- An Engineered Cytidine Deaminase for Biocatalytic Production of a Key Intermediate of the Covid-19 Antiviral Molnupiravir
-
The Covid-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue recently approved as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of an efficient biocatalyst for the production of N-hydroxy-cytidine through evolutionary adaption of the hydrolytic enzyme cytidine deaminase. This engineered biocatalyst performs >85 000 turnovers in less than 3 h, operates at 180 g/L substrate loading, and benefits from in situ crystallization of the N-hydroxy-cytidine product (85% yield), which can be converted to Molnupiravir by a selective 5′-acylation using Novozym 435.
- Birmingham, William R.,Burke, Ashleigh J.,Charnock, Simon J.,Crawshaw, Rebecca,Finnigan, James D.,Green, Anthony P.,Holgate, Gregory M.,Lovelock, Sarah L.,Muldowney, Mark P.,Rowles, Ian,Thorpe, Thomas W.,Turner, Nicholas J.,Young, Carl,Zhuo, Ying,Zucoloto Da Costa, Bruna
-
supporting information
p. 3761 - 3765
(2022/03/15)
-
- Reactivity and DNA Damage by Independently Generated 2′-Deoxycytidin-N4-yl Radical
-
Oxidative stress produces a variety of radicals in DNA, including pyrimidine nucleobase radicals. The nitrogen-centered DNA radical 2′-deoxycytidin-N4-yl radical (dC·) plays a role in DNA damage mediated by one electron oxidants, such as HOCl and ionizing radiation. However, the reactivity of dC· is not well understood. To reduce this knowledge gap, we photochemically generated dC· from a nitrophenyl oxime nucleoside and within chemically synthesized oligonucleotides from the same precursor. dC· formation is confirmed by transient UV-absorption spectroscopy in laser flash photolysis (LFP) experiments. LFP and duplex DNA cleavage experiments indicate that dC· oxidizes dG. Transient formation of the dG radical cation (dG+?) is observed in LFP experiments. Oxidation of the opposing dG in DNA results in hole transfer when the opposing dG is part of a dGGG sequence. The sequence dependence is attributed to a competition between rapid proton transfer from dG+?to the opposing dC anion formed and hole transfer. Enhanced hole transfer when less acidicO6-methyl-2′-deoxyguanosine is opposite dC· supports this proposal. dC· produces tandem lesions in sequences containing thymidine at the 5′-position by abstracting a hydrogen atom from the thymine methyl group. The corresponding thymidine peroxyl radical completes tandem lesion formation by reacting with the 5′-adjacent nucleotide. As dC· is reduced to dC, its role in the process is traceless and is only detectable because of the ability to independently generate it from a stable precursor. These experiments reveal that dC· oxidizes neighboring nucleotides, resulting in deleterious tandem lesions and hole transfer in appropriate sequences.
- Peng, Haihui,Jie, Jialong,Mortimer, Ifor P.,Ma, Zehan,Su, Hongmei,Greenberg, Marc M.
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p. 14738 - 14747
(2021/09/18)
-
- Thermodynamic Reaction Control of Nucleoside Phosphorolysis
-
Nucleoside analogs represent a class of important drugs for cancer and antiviral treatments. Nucleoside phosphorylases (NPases) catalyze the phosphorolysis of nucleosides and are widely employed for the synthesis of pentose-1-phosphates and nucleoside analogs, which are difficult to access via conventional synthetic methods. However, for the vast majority of nucleosides, it has been observed that either no or incomplete conversion of the starting materials is achieved in NPase-catalyzed reactions. For some substrates, it has been shown that these reactions are reversible equilibrium reactions that adhere to the law of mass action. In this contribution, we broadly demonstrate that nucleoside phosphorolysis is a thermodynamically controlled endothermic reaction that proceeds to a reaction equilibrium dictated by the substrate-specific equilibrium constant of phosphorolysis, irrespective of the type or amount of NPase used, as shown by several examples. Furthermore, we explored the temperature-dependency of nucleoside phosphorolysis equilibrium states and provide the apparent transformed reaction enthalpy and apparent transformed reaction entropy for 24 nucleosides, confirming that these conversions are thermodynamically controlled endothermic reactions. This data allows calculation of the Gibbs free energy and, consequently, the equilibrium constant of phosphorolysis at any given reaction temperature. Overall, our investigations revealed that pyrimidine nucleosides are generally more susceptible to phosphorolysis than purine nucleosides. The data disclosed in this work allow the accurate prediction of phosphorolysis or transglycosylation yields for a range of pyrimidine and purine nucleosides and thus serve to empower further research in the field of nucleoside biocatalysis. (Figure presented.).
- Kaspar, Felix,Giessmann, Robert T.,Neubauer, Peter,Wagner, Anke,Gimpel, Matthias
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supporting information
p. 867 - 876
(2020/01/24)
-
- Novel Use
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The invention relates to the use of an amine masked moiety in a method of enzymatic nucleic acid synthesis. The invention also relates to said amine masked moieties per se and a process for preparing nucleotide triphosphates comprising said amine masked moieties.
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-
Paragraph 0020
(2019/05/24)
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- Dehalogenation of Halogenated Nucleobases and Nucleosides by Organoselenium Compounds
-
Halogenated nucleosides, such as 5-iodo-2′-deoxyuridine and 5-iodo-2′-deoxycytidine, are incorporated into the DNA of replicating cells to facilitate DNA single-strand breaks and intra- or interstrand crosslinks upon UV irradiation. In this work, it is shown that the naphthyl-based organoselenium compounds can mediate the dehalogenation of halogenated pyrimidine-based nucleosides, such as 5-X-2′-deoxyuridine and 5-X-2′-deoxycytidine (X=Br or I). The rate of deiodination was found to be significantly higher than that of the debromination for both nucleosides. Furthermore, the deiodination of iodo-cytidines was found to be faster than that of iodo-uridines. The initial rates of the deiodinations of 5-iodocytosine and 5-iodouracil indicated that the nature of the sugar moiety influences the kinetics of the deiodination. For both the nucleobases and nucleosides, the deiodination and debromination reactions follow a halogen-bond-mediated and addition/elimination pathway, respectively.
