Novel synthesis of nucleoside 5'-phosphoramidates through reaction of nucleoside triphosphates with amines mediated by trimethylsilyl chloride

Reaction of nucleoside triphosphates (NTPs) with amines in pyridine mediated by trimethylsilyl chloride produced nucleoside 5'-phosphoramidates in moderate yields without any preprotection of nucleosides and amino acid methyl esters. The reaction pathway is very similar to the mechanism of the RNA capping reaction, DNA or RNA ligation reaction, and catalysis of hydrolases and nucleases involving the formation of covalent enzyme-NMP (nucleoside 5'-monophosphate) intermediates in biological systems, which could provide a valuable clue for the enzymatic reactions.     

Synthesis of tritium-labeled nucleoside 5′-triphosphates and nucleoside 5′-diphosphates

Tritium-labeled nucleoside 5′-triphosphates (NTPs) nucleoside 5′-diphosphates (NDPs) containing the tritium label in positions 8 (in the purine nucleus) and 5 (in the pyrimidine nucleus) have been obtained by the dehalogenation of the corresponding bromine derivatives with gaseous tritium. The dehalogenation of the Br-NTPs and Br-NDPs was carried out at atmospheric pressure in an aqueous alkaline medium using palladium catalysts (5% Pd/BaSO₄ or α-Pd). The possibility of introducing a tritium label into nucleotides of the adenine series by the heterogenecus isotope exchange reaction with gaseous tritium in the presence of 5% Pd/BaSO₄ has been investigated. For the compounds synthesized, the compositions of the eluents used for the chromatographic isolation of the desired products are given. The molar activities of the compounds synthesized were between 370 and 740 TB_q/mole (10–20 kCi/mole).     

A highly selective and sensitive fluorescence sensing system for distinction between diphosphate and nucleoside triphosphates

Among the numerous chemosensors available for diphosphate (P(2)O(7)(4-), PPi) and nucleoside triphosphates (NTPs), only a few can distinguish between PPi and NTPs. Hence, very few bioanalytical applications based on such selective chemosensors have been realized. We have developed a new fluorescence sensing system for distinction between PPi and NTPs based on the combination of two sensors, a binuclear Zn(II) complex (1·2Zn) and boronic acid (BA), in which one chemosensor (1·2Zn) shows signal changes depending on the PPi (or NTP) concentration, and the other (BA) blocks the signal change caused by NTPs; this system enables the distinction of PPi from NTPs and is sensitive to nanomolar concentrations of PPi. The new sensing system has been successfully used for the direct quantification of RNA polymerase activity.     

Cross-coupling reactions of nucleoside triphosphates followed by polymerase incorporation. Construction and applications of base-functionalized nucleic acids

Construction of functionalized nucleic acids (DNA or RNA) via polymerase incorporation of modified nucleoside triphosphates is reviewed and selected applications of the modified nucleic acids are highlighted. The classical multistep approach for the synthesis of modified NTPs by triphosphorylation of modified nucleosides is compared to the novel approach consisting of direct aqueous cross-coupling reactions of unprotected halogenated nucleoside triphosphates. The combination of cross-coupling of NTPs with polymerase incorporation gives an efficient and straightforward two-step synthesis of modified nucleic acids. Primer extension using biotinylated templates followed by separation using streptavidine-coated magnetic beads and DNA duplex denaturation is used for preparation of modified single stranded oligonucleotides. Examples of using this approach for electrochemical DNA labelling and bioanalytical applications are given.     

Monomers for preparation of amide-linked RNA: asymmetric synthesis of all four nucleoside 5'-azido 3'-carboxylic acids

[reaction: see text] Recent discovery of RNA interference as an efficient and naturally occurring mechanism of gene regulation has reinvigorated the interest in chemically modified RNA. For potential in-vivo applications small interfering RNAs require chemical modifications to fine-tune the thermal stability and increase the cellular delivery and potency and in vivo half-life of the RNA duplexes. From this perspective, amides as neutral and hydrophobic internucleoside linkages in RNA are highly interesting modifications for RNA interference. Amides are remarkably good mimics of the phosphodiester backbone of RNA and can be prepared using a relatively straightforward peptide coupling chemistry. However, the progress in the field has been hampered by the shortage of efficient methods to synthesize the monomeric building blocks for such couplings, the nucleoside amino acid equivalents. Herein, we report enantioselective synthesis of 5'-azido 3'-carboxylic acid derivatives of all four natural ribonucleosides. The key transformations in our synthesis are a double asymmetric ene reaction and a stereoselective iodolactonization that form the basic carbon skeleton of the modified ribose. Standard nucleoside synthesis is followed by a short and highly efficient protecting group manipulation to give the enantiomerically pure (>98%) title compounds in 9-10 steps and 15-19% overall yields starting from small achiral molecules. The present results are a significant improvement over our first-generation racemic synthesis and compare favorably with the previously reported synthesis from nucleoside and carbohydrate precursors.     

