Phosphorylation Of Nucleosides Research Articles

Synthesis of 4- and 5-amino-1-(2-deoxy-d-erythro-pentofuranosyl)imidazole nucleosides by chemical and biotransformation methods

(2-Deoxy-d-erythro-pentofuranosyl)imidazole nucleosides have been synthesised by glycosylation of the sodium salt of ethyl 5-aminoimidazole-4-carboxylate with 2-deoxy-3,5-di-O-p-toluoyl-α-d-erythro-pentofuranosyl chloride. Glycosylation of ethyl 5-aminoimidazole-4-carboxylate with 2-deoxy-β-d-erythro-pentofuranose was also achieved enzymically using E. coli, immobilised by ionotropic gelation in an alginate gel, with 2′-deoxyuridine as glycosyl donor. 5-Amino-1-(2-deoxy-β-d-erythro-pentofuranosyl)imidazole-4-carboxylic acid 5′-phosphate and 4-amino-1-(2-deoxy-β-d-erythro-pentofuranosyl)imidazole-5-carboxylic acid 5′-phosphate were synthesised by phosphorylation of the respective nucleosides and examined as inhibitors of phosphoribosylaminoimidazole carboxylase (EC 4.1.1.21) and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (EC 6.3.2.6), which are involved in the de novo biosynthesis of purine nucleotides.

Inhibition of HIV infection of resting peripheral blood lymphocytes by nucleosides.

T cells in the peripheral blood are largely in the resting state and represent a significant pool of potential targets for HIV infection. The protection of these cells from infection is an important goal of nucleoside therapy. Resting PBL may not be protected effectively by such nucleosides as azidothymidine (AZT) since anabolic phosphorylation of thymidine nucleosides is reported to be limited in these cells. In this study we used DNA amplification procedures to follow HIV proviral DNA formation in resting T cells and to determine the ability of azidodeoxythymidine (AZT), dideoxyinosine (ddI), and dideoxycytosine (ddC) to inhibit this process. Experiments confirm that resting PBL synthesize HIV proviral DNA sequences. Drug titrations showed that this synthesis could be inhibited by nucleosides. ddC was the most potent drug, inhibiting transcription at the U5 region. ddI and AZT at similar concentrations (10 microM) did not inhibit. ddI in the concentration range of 10-100 microM was able to inhibit production of transcripts containing U3 and env sequences in resting cells. Similar inhibition levels were accomplished by AZT at 10-100-fold lower drug concentrations. These results demonstrate that resting T cells can be protected from HIV infection by nucleosides and that thymidine nucleosides are effective inhibitors despite the limited potential for anabolic phosphorylation.

A novel non-radioactive method for detection of nucleoside analog phosphorylation by 5′-nucleotidase

Cytosolic 5′-nucleotidase has been implicated in the phosphorylation of certain nucleosides of therapeutic interest. In vitro, IMP and GMP serve as the optimal phosphate donors for this nucleoside phosphotransferase reaction. Existing assays for nucleoside phosphorylation effected by 5′-nucleotidase require a radiolabeled nucleoside as the phosphate acceptor and separation of the substrate-nucleoside from product-nucleotide has been accomplished either by a filter binding method or HPLC. However, detection of the phosphorylation of unlabeled nucleoside by HPLC is difficult since the ultraviolet absorbance of the phosphate donor, IMP, frequently obscures the absorbance of newly formed nucleotide. The use of ribavirin 5′-phosphate (RMP, 1,2,4-triazole-3-carboxamide riboside 5-monophosphate) as the phosphate donor obviates this difficulty since this triazole heterocycle does not significantly absorb at the wavelengths used to detect most nucleoside analogs. Using this procedure, a 5′-nucleotidase activity from the 100 000 × g supernatant fraction of human T-lymphoblasts deficient in adenosine kinase, hypoxanthine-guanine phosphoribosyltransferase, and deoxycytidine kinase, was characterized with regard to structure-activity relationships for certain inosine and guanosine analogs.

