13191-15-6

Undine 5′-O-triphosphate (UTP) is a potent agonist of the purinergic receptor designated P2Y2. UTP is rapidly metabolized in human tissue. To find a compound with similar activity that may be more slowly metabolized, a series of 4-substituted uridine 5′-triphosphates were prepared and evaluated in a P2Y2 receptor second messenger assay. Copyright

We demonstrate that L-ATP: 1) as well as its natural D-enantiomer, acts as a phosphate donor in the reaction catalysed by human deoxycytidine kinase; 2) inhibits human DNA-primase and the ATP-dependent T4 DNA ligase. Thus, the lack of enantioselectivity of the enzymes is more frequent than it was believed a few years ago and we suggest that it would depend on chance more than on an evolutionary strategy. Copyright 1999 by Marcel Dekker, Inc.

Molecular gates within enzymes often play important roles in synchronizing catalytic events. We explored the role of a gate in cytidine-5′-triphosphate synthase (CTPS) from Escherichia coli. This glutamine amidotransferase catalyzes the biosynthesis of CTP from UTP using either L-glutamine or exogenous NH3 as a substrate. Glutamine is hydrolyzed in the glutaminase domain, with GTP acting as a positive allosteric effector, and the nascent NH3 passes through a gate located at the end of a ~25-? tunnel before entering the synthase domain where CTP is generated. Substitution of the gate residue Val 60 by Ala, Cys, Asp, Trp, or Phe using site-directed mutagenesis and subsequent kinetic analyses revealed that V60-substitution impacts glutaminase activity, nucleotide binding, salt-dependent inhibition, and inter-domain NH3 transport. Surprisingly, the increase in steric bulk present in V60F perturbed the local structure consistent with “pinching” the tunnel, thereby revealing processes that synchronize the transfer of NH3 from the glutaminase domain to the synthase domain. V60F had a slightly reduced coupling efficiency at maximal glutaminase activity that was ameliorated by slowing down the glutamine hydrolysis reaction, consistent with a “bottleneck” effect. The inability of V60F to use exogenous NH3 was overcome in the presence of GTP, and more so if CTPS was covalently modified by 6-diazo-5-oxo-L-norleucine. Use of NH2OH by V60F as an alternative bulkier substrate occurred most efficiently when it was concomitant with the glutaminase reaction. Thus, the glutaminase activity and GTP-dependent activation act in concert to open the NH3 gate of CTPS to mediate inter-domain NH3 transport.

A novel method for synthesizing CMP-NeuAc was established. We first confirmed that the putative neuA gene of Haemophilus influenzae, identified by its whole genome sequence project, indeed encodes CMP-NeuAc synthetase (EC 2.7.7.43). The enzyme requires CTP as a cytidylyl donor for cytidylylation of NeuAc. The enzyme was coupled with an enzymatic CTP-generating system from CMP and inorganic polyphosphate as a sole phospho-donor driven by the combination of polyphosphate kinase and CMP kinase, where phosphorylation of CMP is done by the combined activity expressed by both enzymes, and subsequent phosphorylation of CDP by polyphosphate kinase itself occurred efficiently. When CMP-NeuAc synthetase of H. influenzae, polyphosphate kinase, and CMP kinase were added to the reaction mixture containing equimolar concentrations (15 mM) of CMP and NeuAc, and polyphosphate (150 mM in terms of phosphate), CMP-NeuAc was synthesized up to 10 mM in 67% yield.

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The Escherichia coli polyphosphate kinase (PPK) has been known to catalyze the reversible transfer of phosphate molecules between ATP and polyphosphate (poly(P)). It has also been found that the PPK catalyzes the kination of not only ADP but also other nucleoside diphosphates (NDPs) using poly(P) as a phosphate donor, yielding nucleotide triphosphates (NTPs). We used the PPK and poly(P) in place of pyruvate kinase and phosphoenol pyruvate for NTP regeneration followed by synthesis of sugar nucleotides in a cyclic synthesis system for oligosaccharides. It was confirmed that the PPK efficiently catalyzed the UTP regeneration in the cyclic system of N-acetyllactosamine synthesis. This novel activity of PPK enables us to perform the practical synthesis of oligosaccharides.

CTP synthase (CTPS) catalyzes the conversion of UTP to CTP and is a recognized target for the development of anticancer, antiviral, and antiprotozoal agents. Xanthine and related compounds inhibit CTPS activity (IC50 = 0.16-0.58 mM). The presence of an 8-oxo function (i.e., uric acids) enhances inhibition (IC50 = 0.060-0.121 mM). An intact purine ring with anionic character favors inhibition. In general, methylation of the purine does not significantly affect inhibition.

