- Enzymatic synthesis of novel purine nucleosides bearing a chiral benzoxazine fragment
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A series of ribo- and deoxyribonucleosides bearing 2-aminopurine as a nucleobase with 7,8-difluoro- 3,4-dihydro-3-methyl-2H-[1,4]benzoxazine (conjugated directly or through an aminohexanoyl spacer) was synthesized using an enzymatic transglycosylation reaction. Nucleosides 3-6 were resistant to deamination under action of adenosine deaminase (ADA) Escherichia coli and ADA from calf intestine. The antiviral activity of the modified nucleosides was evaluated against herpes simplex virus type 1 (HSV-1, strain L2). It has been shown that at sub-toxic concentrations, nucleoside (S)-4-[2-amino-9-(β-D-ribofuranosyl)-purin-6-yl]-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine exhibit significant antiviral activity (SI?>?32) on the model of HSV-1 in vitro, including an acyclovir-resistant virus strain (HSV-1, strain L2/R).
- Eletskaya, Barbara Z.,Gruzdev, Dmitry A.,Krasnov, Victor P.,Levit, Galina L.,Kostromina, Maria A.,Paramonov, Alexander S.,Kayushin, Alexei L.,Muzyka, Inessa S.,Muravyova, Tatyana I.,Esipov, Roman S.,Andronova, Valeria L.,Galegov, Georgiy A.,Charushin, Valery N.,Miroshnikov, Anatoly I.,Konstantinova, Irina D.
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- Kinetic and conformational studies of adenosine deaminase upon interaction with oxazepam and lorazepam
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Oxazepam and lorazepam inhibit the adenosine deaminase (ADA) differently. In the case of lorazepam temperature increment causes an increase in the inhibition potency whereas higher temperature reduces the inhibitory effect of oxazepam; which proposes the overall profounder structural changes in the case of lorazepam relative to those caused by oxazepam.
- Moosavi-Movahedi,Tehrani, H. Sepassi,Amanlou,Soltani Rad,Hakimelahi,Tsai,Ataie,Saboury,Ahmad,Khalafi-Nezhad,Poursasan,Sharifizadeh
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- 3'-β-ethynyl and 2'-deoxy-3'-β-ethynyl adenosines: First 3'-β- branched-adenosines substrates of adenosine deaminase
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The 3'-C-branched-adenosine and 2'-deoxyadenosine analogues 1-7 were tested as substrate of adenosine deaminase. The 9-(3'-C-ethynyl-β-D-ribo- pentofuranosyl)adenine 1 and its 2'-deoxy analogue 7 were deaminated by the enzyme while the vinyl and ethyl derivatives 2 and 3 were not. The 9-(3'-C- branched-β-D-xylo-pentofuranosyl)adenines 4-6 were deaminated by the deaminase.
- Tritsch, Denis,Jung, Pierre M. J.,Burger, Alain,Biellmann, Jean-Francois
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- Contrasting behavior of conformationally locked carbocyclic nucleosides of adenosine and cytidine as substrates for deaminases
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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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- Nucleolipids of Canonical Purine ?- D -Ribo-Nucleosides: Synthesis and Cytostatic/Cytotoxic Activities Toward Human and Rat Glioblastoma Cells
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We report on the synthesis of two series of canonical purine ?-d-ribonucleoside nucleolipids derived from inosine and adenosine, which have been characterized by elemental analyses, electrospray ionization mass spectrometry (ESI MS) as well as by 1H and 13CNMR, and pH-dependent UV/Vis spectroscopy. A selection of the novel nucleolipids with different lipophilic moieties were first tested on their cytotoxic effect toward human macrophages. Compounds without a significant inhibitory effect on the viability of the macrophages were tested on their cytostatic/cytotoxic effect toward human astrocytoma/oligodendroglioma GOS-3 cells as well as against the rat malignant neuroectodermal BT4Ca cell line. In order to additionally investigate the potential molecular mechanisms involved in the cytotoxic effects of the derivatives on GOS-3 or BT4Ca cells, we evaluated the induction of apoptosis and observed the particular activity of the nucleolipid ethyl 3-{4-hydroxymethyl-2-methyl-6-[6-oxo-1-(3,7,11-trimethyl-dodeca-2,6,10-trienyl)-1,6-dihydro-purin-9-yl]-tetrahydro-furo[3,4-d][1,3]dioxol-2-yl}propionate (8 c) toward both human and rat glioblastoma cell lines invitro. Nucleolipids combat cancer: We report the synthesis of two nucleolipid derivatives from inosine and adenosine with different lipophilic moieties. These have no cytotoxic effect on human macrophages based on invitro side-effect tests but have antiproliferative properties against malignant glioblastoma cell lines.
- Knies, Christine,Hammerbacher, Katharina,Bonaterra, Gabriel A.,Kinscherf, Ralf,Rosemeyer, Helmut
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- Rapid, reliable, and sensitive detection of adenosine deaminase activity by UHPLC-Q-Orbitrap HRMS and its application to inhibitory activity evaluation of traditional Chinese medicines
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Adenosine deaminase (ADA), which is a key enzyme in the metabolism of purine nucleosides, plays important roles in diverse disorders, such as tuberculosis, diabetes, liver disorders, and cancer. Determination of the activities of ADA and its isoenzymes in body fluids has received considerable attention in the diagnosis and treatment of relative diseases. Ultraviolet spectroscopy with adenosine (AD) as a substrate is a classical approach for screening potential ADA inhibitors by measuring the decrease in substrate (AD) at 265 nm or increase in the product (inosine) at 248 nm. However, AD and inosine share a very close maximum absorption wavelength, and the reaction is uncertain and is frequently interfered by the background color of matrix compounds or plant extracts. Thus, the method usually yields false positive or negative results. In this study, a novel, rapid, sensitive, and accurate ultra-high-performance liquid chromatography-Q exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometric (UHPLC-Q-Orbitrap HRMS) method was developed for determining and screening ADA inhibitors by directly determining the deamination product of AD, inosine. A proper separation was achieved for inosine and chlormequat (internal standard) within 2 min via isocratic elution (0.1% formic acid:methanol = 85:15, v/v) at a flow rate of 0.3 mL min?1 on a Waters ACQUITY HSS T3 column (2.1 mm × 100 mm, 1.8 μm) following a simple precipitation of proteins. The intra- and inter-day precisions of the developed method were below 7.17% and 8.99%, respectively. The method exhibited advantages of small total reaction volume (60 μL), short running time (2 min), high sensitivity (lowest limit of quantification of 0.02 μM for inosine), and low cost (small enzyme consumption of 0.007 unit mL?1 for ADA and substrate of 3.74 μM for AD in individual inhibition), and no matrix effects (101.64%–107.12%). Stability results showed that all analytes were stable under the investigated conditions. The developed method was successfully applied to the detection of the inhibitory activity of ADA from traditional Chinese medicines.
