Fluorescent dATP for DNA Synthesis In Vivo.

Fluorescent nucleoside triphosphates are powerful probes of DNA synthesis, but their potential use in living animals has been previously underexplored. Here, we report the synthesis and characterization of 7-deaza-(1,2,3-triazole)-2'-deoxyadenosine-5'-triphosphate (dATP) derivatives of tetramethyl rhodamine ("TAMRA-dATP"), cyanine ("Cy3-dATP"), and boron-dipyrromethene ("BODIPY-dATP"). Upon microinjection into live zebrafish embryos, all three compounds were incorporated into the DNA of dividing cells; however, their impact on embryonic toxicity was highly variable, depending on the exact structure of the dye. TAMRA-EdATP exhibited superior characteristics in terms of its high brightness, low toxicity, and rapid incorporation and depletion kinetics in both a vertebrate (zebrafish) and a nematode (Caenorhabditis elegans). TAMRA-EdATP allows for unprecedented, real-time visualization of DNA replication and chromosome segregation in vivo.

2-Formyl-dATP as Substrate for Polymerase Synthesis of Reactive DNA Bearing an Aldehyde Group in the Minor Groove.

2-Formyl-2'-deoxyadenosine triphosphate (dCHO ATP) was synthesized and tested as a substrate in enzymatic synthesis of DNA modified in the minor groove with a reactive aldehyde group. The multistep synthesis of dCHO ATP was based on the preparation of protected 2-dihydroxyethyl-2'-deoxyadenosine intemediate, which was triphosphorylated and converted to aldehyde through oxidative cleavage. The dCHO ATP triphosphate was a moderate substrate for KOD XL DNA polymerase, and was used for enzymatic synthesis of some sequences using primer extension (PEX). On the other hand, longer sequences (31-mer) with higher number of modifications, or sequences with modifications at adjacent positions did not give full extension. Single-nucleotide extension followed by PEX was used for site-specific incorporation of one aldehyde-linked adenosine into a longer 49-mer sequence. The reactive formyl group was used for cross-linking with peptides and proteins using reductive amination and for fluorescent labelling through oxime formation with an AlexaFluor647-linked hydroxylamine.

Synthesis of nucleotide analogues, EFdA, EdA and EdAP, and the effect of EdAP on hepatitis B virus replication.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) and 4'-ethynyl-2'-deoxyadenosine (EdA) are nucleoside analogues which inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. EdAP, a cyclosaligenyl (cycloSal) phosphate derivative of EdA, inhibits the replication of the influenza A virus. The common structural feature of these compounds is the ethynyl group at the 4'-position. In this study, these nucleoside analogues were prepared by a common synthetic strategy starting from the known 1,2-di-O-acetyl-D-ribofuranose. Biological evaluation of EdAP revealed that this compound reduced hepatitis B virus (HBV) replication dose-dependently without cytotoxicity against host cells tested in this study.

Design, Synthesis, and Biological Evaluation of EdAP, a 4'-Ethynyl-2'-Deoxyadenosine 5'-Monophosphate Analog, as a Potent Influenza a Inhibitor.

Influenza A viruses leading to infectious respiratory diseases cause seasonal epidemics and sometimes periodic global pandemics. Viral polymerase is an attractive target in inhibiting viral replication, and 4'-ethynyladenosine, which has been reported as a highly potent anti-human immunodeficiency virus (HIV) nucleoside derivative, can work as an anti-influenza agent. Herein, we designed and synthesized a 4'-ethynyl-2'-deoxyadenosine 5'-monophosphate analog called EdAP (5). EdAP exhibited potent inhibition against influenza virus multiplication in Madin-Darby canine kidney (MDCK) cells transfected with human α2-6-sialyltransferase (SIAT1) cDNA and did not show any toxicity toward the cells. Surprisingly, this DNA-type nucleic acid analog (5) inhibited the multiplication of influenza A virus, although influenza virus is an RNA virus that does not generate DNA.

6-Aryl-4-amino-pyrimido[4,5-b]indole 2'-deoxyribonucleoside triphosphates (benzo-fused 7-deaza-dATP analogues): Synthesis, fluorescent properties, enzymatic incorporation into DNA and DNA-protein binding study.

