63-37-6Relevant articles and documents
Cell- And Polymerase-Selective Metabolic Labeling of Cellular RNA with 2′-Azidocytidine
Wang, Danyang,Zhang, Yu,Kleiner, Ralph E.
supporting information, p. 14417 - 14421 (2020/10/13)
Metabolic labeling of cellular RNA is a powerful approach to investigate RNA biology. In addition to revealing whole transcriptome dynamics, targeted labeling strategies can be used to study individual RNA subpopulations within complex systems. Here, we describe a strategy for cell- and polymerase-selective RNA labeling with 2′-azidocytidine (2′-AzCyd), a modified nucleoside amenable to bioorthogonal labeling with SPAAC chemistry. In contrast to 2′-OH-containing pyrimidine ribonucleosides, which rely upon uridine-cytidine kinase 2 (UCK2) for activation, 2′-AzCyd is phosphorylated by deoxycytidine kinase (dCK), and we find that expression of dCK mediates cell-selective 2′-AzCyd labeling. Further, 2′-AzCyd is primarily incorporated into rRNA and displays low cytotoxicity and high labeling efficiency. We apply our system to analyze the turnover of rRNA during ribophagy induced by oxidative stress or mTOR inhibition to show that 28S and 18S rRNAs undergo accelerated degradation. Taken together, our work provides a general approach for studying dynamic RNA behavior with cell and polymerase specificity and reveals fundamental insights into nucleotide and nucleic acid metabolism.
Improved synthesis of cytidine diphosphate choline (CDP-choline) via selective phosphorylation
Xia, Ran,Sun, Li-Ping,Chen, Lei-Shan
, p. 358 - 360 (2016/07/06)
An improved, three-step synthesis of cytidine diphosphate choline (CDP-choline) from cytidine was achieved in 68% overall yield. Selective phosphorylation of cytidine was accomplished by the use of morpholinophosphodichloridate to give cytidine-5′-phosphomorpholide, which was condensed with choline phosphate chloride in the presence of a catalytic amount of H2SO4 to give CDP-choline. The intermediates and products could be efficiently purified by recrystallisation, thus avoiding the use of chromatography at all stages. The reaction could be scaled up to 200 g in 64% overall yield, making this route attractive for industrial application.
Fully automated continuous meso-flow synthesis of 5′-nucleotides and deoxynucleotides
Zhu, Chenjie,Tang, Chenglun,Cao, Zhi,He, Wei,Chen, Yong,Chen, Xiaochun,Guo, Kai,Ying, Hanjie
, p. 1575 - 1581 (2015/02/19)
The first continuous meso-flow synthesis of natural and non-natural 5′-nucleotides and deoxynucleotides is described, representing a significant advance over the corresponding in-flask method. By means of this meso-flow technique, a synthesis with time consumption and high-energy consumption becomes facile to generate products with great efficiency. An abbreviated duration, satisfactory output, and mild reaction conditions are expected to be realized under the present procedure.
Immobilized Drosophila melanogaster deoxyribonucleoside kinase (DmdNK) as a high performing biocatalyst for the synthesis of purine arabinonucleotides
Serra, Immacolata,Conti, Silvia,Piskur, Jure,Clausen, Anders R.,Munch-Petersen, Birgitte,Terreni, Marco,Ubiali, Daniela
, p. 563 - 570 (2014/05/20)
Fruit fly (Drosophila melanogaster) deoxyribonucleoside kinase (DmdNK; EC: 2.7.1.145) was characterized for its substrate specificity towards natural and non-natural nucleosides, confirming its potential in the enzymatic synthesis of modified nucleotides. DmdNK was adsorbed on a solid ion exchange support (bearing primary amino groups) achieving an expressed activity >98%. Upon cross-linking with aldehyde dextran, expressed activity was 30-40%. Both biocatalysts (adsorbed or cross-linked) were stable at pH 10 and room temperature for 24 h (about 70% of retained activity). The cross-linked DmdNK preparation was used for the preparative synthesis of arabinosyladenine monophosphate (araA-MP) and fludarabine monophosphate (FaraAMP). Upon optimization of the reaction conditions (50 mM ammonium acetate, substrate/ATP ratio= 1:1.25, 2 mM MgCl2, 378C, pH 8) immobilized DmdNK afforded the title nucleotides with high conversion (>90%), whereas with the soluble enzyme lower conversions were achieved (78-87%). Arabinosyladenine monophosphate was isolated in 95% yield and high purity (96.5%).