- Mondal, Santanu,Mugesh, Govindasamy
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p. 1773 - 1780
(2019/01/10)
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- Tetrazine-mediated bioorthogonal prodrug-prodrug activation
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The selective and biocompatible activation of prodrugs within complex biological systems remains a key challenge in medical chemistry and chemical biology. Herein we report, for the first time, a dual prodrug activation strategy that fully satisfies the principle of bioorthogonality by the symbiotic formation of two active drugs. This dual and traceless prodrug activation strategy takes advantage of the INVDA chemistry of tetrazines (here a prodrug), generating a pyridazine-based miR21 inhibitor and the anti-cancer drug camptothecin and offers a new concept in prodrug activation.
- Neumann, Kevin,Gambardella, Alessia,Lilienkampf, Annamaria,Bradley, Mark
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p. 7198 - 7203
(2018/10/02)
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- Use of Nucleoside Phosphorylases for the Preparation of Purine and Pyrimidine 2′-Deoxynucleosides
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Enzymatic transglycosylation – the transfer of the carbohydrate moiety from one heterocyclic base to another – is being actively developed and applied for the synthesis of practically important nucleosides. This reaction is catalyzed by nucleoside phosphorylases (NPs), which are responsible for reversible phosphorolysis of nucleosides to yield the corresponding heterocyclic bases and monosaccharide 1-phosphates. We found that 7-methyl-2′-deoxyguanosine (7-Me-dGuo) is an efficient and novel donor of the 2-deoxyribose moiety in the enzymatic transglycosylation for the synthesis of purine and pyrimidine 2′-deoxyribonucleosides in excellent yields. Unlike 7-methylguanosine, its 2′-deoxy derivative is dramatically less stable. Fortunately, we have found that 7-methyl-2′-deoxyguanosine hydroiodide may be stored for 24 h in Tris-HCl buffer (pH 7.5) at room temperature without significant decomposition. In order to optimize the reagent ratio, a series of analytical transglycosylation reactions were conducted at ambient temperature. According to HPLC analysis of the transglycosylation reactions, the product 5-ethyl-2′-deoxyuridine (5-Et-dUrd) was obtained in high yield (84–93%) by using a small excess (1.5 and 2.0 equiv.) of 7-Me-dGuo over 5-ethyluracil (5-Et-Ura) and 0.5 equiv. of inorganic phosphate. Thymidine is a less effective precursor of α-d-2-deoxyribofuranose 1-phosphate (dRib-1p) compared to 7-Me-dGuo. We synthesized 2′-deoxyuridine, 5-Et-dUrd, 2′-deoxyadenosine and 2′-deoxyinosine on a semi-preparative scale using the optimized reagent ratio (1.5:1:0.5) in high yields. Unlike other transglycosylation reactions, the synthesis of 2-chloro-2′-deoxyadenosine was performed in a heterogeneous medium because of the poor solubility of the initial 2-chloro-6-aminopurine. Nevertheless, this nucleoside was prepared in good yield. The developed enzymatic procedure for the preparation of 2′-deoxynucleosides may compete with the known chemical approaches. (Figure presented.).
- Drenichev, Mikhail S.,Alexeev, Cyril S.,Kurochkin, Nikolay N.,Mikhailov, Sergey N.
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p. 305 - 312
(2018/01/15)
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- Novel nucleoside protective group and preparation thereof
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The invention relates to a novel nucleoside protective group and a preparation thereof. Concretely, the invention provides a compound with a structure shown in a formula 1, wherein R1 is selected from C1-C6 alkyl or C6-C14 aryl, preferably C1-C4 alkyl or phenyl, such as methyl, ethyl or phenyl; R2 is selected from C1-C6 alkyl or C6-C14 aryl substituted C1-C6 alkyl, preferably C1-C4 alkyl or phenyl substituted C1-C4 alkyl, such as methyl, ethyl, benzyl or phenethyl; X is halogen, and preferably chlorine. In the acidic condition, compared with traditional 4,4'-dismethoxytriphenylmethyl nucleoside protective group, deprotection of the compound is easier.
- -
-
Paragraph 0193-0196
(2018/03/24)
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- Enzymatic synthesis of ribo- and 2′-deoxyribonucleosides from glycofuranosyl phosphates: An approach to facilitate isotopic labeling
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Milligram quantities of α-D-ribofuranosyl 1-phosphate (sodium salt) (αR1P) were prepared by the phosphorolysis of inosine, catalyzed by purine nucleoside phosphorylase (PNPase). The αR1P was isolated by chromatography in >95% purity and characterized by 1H and 13C NMR spectroscopy. Aqueous solutions of αR1P were stable at pH 6.4 and 4 °C for several months. The isolated αR1P was N-glycosylated with different nitrogen bases (adenine, guanine and uracil) using PNPase or uridine phosphorylase (UPase) to give the corresponding ribonucleosides in high yield based on the glycosyl phosphate. This methodology is attractive for the preparation of stable isotopically labeled ribo- and 2′-deoxyribonucleosides because of the ease of product purification and convenient use and recycling of nitrogen bases. The approach eliminates the need for separate reactions to prepare individual furanose-labeled ribonucleosides, since only one ribonucleoside (inosine) needs to be labeled, if desired, in the furanose ring, the latter achieved by a high-yield chemical N-glycosylation. 2′-Deoxyribonucleosides were prepared from 2′-deoxyinosine using the same methodology with minor modifications.
- Zhang, Wenhui,Turney, Toby,Surjancev, Ivana,Serianni, Anthony S.
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p. 125 - 133
(2017/08/08)
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- DEAMINATION OF ORGANOPHOSPHORUS-NUCLEOSIDES
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The invention relates to a new synthethic process for obtaining compounds of formula (I) from compounds of formula (II) by means of cytidine deaminase enzymes.
