- PPN Pyrophosphate: A New Reagent for the Preparation of Nucleoside Triphosphates
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Tris{bis(triphenylphosphoranylidene) ammonium} (PPN) pyrophosphate was accessed via aqueous precipitation and desiccation. The reagent was investigated as a replacement for highly hygroscopic alkylammonium salts in Ludwig-Yoshikawa reactions for the preparation of nucleoside-5′-triphosphates.
- Korhonen, Heidi J.,Bolt, Hannah L.,Vicente-Gines, Leyre,Perks, Daniel C.,Hodgson, David R. W.
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Read Online
- MODIFIED NUCLEOTIDES AND USES THEREOF
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Disclosed herein, inter alia, are compounds, modified nucleotides, compositions, and methods of using the same.
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Paragraph 0398; 0413-0415; 0432-0436
(2021/10/30)
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- REVERSIBLE TERMINATORS FOR DNA SEQUENCING AND METHODS OF USING THE SAME
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The present disclosure provides methods of sequencing polynucleotides and compounds, compositions for sequencing of polynucleotides, and synthesis of such compositions. The chemical compounds include nucleotides and their analogs which possess a sugar moiety comprising a cleavable chemical group capping the 3'-OH group and a base, but without covalently bounded dye. The cleavable chemical group is reactive to form covalent bond(s) with a dye used to confirm the presence of the expected base-pairing. The cleavable chemical group capping the 3'OH group can be removed together with the covalently bounded dye. Furthermore, after the cleavable chemical group is cleaved, the free 3'-OH group can be active in continued elongation. Example chemical compounds according to the present disclosure are shown as Formulas (II) and (V).
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Paragraph 00189-00191
(2021/04/10)
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- Method for synthesizing nucleotide or nucleotide analogue
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The invention provides a method for synthesizing nucleotide or nucleotide analogue. The method includes combining nucleoside with phosphate. The pyrophosphate or tripolyphosphate undergoes nucleophilic substitution reaction under the catalysis of a phase transfer catalyst to obtain said nucleotide or nucleotide analogue, wherein said nucleoside has the structure represented by formula (I) or a stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug thereof shown in formula (I).
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Paragraph 0071-0094
(2021/10/20)
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- Lipophilic Triphosphate Prodrugs of Various Nucleoside Analogues
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The antiviral efficacy of many nucleoside analogues is strongly dependent on their intracellular activation by host cellular kinases to yield ultimately the bioactive nucleoside analogue triphosphates (NTP). The metabolic conversion of nucleoside analogues into their triphosphates often proceeds insufficiently. We developed a nucleoside triphosphate (NTP) delivery system (the TriPPPro approach), in which the γ-phosphate is covalently modified by two different biodegradable masking units, one is the acyloxybenzyl (AB) moiety and the other is the alkoxycarbonyloxybenzyl (ACB) group. Such compounds formed NTPs with high selectivity by an enzyme-triggered mechanism in human T-lymphocyte CEM cell extracts loosing first the AB moiety, followed by the ACB group. This enables the bypass of all steps of the intracellular phosphorylation. This approach was applied here to convert some modestly active or even inactive nucleoside analogues into powerful biologically active metabolites. Potent antiviral activity profiles were obtained depending on the lipophilicity of the TriPPPro-NTP prodrugs against HIV-1 and HIV-2 replication in cultures of infected wild-type CD4+ CEM T-cells and more importantly in thymidine kinase-deficient CD4+ T-cells (CEM/TK-). This TriPPPro strategy offers high potential for future antiviral and antitumoral chemotherapies.
- Jia, Xiao,Schols, Dominique,Meier, Chris
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p. 6991 - 7007
(2020/08/14)
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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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- 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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- Synthetic method of nucleoside tetraphosphate
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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.
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Paragraph 0041; 0048-0052; 0053; 0055
(2019/02/04)
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- Chemical and enzymatic characterization of recombinant rabbit muscle pyruvate kinase
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The stepwise synthesis of thymidine triphosphate (TTP) requires a kinase for phosphorylation in the last step. Because pyruvate kinase (PK) using phosphoenolpyruvate (PEP) as substrate can regenerate adenosine triphosphate and phosphorylate thymidine diphosphate as well, we chose this enzyme for the synthesis of TTP via an enzymatic cascade reaction. The metalloenzyme PK shows pronounced promiscuity and therefore fits well to the conditions of this reaction. PK commonly used today is isolated from rabbit muscle. We cloned and expressed the respective open reading frame in Escherichia coli, purified, and characterized the His-tagged recombinant enzyme. The enzyme has an activity optimum at 37 °C and in the pH range from 7.4 to 7.8. Km constants conformed well with the isolated native enzyme for adenosine diphosphate (ADP) to 0.37±0.02 mm and for PEP to 0.07±0.01 mm. The recombinant enzyme shows the following range in its substrate specificity: ADP>dADP>dGDP>dCDP>thymidine diphosphate (TDP). It allows the phosphorylation of TDP to TTP in high yield (up to 95 % ). The metal ions Mg2+ and K+ are necessary for full enzymatic activity. The addition of transition metal ions such as Mn2+, Cu2+, Co2+, and Ni2+ reduces activity. Storage of the enzyme at -20 °C retains full activity.