- Qi, Shenglan,Guan, Huida,Deng, Gang,Yang, Tao,Cheng, Xuemei,Liu, Wei,Liu, Ping,Wang, Changhong
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- Determination of adenosine deaminase activity in dried blood spots by a nonradiochemical assay using reversed-phase high-performance liquid chromatography
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Adenosine deaminase (ADA) deficiency is a rare metabolic disease causing severe combined immunodeficiency (SCID). An assay to determine ADA activity in dried blood spots was developed using reversed-phase HPLC. The assay was linear with reaction times up to at least 4 hours, and protein concentrations up to at least 2.2 mg/ml. The intra-assay CV and the inter-assay CV for the complete assay was 3.5 and 8.4%, respectively. The ADA activity in a control blood spot, stored at 4°C, remained stable for at least one year. Only a slightly decreased ADA activity (35 ± 13 nmol/mg/h, n = 4) was observed in heterozygotes for a c.704G > A mutation in the ADA gene when compared to that observed in controls (41 ± 13 nmol/mg/h, n = 108). In addition, increased ADA activity as found in a rare form of congenital anemia can be assessed, as observed in a bloodspot from a patient diagnosed with Diamond Blackfan anemia (ADA activity 150 nmol/mg/h). Copyright
- Van Kuilenburg,Zoetekouw,Meijer,Kuijpers
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- Synthesis of 13C-labeled 5-aminoimidazole-4-carboxamide-1-β-D-[13C5] ribofuranosyl 5′-monophosphate
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5-Aminoimidazole-4-carboxamide-1-β-D-[13C5] ribofuranosyl 5′-monophosphate ([13C5 ribose] AICAR-PO3H2) (6) has been synthesized from [13C5]adenosine. Incorporation of the mass-label into [13C5 ribose] AICAR-PO3H2 provides a useful standard to aid in metabolite identification and quantification in monitoring metabolic pathways. A synthetic route to the 13C-labeled compound has not been previously reported. Our method employs a hybrid enzymatic, and chemical synthesis approach that applies an enzymatic conversion from adenosine to inosine followed by a ring-cleavage of the protected inosine. A direct phosphorylation of the resulting 2′,3′-isopropylidine acadesine (5) was developed to yield the title compound in 99% purity following ion exchange chromatography.
- Zarkin, Allison K.,Elkins, Phyllis D.,Gilbert, Amanda,Jester, Teresa L.,Seltzman, Herbert H.
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- Simultaneous detection of ATP and GTP by covalently linked fluorescent ribonucleopeptide sensors
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A noncovalent RNA complex embedding an aptamer function and a fluorophore-labeled peptide affords a fluorescent ribonucleopeptide (RNP) framework for constructing fluorescent sensors. By taking an advantage of the noncovalent properties of the RNP complex, the ligand-binding and fluorescence characteristics of the fluorescent RNP can be independently tuned by taking advantage of the nature of the RNA and peptide subunits, respectively. Fluorescent sensors tailored for given measurement conditions, such as a detection wavelength and a detection concentration range for a ligand of interest can be easily identified by screening of fluorescent RNP libraries. The noncovalent configuration of a RNP becomes a disadvantage when the sensor is to be utilized at very low concentrations or when multiple sensors are applied to the same solution. Here, we report a strategy to convert a fluorescent RNP sensor in the noncovalent configuration into a covalently linked stable fluorescent RNP sensor. This covalently linked fluorescent RNP sensor enabled ligand detection at a low sensor concentration, even in cell extracts. Furthermore, application of both ATP and GTP sensors enabled simultaneous detection of ATP and GTP by monitoring each wavelength corresponding to the respective sensor. Importantly, when a fluorescein-modified ATP sensor and a pyrene-modified GTP sensor were co-incubated in the same solution, the ATP sensor responded at 535 nm only to changes in the concentration of ATP, whereas the GTP sensor detected GTP at 390 nm without any effect on the ATP sensor. Finally, simultaneous monitoring by these sensors enabled real-time measurement of adenosine deaminase enzyme reactions.
- Nakano, Shun,Fukuda, Masatora,Tamura, Tomoki,Sakaguchi, Reiko,Nakata, Eiji,Morii, Takashi
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- L-nucleoside analogues as potential antimalarials that selectively target Plasmodium falciparum adenosine deaminase
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The L-stereoisomer analogues of D-coformycin selectively inhibited P. falciparum adenosine deaminase (ADA) in the picomolar range (L-isocoformycin, K(i) 7 pM; L-coformycin, K(i) 250 pM). While the L-nucleoside analogues, L- adenosine, 2,6-diamino-9-(L-ribofuranosyl)purine and 4-amino- 1 -(L- ribofuranosyl)pyrazolo[3,4-d]-pyrimidine were selectively deaminated by P. falciparum ADA, L-thioinosine and L-thioguanosine were not. This is the first example of 'non-physiological' L-nucleosides that serve as either substrates or inhibitors of malarial ADA and are not utilised by mammalian ADA.
- Brown, David M.,Netting, Andrew G.,Chun, Byoung K.,Choi, Yongseok,Chu, Chung K.,Gero, Annette M.
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- Prebiotic Photochemical Coproduction of Purine Ribo- And Deoxyribonucleosides
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The hypothesis that life on Earth may have started with a heterogeneous nucleic acid genetic system including both RNA and DNA has attracted broad interest. The recent finding that two RNA subunits (cytidine, C, and uridine, U) and two DNA subunits (deoxyadenosine, dA, and deoxyinosine, dI) can be coproduced in the same reaction network, compatible with a consistent geological scenario, supports this theory. However, a prebiotically plausible synthesis of the missing units (purine ribonucleosides and pyrimidine deoxyribonucleosides) in a unified reaction network remains elusive. Herein, we disclose a strictly stereoselective and furanosyl-selective synthesis of purine ribonucleosides (adenosine, A, and inosine, I) and purine deoxynucleosides (dA and dI), alongside one another, via a key photochemical reaction of thioanhydroadenosine with sulfite in alkaline solution (pH 8-10). Mechanistic studies suggest an unexpected recombination of sulfite and nucleoside alkyl radicals underpins the formation of the ribo C2′-O bond. The coproduction of A, I, dA, and dI from a common intermediate, and under conditions likely to have prevailed in at least some primordial locales, is suggestive of the potential coexistence of RNA and DNA building blocks at the dawn of life.
