- Nucleoside diphosphate kinase and the activation of antiviral phosphonate analogs of nucleotides: Binding mode and phosphorylation of tenofovir derivatives
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Tenofovir is an acyclic phosphonate analog of deoxyadenylate used in AIDS and hepatitis B therapy. We find that tenofovir diphosphate, its active form, can be produced by human nucleoside diphosphate kinase (NDPK), but with low efficiency, and that creati
- Koch, Kerstin,Chen, Yuxing,Feng, Joy Y.,Borroto-Esoda, Katyna,Deville-Bonne, Dominique,Janin, Joel,Morera, Solange
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- Class II ribonucleotide reductases catalyze carbon-cobalt bond reformation on every turnover
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Ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the reduction of nucleotides to deoxynucleotides with concomitant oxidation of two cysteines within the active site to a disulfide. RTPR requires adenosylcobalamin (AdoCbl) as a cofactor and as a radical chain initiator. Catalysis is initiated by homolysis of the carbon-cobalt bond of AdoCbl to yield cob(II)alamin, 5'-deoxyadenosine, and a protein-based thiyl radical. The turnover numbers for ATP with its allosteric effector dGTP, and for CTP with its allosteric effector dATP, are both 2 s-1. The rate- limiting step for turnover in the steady state is re-reduction of the oxidized form of the protein, a conformational change, or both. Under conditions where [RTPR] >> [AdoCbl], the rates of ATP and CTP reduction do not vary linearly with [AdoCbl] but instead exhibit saturation behavior with turnover numbers of 10 s-1 (ATP) and 8.5 s-1 (CTP). This result suggests that dissociation of AdoCbl, which requires carbon-cobalt bond reformation, follows nucleotide reduction, but precedes the rate-limiting step in catalysis. A presteady-state analysis of the ATP reduction (using rapid chemical quench methods) in the presence of [5'-3H]-AdoCbl reveals formation of product dATP at a k(obs) of 55 ± 10 s-1 and tritium washout from [5'- 3H]-AdoCbl at 0.6 s-1. The rate of washout is approximately equivalent to the rate of washout of 3H in the absence of substrate. Measurement of the ratio of 3H2O:dATP over time reveals that washout of 3H occurs at the end of each turnover. Production of 3H2O requires reformation of the carbon- cobalt bond. These steady-state and presteady-state data suggest that carbon- cobalt bond reformation and dissociation of AdoCbl into solution accompany each turnover and that the radical chain length of the RTPR-catalyzed nucleotide reduction is approximately one.
- Licht, Stuart S.,Lawrence, Christopher C.,Stubbe, JoAnne
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- Preparative 2'-reduction of ATP catalyzed by ribonucleotide reductase purified by liquid-liquid extraction.
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Recombinant Lactobacillus leichmannii ribonucleosidetriphosphate reductase (RTPR, E.C.1.17.4.2) constitutively expressed by E. coli HB101 pSQUIRE has been purified from sonicated cell material in a one-step procedure by PEG 4000 (16% (w/w))/phosphate (7% (w/w)) liquid-liquid extraction. A high yield of 75.1% RTPR in the top phase and a partitioning of 8.5:1 between total RTPR activity in top and bottom phase were obtained in this preparative system. The RTPR-containing top phase was used to reduce ATP in the 2'-position on a gram scale with high final conversion and yield proving the ribonucleotide reductase approach feasible for the preparative synthesis of 2'-deoxyribonucleotides. High concentrations of sodium acetate in the reaction served to substitute for allosteric effectors of RTPR. 1,4-Dithio-DL-threitol was used as an artificial reducing agent for RTPR.
- Brunella,Abrantes,Ghisalba
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- P(V) Reagents for the Scalable Synthesis of Natural and Modified Nucleoside Triphosphates
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Natural and modified nucleoside triphosphates impact nearly every major aspect of healthcare research from DNA sequencing to drug discovery. However, a scalable synthetic route to these molecules has long been hindered by the need for purification by high performance liquid chromatography (HPLC). Here, we describe a fundamentally different approach that uses a novel P(V) pyrene pyrophosphate reagent to generate derivatives that are purified by silica gel chromatography and converted to the desired compounds on scales vastly exceeding those achievable by HPLC. The power of this approach is demonstrated through the synthesis of a broad range of natural and unnatural nucleoside triphosphates (dNTPs and xNTPs) using protocols that are efficient, inexpensive, and operationally straightforward.
- Liao, Jen-Yu,Bala, Saikat,Ngor, Arlene K.,Yik, Eric J.,Chaput, John C.
