective and compatible with many functional groups present in the reactants.     

An Efficient Protection-Free One-Pot Chemical Synthesis of Modified Nucleoside-5′-Triphosphates

A simple, reliable, and an efficient “one-pot, three step” chemical method for the synthesis of modified nucleoside triphosphates such as 5-methylcytidine-5′-triphosphate (5-MeCTP), pseudouridine-5′-triphosphate (pseudoUTP) and N¹-methylpseudouridine-5′-triphosphate (N¹-methylpseudoUTP) starting from the corresponding nucleoside is described. The overall reaction involves the monophosphorylation of nucleoside, followed by the reaction with pyrophosphate and subsequent hydrolysis of the cyclic intermediate to furnish the corresponding NTP in moderate yields with high purity (>99.5%).     

Selective Inhibition of DNA Chain Elongation Catalyzed by DNA Polymerases

Abstract

The results of series of works on the properties of a large number of nucleoside 5′-triphosphates analogs in the reaction catalyzed by several DNA polymerases are summarized.

Molecular mechanisms of substrate selection by DNA polymerases are not studied in detail. Therefore we have undertaken a comparative analysis of DNA polymerases from different sources employing nucleoside 5′-triphosphate analogs capable of incorporating into DNA chains terminating these chains elongation. Synthesis of a large line of nucleoside 5′-triphosphate analogs with substitution at the sugar residue has been performed. DNA polymerases have been isolated, and the synthesis of DNA has been studied using phage M13 DNA or phage MS2 RNA with synthetic deoxyoligonucleoti-de primers. The molecular mechanism of the substrate action has been determined by PAG electrophoresis of the reaction     

Significance of accumulation of the alarmone (p)ppGpp in chloroplasts for controlling photosynthesis and metabolite balance during nitrogen starvation in Arabidopsis

Photosynthesis Research - The regulatory nucleotides, guanosine 5′-triphosphate 3′-diphosphate (pppGpp) and guanosine 5′-diphosphate 3′-diphosphate (ppGpp), were originally...     

Partial purification and characterization of thymidylate kinase from embryonic chick liver.

Thymidylate kinase from the livers of 18-day-old chick embryos was concentrated 423-fold. The purification procedure included acid precipitation, ammonium sulfate fractionation, gel filtration on Sephadex G-100 and G-75 Super Fine, and ion-exchange chromatography on Diethylaminoethyl Sephadex A-50. This enzyme was found to be very labile but could be stabilized for long periods of time by its substrate (thymidine 5′-monophosphate) in the presence of 2-mercaptoethanol. Enzymes responsible for the formation of thymidine 5′-diphosphate and thymidine 5′-triphosphate, respectively, were separated during fractionation procedures. Thymidylate kinase from chick embryo liver was found to be a single protein having a molecular weight of approximately 46,000, Michaelis constant approximately 8 × 10^?5m, and a broad pH optimum between 6.6 and 8.6. A 2–3 mm requirement of Mg²⁺ above the adenosine 5′-triphosphate concentration was shown to be necessary for maximum enzyme activity. The enzyme appears to be competitively inhibited by thymidine, thymidine 5′-diphosphate, and thymidine 5′-triphosphate and noncompetitively inhibited by adenosine 5′-diphosphate.Thymidylate kinase enzymes isolated from two stages of developing embryonic liver and adult chick liver were shown to be identical.     

Trifluoromethyldiazirine-Containing dUTP: Synthesis and Application in DNA/Protein Crosslinking

Abstract

The 5-[N-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoyl)-3-aminoallyl]-2′-deoxyuridine-5′-triphosphate was synthesized via acylation of 5-aminoallyl-2′-deoxyuridine-5′-triphosphate with 4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoate N-hydroxysuccinimide. It was used for the preparation of 30 bp ATFMD-DNA coding for promoter sequence. UV-Irradiation (365 nm) of the specific complex of this duplex and E. coli RNA polymerase leads to the effective crosslinking DNA with all protein subunits.     