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Page/Page column 29
(2016/10/24)
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- Method for preparing 2'-deoxyuridine
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The invention discloses a method for preparing 2'-deoxyuridine shown in a preparation formula (IV) (please see the formula in the description). The method comprises the following steps that uridine shown in the formula (I) (please see the formula in the description) and a dehydrating agent are mixed in a solvent according to the following chemical equation, and uridine dehydrated matter shown in the formula (II) (please see the formula in the description) is generated under the catalyzing action of inorganic alkali; halogen hydride is added, and uridine halide shown in the formula (III) (please see the formula in the description) is generated through a halogenation reaction; hydrogen is introduced, and 2'-deoxyuridine shown in the formula (IV) is generated through a reduction reaction. According to the method for preparing 2'-deoxyuridine shown in the formula (IV), little harm is generated to the environment and the human body, and generated waste can be recycled; in addition, the one-pot preparation method is adopted, operation is easy, the labor cost is low, equipment investment is low, the production cycle is significantly shortened, and the method is suitable for industrialized mass production.
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-
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- PRODUCTION METHOD OF NUCLEOSIDE COMPOUND
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PROBLEM TO BE SOLVED: To provide a production method of a nucleoside compound by which an isotopic labeled nucleoside compound can be produced efficiently. SOLUTION: A production method of a nucleoside compound comprises obtaining a target nucleoside compound by the base exchange reaction of a raw material nucleoside compound and a base in the solution containing a phosphoric acid ion by a nucleoside phosphorylase, wherein the target nucleoside compound is labeled with a stable isotope or a radioisotope. COPYRIGHT: (C)2015,JPOandINPIT
- -
-
Paragraph 0032-0033
(2017/03/24)
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- Selenium-Mediated Dehalogenation of Halogenated Nucleosides and its Relevance to the DNA Repair Pathway
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Halogenated nucleosides can be incorporated into the newly synthesized DNA of replicating cells and therefore are commonly used in the detection of proliferating cells in living tissues. Dehalogenation of these modified nucleosides is one of the key pathways involved in DNA repair mediated by the uracil-DNA glycosylase. Herein, we report the first example of a selenium-mediated dehalogenation of halogenated nucleosides. We also show that the mechanism for the debromination is remarkably different from that of deiodination and that the presence of a ribose or deoxyribose moiety in the nucleosides facilitates the deiodination. The results described herein should help in understanding the metabolism of halogenated nucleosides in DNA and RNA. Saying goodbye to I: Selenium compounds are employed to mediate the dehalogenation of halogenated nucleobases and nucleosides in aqueous media under physiological conditions. These results may be important for the development of novel reagents for DNA modification and repair and suggest that Se derivatives may play a broader role in the metabolism of halogenated organic compounds in biology.
- Mondal, Santanu,Manna, Debasish,Mugesh, Govindasamy
-
supporting information
p. 9298 - 9302
(2015/08/06)
-
- Characterization of a novel resistance-related deoxycytidine deaminase from Brassica oleracea var. capitata
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Brassica oleracea deoxycytidine deaminase (BoDCD), a deoxycytidine deaminase (DCD, EC 3.5.4.14) enzyme, is known to play an important role in the Trichoderma harzianum ETS 323 mediated resistance mechanism in young leaves of B. oleracea var. capitata during Rhizoctonia solani infection. BoDCD potentially neutralizes cytotoxic products of host lipoxygenase activity, and thereby BoDCD restricts the hypersensitivity-related programmed cell death induced in plants during the initial stages of infection. To determine the biochemical characteristics and to partially elucidate the designated functional properties of BoDCD, the enzyme was cloned into an Escherichia coli expression system, and its potential to neutralize the toxic analogues of 2′-deoxycytidine (dC) was examined. BoDCD transformants of E. coli cells were found to be resistant to 2′-deoxycytidine analogues at all of the concentrations tested. The BoDCD enzyme was also overexpressed as a histidine-tagged protein and purified using nickel chelating affinity chromatography. The molecular weight of BoDCD was determined to be 20.8 kDa as visualized by SDS-PAGE. The substrate specificity and other kinetic properties show that BoDCD is more active in neutralizing cytotoxic cytosine β-d-arabinofuranoside than in deaminating 2′-deoxycytinde to 2′-deoxyuridine in nucleic acids or in metabolizing cytidine to uridine. The optimal temperature and pH of the enzyme were 27 C and 7.5. The Km and Vmax values of BoDCD were, respectively, 91.3 μM and 1.475 mM for its natural substrate 2′-deoxycytidine and 63 μM and 2.072 mM for cytosine β-d-arabinofuranoside. The phenomenon of neutralization of cytotoxic dC analogues by BoDCD is discussed in detail on the basis of enzyme biochemical properties.
- Shibu, Marthandam Asokan,Yang, Hsueh-Hui,Lo, Chaur-Tsuen,Lin, Hong-Shin,Liu, Shu-Ying,Peng, Kou-Cheng
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p. 1796 - 1801
(2014/03/21)
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- Deamination, oxidation, and C-C bond cleavage reactivity of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine
-
Three new cytosine derived DNA modifications, 5-hydroxymethyl-2′- deoxycytidine (hmdC), 5-formyl-2′-deoxycytidine (fdC) and 5-carboxy-2′-deoxycytidine (cadC) were recently discovered in mammalian DNA, particularly in stem cell DNA. Their function is currently not clear, but it is assumed that in stem cells they might be intermediates of an active demethylation process. This process may involve base excision repair, C-C bond cleaving reactions or deamination of hmdC to 5-hydroxymethyl-2′- deoxyuridine (hmdU). Here we report chemical studies that enlighten the chemical reactivity of the new cytosine nucleobases. We investigated their sensitivity toward oxidation and deamination and we studied the C-C bond cleaving reactivity of hmdC, fdC, and cadC in the absence and presence of thiols as biologically relevant (organo)catalysts. We show that hmdC is in comparison to mdC rapidly oxidized to fdC already in the presence of air. In contrast, deamination reactions were found to occur only to a minor extent. The C-C bond cleavage reactions require the presence of high concentration of thiols and are acid catalyzed. While hmdC dehydroxymethylates very slowly, fdC and especially cadC react considerably faster to dC. Thiols are active site residues in many DNA modifiying enzymes indicating that such enzymes could play a role in an alternative active DNA demethylation mechanism via deformylation of fdC or decarboxylation of cadC. Quantum-chemical calculations support the catalytic influence of a thiol on the C-C bond cleavage.