- Xu, Jianfeng,Green, Nicholas J.,Russell, David A.,Liu, Ziwei,Sutherland, John D.
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supporting information
p. 14482 - 14486
(2021/09/18)
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- Biochemical characterization of a recombinant acid phosphatase from Acinetobacter baumannii
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Genomic sequence analysis of Acinetobacter baumannii revealed the presence of a putative Acid Phosphatase (AcpA; EC 3.1.3.2). A plasmid construct was made, and recombinant protein (rAcpA) was expressed in E. coli. PAGE analysis (carried out under denaturing/ reducing conditions) of nickel-affinity purified protein revealed the presence of a nearhomogeneous band of approximately 37 kDa. The identity of the 37 kDa species was verified as rAcpA by proteomic analysis with a molecular mass of 34.6 kDa from the deduced sequence. The dependence of substrate hydrolysis on pH was broad with an optimum observed at 6.0. Kinetic analysis revealed relatively high affinity for PNPP (Km = 90 μM) with Vmax, kcat, and Kcat/Km values of 19.2 pmoles s-1, 4.80 s-1(calculated on the basis of 37 kDa), and 5.30 × 104 M-1s-1, respectively. Sensitivity to a variety of reagents, i.e., detergents, reducing, and chelating agents as well as classic acid phosphatase inhibitors was examined in addition to assessment of hydrolysis of a number of phosphorylated compounds. Removal of phosphate from different phosphorylated compounds is supportive of broad, i.e., 'nonspecific' substrate specificity; although, the enzyme appears to prefer phosphotyrosine and/or peptides containing phosphotyrosine in comparison to serine and threonine. Examination of the primary sequence indicated the absence of signature sequences characteristic of Type A, B, and C nonspecific bacterial acid phosphatases.
- Smiley-Moreno, Elizabeth,Smith, Douglas,Yu, Jieh-Juen,Cao, Phuong,Arulanandam, Bernard P.,Chambers, James P.
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- An enzymatic flow-based preparative route to vidarabine
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The bi-enzymatic synthesis of the antiviral drug vidarabine (arabinosyladenine, ara-A), catalyzed by uridine phosphorylase from Clostridium perfringens (CpUP) and a purine nucleoside phosphorylase fromAeromonas hydrophila (AhPNP), was re-designed under continuous-flow conditions. Glyoxyl-agarose and EziGTM1 (Opal) were used as immobilization carriers for carrying out this preparative biotransformation. Upon setting-up reaction parameters (substrate concentration and molar ratio, temperature, pressure, residence time), 1 g of vidarabine was obtained in 55% isolated yield and >99% purity by simply running the flow reactor for 1 week and then collecting (by filtration) the nucleoside precipitated out of the exiting flow. Taking into account the substrate specificity of CpUP and AhPNP, the results obtained pave the way to the use of the CpUP/AhPNP-based bioreactor for the preparation of other purine nucleosides.
- Annunziata, Francesca,Bavaro, Teodora,Calleri, Enrica,Conti, Paola,Pinto, Andrea,Previtali, Clelia,Rinaldi, Francesca,Speranza, Giovanna,Tamborini, Lucia,Terreni, Marco,Ubiali, Daniela
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- Thermodynamic Reaction Control of Nucleoside Phosphorolysis
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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)
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- Fluorescing Isofunctional Ribonucleosides: Assessing Adenosine Deaminase Activity and Inhibition
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The enzymatic conversion of isothiazolo[4,3-d]pyrimidine-based adenosine (tzA) and 2-aminoadenosine (tz2-AA) analogues to the corresponding isothiazolo[4,3-d]pyrimidine-based inosine (tzI) and guanosine (tzG) derivatives is evaluated and compared to the conversion of native adenosine to inosine. Henri–Michaelis–Menten analyses provides the foundation for a high-throughput screening assay, and the efficacy of the assay is showcased by fluorescence-based analysis of tzA conversion to tzI in the presence of known and newly synthesized inhibitors.
- Ludford, Paul T.,Rovira, Alexander R.,Fin, Andrea,Tor, Yitzhak
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p. 718 - 726
(2019/03/05)
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- SYNTHESIS AND STRUCTURE OF HIGH POTENCY RNA THERAPEUTICS
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This invention provides expressible polynucleotides, which can express a target protein or polypeptide. Synthetic mRNA constructs for producing a protein or polypeptide can contain one or more 5′ UTRs, where a 5′ UTR may be expressed by a gene of a plant. In some embodiments, a 5′ UTR may be expressed by a gene of a member of Arabidopsis genus. The synthetic mRNA constructs can be used as pharmaceutical agents for expressing a target protein or polypeptide in vivo.
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- Structure-Guided Tuning of a Selectivity Switch towards Ribonucleosides in Trypanosoma brucei Purine Nucleoside 2′-Deoxyribosyltransferase
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The use of nucleoside 2′-deoxyribosyltransferases (NDTs) as biocatalysts for the industrial synthesis of nucleoside analogues is often hindered by their strict preference for 2′-deoxyribonucleosides. It is shown herein that a highly versatile purine NDT from Trypanosoma brucei (TbPDT) can also accept ribonucleosides as substrates; this is most likely because of the distinct role played by Asn53 at a position that is usually occupied by Asp in other NDTs. Moreover, this unusual activity was improved about threefold by introducing a single amino acid replacement at position 5, following a structure-guided approach. Biophysical and biochemical characterization revealed that the TbPDTY5F variant is a homodimer that displays maximum activity at 50 °C and pH 6.5 and shows a remarkably high melting temperature of 69 °C. Substrate specificity studies demonstrate that 6-oxopurine ribonucleosides are the best donors (inosine>guanosine?adenosine), whereas no significant preferences exist between 6-aminopurines and 6-oxopurines as base acceptors. In contrast, no transferase activity could be detected on xanthine and 7-deazapurines. TbPDTY5F was successfully employed in the synthesis of a wide range of modified ribonucleosides containing different purine analogues.
- Del Arco, Jon,Mills, Alberto,Gago, Federico,Fernández-Lucas, Jesús
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p. 2996 - 3000
(2019/11/11)
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- Abiotic synthesis of purine and pyrimidine ribonucleosides in aqueous microdroplets
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Aqueous microdroplets (a nucleobase (uracil, adenine, cytosine, or hypoxanthine) are electrosprayed from a capillary at +5 kV into a mass spectrometer at room temperature and 1 atm pressure with 3 mM divalent magnesium ion (Mg2+) as a catalyst. Mass spectra show the formation of ribonucleosides that comprise a four-letter alphabet of RNA with a yield of 2.5% of uridine (U), 2.5% of adenosine (A), 0.7% of cytidine (C), and 1.7% of inosine (I) during the flight time of ～50 μs. In the case of uridine, no catalyst is required. An aqueous solution containing guanine cannot be generated under the same conditions given the extreme insolubility of guanine in water. However, inosine can base pair with cytidine and thus substitute for guanosine. Thus, a full set of ribonucleosides to generate the purine–pyrimidine base pairs A-U and I-C are spontaneously generated in aqueous microdroplets under similar mild conditions.