Practical Synthesis of 2'-5'-Linked Oligoadenylates (2-5A Oligomers)

A general, facile synthesis of 2'-5'-linked oligonucleotides (2-5A oligomers ) has been achieved based on the second-order regioselective protection of adenosine, one-pot formation of the 2'5' internucleotide linkage, and O-selective phosphorylation of N-unblocked nucleosides. Standard t- butyldimethylsilylation of 5'-O-p-methoxytrityladenosine followed by careful recrystallization from a mixture of triethylamine , methanol, ethyl acetate and ether (4 : 4 : 5 : 100 v/v) gives the 3',5'-di-O-protected adenosine in high yield. Magnesium alkoxide -mediated condensation of the 2'-O-free adenosine with o-chlorophenyl p-nitrophenyl phosphorochloridate followed by 2',3'-di-O-t-butyldimethylsilyladenosine produces the N-free and fully O-protected adenylyl (2'-5')adenosine. The resulting adenylyl dimer, after removal of the 5'-O-trityl protector, is elongated to the protected trimeric compound through a similar reaction sequence. Deprotection of the product furnishes the 2-5A core. Condensation of the 5?-0-detritylated core and bis (2,2,2-trichloroethyl) phosphorochloridite assisted by 2,6-lutidine and subsequent oxidation with aqueous iodine produces, after deblocking, 2-5A 5'-monophosphate (p5'A2'p5'pA2'p5'A). The 2-5A 5'-monophosphate is converted into 2-5A 5'-triphosphate (ppp5'A2'p5'A2'p5'A) by reaction with N,N'-carbonyldiimidazole in the presence of triethylamine followed by tributylammonium diphosphate. This procedure allows ready synthesis of 2-5A oligomers and related compounds on a multigram scale.

Mutant varicella-zoster virus thymidine kinase: correlation of clinical resistance and enzyme impairment.

Varicella-zoster virus (VZV) encodes a thymidine kinase (EC 2.7.2.21) which phosphorylates several antiviral nucleoside analogs, including acyclovir (ACV). A mutation in the VZV thymidine kinase coding sequence, resulting in an arginine-to-glutamine substitution at amino acid residue 130 (R130Q), is associated with clinical resistance to ACV. We have expressed the wild-type and the mutant enzymes in bacteria and have studied the kinetic characteristics of the purified enzymes. The arginine-to-glutamine substitution resulted in decreased catalytic activity and altered substrate specificity. The most striking effect was a decrease in the rates of nucleoside phosphorylation to less than 2% of the rates with the wild-type enzyme. This was accompanied by increased apparent Km values for thymidine and deoxycytidine. ACV was not detectably phosphorylated by the R130Q enzyme but still competed with thymidine for the enzyme. The inability of the R130Q enzyme to catalyze the phosphorylation of ACV correlates with resistance to ACV noted with a clinical isolate of VZV.

Novel P 1,P 2-substituted phosphonate analogues of 2′-deoxyadenosine and 2′-deoxythymidine 5′-triphosphates

P 1, P 2-Substituted methylene, fluoromethylene, and difluoromethylene analogues of the deoxynucleotides 2′-deoxyadenosine 5′-triphosphate and thymidine 5′-triphosphate have been prepared. by reaction between the bisphosphonic acid and the appropriate 5′- O-tosyl deoxynucleoside giving analogues of the 2′-deoxynucleoside 5′-diphosphate. The γ-phosphate is added subsequently either via activation of P-2 of the dNDP as its morpholidate followed by reaction with inorganic phosphates or using a novel, general reaction for the phosphorylation of nucleoside 5′-diphosphates employing p-nitrobenzyl phosphoromorpholidate.

Nucleic acid related compounds. 65. New syntheses of 1-(.beta.-D-arabinofuranosyl)-5(E)-(2-iodovinyl)uracil (IVAraU) from vinylsilane precursors. Radioiodine uptake as a marker for thymidine kinase herpes viral infections