- Schiesser, Stefan,Pfaffeneder, Toni,Sadeghian, Keyarash,Hackner, Benjamin,Steigenberger, Barbara,Schroeder, Arne S.,Steinbacher, Jessica,Kashiwazaki, Gengo,Hoefner, Georg,Wanner, Klaus T.,Ochsenfeld, Christian,Carell, Thomas
-
supporting information
p. 14593 - 14599
(2013/10/22)
-
- Developing a collection of immobilized nucleoside phosphorylases for the preparation of nucleoside analogues: Enzymatic synthesis of arabinosyladenine and 2',3'-dideoxyinosine
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The use of nucleoside phosphorylases (NPs; EC 2.4.2.n) represents a convenient alternative to the chemical route for the synthesis of natural and modified nucleosides. We purified four recombinantly expressed nucleoside phosphorylases from the bacterial pathogens Citrobacter koseri, Clostridium perfringens, and Streptococcus pyogenes (CkPNPI, CkPNPII, CpUP, SpUP) and their substrate specificity was investigated towards either natural pyrimidine or purine nucleosides and some analogues, namely, arabinosyladenine (araA) and 2',3'-dideoxyinosine (ddI). A 2-3 % activity towards these latter compounds (compared to the natural substrates) was observed. Enzyme activities were compared to the specificities obtained for the enzymes pyrimidine nucleoside phosphorylase from Bacillus subtilis (BsPyNP) and purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNPII) previously reported by some of the authors. The enzymes displaying the suitable specificity for the synthesis of araA and ddI were immobilized on aldehyde-agarose. The immobilized preparations were highly stable at alkaline pH and in the presence of methanol or acetonitrile as cosolvent. They were used in the synthesis of araA and ddI by a one-pot, bienzymatic transglycosylation achieving 74 and 44 % conversion, respectively. Something different: Nucleoside phosphorylases are a convenient alternative to the chemical route for the synthesis of natural and modified nucleosides. Four new nucleoside phosphorylases have been prepared, characterized, and tested for their use in biocatalyzed syntheses of araA and ddI (see scheme). A generally applicable immobilization technique has been found to provide active and stable biocatalysts.
- Serra, Immacolata,Ubiali, Daniela,Piskur, Jure,Christoffersen, Stig,Lewkowicz, Elizabeth S.,Iribarren, Adolfo M.,Albertini, Alessandra M.,Terreni, Marco
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p. 157 - 165
(2013/04/24)
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- NOVEL 3′-DEOXY-3′-METHYLIDENE- -L-NUCLEOSIDES
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The present invention includes novel 3′-deoxy-3′-methylidene-β-L-nucleosides, pharmaceutical composition comprising such compounds, as well as the methods to treat or to prevent viral infections and in particular HBV and/or HIV infections. In accordance with the present invention, there are provided compounds represented by Formula (I), wherein B is selected from A1 and A2;
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Page/Page column 35
(2011/07/07)
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- Contrasting behavior of conformationally locked carbocyclic nucleosides of adenosine and cytidine as substrates for deaminases
-
In addition to the already known differences between adenosine deaminase (ADA) and cytidine deaminase (CDA) in terms of their tertiary structure, the sphere of Zn+2 coordination, and their reverse stereochemical preference, we present evidence that the enzymes also differ significantly in terms of the North/South conformational preferences for their substrates and the extent to which the lack of the O(4') oxygen affects the kinetics of the enzymatic deamination of carbocyclic substrates. The carbocyclic nucleoside substrates used in this study have either a flexible cyclopentane ring or a rigid bicyclo[3.1.0]hexane scaffold.
- Marquez, Victor E.,Schroeder, Gottfried K.,Ludek, Olaf R.,Siddiqui, Maqbool A.,Ezzitouni, Abdallah,Wolfenden, Richard
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experimental part
p. 614 - 632
(2010/08/19)
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- Chemoenzymatic preparation of nucleosides from furanoses
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Chemoenzymatic preparation of ribose, deoxyribose and arabinose 5-phosphates was accomplished. These compounds were tested as starting materials in the enzymatic preparation of natural and modified purine and pyrimidine nucleosides, using an overexpressed Escherichia coli phosphopentomutase.
- Taverna-Porro, Marisa,Bouvier, Leon A.,Pereira, Claudio A.,Montserrat, Javier M.,Iribarren, Adolfo M.
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p. 2642 - 2645
(2008/09/19)
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- Protecting groups transfer: Unusual method of removal of tr and TBDMS groups by transetherification
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The triphenylmethyl (Tr) group undergoes a transfer (transetherification or disproportionation) between the molecules of 5'-O-Tr-2'-deoxynucleosides in a process mediated by anhydrous sulfates of Cu+2, Fe+2, or Ni+2 to yield mixtures of 3',5'-bis-O-Tr and 3'-O-Tr products. If phenylmethanol is present in a reaction medium, detritylation results with concomitant formation of phenylmethyl triphenylmethyl ether. The behavior of t-butyldimethylsilyl (TBDMS) group in 5'-O-TBDMS-2'-deoxynucleosides is exactly the same. Such type of transetherifications was not observed before for the O-Tr and O-TBDMS groups. Copyright Taylor & Francis Group, LLC.