- Nam, Inho,Nam, Hong Gil,Zare, Richard N.
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- The Chemoenzymatic Synthesis of 2-Chloro- and 2-Fluorocordycepins
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Two approaches to the chemoenzymatic synthesis of 2-fluorocordycepin and 2-chlorocordycepin were studied: (i) the use of 3′-deoxyadenosine (cordycepin) and 3′-deoxyinosine (3′dIno) as donors of 3-deoxy- d -ribofuranose in the transglycosylation of 2-fluoro- (2F Ade) and 2-chloroadenine (2Cl Ade) catalyzed by the recombinant E. coli purine nucleoside phosphorylase (PNP), and (ii) the use of 2-fluoroadenosine and 3′-deoxyinosine as substrates of the cross-glycosylation and PNP as a biocatalyst. An efficient method for 3′-deoxyinosine synthesis starting from inosine was developed. However, the very poor solubility of 2Cl Ade and 2F Ade is the limiting factor of the first approach. The second approach enables this problem to be overcome and it appears to be advantageous over the former approach from the viewpoint of practical synthesis of the title nucleosides. The 3-deoxy-α- d -ribofuranose-1-phosphate intermediary formed in the 3′dIno phosphorolysis by PNP was found to be the weak and marginal substrate of E. coli thymidine (TP) and uridine (UP) phosphorylases, respectively. Finally, one-pot cascade transformation of 3-deoxy- d -ribose in cordycepin in the presence of adenine and E. coli ribokinase, phosphopentomutase, and PNP was tested and cordycepin formation in ca. 3.4% yield was proved.
- Denisova, Alexandra O.,Tokunova, Yulia A.,Fateev, Ilja V.,Breslav, Alexandra A.,Leonov, Vladimir N.,Dorofeeva, Elena V.,Lutonina, Olga I.,Muzyka, Inessa S.,Esipov, Roman S.,Kayushin, Alexey L.,Konstantinova, Irina D.,Miroshnikov, Anatoly I.,Stepchenko, Vladimir A.,Mikhailopulo, Igor A.
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p. 4853 - 4860
(2017/10/06)
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- Recognition of Artificial Nucleobases by E. coli Purine Nucleoside Phosphorylase versus its Ser90Ala Mutant in the Synthesis of Base-Modified Nucleosides
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A wide range of natural purine analogues was used as probe to assess the mechanism of recognition by the wild-type (WT) E. coli purine nucleoside phosphorylase (PNP) versus its Ser90Ala mutant. The results were analyzed from viewpoint of the role of the Ser90 residue and the structural features of the bases. It was found that the Ser90 residue of the PNP 1) plays an important role in the binding and activation of 8-aza-7-deazapurines in the synthesis of their nucleosides, 2) participates in the binding of α-D-pentofuranose-1-phosphates at the catalytic site of the PNP, and 3) catalyzes the dephosphorylation of intermediary formed 2-deoxy-α-D-ribofuranose-1-phosphate in the trans-2-deoxyribosylation reaction. 5-Aza-7-deazaguanine manifested excellent substrate activity for both enzymes, 8-amino-7-thiaguanine and 2-aminobenzothiazole showed no substrate activity for both enzymes. On the contrary, the 2-amino derivatives of benzimidazole and benzoxazole are substrates and are converted into the N1- and unusual N2-glycosides, respectively. 9-Deaza-5-iodoxanthine showed moderate inhibitory activity of the WT E. coli PNP, whereas 9-deazaxanthine and its 2′-deoxyriboside are weak inhibitors. How does it work? The substrate and inhibitory properties of a wide range of artificial bases for the wild-type E. coli purine nucleoside phosphorylase (PNP) versus its Ser90Ala mutant were studied to evaluate the mechanism of recognition by PNP and the role of various electronic and structural features in this process. The PNP recognized a broad palette of bases consisting of a number of dissimilar fragments determining its ability to interact with the Asp204 and Ser90 residues (see scheme).
- Fateev, Ilja V.,Kharitonova, Maria I.,Antonov, Konstantin V.,Konstantinova, Irina D.,Stepanenko, Vasily N.,Esipov, Roman S.,Seela, Frank,Temburnikar, Kartik W.,Seley-Radtke, Katherine L.,Stepchenko, Vladimir A.,Sokolov, Yuri A.,Miroshnikov, Anatoly I.,Mikhailopulo, Igor A.
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p. 13401 - 13419
(2015/09/15)
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- Characterization of inosine-uridine nucleoside hydrolase (RihC) from Escherichia coli
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A non-specific nucleoside hydrolase from Escherichia coli (RihC) has been cloned, overexpressed, and purified to greater than 95% homogeneity. Size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis show that the protein exists as a homodimer. The enzyme showed significant activity against the standard ribonucleosides with uridine, xanthosine, and inosine having the greatest activity. The Michaelis constants were relatively constant for uridine, cytidine, inosine, adenosine, xanthosine, and ribothymidine at approximately 480 μM. No activity was exhibited against 2′-OH and 3′-OH deoxynucleosides. Nucleosides in which additional groups have been added to the exocyclic N6 amino group also exhibited no activity. Nucleosides lacking the 5′-OH group or with the 2′-OH group in the arabino configuration exhibited greatly reduced activity. Purine nucleosides and pyrimidine nucleosides in which the N7 or N3 nitrogens respectively were replaced with carbon also had no activity.