(Trimethylsilyl)acetylene was coupled with 1-(2,3,5-tri-O-acetyl-beta-D- arabinofuranosyl)-5-iodouracil to give 1- (2,3,5-tri-O-acetyl-beta-D-arabinofuranosyl)-5-[2-(trimethylsilyl)eth yny l] uracil. Lindlar hydrogenation of 4 gave 1-(2,3,4-tri-O-acetyl-beta-D-arabinofuranosyl)-5(Z)-[2- (trimethylsilyl)vinyl]uracil. Treatment of 5 with iodine monochloride (or sodium iodide/phenyliodine(III) dichloride) in benzene gave 1-(2,3,5-tri-O-acetyl-beta-D-arabinofuranosyl)-5(E)-(2-iodovinyl)uracil (7), whereas polar solvents favored the (Z)-iodovinyl isomer 8. Deacetylation of 7 gave 1-(beta-D-arabinofuranosyl)-5(E)-(2-iodovinyl)uracil (IVAraU, 9). A microscale in situ synthesis with Na*I gave [*I]IVAraU. Treatment of HSV-infected cells with [125I]IVAraU resulted in virus-dependent uptake associated with nucleoside phosphorylation by wild type or acyclovir-resistant DNA polymerase mutants (but not with TK-HSV-1 mutants). Uptake was virus-inoculum dependent and was detectable within 4 h postinfection. The process was not completely reversible. Virus-specified uptake of [125I]IVAraU may allow automated in vitro detection of HSV isolates.

Solution conformations of nucleoside analogues exhibiting antiviral activity against human immunodeficiency virus

The molecular-conformational basis for HIV-1 antiviral activity of dideoxynucleoside analogues is unknown. A recent proposal by van Roey [1] that furanose sugar puckering in the C2′ -endo family (namely C3′ -exo) may account for the enhanced anti-HIV-1 activity of azidothymidine (AZT), dideoxythymidine (ddT) and dideoxycytidine (ddC) has been tested by conformational analysis of these and related agents, using laser Raman spectroscopy of their solutions and crystal structures. The results show that nucleoside analogues exhibiting anti-HIV-1 activity, including AZT, ddT and ddC, exist in solution with C3′ -endo as the predominating sugar pucker. The C3′ -endo solution conformations differ fundamentally from the C3′ -exo conformations observed in the corresponding crystal structures. Accordingly, the crystal conformation cannot be responsible for enhanced recognition of these agents, either by nucleoside kinase or reverse transcriptase, as a mechanism to explain antiviral activity. The present findings suggest that C3′ -endo sugear pucker, rather than C3′ -exo pucker, or other puckers of the C2′ -endo family, is more probably the required conformation for antivaral activity.The present work also shows that nucleoside phosphorylation does not, in general, change the preferred solution conformation of a nucleoside. Therefore, C3′ -endo sugar pucker is likely to be the preferred conformation for both nucleoside kinase and reverse transcriptase recognition. In this study, the list of thymidine nucleoside conformation markers available from Raman spectra is extended and additional group frequency assignments for C3′ -azido, C3′ -deoxy and related nucleoside derivatives are provided.

32P-Postabelling of alkylated thymidines using Epstein-Barr virus encoded thymidin kinase

Alkylated nucleotides have been detected by 32P-postlabelling using the enzyme T4 polynucleotide kinase which phosphorylates the 3'-mononucleotides to give the 3',[5'-32P]bisphosphates. These may then be separated by two-dimensional TLC as the bisphosphates or the [5'-32P]monophosphates. We describe here an alternative approach using the Epstein-Barr virus (EBV) encoded thymidine kinase (TK) to directly phosphorylate adducted nucleosides to give the [5'-32P]monophosphates. Using a series of methyl, ethyl and butyl thymidines EBV-encoded TK was shown to phosphorylate a wide range of adducted thymidines with varying degrees of labelling efficiency; N3-methyl thymidine was labelled with the highest efficiency and O4-ethyl thymidine the lowest. Whereas O4-methyl thymidine was labelled at a higher efficiency than O2-methyl thymidine, O4-ethyl and O4-butyl thymidines were labelled at a much lower efficiency than the corresponding O2-alkyl thymidines. Labelling efficiency increased with pH in the range pH 7 to pH 9, but the relative labelling efficiency was ATP independent. This direct phosphorylation of adducted nucleosides offers an alternative approach to the detection of alkylated residues in DNA which may complement current postlabelling procedures.

Specific Effects of 4-Morpholine-N,N'-dicyclohexylcarboxamidine as a Catalyst for the Alkylation and Phosphorylation of Ribonucleosides by a Two-step Phosphorylating Agent, MTBO: Its Application to the Synthesis of Cytokinin-like Nucleotides.