- Cabral, Nadia L. D.,Thiessen, Luciano Hoeltgebaum,Doboszewski, Bogdan
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p. 931 - 948
(2008/12/21)
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- Importance of 3′-hydroxyl group of the nucleosides for the reactivity of thymidine phosphorylase from Escherichia coli
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Thymidine phosphorylase in phosphate buffer catalyzed the conversion of thymidine to unnatural nucleosides. The 3′-OH, but not the 5′-OH of ribosyl moiety is necessary to be recognized as a substrate. Thus 3′-deoxythymidine could not convert to 5-fluorouracil-2′,3′- dideoxyribose. However, 5′-deoxythymidine was converted to 5-fluorouracil-2′,5′-dideoxyribose. Copyright
- Hatano, Akihiko,Harano, Aiko,Kirihara, Masayuki
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p. 232 - 233
(2007/10/03)
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- Synthesis of C-5 substituted nucleosides via palladium-catalyzed coupling of dienes and amines
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The palladium-catalyzed coupling of C-5 iodopyrimidine nucleosides; 1,2-, 1,3-, or 1,ω-dienes; and amines provides a novel and efficient method for the preparation of a wide variety of C-5 aminoalkyl-substituted nucleosides. Adding certain Lewis acids, particularly zinc salts, improves the yields significantly. Secondary amines are the most effective amines for this process. Acyclic and cyclic dienes have been successfully employed. Protection of the 3′- and 5′-hydroxyl groups of iodouridine is required in order to obtain good yields when the coupling process is carried out on 1,3-dienes or long chain or branched non-conjugated dienes.
- Larock, Richard C.,Wang, Yao,Dong, Xiaoyang,Yao, Tuanli
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p. 11427 - 11439
(2007/10/03)
-
- Preparation of deoxynucleosides
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Methods for preparing deoxynucleosides from their corresponding ribonucleosides by forming 3-tert-butylphenoxythiocarbonylderivatives of the ribonucleosides and subsequently effecting radical deoxygenation reactions at the carbon atoms to be deoxygenated.
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Page column 16
(2008/06/13)
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- Methods of manufacture of 2'-deoxy-beta-L-nucleosides
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The present invention relates to the synthesis of 2′-deoxy-β-L-thymidine, 2′-deoxy-β-L-uridine and 2′-deoxy-β-L-cytidine, and their derivatives, such as the 3′-O-acyl or 3′,5′-O-diacyl prodrugs, including the 3′-O-L-aminoacyl and 3′,5′-O-L-diaminoacyl prodrugs, and particularly the 3′-O-L-valinyl and 3′,5′-O-L-divalinyl prodrugs.
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Page/Page column 23
(2008/06/13)
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- Resistance towards exonucleases of dinucleotides with stereochemically altered internucleotide phosphate bonds
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Kinetic constants for the hydrolytic susceptibility of the internucleotide phosphate bond in normal dinucleotides [e.g., 2′-deoxycytidylyl- (3′>5′)-2′-deoxyuridine (dCpdU) and 2′-deoxyadenylyl- (3′→5′)-2′-deoxycytidine (dApdC)] and isomeric dinucleotides
- Nair, Vasu,Pal, Suresh
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p. 289 - 291
(2007/10/03)
-
- Guanidinium functionalized oligonucleotides and method/synthesis
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The present invention provides oligomers which are specifically hybridizable with a selected sequence of RNA or DNA wherein at least one of the nucleoside moieties of the oligomer is modified to include a guanidinium group. These oligomers are useful for diagnostic, therapeutic and investigative purposes.
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-
-
- Independent generation and study of 5,6-dihydro-2′-deoxyuridin-6-yl, a member of the major family of reactive intermediates formed in DNA from the effects of γ-radiolysis
-
Nucleobase radicals are the major family of reactive intermediates formed when nucleic acids are exposed to γ-radiolysis. Elucidation of their reactivity is complicated by the formation of multiple species randomly throughout the biopolymers. 5,6-Dihydro-2′-deoxyuridin-6-yl (1) was generated upon photolysis (350 nm) of the respective tert-butyl ketone (2). The radical abstracts hydrogen atoms from β-mercaptoethanol (k = 8.8 ± 0.5 × 106 M-1 s-1) and 2,5-dimethyltetrahydrofuran (k = 31 ± 2.5 M-1 s-1). The latter was used as a model for the 2-deoxyribose component of DNA. The major product formed in the presence of O2 was 6-hydroxy-5,6-dihydro-2′-deoxyuridine (11), which is believed to be formed directly from the peroxy precursor and not via elimination of superoxide. Small amounts of 2-deoxyribonolactone (13) were also formed under aerobic conditions. This product is believed to result from intramolecular hydrogen atom abstraction by the C6-peroxyl radical (14) and suggests that γ-radiolysis may indirectly result in oxidation of the C1′-position of nucleotides, despite the inaccessibility of this hydrogen in duplex DNA.
- Carter, K. Nolan,Greenberg, Marc M.
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p. 4275 - 4280
(2007/10/03)
-
- A facile method for deprotection of trityl ethers using column chromatography
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A mild, efficient and inexpensive detritylation method is reported that uses trifluoroacetic acid on a silica gel column to obtain pure, detritylated compounds in one-step. This method is applicable to acid stable as well as acid sensitive compounds with only slight alterations in the procedure. Nineteen examples are given.