Four 6-substituted 4-amino-pyrimido[4,5-b]indole 2'-deoxyribonucleoside triphosphates (dA(BX)TPs) were prepared by glycosylation of 4,6-dichloropyrimidoindole followed by ammonolysis, cross-coupling and triphosphorylation. They were found to be moderate to good substrates for DNA polymerases in primer extension. They also exerted fluorescence with emission maxima 335-378nm. When incorporated to oligonucleotide probes, they did not show significant mismatch discrimination but the 6-benzofuryl 4-amino-pyrimido[4,5-b]indole nucleotide displayed a useful sensitivity to protein binding in experiment with SSB protein.

Concise and efficient synthesis of 3'-O-tetraphosphates of 2'-deoxyadenosine and 2'-deoxycytidine.

We describe concise and efficient synthesis of biologically very important 3'-O-tetraphosphates namely 2'-deoxyadenosine-3'-O-tetraphosphate (2'-d-3'-A4P) and 2'-deoxycytidine-3'-O-tetra-phosphate (2'-d-3'-C4P). N(6)-benzoyl-5'-O-levulinoyl-2'-deoxyadenosine was converted into N(6)-benzoyl-5'-O-levulinoyl-2'-deoxyadenosine-3'-O-tetraphosphate in 87% yield using a one-pot synthetic methodology. One-step concurrent deprotection of N(6)-benzoyl and 5'-O-levulinoyl groups using concentrated aqueous ammonia resulted 2'-d-3'-A4P in 74% yield. The same synthetic strategy was successfully employed to convert N(4)-benzoyl-5'-O-levulinoyl-2'-deoxycytidine into 2'-d-3'-C4P in 68% yield.

Gram-scale chemical synthesis of 2'-deoxynucleoside-5'-o-triphosphates.

A simple, straightforward, reliable, and efficient method for the chemical synthesis of sodium salt of 2'-deoxynucleoside-5'-O-triphosphates (dNTPs), starting from the corresponding nucleoside, is described. This improved "one-pot, three-step" synthetic strategy involves the monophosphorylation of nucleoside, followed by reaction with tributylammonium pyrophosphate and hydrolysis of the resulting cyclic intermediate to provide the corresponding dNTP in good yields (65% to 70%). It is noteworthy that the protocol holds good for both the purine deoxynucleotides, such as 2'-deoxyguanosine-5'-O-triphosphate (dGTP) and 2'-deoxyadenosine-5'-O-triphosphate (dATP), and pyrimidine deoxynucleotides, such as 2'-deoxycytidine-5'-O-triphosphate (dCTP), thymidine-5'-O-triphosphate (TTP), and 2'-deoxyuridine-5'-O-triphosphate (dUTP).

Pre-steady state kinetic analysis of cyclobutyl derivatives of 2'-deoxyadenosine 5'-triphosphate as inhibitors of HIV-1 reverse transcriptase.

Pre-steady state kinetic analysis was utilized for biochemical evaluation of a series of cyclobutyl adenosine nucleotide analogs with HIV-1 RT(WT). The phosphonyl-diphosphate form of the cyclobutyl nucleotide, 5, was the most efficiently incorporated of the series. Nucleotide 5 was fourfold more efficiently incorporated than the FDA approved TFV-DP by RT(WT). The kinetics of incorporation for 5 using the drug resistant mutant enzyme K65R was also determined. Compound 5 was threefold more efficiently incorporated compared to TFV-DP with RT(K65R). These results demonstrate cyclobutyl adenosine analogs can act as substrates for incorporation by HIV-1 RT and be a potential scaffold for HIV inhibitors.

The synthesis of 2'-methylseleno adenosine and guanosine 5'-triphosphates.

Modified nucleoside triphosphates (NTPs) represent powerful building blocks to generate nucleic acids with novel properties by enzymatic synthesis. We have recently demonstrated the access to 2'-SeCH(3)-uridine and 2'-SeCH(3)-cytidine derivatized RNAs for applications in RNA crystallography, using the corresponding nucleoside triphosphates and distinct mutants of T7 RNA polymerase. In the present note, we introduce the chemical synthesis of the novel 2'-methylseleno-2'-deoxyadenosine and -guanosine 5'-triphosphates (2'-SeCH(3)-ATP and 2'-SeCH(3)-GTP) that represent further candidates for the enzymatic RNA synthesis with engineered RNA polymerases.

Optimization of pyrosequencing reads by superior successive incorporation efficiency of improved 2'-deoxyadenosine-5'-triphosphate analogs.