An examination of amines as catalysts for the first-step phosphorylation of nucleosides by a two-step phosphorylating agent, 2-methylthio-4H-1,3,2-benzodioxaphosphorin 2-oxide (MTBO, 2), revealed that, in the presence of 4-morpholine-N,N′-dicyclohexylcarboxamidine (MDC, 3) as the catalyst, MTBO phosphorylates and o-hydroxybenzylates adenosine borate complex (1) to give 1-(o-hydroxybenzyl)adenosine 5′-S-methyl phosphorothiolate (5), with subsequent Dimroth rearrangement to give N6-(o-hydroxybenzyl)adenosine 5′-S′-methyl phosphorothiolate (6).The guanosine borate complex (7) gave 1-(o-hydroxybenzyl)guanosine 5′-S-methyl phosphorothiolate (8) under the same conditions.MTBO phosphorylated N6-benzyladenosine and N6-allyladenosine borate complexes (9a and b) in the presence of n-propylamine (11) to give their 5′-S-methyl phosphorothiolates (10a and b), respectively.Thus, the 5′-S-methyl phosphorothiolates of cytokinin-like nucleosides were synthesized by the use of different amine catalysts from both intact and...

ChemInform Abstract: Organometallic Methodologies for Nucleic Acid Synthesis

A new, general preparation of 2'-5'-linked oligoadenylates has been developed, which relies on: (1) chemoselective 0- phosphorylation of N-unblocked nucleosides via hydroxyl activation by Grignard reagents, (2) one-pot construction of internucleotide linkage using bifunctional phosphorylating agents, and (3) selective production of a 3',5'-di-O-protected adenosine. This solution-phase synthesis allows large-scale preparation of a wide range of related oligomers. Palladium chemistry coupled with allylic protection of functional groups leads to the development of an efficient solid-phase synthesis of DNA oligomers via a phosphoramidite approach. Ally1 groups on internucleotide linkages and allyloxycarbonyl groups on amino moieties of nucleobases are removable all at once on the solid supports by exposure to a palladium(0) complex and nucleophiles. This procedure has been utilized for synthesis of a DNA 60mer of unprecedented high purity. In addition, this marks the realization of the first efficient preparation of solid-anchored DNA oligomers. Oligonucleotides are one of the most significant classes of compounds from both the academic and industrial point of view. The chemical synthesis, however, poses an array of problems in the transformation and isolation of products, and high overall efficiency is secured only by using a combination of truly appropriate methods. Organometallic chemistry, though exerting general intense influence on contemporary organic synthesis, has seldom been used in nucleic acids synthesis, perhaps because of the multifunctional, polar nature of the substrates. We demonstrate here the potent utility of metallo-organic methodologies in this area by describing a solution-phase synthesis of some short-chain oligomers having unusual structures and a solid-phase preparation of a long-sequence DNA.

Inhibitors of IMP dehydrogenase stimulate the phosphorylation of the antiviral nucleoside 2′,3′-dideoxyguanosine

The inosinate dehydrogenase (IMPD) inhibitors ribavirin, tiazofurin and mycophenolic acid were found to stimulate by as much as 20-fold the anabolism of the anti-HIV agent 2′,3′dideoxyguanosine to its 5′-diphosphate (ddGDP) in a human T-cell culture system (Molt-4 cells). Stimulation of the further conversion to ddGTP (the active form of the drug) was lesser in magnitude but still highly significant (up to 4-fold at appropriate concentrations of ribavirin or tiazofurin). In parallel with these increases, the inhibitors also produced increases of up to 35-fold in IMP levels. These results support the proposal that the initial phosphorylation of ddGuo is catalyzed by a phosphotransferase (5′-nucleotidase) which utilizes IMP as its phosphate donor (Johnson and Fridland, [1989] Molec. Pharmacol. 36, 291–295). Concomitant with this increase in 5′-phosphorylation of ddGuo, an increase in its anti-HIV activity of up to 6.5-fold was observed when this agent was combined with ribavirin (5μM) in the CEM cell assay system.