- Pathak, Ashish K.,Pathak, Vibha,Seitz, Lainne E.,Tiwari, Kamal N.,Akhtar, Mohammad S.,Reynolds, Robert C
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p. 7755 - 7757
(2007/10/03)
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- Products of the reaction between a diazoate derivative of 2′-deoxycytidine and L-Lysine and its implication for DNA - Nucleoprotein cross-linking by NO or HNO2
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Recently, we have reported that a stable diazoate intermediate (dCyd-diazoate) is produced upon the reaction of dCyd with nitrous acid and nitric oxide [Suzuki, T., Nakamura, T., Yamada, M., Ide, H., Kanaori, K., Tajima, K., Morii, T., and Makino, K. (1999) Biochemistry 38, 7151-7158]. In this work, the reaction of dCyd-diazoate with L-Lys was investigated. When 0.4 mM dCyd-diazoate was incubated with 10 mM L-Lys in sodium phosphate buffer (pH 7.4) at 37 °C, two unknown products were formed in addition to dUrd. By spectrometric measurements, the products were identified as dCyd-Lys adducts with C4(dCyd)-Nα(Lys) and C4(dCyd)-N∈(Lys) linkages (abbreviated as dCyd-αLys and dCyd-∈Lys, respectively). The yields at the reaction time of 72 h were 28.0% dCyd-αLys, 13.4% dCyd-∈Lys, and 11.1% dUrd with 33.9% unreacted dCyd-diazoate. When 0.4 mM dCyd-diazoate was incubated with 22 mg/mL poly(L-Lys) at pH 7.4 and 37 °C for 24 h, 82% of the free dCyd-diazoate disappeared, indicating adduct formation with the polymer. At pH 7.4 and 37 °C, dCyd-αLys and dCyd-∈Lys were fairly stable and gave rise to no product after incubation for 7 days. At pH 4.0 and 70 °C, both adducts disappeared with the same first-order rate constant of 1.7 x 10-6 s-1 (t1/2 = 110 h), which was ～1/3 of that of dCyd. These results suggest that if dCyd-diazoate is formed in DNA in vivo, it may react with free L-Lys and the side chain of L-Lys in nucleoproteins, resulting in stable adducts and DNA-protein cross-links, respectively.
- Suzuki, Toshinori,Yamada, Masaki,Nakamura, Takanori,Ide, Hiroshi,Kanaori, Kenji,Tajima, Kunihiko,Morii, Takashi,Makino, Keisuke
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p. 1223 - 1227
(2007/10/03)
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- Synthesis of pyrimidine 2′-deoxy ribonucleosides branched at the 2′-position via radical atom-transfer cyclization reaction with a vinylsilyl group as a radical-acceptor tether
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Recently, we developed a regio- and stereoselective method for introducing a vinyl group at the position β to a hydroxyl group in halohydrins or α-phenylselenoalkanols via a radical atom-transfer cyclization reaction with a vinylsilyl group as a temporary connecting radical-acceptor tether. The synthesis of 2′-deoxy-2′-C-vinyl- and 2′-deoxy-2′-C-hydroxymethyluridines (7 and 8, respectively) and the corresponding 2′-deoxycytidine congeners (10 and 11, respectively), which were designed as potential antitumor and/or antiviral agents, was achieved using this radical atom-transfer cyclization as the key step. When the 2′-deoxy-2′-iodo-5′-O-monomethoxytrityl (MMTr) uridine derivative 19a, bearing a vinylsilyl group at the 3′-hydroxyl group, was heated with (Me3Sn)2 and AIBN in benzene, the corresponding radical atom-transfer product was generated, which in turn was successively treated with tetrabutylammonium fluoride and TBSCI/imidazole to give the desired 2′-deoxy-5′-O-MMTr-3′-O-TBS-2′-C-vinyluridine (25). Compound 25 was successfully converted into the target 2′-deoxy-2′-branched pyrimidine ribonucleosides 7, 8, 10, and 11.
- Sukeda,Shuto,Sugimoto,Ichikawa,Matsuda
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p. 8988 - 8996
(2007/10/03)
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- Investigation of the kinetics of degradation of hexopyranosylated cytosine nucleosides using liquid chromatography
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Liquid chromatography was used to follow the degradation of hexopyranosylated cytosine nucleosides in buffers of acid, neutral and alkaline pH and of constant ionic strength. The compounds were found to degrade by hydrolysis to cytosine and/or by deamination to the corresponding uracil nucleosides. Degradation in acid is influenced by the number of sugar hydroxyl groups, presence of sugar double bonds and the type of anomer. Stability of some of the compounds was compared with that of related thymine nucleosides. Temperature studies support a unimolecular mechanism of hydrolysis at pH 1.22.
- Thoithi, Grace N.,Van Schepdael, Ann,Busson, Roger,Janssen, Gerard,Van Aerschot, Arthur,Herdewijn, Piet,Roets, Eugene,Hoogmartens, Jos
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p. 189 - 203
(2007/10/03)
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- Pyrimidine nucleotidases/phosphotransferases from human erythrocyte
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Two cytoplasmic pyrimidine 5'-nucleotidase have been purified from human erythrocytes to homogeneity and partially characterized. The two enzymes, indicated as PN-I and PN-II, preferentially hydrolyse pyrimidine 5'- monophosphates and 3'-monophosphates, respectively. The kinetic analysis demonstrate that pyrimidine 5'-nucleotidases, in the presence of suitable nucleoside substrates, can operate as phosphotransferases by transferring phosphate to various nucleoside acceptors, including nucleoside analogues known as important drugs widely used in chemotherapy.
- Amici,Emanuelli,Raffaelli,Ruggieri,Magni
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p. 853 - 855
(2007/10/03)
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- Debromination of 8-bromo-2'-deoxyguanosine by methylene blue and visible light
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Debromination of 8-bromo-2'-deoxyguanosine was accomplished in high yield under neutral conditions in aqueous methanol by irradiating with visible light in the presence of methylene blue as a sensitizer and triethylamine as an electron donor. The method can be extended for the debromination of other bromoaromatic compounds.
- Venkatarangan, Lata,Yang, Dan-Hui,Epling, Gary A.,Basu, Ashis K.
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p. 1441 - 1444
(2007/10/03)
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- Photochemical halogen-exchange reaction of 5-iodouracil-containing oligonucleotides
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Photoreactions of 5-iododeoxyuridine (d(I)U) and d(I)U-containing oligonucleotides in aqueous solutions in the presence of various inorganic salts have been investigated. In the presence of NaCl and NaBr, d(I)U and d(I)U-containing oligonucleotides undergo an efficient photochemical halogen-exchange reaction to give d(Cl)U and d(Br)U, respectively.