Conventional pyrosequencing using 2'-deoxyadenosine-5'-O-(1-thiotriphosphate) (dATPαS) is problematic due to the high cost of the substrate (dATPαS) and deterioration in the accuracy of incorporation to read through poly(T) regions. One reason for these problems is that dATPαS has a sulfur on the α-phosphate and also has isomers (Sp and Rp). To solve these problems, 11 nucleotide substrates, which could replace dATPαS in pyrosequencing, were newly synthesized. All substrates were modified on the seventh or eighth position of the adenine base from normal dATP. We found that the substrate that had an ethenyl-linked modified group on the seventh position of the adenine base had low activity in the luciferase reaction and high incorporation efficiency with the thymine base. One substrate in particular had 10-fold better incorporation efficiency than dATPαS. The new nucleotide substrate satisfied all conditions as a replacement of dATPαS.

Quantification of recombinant and platelet P2Y(1) receptors utilizing a [(125)I]-labeled high-affinity antagonist 2-iodo-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate ([(125)I]MRS2500).

The ADP-activated P2Y(1) receptor is broadly expressed and plays a crucial role in ADP-promoted platelet aggregation. We previously synthesized 2-iodo-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate (MRS2500), as a selective, high-affinity, competitive antagonist of this receptor. Here we report utilization of a trimethylstannyl precursor molecule for the multi-step radiochemical synthesis of a [(125)I]-labeled form of MRS2500. [(125)I]MRS2500 bound selectively to Sf9 insect cell membranes expressing the human P2Y(1) receptor but did not specifically bind to membranes isolated from empty vector-infected cells. Binding of [(125)I]MRS2500 to P2Y(1) receptors was saturable with a Kd of 1.2nM. Known agonists and antagonists of the P2Y(1) receptor inhibited [(125)I]MRS2500 binding to P2Y(1) receptor-expressing membranes with potencies in agreement with those previously observed in functional assays of this receptor. A high-affinity binding site for [(125)I]MRS2500 also was observed on intact human platelets (Kd=0.61nM) and mouse platelets (Kd=1.20nM) that exhibited the pharmacological selectivity of the P2Y(1) receptor. The densities of sites observed were 151 sites/platelet and 229 sites/platelet in human and mouse platelets, respectively. In contrast, specific binding was not observed in platelets isolated from P2Y(1) receptor(-/-) mice. Taken together, these data illustrate the synthesis and characterization of a novel P2Y(1) receptor radioligand and its utility for examining P2Y(1) receptors natively expressed on human and mouse platelets.

Chemical synthesis, DNA incorporation and biological study of a new photocleavable 2'-deoxyadenosine mimic.

The phototriggered cleavage of chemical bonds has found numerous applications in biology, particularly in the field of gene sequencing through photoinduced DNA strand scission. However, only a small number of modified nucleosides that are able to cleave DNA at selected positions have been reported in the literature. Herein, we show that a new photoactivable deoxyadenosine analogue, 3-nitro-3-deaza-2'-deoxyadenosine (d(3-NiA)), was able to induce DNA backbone breakage upon irradiation (lambda > 320 nm). The d(3-NiA) nucleoside was chemically incorporated at desired positions into 40-mer oligonucleotides as a phosphoramidite monomer and subsequent hybridization studies confirmed that the resulting modified duplexes display a behaviour that is close to that of the related natural sequence. Enzymatic action of the Klenow fragment exonuclease free revealed the preferential incorporation of dAMP opposite the 3-NiA base. On the other hand, incorporation of the analogous 3-NiA triphosphate to a primer revealed high enzyme efficiency and selectivity for insertion opposite thymine. Furthermore, only the enzymatically synthesized base pair 3-NiA:T was a substrate for further extension by the enzyme. All the hybridization and enzymatic data indicate that this new photoactivable 3-NiA triphosphate can be considered as a photochemically cleavable dATP analogue.

Alpha,beta-difluoromethylene deoxynucleoside 5'-triphosphates: a convenient synthesis of useful probes for DNA polymerase beta structure and function.

Alpha,beta-difluoromethylene deoxynucleoside 5'-triphosphates (dNTPs, N = A or C) are advantageously obtained via phosphorylation of corresponding dNDP analogues using catalytic ATP, PEP, nucleoside diphosphate kinase, and pyruvate kinase. DNA pol beta K(d) values for the alpha,beta-CF(2) and unmodified dNTPs, alpha,beta-NH dUTP, and the alpha,beta-CH(2) analogues of dATP and dGTP are discussed in relation to the conformations of alpha,beta-CF(2) dTTP versus alpha,beta-NH dUTP bound into the enzyme active site.