PDE7A1 hydrolyzes cCMP
Monzel, Maike,Kuhn, Maike,B?hre, Heike,Seifert, Roland,Schneider, Erich H.
, p. 3469 - 3474 (2015/03/31)
The degradation and biological role of the cyclic pyrimidine nucleotide cCMP is largely elusive. We investigated nucleoside 3′,5′-cyclic monophosphate (cNMP) specificity of six different recombinant phosphodiesterases (PDEs) by using a highly-sensitive HPLC-MS/MS detection method. PDE7A1 was the only enzyme that hydrolyzed significant amounts of cCMP. Enzyme kinetic studies using purified GST-tagged truncated PDE7A1 revealed a cCMP KM value of 135 ± 19 μM. The Vmax for cCMP hydrolysis reached 745 ± 27 nmol/(min mg), which is about 6-fold higher than the corresponding velocity for adenosine 3′,5′-cyclic monophosphate (cAMP) degradation. In summary, PDE7A is a high-speed and low-affinity PDE for cCMP.
The reaction of activated RNA species with aqueous fluoride ion: A convenient synthesis of nucleotide 5′-phosphorofluoridates and a note on the mechanism
Aldersley, Michael F.,Joshi, Prakash C.,Schwartz, Herbert M.,Kirby, Anthony J.
, p. 1464 - 1466 (2014/03/21)
The chemistry of 5′-phosphorimidazolides of ribonucleosides is extended to include their reaction with alkali metal fluorides in aqueous solution. High yields of 5′-phosphorofluoridates are formed, especially with potassium fluoride, but no detectable oligomerization products were formed. A combination of HPLC, mass spectrometry, synthesis, kinetics, and NMR confirms the identities of the products. Judicious control of pH leads to higher yields in shorter reaction times. This new methodology contrasts favorably with other synthetic routes involving non-aqueous chemistry or aqueous chemistry with a nucleotide triphosphate.
Biosynthetic origin and mechanism of formation of the aminoribosyl moiety of peptidyl nucleoside antibiotics
Chi, Xiuling,Pahari, Pallab,Nonaka, Koichi,Van Lanen, Steven G.
supporting information; experimental part, p. 14452 - 14459 (2011/11/04)
Several peptidyl nucleoside antibiotics that inhibit bacterial translocase I involved in peptidoglycan cell wall biosynthesis contain an aminoribosyl moiety, an unusual sugar appendage in natural products. We present here the delineation of the biosynthetic pathway for this moiety upon in vitro characterization of four enzymes (LipM-P) that are functionally assigned as (i) LipO, an l-methionine:uridine-5′-aldehyde aminotransferase; (ii) LipP, a 5′-amino-5′-deoxyuridine phosphorylase; (iii) LipM, a UTP:5-amino-5-deoxy-α-d-ribose-1-phosphate uridylyltransferase; and (iv) LipN, a 5-amino-5-deoxyribosyltransferase. The cumulative results reveal a unique ribosylation pathway that is highlighted by, among other features, uridine-5′-monophosphate as the source of the sugar, a phosphorylase strategy to generate a sugar-1-phosphate, and a primary amine-requiring nucleotidylyltransferase that generates the NDP-sugar donor.