- Kawai, Kiyohiko,Saito, Isao,Sugiyama, Hiroshi
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p. 5721 - 5724
(2007/10/03)
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- Synthesis and characterization of isotopically enriched pyrimidine deoxynucleoside oxidation damage products
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Oxidative damage to DNA is an established source of genomic instability. In this paper, we describe the synthesis and characterization of several pyrimidine deoxynucleoside oxidation damage products, enriched with stable isotopes. These products include the 2'-deoxynucleoside derivatives of 5- (hydroxymethyl)uracil, 5-formyluracil, 5-hydroxyuracil, 5-(hydroxymethyl)- cytosine, 5-formylcytosine, and 5-hydroxycytosine. The common precursor is 2'-deoxy-2''-deutero[1,3-15N]uridine. Additional stable isotopes are added during functional group conversions. Characterization of these derivatives includes mass spectrometry and 1H and 15N NMR spectroscopy. Proton and nitrogen NMR studies reported here allow an examination of the influence of the modification on sugar conformation and tautomeric equilibrium, properties likely to be important in understanding the biological consequences of these DNA damage products.
- LaFrancois, Christopher J.,Fujimoto, June,Sowers, Lawrence C.
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- The phototransformation of 5-tert-butyl-2′-deoxyuridine: An approach to the synthesis of new 1,2-dihydrocyclobuta[d]pyrimidin-2-ones
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A new type of 2-oxo-1,2-dihydrocyclobuta[d]pyrimidin-1-yl-2′-deoxynucleoside 2a is obtained in 52% yield upon irradiation of aqueous solutions of 5-tert-butyl-2′-deoxyuridine 1a with short wavelength (254 nm) UV light. The identity and structure of the photoproduct is unequivocally established by 1H and 13C NMR (including a 2D INADEQUATE experiment), mass and UV spectroscopy. This new phototransformation represents an alternative route for the photolysis of 5-alkyl-substituted uracil derivatives where the side chain has more than one carbon atom. A mechanism for the phototransformation of compound 1a into 2a is proposed, and this requires the creation of a cyclobutanol-type intermediate (type-II photoprocess). A general pathway of UV-induced photolysis of 5-alkyl-substituted uracil derivatives (other than 5-methyl substituted) into 1,2-dihydrocyclobuta[d]pyrimidin-2-ones and photohydrated uracils is proposed.
- Basnak, Ivan,McKinnell, Denise,Spencer, Neil,Balkan, Ayla,Ashton, Peter R.,Walker, Richard T.
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p. 121 - 125
(2007/10/03)
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- Chemical synthesis of 2', 3'-dideoxycytidine
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Method for synthesis of 2',3'-dideoxycytidine by providing 5'-silylated-2',3'-dideoxyuridine as an intermediate.
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- Antiviral agents
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Nucleoside compounds of the formula STR1 wherein: B is a purine or a pyrimidine; X and X' are H, OH or F, provided that at least one is H; Y and Y' are H, OH, OCH3 or F, provided that at least one is H; Y' and Z together form a cyclic phosphate ester, provided that Y is H; or Z is STR2 where n is zero, one, two or three; and Z' is N3 or OCH3 ; provided that when X' and Y' are OH and Z' is N3, B is not cytosine, and when X' and Y' are OH and Z' is OCH3, B is not uracil, adenine or cytosine; and the pharmaceutically acceptable esters, ethers and salts thereof, have been found to have potent antiviral activity with a high therapeutic ratio.
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- Selective alkylation of pyrimidyl dianions II: Synthesis, characterization, and comparative reactivity of 3′, 5′-o-bis- tetrahydropyranyl, trimethylsilyl and tert-butyldimethylsilyl derivatives of 5-bromo-2′-deoxyuridine
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Three compounds which can be used as precursors for thymidine synthesis via methylation at the 5 position, including 3′, 5′-o-bis(tetrahydropyranyl)-5-bromo-2′-deoxyuridine 1a, 3′, 5′-o-bis-(trimethylsily)-5-bromo-2′-deoxyuridine 1b, and 3′, 5′-o-bis-(t-butyldimethylsilyl)-5-bromo-2′-deoxyuridine 1c, were prepared, isolated and characterized by spectroscopic methods. Alkylation with methyl iodide using an organo-lithium reagent at low temperature produced 72%, 41% and 74% of thymidine 6, respectively. Tetrahydropyranyl and t-butyldimethylsilyl ethers are found to be better precursors for introduction of a methyl group at the 5 position.
- Alauddin, Mian M.,Conti, Peter S.
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p. 1699 - 1706
(2007/10/02)
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- Synthesis of 2'-deoxypyrimidine nucleosides
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A method for the preparation of 2'-deoxynucleosides and 2',3'-dideoxy-2',3'-didehydronucleosides that includes the step of reacting a nucleoside having hydroxyl groups in the 2' and 3' positions with a mixture of acyl bromide or chloride and HX, wherein X is Br or Cl, at moderate temperature, to give a haloacyl nucleoside derivative that can be deprotected and reduced to form the desired compound.
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- Facile synthesis of thymidine derivatives by cross-coupling of 5-halogenouridine derivatives with trimethylaluminum
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An efficient method for the introduction of a methyl group in the 5-position of uridine derivatives is described. This method involves three steps: protection of 5-halogenouridines 4 and 5 with hexamethyldisilazane, a palladium-catalyzed cross-coupling of the pertrimethylsilylated nucleosides with trimethylaluminum, and subsequent deprotection to afford the corresponding thymidine derivatives 6 in high overall yields.