- Arivett, Brock,Farone, Mary,Masiragani, Ranjith,Burden, Andrew,Judge, Shelby,Osinloye, Adedoyin,Minici, Claudia,Degano, Massimo,Robinson, Matthew,Kline, Paul
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p. 656 - 662
(2014/03/21)
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- 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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- Deamination of 6-aminodeoxyfutalosine in menaquinone biosynthesis by distantly related enzymes
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Proteins of unknown function belonging to cog1816 and cog0402 were characterized. Sav2595 from Steptomyces avermitilis MA-4680, Acel0264 from Acidothermus cellulolyticus 11B, Nis0429 from Nitratiruptor sp. SB155-2 and Dr0824 from Deinococcus radiodurans R1 were cloned, purified, and their substrate profiles determined. These enzymes were previously incorrectly annotated as adenosine deaminases or chlorohydrolases. It was shown here that these enzymes actually deaminate 6-aminodeoxyfutalosine. The deamination of 6-aminodeoxyfutalosine is part of an alternative menaquinone biosynthetic pathway that involves the formation of futalosine. 6-Aminodeoxyfutalosine is deaminated by these enzymes with catalytic efficiencies greater than 10 5 M-1 s-1, Km values of 0.9-6.0 μM, and kcat values of 1.2-8.6 s-1. Adenosine, 2′-deoxyadenosine, thiomethyladenosine, and S-adenosylhomocysteine are deaminated at least an order of magnitude slower than 6-aminodeoxyfutalosine. The crystal structure of Nis0429 was determined and the substrate, 6-aminodeoxyfutalosine, was positioned in the active site on the basis of the presence of adventitiously bound benzoic acid. In this model, Ser-145 interacts with the carboxylate moiety of the substrate. The structure of Dr0824 was also determined, but a collapsed active site pocket prevented docking of substrates. A computational model of Sav2595 was built on the basis of the crystal structure of adenosine deaminase and substrates were docked. The model predicted a conserved arginine after β-strand 1 to be partially responsible for the substrate specificity of Sav2595.
- Goble, Alissa M.,Toro, Rafael,Li, Xu,Ornelas, Argentina,Fan, Hao,Eswaramoorthy, Subramaniam,Patskovsky, Yury,Hillerich, Brandan,Seidel, Ron,Sali, Andrej,Shoichet, Brian K.,Almo, Steven C.,Swaminathan, Subramanyam,Tanner, Martin E.,Raushel, Frank M.
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p. 6525 - 6536
(2013/10/01)
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- Enzymatic production of 5′-inosinic acid by a newly synthesised acid phosphatase/phosphotransferase
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5′-Nucleotides including 5′-inosinic acid have characteristic taste and important application in various foods as flavour potentiators. The selective nucleoside acid phosphatase/phosphotransferase (AP/PTase) can catalyse the synthesis of 5′-nucleotides by transfer of phosphate groups. In this study, a 747-bp gene encoding AP/PTase from Escherichia blattae was synthesised. After expression, the recombinant AP/PTase was purified using nickel-NTA. The optimal temperature and pH of this enzyme were 30°C and 5.0, respectively. The activity was partially inhibited by metal ions such as Hg2+, Ag+ and Cu2+, but not by chelating reagents such as EDTA. The values of Km and Vmax for inosine were 40 mM and 3.5 U/mg, respectively. Using this purified enzyme, 16.83 mM of 5′-IMP was synthesised from 37 mM of inosine and the molar yield reached 45.5%. Homology modelling and docking simulation were discussed.
- Liu, Zhi-Qiang,Zhang, Ling,Sun, Li-Hui,Li, Xiao-Jun,Wan, Nan-Wei,Zheng, Yu-Guo
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experimental part
p. 948 - 956
(2012/08/14)
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- Synthesis and enzymatic deprotection of biodegradably protected dinucleoside-2′,5′-monophosphates: 3-(Acetyloxy)-2,2- bis(ethoxycarbonyl)propyl phosphoesters of 3′-O-(acyloxymethyl)adenylyl- 2′,5′-adenosines
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As a first step towards a viable prodrug strategy for short oligoribonucleotides, such as 2-5A and its congeners, adenylyl-2′, 5′-adenosines bearing a 3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl group at the phosphate moiety, and an (acetyloxy)methyl- or a (pivaloyloxy)methyl- protected 3′-OH group of the 2′-linked nucleoside have been prepared. The enzyme-triggered removal of these protecting groups by hog liver carboxyesterase at pH 7.5 and 37° has been studied. The (acetyloxy)methyl group turned out to be too labile for the 3′-O-protection, being removed faster than the phosphate-protecting group, which results in 2′,5′- to 3′,5′-isomerization of the internucleosidic phosphoester linkage. In addition, the starting material was unexpectedly converted to the 5′-O-acetylated derivative. (Pivaloyloxy)methyl group appears more appropriate for the purpose. The fully deprotected 2′,5′-ApA was accumulated as a main product, although, even in this case, the isomerization of the starting material takes place.
- Kiuru, Emilia,Ora, Mikko,Beigelman, Leonid,Blatt, Lawrence,Loennberg, Harri
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experimental part
p. 266 - 286
(2011/10/05)
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- Simple method for fast deprotection of nucleosides by triethylamine- catalyzed methanolysis of acetates in aqueous medium
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A straightforward methodology for deacetylation of protected ribonucleosides was developed based on triethylamine-catalyzed solvolysis in aqueous methanol. Reactions are completed in a few minutes under microwave irradiation and the free nucleosides are obtained in high yield after simple evaporation of volatiles. Other important features include the involvement of readily available reagents and the compatibility with diverse functional groups, which make this process very attractive for broad application.
- Meier, Lidiane,Monteiro, Gustavo C.,Baldissera, Rodrigo A.M.,Sa?, Marcus Mandolesi
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body text
p. 859 - 866
(2010/09/11)
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- A secreted enzyme reporter system for MRI
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(Figure Presented) Let's see what comes out: An extracellular enzymatic gene-reporter system for magnetic resonance imaging (MRI) yields strong, reversible contrast changes in response to the expression of secreted alkaline phosphatase (SEAP; see picture). Products of SEAP activity were specifically detected using an iron-oxide-based sensor. The contrast agent is not used up by the enzyme, cell delivery is not required, and multimodal detection is possible.
- Westmeyer, Gil G.,Durocher, Yves,Jasanoff, Alan
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scheme or table
p. 3909 - 3911
(2010/09/05)
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- Floating pharmaceutical composition comprising an active phase and a non-active phase
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The invention concerns a floating pharmaceutical composition consisting of at least a first phase comprising at least a high dose active principle combined with one or several carriers and at least a second phase comprising at least a gas-generating system. The invention also concerns tablets comprising such a pharmaceutical composition and a method for preparing such tablets.
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- Aeromonas hydrophila strains as biocatalysts for transglycosylation
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Microbial transglycosylation is useful as a green alternative in the preparation of purine nucleosides and analogues, especially for those that display pharmacological activities. In a search for new transglycosylation biocatalysts, two Aeromonas hydrophila strains were selected. The substrate specificity of both micro-organisms was studied and, as a result, several nucleoside analogues have been prepared. Among them, ribavirin, a broad spectrum antiviral, and the well-known anti HIV didanosine, were prepared, in 77 and 62% yield using A. hydrophila CECT 4226 and A. hydrophila CECT 4221, respectively. In order to scale-up the processes, the reaction conditions, product purification and biocatalyst preparation were analyzed and optimized.