Thirty years ago it was shown that the non-enzymatic, template-directed polymerization of activated mononucleotides proceeds readily in a homochiral system, but is severely inhibited by the presence of the opposing enantiomer. This finding poses a severe challenge for the spontaneous emergence of RNA-based life, and has led to the suggestion that either RNA was preceded by some other genetic polymer that is not subject to chiral inhibition or chiral symmetry was broken through chemical processes before the origin of RNA-based life. Once an RNA enzyme arose that could catalyse the polymerization of RNA, it would have been possible to distinguish among the two enantiomers, enabling RNA replication and RNA-based evolution to occur. It is commonly thought that the earliest RNA polymerase and its substrates would have been of the same handedness, but this is not necessarily the case. Replicating d- and l-RNA molecules may have emerged together, based on the ability of structured RNAs of one handedness to catalyse the templated polymerization of activated mononucleotides of the opposite handedness. Here we develop such a cross-chiral RNA polymerase, using in vitro evolution starting from a population of random-sequence RNAs. The d-RNA enzyme, consisting of 83 nucleotides, catalyses the joining of l-mono- or oligonucleotide substrates on a complementary l-RNA template, and similar behaviour occurs for the l-enzyme with d-substrates and a dlate. Chiral inhibition is avoided because the 10 6 -fold rate acceleration of the enzyme only pertains to cross-chiral substrates. The enzyme € s activity is sufficient to generate full-length copies of its enantiomer through the templated joining of 11 component oligonucleotides.

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The invention discloses a synthetic method of nucleoside tetraphosphate. The synthetic method comprises the steps of carrying out selective phosphorylation reaction by virtue of nucleoside and a cyclic phosphorylation reagent, and carrying out oxidation and hydrolysis loop opening, so as to obtain nucleoside tetraphosphate. The structure of the cyclic phosphorylation reagent is represented by a formula I (shown in the description). According to the synthetic method, 5'-nucleoside tetraphosphate is selectively generated from nucleoside under the effect of the high-selectivity phosphorylation reagent, and 3'-OH (and 2'-OH) does not need to be protected in the process, namely that the generaiton of 3'(and 2'-)tetraphosphate can be effectively inhibited. Nucleoside tetraphosphate synthesized by virtue of the method has wide use ranges in the biology fields of DNA sequencing, labeling, extension and the like; currently, the selling prices is expensive, a synthetic method is complex, the reaction selectivity is poor; and the synthetic method provided by the invention is good in selectivity and easy in separation and purification, required experimental conditions are simple, and the synthetic processes are all conventional chemical reactions, so that the synthetic method is applicable to large-scale popularization and use.

The wide specificity of 3-phosphoglycerate kinase (PGK) towards its nucleotide substrate is a property that allows contribution of this enzyme to the effective phosphorylation (i.e. activation) of nucleotide-based pro-drugs against HIV. Here, the structural basis of the nucleotide-PGK interaction is characterised in comparison to other kinases, namely pyruvate kinase (PK) and creatine kinase (CK), by enzyme kinetic analysis and structural modelling (docking) studies. The results provided evidence for favouring the purine vs. pyrimidine base containing nucleotides for PGK rather than for PK or CK. This is due to the exceptional ability of PGK in forming the hydrophobic contacts of the nucleotide rings that assures the appropriate positioning of the connected phosphate-chain for catalysis. As for the d-/l-configurations of the nucleotides, the l-forms (both purine and pyrimidine) are well accepted by PGK rather than either by PK or CK. Here again the dominance of the hydrophobic interactions of the l-form of pyrimidines with PGK is underlined in comparison with those of PK or CK. Furthermore, for the l-forms, the absence of the ribose OH-groups with PGK is better tolerated for the purine than for the pyrimidine containing compounds. On the other hand, the positioning of the phosphate-chain is an even more important term for PGK in the case of both purines and pyrimidines with an l-configuration, as deduced from the present kinetic studies with various nucleotide-site mutants of PGK. These characteristics of the kinase-nucleotide interactions can provide a guideline for designing new drugs.

Bacteriophage T5 deoxynucleoside monophosphate kinase (dNMP kinase, EC 2.7.4.13) is shown to catalyze the phosphorylation of both d2CMP and ribonucleotides AMP, GMP, and CMP, but does not phosphorylate UMP. For natural acceptors of the phosphoryl group, k m and k cat were found. The applicability of T5 dNMP kinase as a universal enzyme capable of the phosphorylation of labelled r/dNMP was shown for the synthesis of [α- 32P]rNTP and [α-32P]dNTP.