Organometallic methodologies for nucleic acid synthesis

Abstract

Regulation of purine deoxynucleoside phosphorylation by deoxycytidine kinase from human leukemic blast cells

The kinetics and regulation of nucleoside phosphorylation by highly purified human deoxycytidine kinase from leukemic lymphoblasts were studied. The phosphorylation of purine nucleosides by this enzyme showed sensitivity to the endogenous inhibitors dCTP and UDP three times greater than the phosphorylation of dCyd. Examination of nucleotide pools in human T and B lymphoblasts disclosed that the levels of dCTP and UDP in these cells were sufficient to regulate kinase activity. The enhanced sensitivity of the kinase to dCTP and UDP was related to its reduced ability to interact with purine nucleosides. Comparison of the phosphorylation kinetics for pyrimidine and purine dideoxynucleosides used in antiviral therapy showed that the purine nucleosides were at least 50-fold less efficient as enzyme substrates. These results suggest that the phosphorylation of pharmacologically active purine nucleosides by deoxycytidine kinase is regulated by cellular nucleotide pools.

5'-Фосфорилирование природных нуклеозидов и их модифицированных аналогов интактными клетками Erwinia herbicola: специфичность в отношении донора фосфатной группы и акцептора

5'-Фосфорилирование природных нуклеозидов и их модифицированных аналогов интактными клетками Erwinia herbicola: специфичность в отношении донора фосфатной группы и акцептора

Prebiotic phosphorylation of nucleosides — Preparation of labelled nucleotides

Prebiotic phosphorylation of nucleosides — Preparation of labelled nucleotides

Formate ester formation in amide solutions.

Simple aliphatic alcohols, deoxynucleosides and nucleosides undergo reaction with formamide yielding formate esters. Formate ester formation was observed to occur slowly at 100 degrees C and more rapidly at 130 degrees C. As expected, formate esters were hydrolyzed to the alcohol and formic acid upon heating in aqueous solution. It was proposed to study the possibility that formate esters are formed initially in amide solvents, followed by displacement of formate by dihydrogen phosphate ion to form monophosphate esters. Experiments are described which demonstrate the formation and hydrolysis of formate esters, as well as their lack of reaction with hydrogen phosphate ion. Formate esters are not intermediates in the phosphorylation of nucleosides in formamide. Their formation has been observed and such an esterification is a side reaction during the phosphorylation of nucleosides in formamide.

Mode of action of a new type of UDP-glucose analog against herpesvirus replication.

The mode of action of a new type of UDP-glucose analog against herpes simplex virus type 1 (HSV-1) replication was examined. The analog showed good selectivity and potent activity. At 10 micrograms/ml, P-536 inhibited the formation of infectious HSV-1 by more than 90%, whereas at 100 micrograms/ml it had no cytotoxic effects, as evidenced by phase-contrast microscopy. P-536 showed a wide spectrum of action and was active against HSV-1, adenovirus type 5, vaccinia virus, poliovirus type 1, encephalomyocarditis virus, vesicular stomatitis virus, influenza virus, and measles virus, irrespective of whether these viruses have lipidic envelopes or not. P-536 clearly inhibited protein glycosylation if added at the time when late viral proteins were being synthesized. Moreover, it also interfered with the synthesis of nucleic acids and the phosphorylation of nucleosides. If P-536 was present from the beginning of infection, HSV-1 replication was blocked at an early step and the infected cells continued to synthesize cellular proteins for long periods.

Iodine and Iodine Catalysed Phosphorylation of Nucleosides by Phosphorodiester Derivatives

Abstract Sodium iodide was found to catalyse the phosphorylation of nucleosides by phosphorodiester derivatives such as chlorophosphates, pyrophosphates and triazolides. A similar catalytic effect, but on phosphorochloridates only, was also exerted by iodine.

ChemInform Abstract: Recent Developments in Phosphorylation of Nucleosides

The phosphorylation of nucleosides is a fundamental reaction in the synthesis of oligonucleotides. This article reviews recent results on the phosphorylation of nucleosides in the synthesis of oligonucleotides by the phosphotriester method and the phosphite triester method. The new condensing and phosphorylating reagents for modified phosphotriester method, activated phosphotriester method, and modified phosphite triester method were discussed.