- Nbile, Matas,Terreni, Marco,Lewkowicz, Elizabeth,Iribarren, Adolfo M.
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experimental part
p. 395 - 402
(2011/10/08)
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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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- MW-assisted Er(OTf)3-catalyzed mild cleavage of isopropylidene acetals in Tricky substrates
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Erbium(III) trifluoromethane sulfonate is proposed as a very gentle Lewis acid catalyst in a MW-assisted chemoselective method for the cleavage of isopropylidene acetals in awkward substrates by using pure water as the solvent.
- Procopio, Antonio,Gaspari, Marco,Nardi, Monica,Oliverio, Manuela,Romeo, Roberto
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p. 1961 - 1964
(2008/09/19)
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- Formation of a β-pyrimidine nucleoside by a free pyrimidine base and ribose in a plausible prebiotic reaction
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The problem of β-nucleoside formation under prebiotic conditions represents one of the most significant challenges to the RNA world hypothesis and to many of its proposed precursors. The possibility exists that alternative bases may have come before the contemporary bases (i.e., A, G, C, and U), including bases that more readily form nucleosides. In a search for pyrimidine bases that are able to form nucleosides in plausible prebiotic reactions, it was discovered that the drying and heating of 2-pyrimidinone with ribose produces a β-furanosyl ribonucleoside in approximately 12% yield. At least two other chemical isomers of zebularine are also produced in the condensation reaction. This work represents the first successful synthesis of a pyrimidine nucleoside from a free base and a nonactivated sugar in a plausible prebiotic reaction. A comparison of 2-pyrimidinone with the purine bases that have also been demonstrated to form nucleosides in plausible prebiotic reactions provides insights regarding what chemical features of the bases facilitate glycoside formation in drying-heating reactions. Copyright
- Bean, Heather D.,Sheng, Yinghong,Collins, James P.,Anet, Frank A. L.,Leszczynski, Jerzy,Hud, Nicholas V.
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p. 9556 - 9557
(2008/02/11)
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- Synthesis of 5′-methylthio coformycins: Specific inhibitors for malarial adenosine deaminase
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Transition state theory suggests that enzymatic rate acceleration (k cat/knon) is related to the stabilization of the transition state for a given reaction. Chemically stable analogues of a transition state complex are predicted to convert catalytic energy into binding energy. Because transition state stabilization is a function of catalytic efficiency, differences in substrate specificity can be exploited in the design of tight-binding transition state analogue inhibitors. Coformycin and 2′-deoxycoformycin are natural product transition state analogue inhibitors of adenosine deaminases (ADAs). These compounds mimic the tetrahedral geometry of the ADA transition state and bind with picomolar dissociation constants to enzymes from bovine, human, and protozoan sources. The purine salvage pathway in malaria parasites is unique in that Plasmodium falciparum ADA (PfADA) catalyzes the deamination of both adenosine and 5′-3 methylthioadenosine. In contrast, neither human adenosine deaminase (HsADA) nor the bovine enzyme (BtADA) can deaminate 5′-methylthioadenosine. 5′-Methylthiocoformycin and 5′-methylthio-2′-deoxycoformycin were synthesized to be specific transition state mimics of the P. falciparum enzyme. These analogues inhibited PfADA with dissociation constants of 430 and 790 pM, respectively. Remarkably, they gave no detectable inhibition of the human and bovine enzymes. Adenosine deamination is involved in the essential pathway of purine salvage in P. falciparum, and prior studies have shown that inhibition of purine salvage results in parasite death. Inhibitors of HsADA are known to be toxic to humans, and the availability of parasite-specific ADA inhibitors may prevent this side-effect. The potent and P. falciparum-specti'ic inhibitors described here have potential for development as antimalarials without inhibition of host ADA.
- Tyler, Peter C.,Taylor, Erika A.,Froehlich, Richard F. G.,Schramm, Vern L.
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p. 6872 - 6879
(2008/02/10)
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- High-throughput five minute microwave accelerated glycosylation approach to the synthesis of nucleoside libraries
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The Vorbrueggen glycosylation reaction was adapted into a one-step 5 min/130 °C microwave assisted reaction. Triethanolamine in acetontrile containing 2% water was determined to be optimal for the neutralization of trimethylsilyl inflate allowing for direct MPLC purification of the reaction mixture. When coupled with a NH3/methanol deprotection reaction, a high-throughput method of nucleoside library synthesis was enabled. The method was demonstrated by examining the ribosylation of 48 nitrogen containing heteroaromatic bases that included 25 purines, four pyrazolopyrimidines, two 8-azapurines, one 2-azapurine, two imidazopyridines, two benzimidazoles, three imidazoles, three 1,2,4-triazoles, two pyrimidines, two 3-deazapyrimidines, one quinazolinedione, and one alloxazine. Of these, 32 yielded single regioisomer products, and six resulted in separable mixtures. Seven examples provided inseparable regioisomer mixtures of -two to three compounds (16 nucleosides), and three examples failed to yield isolable products. For the 45 single isomers isolated, the average two-step overall yield ± SD was 26 ± 16%, and the average purity ± SD was 95 ± 6%. A total of 58 different nucleosides were prepared of which 15 had not previously been accessed directly from glycosylation/deprotection of a readily available base.
- Bookser, Brett C.,Raffaele, Nicholas B.
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p. 173 - 179
(2007/10/03)
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- QSAR Analysis for ADA upon Interaction with a Series of Adenine Derivatives as Inhibitors
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The kinetic parameters of adenosine deaminase such as Km and Ki were determined in the absence and presence of adenine derivatives (R1- R24) in sodium phosphate buffer (50 mM; pH 7.5) solution at 27°C. These kinetic parameters were used for QSAR analysis. As such, we found some theoretical descriptors to which the binding affinity of adenosine deaminase (ADA) towards several adenine nucleosides as inhibitors is correlated. QSAR analysis has revealed that binding affinity of the adenine nucleosides upon interaction with ADA depends on the molecular volume, dipole moment of the molecule, electric charge around the N1 atom, and the highest of positive charge for the related molecules.