Synthesis of oligoribonucleotides with phosphonate-modified linkages
Pav, Ondej,Koiova, Ivana,Barvik, Ivan,Pohl, Radek,Budinsky, Milo,Rosenberg, Ivan
supporting information; experimental part, p. 6120 - 6126 (2011/10/10)
Solid phase synthesis of phosphonate-modified oligoribonucleotides using 2′-O-benzoyloxymethoxymethyl protected monomers is presented in both 3′→5′ and 5′→3′ directions. Hybridisation properties and enzymatic stability of oligoribonucleotides modified by regioisomeric 3′- and 5′-phosphonate linkages are evaluated. The introduction of the 5′-phosphonate units resulted in moderate destabilisation of the RNA/RNA duplexes (ΔTm -1.8 °C/mod.), whereas the introduction of the 3′-phosphonate units resulted in considerable destabilisation of the duplexes (ΔTm -5.7 °C/mod.). Molecular dynamics simulations have been used to explain this behaviour. Both types of phosphonate linkages exhibited remarkable resistance in the presence of ribonuclease A, phosphodiesterase I and phosphodiesterase II.
Deciphering the Late Biosynthetic Steps of Antimalarial Compound FR-900098
Johannes, Tyler W.,DeSieno, Matthew A.,Griffin, Benjamin M.,Thomas, Paul M.,Kelleher, Neil L.,Metcalf, William W.,Zhao, Huimin
experimental part, p. 57 - 64 (2010/08/06)
FR-900098 is a potent chemotherapeutic agent for the treatment of malaria. Here we report the heterologous production of this compound in Escherichia coli by reconstructing the entire biosynthetic pathway using a three-plasmid system. Based on this system, whole-cell feeding assays in combination with in vitro enzymatic activity assays reveal an unusual functional role of nucleotide conjugation and lead to the complete elucidation of the previously unassigned late biosynthetic steps. These studies also suggest a biosynthetic route to a second phosphonate antibiotic, FR-33289. A thorough understanding of the FR-900098 biosynthetic pathway now opens possibilities for metabolic engineering in E. coli to increase production of the antimalarial antibiotic and combinatorial biosynthesis to generate novel derivatives of FR-900098.
Evaluation of the role of three candidate human kinases in the conversion of the hepatitis C virus inhibitor 2′-C-methyl-cytidine to its 5′-monophosphate metabolite
Golitsina, Nina L.,Danehy Jr., Francis T.,Fellows, Ross,Cretton-Scott, Erika,Standring, David N.
experimental part, p. 470 - 481 (2010/12/19)
Nucleoside analogs are effective inhibitors of the hepatitis C virus (HCV) in the clinical setting. One such molecule, 2′-C-methyl-cytidine (2′-MeC), entered clinical development as NM283, a valine ester prodrug form of 2′-MeC possessing improved oral bioavailability. To be active against HCV, 2′-MeC must be converted to 2′-MeC triphosphate which inhibits NS5B, the HCV RNA-dependent RNA polymerase. Conversion of 2′-MeC to 2′-MeC monophosphate is the first step in 2′-MeC triphosphate production and is thought to be the rate-limiting step. Here we investigate which of three possible enzymes, deoxycytidine kinase (dCK), uridine-cytidine kinase 1 (UCK1), or uridine-cytidine kinase 2 (UCK2), mediate this first phosphorylation step. Purified recombinant enzymes UCK2 and dCK, but not UCK1, could phosphorylate 2′-MeC in vitro. However, siRNA knockdown experiments in three human cell lines (HeLa, Huh7 and HepG2) defined UCK2 and not dCK as the key kinase for the formation of 2′-MeC monophosphate in cultured human cells. These results underscore the importance of confirming enzymatic kinase data with appropriate cell-based assays. Finally, we present data suggesting that inefficient phosphorylation by UCK2 likely limits the antiviral activity of 2′-MeC against HCV. This paves the way for the use of a nucleotide prodrug approach to overcome this limitation.