PREPARATION OF OLIGODEOXYNUCLEOTIDE 5′-TRIPHOSPHATES USING SOLID SUPPORT APPROACH

We report a synthetic procedure for conversion of oligonucleotides to their 5′-triphosphate derivatives with moderate yield. The oligonucleotides were synthesized on solid support using standard phosphoramidite protocols. The DMT protection group was removed and the 5′-OH was phosphitylated using 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one followed by reaction with tributyammonium pyrophosphate and iodine oxidation. After subsequent removal from support and complete deprotection, the products were isolated by anion-exchange HPLC chromatography. Structures of several 5′-triphosphate derivatives have been proven by phosphorus NMR, Mass-spectrometry and by HPLC comparison with authentic samples.     

Selective high metabolic lability of uridine triphosphate in response to glucosamine feeding of untransformed and polyoma virus-transformed hamster fibroblasts

Derepression of hexose transport in a line of Syrian hamster fibroblasts (Nil) and polyoma-transformed (PyNil) hamster fibroblasts is obtained when cells are either starved for glucose or fed with fructose as the only hexose source. D-glucosamine feeding of these cells does not alter the repressed state with regard to hexose transport. High, derepressed rates of galactose transport were changed to low, repressed rates, within 18 hours of refeeding glucose-starved cells with D-glucosamine as the only hexose source. Nil and PyNil cells, when cultured in the presence of D-glucosamine, undergo rapid reductions in total cellular uridine 5′-triphosphate (UTP) pool sizes. By contrast, the total cellular pools of adenosine 5′-triphosphate, guanosine 5′-triphosphate, and cytosine 5′-triphosphate (ATP, GTP, and CTP) were only moderately affected by the treatment of the cells with glucosamine. The metabolic drain of the UTP pools in PyNil cells was much more pronounced than in the untransformed cells. The larger and more rapid metabolic lability of UTP pools in the transformed cells may be the primary reason for the selective toxicity of glucosamine on tumor cells. A comparison of the effects of glucosamine on hexose-starved Nil and PyNil cells demonstrated that only the untransformed cells were able to utilize glucosamine to increase the hexose starvation-depleted pools of all nucleoside triphosphates. Accumulation of UDP-glucosamine and UDP-N-acetylglucosamine followed the reduction in the UTP pools. Inhibition of protein synthesis by cycloheximide during glucosamine feeding led to higher levels of UDP-glucosamine and UDP-N-acetylglucosamine accumulation. It is suggested that the drain of UTP pools during glucosamine treatment proceeds through the formation of the UDP-aminosugars which turn over due to the action of intracellular UDP-aminosugar pyrophosphatase activities.     

Carborane-linked 2′-deoxyuridine 5′-O-triphosphate as building block for polymerase synthesis of carborane-modified DNA

5-[(p-Carborane-2-yl)ethynyl]-2′-deoxyuridine 5′-O-triphosphate was synthesized and used as a good substrate in enzymatic construction of carborane-modified DNA or oligonucleotides containing up to 21 carborane moieties in primer extension reactions by DNA polymerases.     

NOVEL NUCLEOSIDE TRIPHOSPHATE ANALOGS FOR THE ENZYMATIC LABELING OF NUCLEIC ACIDS

We have evaluated several novel nucleotide analogs suitable for enzymatic labeling of nucleic acid targets for a variety of array-based assays. Two new reagents in particular, a C4-labeled 1-(2′,3′-dideoxy-β-D-ribofuranosyl) imid- azole-4-carboxamide 5′-triphosphate 5 and an N1-labeled 5-(β-D- ribofuranosyl)-2,4(1H,3H)-pyrimidinedione 5′-triphosphate 3, were found to be excellent substrates for labeling with terminal deoxynucleotidyl transferase and T7 RNA polymerase, respectively.