- Moosavi-Movahedi,Safarian,Hakimelahi,Ataei,Ajloo,Panjehpour,Riahi,Mousavi,Mardanyan,Soltani,Khalafi-Nezhad,Sharghi,Moghadamnia,Saboury
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p. 613 - 624
(2007/10/03)
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- New nucleoside-based polymeric supports for the solid phase synthesis of ribose-modified nucleoside analogues
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New solid supports, linking protected pyrimidine and purine nucleoside derivatives through the nucleobase, have been prepared. The support, incorporating a suitable derivative of 2′-azido, 2′-deoxyuridine, allowed the simple and efficient solid-phase synthesis of ribose-modified nucleoside and nucleic acid analogues, particularly of aminoacyl derivatives of 2′-deoxy, 2′-amino-uridine, following methodologies well established in peptide and oligonucleotide chemistry.
- De Napoli, Lorenzo,Di Fabio, Giovanni,D'Onofrio, Jennifer,Montesarchio, Daniela
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p. 1975 - 1979
(2007/10/03)
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- Improving the pyrophosphate-inosine phosphotransferase activity of Escherichia blattae acid phosphatase by sequential site-directed mutagenesis
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Escherichia blattae acid phosphatase/phosphotransferase (EB-AP/PTase) exhibits C-5′-position selective pyrophosphate-nucleoside phosphotransferase activity in addition to its intrinsic phosphatase. Improvement of its phosphotransferase activity was investigated by sequential site-directed mutagenesis. By comparing the primary structures of higher 5′-inosinic acid (5′-IMP) productivity and lower 5′-IMP productivity acid phosphatase/phosphotransferase, candidate residues of substitution were selected. Then a total of 11 amino acid substitutions were made with sequential substitutions. As the number of substituted amino acid residues increased, the 5′-IMP productivity of the mutant enzyme increased, and the activity of the 11 mutant phosphotransferases of EB-AP/PTase reached the same level as that of Morganella morganii AP/PTase. This result shows that Leu63, Ala65, Glu66, Asn69, Ser71, Asp116, Thr135, and Glu136, whose relevance was not directly established by structural analysis alone, also plays an important role in the phosphotransferase activity of EB-AP/PTase.
- Mihara, Yasuhiro,Ishikawa, Kohki,Suzuki, Ei-Ichiro,Asano, Yasuhisa
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p. 1046 - 1050
(2007/10/03)
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- Phosphorylation and dephosphorylation of polyhydroxy compounds by class A bacterial acid phosphatases
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Nonspecific acid phosphatases share a conserved active site with mammalian glucose-6-phosphatases (G6Pase). In this work we examined the kinetics of the phosphorylation of glucose and dephosphorylation of glucose-6-phosphate (G6P) catalysed by the acid phosphatases from Shigella flexneri (PhoN-Sf) and Salmonella enterica (PhoN-Se). PhoN-Sf is able to phosphorylate glucose regiospecifically to G6P, glucose-1-phosphate is not formed. The Km for glucose using pyrophosphate (PPi) as a phosphate donor is 5.3 mM at pH 6.0. This value is not significantly affected by pH in the pH region 4-6. The Km value for G6P by contrast is much lower (0.02 mM). Our experiments show these bacterial acid phosphatases form a good model for G6Pase. We also studied the phosphorylation of inosine to inosine monophosphate (IMP) using PPi as the phosphate donor. PhoN-Sf regiospecifically phosphorylates inosine to inosine-5′-monophosphate whereas PhoN-Se produces both 5′IMP and 3′IMP. The data show that during catalysis an activated phospho-enzyme intermediate is formed that is able to transfer its phosphate group to water, glucose or inosine. A general mechanism is presented of the phosphorylation and dephosphorylation reaction catalysed by the acid phosphatases. Considering the nature of the substrates that are phosphorylated it is likely that this class of enzyme is able to phosphorylate a wide range of hydroxy compounds.
- Tanaka, Naoko,Hasan, Zulfiqar,Hartog, Aloysius F.,Van Herk, Teunie,Wever, Ron
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p. 2833 - 2839
(2007/10/03)
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- Anti-HCV nucleoside derivatives
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The present invention comprises novel and known purine and pyrimidine nucleoside derivatives which have been discovered to be active against hepatitis C virus (HCV). The use of these derivatives for the treatment of HCV infection is claimed as are the novel nucleoside derivatives disclosed herein.
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- Is the anomeric effect an important factor in the rate of adenosine deaminase catalyzed hydrolysis of purine nucleosides? A direct comparison of nucleoside analogues constructed on ribose and carbocyclic templates with equivalent heterocyclic bases selected to promote hydration.
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The aglycone of (North)-methanocarbadeoxyadenosine [(N)-MCdA, (5)], a relatively weak substrate for adenosine deaminase (ADA)-relative rate of deamination ca. 100 times lower than adenosine-was modified with substitutions at positions 6 (6-fluoro, compound 6) and 8 (8-aza, compound 7) with the intent to improve the level of hydration and hence hydrolysis by ADA. In these substrates the fused cyclopropane moiety constrains the cyclopentane ring to mimic the conformation of a furanose sugar in the North hemisphere of the pseudorotational cycle, which matches the conformation of the ribose ring of adenosine in complex with ADA. The order of susceptibility to ADA hydrolysis was adenosine>>(N)-MCdA (5) approximately equal to(N)-6F-MCdP (6)>(N)-8-aza-MCdA (7). Despite the known fact that 8-azaadenosine is hydrolyzed twice as fast as adenosine, the corresponding carbocyclic analogue 7 was hydrolyzed at approximately half the rate of the parent 5. These results argue in favor of the critical role of the O(4') oxygen atom and its associated anomeric effect in assisting hydrolysis by ADA.
- Hernandez, Susana,Ford Jr., Harry,Marquez, Victor E
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p. 2723 - 2730
(2007/10/03)
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- Stability of disodium salt of inosine phosphate in aqueous solutions
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The HPLC method for the separation of the disodium salt of inosine phosphate (PIN) and the product of its transformation, inosine (IN) and hypoxanthine (HP) were developed and validated. The hydrolysis kinetics of disodium salt of inosine phosphate was studied in aqueous solution at 353 K over a pH range of 0,45-12,13.
- Jelinska,Lewandowski
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p. 331 - 335
(2007/10/03)
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- Lipid esters of nucleoside monophosphates and their use as immunosuppressive drugs
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The present invention is directed to new nucleoside monophosphate derivatives of lipid ester residues of general formula (I) wherein R1 represents an optionally substituted alkyl chain having 1-20 carbon atoms; R2 represents hydrogen, an optionally substituted alkyl chain having 1-20 carbon atoms; R3, R4 and R5 represent hydrogen, hydroxy, azido, amino, cyano, or halogen; X represents a valence dash, oxygen, sulfur, a sulfinyl or sulfonyl group; Y represents a valence dash, an oxygen or sulfur atom; B represents a purine and/or pyrimidine base; with the proviso that at least one of the residues R3 or R5 is hydrogen; to their tautomers and their physiologically acceptable salts of inorganic and organic acids and/or bases, as well as to processes for their preparation, and to drugs containing said compounds.