Convenient synthesis of nucleoside borane diphosphate analogues: deoxy- and ribonucleoside 5'-P(alpha)-boranodiphosphates

The nucleoside boranophosphates, having one of the nonbridging phosphate oxygens substituted with a borane (BH(3)) group, have shown potential therapeutical applications as aptamers, antisense agents, and antiviral prodrugs. An oxathiaphospholane approach, which does not require exocyclic amine protection of the nucleobase, has been successfully developed to efficiently synthesize 5'-P(alpha)-boranodiphosphates of 2'-deoxythymidine, adenosine, guanosine, and uridine. The approach involves a key intermediate, the borane complex of nucleoside 5'-O-1,3,2-oxathiaphospholane 16, that undergoes a ring-opening reaction catalyzed by 1,4-diazabicyclo[5.4.0]-undec-7-ene to form the protected nucleoside 5'-P(alpha)-boranodiphosphate 18. Treatment of 18 with ammonium hydroxide yielded diastereoisomeric mixtures of nucleoside 5'-P(alpha)-boranodiphosphates 5. This oxathiaphospholane approach ensures the availability of nucleoside 5'-P(alpha)-boranodiphosphate analogues needed for antiviral drug research.     

Glycerol nucleoside triphosphates: synthesis and polymerase substrate activities

[Structure: see text] The synthesis of (S)-glycerol nucleoside triphosphates (gNTPs) and the analysis of their substrate activities for enzymatic polymerization is described. NTPs with simplified carbohydrate backbones such as the tNTPs (alpha-L-threose-NTPs) are polymerase substrates and offer the potential to create non-natural aptamer sequences with simplified backbones through enzymatic means. The acyclic (S)-GNA was modeled after the shortened alpha-threofuranosyl backbone. Here we describe the synthesis of (S)-glycerol NTPs and initial enzymatic testing of this further simplified nucleic acid backbone.     

Nucleotide Analogues Bearing a C2′ or C3′-Stereogenic All-Carbon Quaternary Center as SARS-CoV-2 RdRp Inhibitors

The design of novel nucleoside triphosphate (NTP) analogues bearing an all-carbon quaternary center at C2′ or C3′ is described. The construction of this all-carbon stereogenic center involves the use of an intramoleculer photoredox-catalyzed reaction. The nucleoside analogues (NA) hydroxyl functional group at C2′ was generated by diastereoselective epoxidation. In addition, highly enantioselective and diastereoselective Mukaiyama aldol reactions, diastereoselective N-glycosylations and regioselective triphosphorylation reactions were employed to synthesize the novel NTPs. Two of these compounds are inhibitors of the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2, the causal virus of COVID-19.     

RNA synthetic activity of glutamate dehydrogenase

The activity of glutamate dehydrogenase (GDH), an important enzyme in carbon and nitrogen metabolism, is routinely assayed by photometry. The RNA synthetic activity of the enzyme provides new technologies for assaying its activity. The enzyme was made to synthesize RNAs in the absence of DNA and total RNA but with different mixes of the four nucleoside triphosphates (NTPs) in order to investigate the RNA characteristics. RNase VI (hydrolyzes base-paired residues) digested the poly (U,A) RNA completely because the U and A residues were evenly distributed to produce many base-paired regions. Therefore, the synthesis of RNA by GDH was by random addition of NTPs. The RNA synthetic activity of the enzyme was at least 50-fold more active in the deamination than in the amination direction, thus providing a robust technology for assay of the enzyme's activity. cDNAs prepared from the RNAs were subjected to restriction fragment differential display polymerase chain reaction analyses. Sequencing of the cDNA fragments showed that some of the RNA synthesized by GDH shared sequence homology with total RNA. Database searches showed that the RNA fragments shared sequence homologies with the G proteins, adenosine triphosphatase, calmodulin, phosphoenol pyruvate (PEP) carboxylase, and PEP carboxykinase, thus explaining the molecular mode of their functions in signal transduction.     