- Hirota,Kitade,Kanbe,Isobe,Maki
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p. 213 - 215
(2007/10/02)
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- One-electron-reduction potentials of pyrimidine bases, nucleosides, and nucleotides in aqueous solution. Consequences for DNA redox chemistry
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The reduction potentials in aqueous solution of the pyrimidine bases, nucleosides, and nucleotides of uracil (U) and thymine (T) were determined using the technique of pulse radiolysis with time-resolved spectrophotometric detection. The electron adducts of U and T were found to undergo reversible electron exchange with a series of ring-substituted N-methylpyridinium cations with known reduction potential. From the concentrations of the pyrimidine electron adducts and the reduced N-methylpyridinium compounds at electron-transfer equilibrium, the thermodynamical equilibrium constants were obtained and from these the reduction potentials. The results show U and T and their nucleosides and nucleotides to have very similar reduction potentials, ～ -1.1 V/NHE at pH 8, i.e., the effect of methylation at C5, C6, or of substitution at N1 is small, ≤0.1 V. In the case of cytosine (C) the electron adduct is protonated (probably at N3), even up to pH 13. The protonated adduct (C(H)?) undergoes a reversible electron transfer with the N-methylpyridinium cations. This is accompanied in one direction by transfer of a proton but by that of a water molecule in the other direction. As a result of the protonation of the electron adduct, the effective ease of reduction of C in aqueous solution is similar to that of U and T. It is suggested that in DNA the tendency for C?- to be protonated (by its complementary base G) is larger by ≥10 orders of magnitude than that for protonation of T?- by its complementary base A. This results in C and not T being the most easily reduced base in DNA. A further consequence is that lack of neutralization by intrapair proton transfer of T?- enables the irreversible extra-pair protonation on C6 of the radical anion to take place.
- Steenken,Telo,Novais,Candeias
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p. 4701 - 4709
(2007/10/02)
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- Ionization of purine nucleosides and nucleotides and their components by 193-nm laser photolysis in aqueous solution: Model studies for oxidative damage of DNA 1
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The effect of 20-ns pulses of 193-nm laser light on aqueous solutions of purine bases, (2′-deoxy)nucleosides, and (2′-deoxy)nucleotides was investigated, and monophotonic ionization was observed. Although (deoxy)ribose and (deoxy)ribose phosphates are also ionized by 193-nm light, the photoionization of the (deoxy)nucleosides and -tides takes place predominantly (90%) at the purine moiety, due to the much higher extinction coefficients at 193 nm of the bases as compared to the (deoxy)ribose phosphates. The quantum yields of photoionization (φPl) of the purines are in the range 0.01 to 0.08, based on φ(Cl-) at 193 nm of 0.46. As shown by comparison with data obtained from pulse radiolysis, the ionized purines, i.e., the radical cations, deprotonate in neutral solution, yielding neutral radicals. The radical cation of 1-methylguanosine, produced by photoionization in oxygen-saturated aqueous solution, deprotonates with the rate constant 3.5 × 105 s-1. In the absence of oxygen, the hydrated electrons resulting from the photoionization react with the untransformed purine derivatives to yield the corresponding radical anions. As these are rapidly protonated by water (as concluded from pulse radiolysis), the photoionization in deaerated neutral solution results in two different neutral radicals: a deprotonated radical cation and a protonated radical anion.
- Candeias,Steenken
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p. 699 - 704
(2007/10/02)
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- Engineering Tethered DNA Molecules by the Convertible Nucleoside Approach
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Non-natural functional groups, tethered to DNA, provide a chemical handle for the site-specific attachment of reporter and effector elements.Herein we report a general strategy for the synthesis of oligodeoxynucleotides bearing tethered functionality (functionally tethered oligonucleotides, FTOs).In this approach, the convertible nucleoside 4-O-(2,4,6-trimethylphenyl)-2'-deoxyuridine (TMP-dU) is introduced site-specifically into DNA during automated synthesis.Upon treatment with aqueous amines, the TMP-dU moiety undergoes nucleophilic substitution to yield an N4-alkyl-dC nucleoside - the DNA product is a dC-tethered FTO.Since the tether structure is solely determined by choice of amine used in the deprotection/conversion reaction, this convertible nucleoside approach permits a wide variety of FTOs to be synthesized from a single precursor.
- MacMillan, Andrew M.,Verdine, Gregory L.
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p. 2603 - 2616
(2007/10/02)
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- Palladium-Mediated Synthesis of C-5 Pyrimidine Nucleoside Thioethers from Disulfides and Mercurinucleosides
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Thioether-linked side chains can be created at C-5 of pyrimidine nucleosides via a palladium-mediated reaction of mercurated nucleosides with organic disulfides. 5-(Chloromercuri)-2'-deoxyuridine reacts with butyl disulfide, phenyl disulfide, dimethyl 3,3'-dithiopropionate, and N,N'-bis(trifluoroacetyl)cystamine to yield respectively 5-(1-thiapentyl)-2'-deoxyuridine, 5-(phenylthio)-2'-deoxyuridine, 5--2'-deoxyuridine, and 5--2'-deoxyuridine in yields ranging from 46 to 73percent.Other mercurated nucleosides, including 5-(chloromercuri)-2'-deoxycytidine, 5-(chloromercuri)cytidine, and 5-(chloromercuri)tubercidin react with N,N'-bis(trifluoroacetyl)cystamine and lithium-palladium chloride in methanol to yield the corresponding coupled products, but the yields are much lower (5-10percent).The nucleoside coupling reaction is complicated by competing side reactions between disulfides and Pd2+, which remain to be elucidated
- Bergstrom, Donald E.,Beal, Peter,Jenson, Jeffrey,Lin, Xiaoping
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p. 5598 - 5602
(2007/10/02)
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- A Practical Synthesis of 2'-Deoxyuridine from Uridine
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An efficient and improved synthetic method for 2'-deoxyuridine from uridine has been developed.
- Huang, Haoqlang,Chu, Chung K.
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p. 1039 - 1046
(2007/10/02)
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- REGIOSELECTIVE DEPROTECTION OF 3',5'-O-ACYLATED PYRIMIDINE NUCLEOSIDES BY LIPASE AND ESTERASE
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A lipase was found to catalyze the regioselective hydrolysis at the secondary hydroxyl group of 2'-deoxy 3',5'-di O-hexanoyl pyrimidine nuclosides, whereas a protease catalyzes that at the primary hydroxyl group.
- Uemura, Atsuhiko,Nozaki, Kenji,Yamashita, Jun-ichi,Yasumoto, Mitsugi
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p. 3819 - 3820
(2007/10/02)
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