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- Conformationally restricted nucleosides. The reaction of adenosine deaminase with substrates built on a bicyclo[3.1.0]hexane template
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Adenosine deaminase (ADA) can discriminate between two distinct (North and South), conformationally rigid substrate conformers. (N)-methanocarba- 2'dA (4) is deaminated 100 times faster than the antipodal (S)-methanocarba- 2'dA (5), whereas a nonrigid analogue, aristeromycin (6), is deaminated at an intermediate rate. These results are in agreement with crystallographic data from ADA-ribonucleoside complexes showing the furanose ring of the bound purine in a C3'-endo (North) conformation. The data presented here suggests that 4 and 5 are useful probes to ascertain conformational preferences by purine metabolizing enzymes.
- Marquez, Victor E.,Russ, Pamela,Alonso, Randolph,Siddiqui, Maqbool A.,Shin, Kye-Jung,George, Clifford,Nicklaus, Marc C.,Dai, Fang,Ford Jr., Harry
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p. 521 - 530
(2007/10/03)
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- Synthesis of conformationally restricted carbocyclic nucleosides: The role of the O(4')-atom in the key hydration step of adenosine deaminase
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Conformationally restricted carbocyclic nucleosides with either a northern(N)-type conformation, i.e., N-type 2'-deoxy-methanocarba-adenosine 8 ((N)MCdAdo) or a southern(S)-type conformation, i.e. S-type 2'-deoxy- methanocarba-adenosine 9, ((S)MCdAdo), were used as substrates for adenosine deaminase (ADA) to assess the enzyme's preference for a fixed conformation relative to the flexible conformation represented by the carbocyclic nucleoside aristeromycin (10). Further comparison between the rates of deamination of these compounds with those of the two natural substrates adenosine (Ado: 1) and 2'-deoxyadenosine (dAdo; 2), as well as with that of the conformationally locked nucleoside LNA-Ado (11), which, like the natural substrates, has a furanose O(4') atom, helped differentiate between the roles of the O(4') anomeric effect and sugar conformation in controlling the rates of deamination by ADA. Differences in rates of deamination as large as 10000 can be attributed to the combined effect of the Q(4') atom and the enzyme's preference for an N-type conformation. The hypothesis proposed is that ADA's preference for N-type substrates is not arbitrary; it is rather the direct consequence of the conformationally dependent O(4') anomeric effect, which is more efficient in N-type conformers in promoting the formation of a covalent hydrate at the active site of the enzyme. The formation of a covalent hydrate at the active site of ADA precedes deamination. A new and efficient synthesis of the important carbobicyclic template 14a, a useful intermediate for the synthesis of (N)MCdAdo (8) and other conformationally restricted nucleosides is also reported.
- Marquez, Victor E.,Russ, Pamela,Alonso, Randolph,Siddiqui, Maqbool A.,Hernandez, Susana,George, Clifford,Nicklaus, Marc C.,Dai, Fang,Ford Jr., Harry
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p. 2119 - 2129
(2007/10/03)
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- A new synthesis of oxanosine and 2'-deoxyoxanosine
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An easy and more efficient synthesis of oxanosine and 2'-deoxyoxanosine has been developed, a key step in the reported synthesis is a new photochemical transformation by UV irradiation of 1-hydroxy derivatives of inosine.
- De Napoli, Lorenzo,Di Fabio, Giovanni,Messere, Anna,Montesarchio, Daniela,Piccialli, Gennaro,Varra, Michela
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p. 7397 - 7400
(2007/10/03)
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- High pressure in enzyme catalyzed organic reactions
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Adenosine deaminase and lipases catalyzed hydrolysis under high pressure were described. The hydrolysis by both enzymes was facilitated by high pressures. This technique was used for the kinetical resolution of racemic highly modified nucleosides having biological activities.
- Katagiri, Nobuya,Yamaguchi, Masahiko,Kaneko, Chikara
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p. 1023 - 1043
(2007/10/03)
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- 1,1,1,3,3,3-Hexafluoro-2-propanol for the Removal of the 4,4'-Dimethoxytrityl Protecting Group from the 5'-Hydroxyl of Acid-Sensitive Nucleosides and Nucleotides
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1,1,1,3,3,3-Hexafluoro-2-propanol is introduced as a suitable reagent and solvent for the detritylation of 5'-O-(4,4'-dimethoxytrityl)-nucleosides and -deoxy- nucleosides, especially those that are susceptible to N-glycosyl cleavage under more strongly acidic conditions.
- Leonard, Nelson J.,Neelima
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p. 7833 - 7836
(2007/10/02)
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- Reactions of Nitric Oxide with Amines in the Presence of Dioxygen
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Nitric oxide (NO), a multifaceted bioregulatory agent and an environmental pollutant, can effectively convert aromatic amines to the corresponding triazenes under aerobic conditions, but not under anaerobic conditions.Nucleic acid bases and nucleosides are also determinated via hydrolysis of the diazonium ion products with exposure to aerobic NO solution.A peroxynitrite radical or nitrogen dioxide is suggested to be the ultimate reactive species.
- Nagano, Tetsuo,Takizawa, Hiromasa,Hirobe, Masaaki
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p. 8239 - 8242
(2007/10/02)
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- AMP Deaminase as a Novel Practical Catalyst in the Synthesis of 6-Oxopurine Ribosides and Their Analogs
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Adenylic acid deaminase from Aspergillus niger (AMP deaminase; AMPDA; EC 3.5.4.6) has beenintroduced as a novel practical catalyst in the synthesis of 6-oxopurine riboside and their analogs.This enzyme has a very broad substrate specificity and has been used on a preparative scale for deamination of several derivatives of adenosine including phosphorylated, cyclic, carbocyclic as well as cyclic analogs.In addition, AMPDA catalyzes dechlorination and demethoxylation of the purine ribosides.Overall substrate specificity of AMPDA is much broader than that of adenosine deaminase which can also be used for the synthesis of 6-oxopurine ribosides.Although the stereoselectivity of AMPDA is modest, this enzyme has successfully been used in the synthesis of a novel antiviral agent, carbovir phosphonate (14), after the carbocyclic component was resolved via lipase-catalyzed hydrolysis or acylation.
- Margolin, Alexey L.,Borcherding, David R.,Wolf-Kugel, Dominique,Margolin, Nara
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p. 7214 - 7218
(2007/10/02)
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