- Boehme, Christian,Bieber, Frank,Linnemann, Julia,Breitling, Reinhard,Lorkowski, Stefan,Reissmann, Siegmund
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supporting information
p. 695 - 701
(2013/09/02)
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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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experimental part
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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- CHEMICALLY MODIFIED NUCLEOSIDE 5'-TRIPHOSPHATES FOR THERMALLY INITIATED AMPLIFICATION OF NUCLEIC ACID
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Provided herein are methods and compositions for nucleic acid amplification. These methods involve the use of 3'-substituted nucleoside 5'-triphosphates or 3'-substituted terminated primers in nucleic acid amplification reactions. In certain aspects, the methods are accomplished by use of 3'-substituted NTPs and/or 3'-substituted terminated primers which provide utility in nucleic acid amplification. In preferred embodiments, the NTPs and/or primers are substituted at the 3'-position with particular heat labile chemical groups such as ethers, esters or carbonate esters.
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Page/Page column 61-62
(2010/01/07)
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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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- Selective diphosphorylation, dithiodiphosphorylation, triphosphorylation, and trithiotriphosphorylation of unprotected carbohydrates and nucleosides
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(Chemical Equation Presented) Aminomethyl polystyrene resin-bound linkers of p-acetoxybenzyl alcohol were subjected to reactions with diphosphitylating and triphosphitylating reagents to yield the corresponding polymer-bound diphosphitylating and triphosphitylating reagents, respectively. A number of unprotected carbohydrates and nucleosides were reacted with the polymer-bound reagents. Oxidation with tert-butyl hydroperoxide or sulfurization with Beaucage's reagent, followed by removal of cyanoethoxy group with DBU and the acidic cleavage, respectively, afforded only one type of monosubstituted nucleoside and carbohydrate diphosphates, dithiodiphosphates, triphosphates, and trithiotriphosphates with high regioselectivity.
- Ahmadibeni, Yousef,Parang, Keykavous
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p. 5589 - 5592
(2007/10/03)
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- Pyridoxal-catalyzed release of nucleotides
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The synthesis of phosphoric esters linking the 5' alcohol of thymidine to the alcohol moiety of L-serine derivatives is reported, together with the pyridoxal-catalyzed β-elimination reaction releasing thymidylate from such compounds. The process was extended to the release of thymidine di- and tri- phosphate.
- Pochet, Sylvie,Beaussire, Jean-Jacques,Dugue, Laurence,Marliere, Philippe
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p. 1019 - 1020
(2007/10/03)
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- Reaction of Trifluoroacetic Anhydride with Thymidine 5'-Phosphate
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The interaction of thymidine 5'-phosphate with trifluoroacetic anhydride in an aprotic solvent in the presence of a base is shown to give a mixed anhydride of thymidine 5'-phosphoric and trifluoroacetic acids.The anhydride reacts readily with high yield with various nucleophilic agents (morpholine, p-chlorophenol, pyrophosphate) to form nucleotide derivatives after pretreatment with nucleophilic catalysts (N-methylimidazole, 4-dimethylaminopyridine).Without these catalysts, nucleotide derivatives are not synthesized.A scheme of the reactions is proposed.Key words: trifluoroacetic anhydride, thymidine 5'-phosphate.
- Bogachev, V. S
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p. 182 - 186
(2007/10/02)
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- Rapid and Efficient Synthesis of Nucleoside 5'-O-(1-Thiotriphosphates), 5'-Triphosphates and 2',3'-Cyclophosphorothioates Using 2-Chloro-4H-1,3,2-benzodioxaphosphorin-4-one
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2-Chloro-4H-1,3,2-benzodioxaphosphorin-4-one phosphitylates the 5'-hydroxy group of a nucleoside to form an intermediate (2), which on subsequent reaction with pyrophosphate produces, in a double displacement process, a P2,P3-dioxo-P1-5'-nucleosidylcyclotriphophite (3).Oxidation with sulfur gives a nucleoside 5'-(1-thiocyclotriphosphate) (4), which is hydrolyzed to the diastereomeric mixture of a nucleoside 5'-O-(1-thiotriphosphate) (5).Alternatively, 3 can be oxidized with iodine/water to furnish nucleoside 5'-triphosphates 6.This reagent can also be applied to the synthesis of nucleoside 2',3'-cyclic phosphorothioates.Protection of nucleobase functional groups is not required.
- Ludwig, Janos,Eckstein, Fritz
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p. 631 - 635
(2007/10/02)
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