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supporting information
p. 13286 - 13289
(2019/09/04)
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- COMPOSITIONS AND METHODS FOR SYNTHESIS OF PHOSPHORYLATED MOLECULES
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The invention provides compositions and methods for synthesis of phosphorylated organic compounds, including nucleoside triphosphates.
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Page/Page column 86; 88; 89; 94; 97
(2019/10/29)
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- REVERSIBLY PROTECTED NUCLEOTIDE REAGENTS WITH HIGH THERMAL STABILITY
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The present invention provides for stable nucleotide reagents used for nucleic acid amplification by PCR and RT-PCR (Reverse Transcriptase-PCR) that comprises modified nucleoside polyphosphates. The present invention also provides for methods for using the modified nucleoside polyphosphates for detecting the presence or absence of a target nucleic acid sequence in a sample in an amplification reaction.
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Page/Page column 22
(2018/07/29)
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- An improved protection-free one-pot chemical synthesis of 2′-deoxynucleoside-5′-triphosphates
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□ A facile, straightforward, reliable, and an efficient method for the gram-scale chemical synthesis of both purine deoxynucleotides such as 2 ′-deoxyguanosine-5 ′-triphosphate (dGTP) and 2 ′- deoxyadenosine-5′-triphosphate (dATP) and pyrimidine deoxynucleotides such as 2 ′-deoxycytidine- 5 ′-triphosphate (dCTP), thymidine-5 ′-triphosphate (TTP), and 2 ′-deoxyuridine-5 ′-triphosphate (dUTP) starting from the corresponding nucleoside is described. This improved "one-pot, three step"Ludwig synthetic strategy involves the monophosphorylation of nucleoside followed by reaction with tributylammonium pyrophosphate and hydrolysis of the resulting cyclic intermediate to provide the corresponding dNTP in good yields (65%-70%). Copyright Taylor and Francis Group, LLC.
- Kore, Anilkumar R.,Shanmugasundaram, Muthian,Senthilvelan, Annamalai,Srinivasan, Balasubramanian
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p. 423 - 431
(2012/08/14)
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- Nucleotide promiscuity of 3-phosphoglycerate kinase is in focus: Implications for the design of better anti-HIV analogues
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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.
- Varga, Andrea,Chaloin, Laurent,Sagi, Gyula,Sendula, Robert,Graczer, Eva,Liliom, Karoly,Zavodszky, Peter,Lionne, Corinne,Vas, Maria
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experimental part
p. 1863 - 1873
(2012/04/17)
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- Substrate specificity of T5 bacteriophage deoxyribonucleoside monophosphate kinase and its application for the synthesis of [α-32P]d/rNTP
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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.
- Skoblov,Mikoulinskaia,Taran,Miroshnikov,Feofanov,Skoblov
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experimental part
p. 734 - 738
(2010/08/07)
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- A kinetic study of the rat liver adenosine kinase reverse reaction
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Adenosine kinase is an enzyme catalyzing the reaction: adenosine + ATP → AMP + ADP. We studied some biochemical properties not hitherto investigated and demonstrated that the reaction can be easily reversed when coupled with adenosine deaminase, which transforms adenosine into inosine and ammonia. The overall reaction is: AMP + ADP → ATP + inosine + NH3. The exoergonic ADA reaction shifts the equilibrium and fills the energy gap necessary for synthesis of ATP. This reaction could be used by cells under particular conditions of energy deficiency and, together with myokinase activity, may help to restore physiological ATP levels. Copyright Taylor & Francis Group, LLC.
- Vannoni,Giglioni,Santoro,Aceto,Marinello,Leoncini
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p. 872 - 875
(2008/12/21)
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- Derivatives of L-adenosine and L-guanosine as substrates for human deoxycytidine kinase
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A series of analogues of L-adenosine and of L-guanosine, including β- L-dA, β-L-Ado, β-L-araA, and β-L-dG, have been shown to be substrates of human deoxycytidine kinase thus demonstrating the complete lack of enantioselectivity of this enzyme.
- Gaubert,Gosselin,Imbach,Eriksson,Maury
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p. 857 - 860
(2007/10/03)
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- TRITYLOXYETHYLAMINO GROUP FOR THE PROTECTION OF PHOSPHORYL GROUP IN OLIGONUCLEOTIDE SYNTHESIS
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Trityloxyethylamino group was used for the protection of 5'-phosphoryl group of deoxyribonucleoside.This group is not only protecting group of phosphate also the intermediate for the synthesis of d-ppA and d-pppA.
- Tanaka, Toshiki,Yamada, Yasuki,Ikehara, Morio
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p. 5641 - 5644
(2007/10/02)
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