β-D-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) is a potent specific inhibitor of hepatitis C virus (HCV) RNA synthesis in Huh-7 replicon cells. To inhibit the HCV NS5B RNA polymerase, PSI-6130 must be phosphorylated to the 5′-triphosphate form. The phosphorylation of PSI-6130 and inhibition of HCV NS5B were investigated. The phosphorylation of PSI-6130 by recombinant human 2′-deoxycytidine kinase (dCK) and uridine-cytidine kinase 1 (UCK-1) was measured by using a coupled spectrophotometric reaction. PSI-6130 was shown to be a substrate for purified dCK, with a Km of 81 μM and a kcat of 0.007 s -1, but was not a substrate for UCK-1. PSI-6130 monophosphate (PSI-6130-MP) was efficiently phosphorylated to the diphosphate and subsequently to the triphosphate by recombinant human UMP-CMP kinase and nucleoside diphosphate kinase, respectively. The inhibition of wild-type and mutated (S282T) HCV NS5B RNA polymerases was studied. The steady-state inhibition constant (Ki) for PSI-6130 triphosphate (PSI-6130-TP) with the wild-type enzyme was 4.3 μM. Similar results were obtained with 2′-C-methyladenosine triphosphate (Ki = 1.5 μM) and 2′-C-methylcytidine triphosphate (Ki = 1.6 μM). NS5B with the S282T mutation, which is known to confer resistance to 2′-C- methyladenosine, was inhibited by PSI-6130-TP as efficiently as the wild type. Incorporation of PSI-6130-MP into RNA catalyzed by purified NS5B RNA polymerase resulted in chain termination. Copyright

A novel method for the preparation of nucleoside triphosphates has been developed. The strategy employs a highly reactive pyrrolidinium phosphoramidate zwitterion intermediate that undergoes efficient coupling with tris(tetra-n-butylammonium) hydrogen pyrophosphate to generate nucleoside triphosphate.

Cidofovir [CDV; (S)-1-(3-hydroxy-2-phosphonomethoxyethyl)-cytosine] is an acyclic nucleotide analog with potent and selective in vitro and in vivo activities against a broad spectrum of herpesviruses and other DNA viruses. We studied the mechanism of enzymatic synthesis of CDV diphosphate, the putative antiviral metabolite of CDV. The phosphorylation is two-step process catalyzed by several enzymes. An enzymatic activity phosphorylating CDV to its monophosphate derivative was purified from human liver and identified as pyrimidine nucleoside monophosphate kinase (EC 2.7.4.14.). CDV (K(m) = 2.10 ± 0.18 mM and V(max) = 1.10 ± 0.05 μmol/min/mg) was found to be a substantially weaker substrate for purified enzyme than CMP, UMP, or dCMP. Pyrimidine nucleoside monophosphate kinase was used for preparative enzymatic synthesis of CDV monophosphate. Pyruvate kinase (EC 2.7.1.40), creatine kinase (EC 2.7.3.2), and nucleoside diphosphate kinase (EC 2.7.4.6) were found to catalyze CDV diphosphate synthesis from CDV monophosphate, whereas phosphoglycerate kinase (EC 2.7.2.3) and succinyl-CoA synthetase (EC 6.2.1.4) did not. Based on V(max)/K(m) (phosphorylation efficiency) values determined with enzymes purified from human sources, the most efficient phosphorylation of CDV monophosphate is catalyzed by pyruvate kinase. After infection of human lung fibroblasts with cytomegalovirus, the intracellular activities of pyrimidine nucleoside monophosphate kinase, pyruvate kinase, creatine kinase, and nucleoside diphosphate kinase increased 2-, 1.3-, 3-, and 5-fold, respectively. The metabolism of [3H]CDV in mock- and cytomegalovirus-infected cells was examined. The intracellular levels of CDV monophosphate and CDV diphosphate increased ~20- and 8-fold, respectively, in cytomegalovirus-infected cells, presumably due to the stimulation of CDV uptake and higher activities of phosphorylating enzymes.

5'-Polyphosphates of mono- and oligonucleotides were prepared from the corresponding 5'-monophosphates with phosphorotriimidazolide or phosphorotribenzimidazolide mediated by Mg(II) or Mn(II) ion in aqueous solution.

The preparative formation of cytidine 5'-triphosphate by an enzymatic process with adenylate kinase and pyruvate kinase is described.The enzymes may either be used free, in the "MEEC" technique, or immobilized on a synthetic copolymer of vinyl acetate and N,N'-divinylethyleneurea ("VA-Epoxy").Cytidine 5'-monophosphosialate synthase (preferable isolated from calf brain) and inorganic pyrophosphatase were used to prepare the activated neuraminic acid cytidine 5'-monophosphosialate (CMP-Neu5Ac)from CTP and neuraminic acid in good yield.Finally, both processes could be integrated and led to an in situ approach which, owing to moderate yields, requires further development.