Synthesis and properties of 5-cyano-substituted nucleoside analog with a donor-donor-acceptor hydrogen-bonding pattern

6-Aminopyridin-2-ones form Watson-Crick pairs with complementary purine analogues to add a third nucleobase pair to DNA and RNA, if an electron-withdrawing group at position 5 slows oxidation and epimerization. In previous work with a nucleoside analogue trivially named dZ, the electron withdrawing unit was a nitro group. Here, we describe an analogue of dZ (cyano-dZ) having a cyano group instead of a nitro group, including its synthesis, pK(a), rates of acid-catalyzed epimerization, and enzymatic incorporation.     

Hydrophilic interaction chromatography of nucleoside triphosphates with temperature as a separation parameter

Eight deoxynucleoside triphosphates (dNTPs) and nucleoside triphosphates (NTPs): ATP, CTP, GTP, UTP, dATP, dCTP, dGTP and dTTP, were separated with two 15 cm ZIC-pHILIC columns coupled in series, using LC-UV instrumentation. The polymer-based ZIC-pHILIC column gave significantly better separations and peak shape than a silica-based ZIC-HILIC column. Better separations were obtained with isocratic elution as compared to gradient elution. The temperature markedly affected the selectivity and could be used to fine tune separation. The analysis time was also affected by temperature, as lower temperatures surprisingly reduced the retention of the nucleotides. dNTP/NTP standards could be separated in 35 min with a flow rate of 200 μL/min. In Escherichia coli cell culture samples dNTP/NTPs could be selectively separated in 7 0min using a flow rate of 100 μL/min.     

Synthetic utility of an isolable nucleoside phosphonium salt

The reaction of O6-benzyl-3',5'-bis- O-( tert-butyldimethylsilyl)-2'-deoxyxanthosine with 1H-benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) yielded the nucleoside C-2 tris(dimethylamino)phosphonium hexafluorophosphate salt as a stable, isolable species. This is in contrast to reactions of inosine nucleosides with BOP, where the in situ formed phosphonium salts undergo subsequent reaction to yield O6-(benzotriazol-1-yl)inosine derivatives. The phosphonium salt obtained from the 2'-deoxyxanthosine derivative can be effectively used to synthesize N2-modified 2'-deoxyguanosine analogues. Using this salt, a new synthesis of an acrolein-2'-deoxyguanosine adduct has also been accomplished.     

Convenient synthesis of nucleoside 5'-triphosphates for RNA transcription

By generating a selective phosphitylating reagent in situ, nucleoside 5'-triphosphates can be conveniently synthesized in one pot. This novel strategy without nucleoside protection has been developed to largely simplify synthesis of the nucleoside triphosphates. This demonstrated principle can be applied to the 5'-triphosphate synthesis of both native and modified nucleosides.     

Bisphosphonate derivatives of nucleoside antimetabolites: hydrolytic stability and hydroxyapatite adsorption of 5'-beta,gamma-methylene and 5'-beta,gamma-(1-hydroxyethylidene) triphosphates of 5-fluorouridine and ara-cytidine

Kinetics of the hydrolytic reactions of four bisphosphonate derivatives of nucleoside antimetabolites, viz., 5-fluorouridine 5'-beta,gamma-(1-hydroxyethylidene) triphosphate ( 4), 5-fluorouridine 5'-beta,gamma-methylene triphosphate ( 5), ara-cytidine 5'-beta,gamma-(1-hydroxyethylidene) triphosphate ( 6), and ara-cytidine 5'-beta,gamma-methylene triphosphate ( 7), have been studied over a wide pH range (pH 1.0-8.5) at 90 degrees C. With each compound, the disappearance of the starting material was accompanied by formation of the corresponding nucleoside 5'-monophosphate, the reaction being up to 2 orders of magnitude faster with the beta,gamma-(1-hydroxyethylidene) derivatives ( 4, 6) than with their beta,gamma-methylene counterparts ( 5, 7). With compound 7, deamination of the cytosine base competed with the phosphate hydrolysis at pH 3-6. The measurements at 37 degrees C (pH 7.4) in the absence and presence of divalent alkaline earth metal ions (Mg (2+) and Ca (2+)) showed no sign of metal ion catalysis. Under these conditions, the initial product, nucleoside 5'-monophosphate, underwent rapid dephosphorylation to the corresponding nucleoside. Hydrolysis of the beta,gamma-methylene derivatives ( 5, 7) to the corresponding nucleoside 5'-monophosphates was markedly faster in mouse serum than in aqueous buffer (pH 7.4), the rate-acceleration being 5600- and 3150-fold with 5 and 7, respectively. In human serum, the accelerations were 800- and 450-fold compared to buffer. In striking contrast, the beta,gamma-(1-hydroxyethylidene) derivatives did not experience a similar decrease in hydrolytic stability. The stability in human serum was comparable to that in aqueous buffer (tau 1/2 = 17 and 33 h with 4 and 6, respectively), and on going to mouse serum, a 2- to 4-fold acceleration was observed. To elucidate the mineral-binding properties of 4- 7, their retention on a hydroxyapatite column was studied and compared to that of zoledronate ( 1a) and nucleoside mono-, di-, and triphosphates.     

Synthesis of 5'-amino-5'-phosphonate analogues of pyrimidine nucleoside monophosphates

The 5'-amino-5'-phosphono derivatives of cytidine, cytosine arabinoside (ara-C), and uridine have been prepared via the corresponding nucleoside aldehydes. Phosphite addition to imines derived from the nucleoside aldehydes and p-methoxybenzylamine was efficient, and use of this amine allowed cleavage of the products to the parent amino phosphonic acids. The phosphite additions proved to be diastereoselective, with the cytidine and uridine derivatives favoring the 5'S stereochemistry and the ara-C derivative favoring the 5'R isomer. The stereochemistry of one cytidine derivative was established by single-crystal diffraction analysis, and detailed comparisons of the (13)C NMR data allowed assignments of the other amino phosphonates.     

Acetyl derivatives of nucleoside 5′-triphosphates,I

Acetyl derivatives of nucleoside 5′-phosphates and nucleoside 5′-triphosphates (both ribo and deoxyribo series) were prepared by accomplishing partial (O-) and full (N, O-) acetylation of the basic compounds with acetic anhydride in pyridine medium.     

Preparation of a mixture of nucleoside triphosphates suitable for use in synthesis of nucleotide phosphate sugars from ribonucleic acid using nuclease P1, a mixture of nucleoside monophosphokinases and acetate kinase

This paper (1) describes the enzymatic synthesis of a mixture of adenosine, guanosine, cytidine, and uridine triphosphates (ATP, GTP, CTP, and UTP) from ribonucleic acid (RNA). RNA was hydrolyzed by nuclease P1 to a mixture of 5'-nucleoside monophosphates. This mixture was converted to the nucleoside triphosphates using a mixture of nucleoside monophosphate kinases and acetate kinase, with acetyl phosphate as the ultimate phosphoryl donor. The nucleoside monophosphokinases were extracted from brewer's yeast in a four-step procedure. The specific activity of the yeast enzyme preparation after gel permeation chromatography was sufficiently high that the yeast kinases could be immobilized in volumes that were practical for laboratory scale syntheses. Conversions from NMP to NTP in a mixture containing 0.34 mol of total nucleoside phosphates were: ATP, 90%; GTP, 90%; CTP, 60%; and UTP, 40%.     

Metabolic stability of the nucleoside transport system of Novikoff rat hepatoma cells

Rates of transport of uridine and thymidine, estimated with a rapid sampling technique, did not change with culture age. Inhibition of cellular RNA and protein synthesis for periods up to 6 h, did not lead to a loss of nucleoside transport activity. Mild treatment of cell suspensions with trypsin or neuraminidase had no effect on the kinetics of thymidine transport. Thus we conclude, contrary to previous reports, that nucleoside transporters are metabolically stable and that the decreases in nucleoside uptake rates observed with decreased protein synthesis reflect loss of nucleoside kinase activities. These kinases (which have narrow substrate specificity) rather than the membrane-associated, transport apparatus (which has broad substrate specificity) are the most likely sites for regulation of nucleoside uptake.