Ultraviolet crosslinking of DNA protein complexes via 8-azidoadenine.

The synthesis of 8-azido-2'-deoxyadenosine-5'-triphosphate is described. The photoreactive dATP analog was characterized by thin layer chromatography and UV spectroscopy. Its photoreactivity upon UV irradiation was studied. After incorporation of this dATP analog by nick translation into DNA containing the tet operator sequence the investigation of the interactions between tet operator DNA and Tet repressor becomes possible. Photocrosslinking of protein to DNA was demonstrated by the reduced migration of the DNA protein crosslinks in SDS polyacrylamide gel electrophoresis.

Synthesis of 8-bromo-, 8-methyl- and 8-phenyl-dATP and their polymerase incorporation into DNA.

dATP derivatives bearing Br, Me or Ph groups in position 8 were prepared and tested as substrates for DNA polymerases to show that 8-Br-dATP and 8-Me-dATP were efficiently incorporated, while 8-Ph-dATP was a poor substrate due to its bulky Ph group.

Termination of DNA synthesis by N6-alkylated, not 3'-O-alkylated, photocleavable 2'-deoxyadenosine triphosphates.

The Human Genome Project has facilitated the sequencing of many species, yet the current Sanger method is too expensive, labor intensive and time consuming to accomplish medical resequencing of human genomes en masse. Of the 'next-generation' technologies, cyclic reversible termination (CRT) is a promising method with the goal of producing accurate sequence information at a fraction of the cost and effort. The foundation of this approach is the reversible terminator (RT), its chemical and biological properties of which directly impact the performance of the sequencing technology. Here, we have discovered a novel paradigm in RT chemistry, the attachment of a photocleavable, 2-nitrobenzyl group to the N(6)-position of 2'-deoxyadenosine triphosphate (dATP), which, upon incorporation, terminates DNA synthesis. The 3'-OH group of the N(6)-(2-nitrobenzyl)-dATP remains unblocked, providing favorable incorporation and termination properties for several commercially available DNA polymerases while maintaining good discrimination against mismatch incorporations. Upon removal of the 2-nitrobenzyl group with UV light, the natural nucleotide is restored without molecular scarring. A five-base experiment, illustrating the exquisite, stepwise addition through a homopolymer repeat, demonstrates the applicability of the N(6)-(2-nitrobenzyl)-dATP as an ideal RT for CRT sequencing.

Recognition of nucleotide analogs containing the 7,8-dihydro-8-oxo structure by the human MTH1 protein.

The MTH1 protein catalyzes hydrolysis of oxidatively damaged purine nucleotides including 8-hydroxy-dGTP to the monophosphates. The MTH1 protein seems to act as an important defense system against mutagenesis, carcinogenesis, and cell death induced by oxidized purine nucleotides. We previously reported that the functional groups at the 2- and 6-positions of the purine ring affect the recognition by the human MTH1 protein. 8-Hydroxy-dGTP and 8-hydroxy-dATP are substrates of MTH1, and both have the "7,8-dihydro-8-oxo structure." In this study, three nucleotide analogs containing this motif were examined. A synthetic purine analog containing the 7,8-dihydro-8-oxo structure and the 2-amino function (dJTP) was hydrolyzed to the monophosphate with high efficiency by MTH1. On the other hand, two analogs that lack the two-ring system of their bases [formamidopyrimidine-dGTP (FAPY-dGTP) and 2-OH-dYTP] were poor substrates. FAPY-dGTP is a mixture of conformers and was hydrolyzed more than ten-fold less efficiently than 8-hydroxy-dGTP. These results clarify the effects of the 2-amino group and the two-ring system of the purine base on the recognition by the human MTH1 protein.

[32P]2-iodo-N6-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate ([32P]MRS2500), a novel radioligand for quantification of native P2Y1 receptors.

Analysis of the P2Y family of nucleotide-activated G-protein-coupled receptors has been compromised by the lack of selective high-affinity, high-specific-radioactivity radioligands. We have pursued quantification of the P2Y1 receptor through the development of a series of selective P2Y1 receptor antagonists. Recently, we synthesized 2-iodo-N6-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bisphosphate (MRS2500), a selective, competitive antagonist that exhibits a Ki of 0.8 nM in competition-binding assays with [3H]MRS2279. A 3'-monophosphate precursor molecule, MRS2608, was radiolabeled at the 5' position with 32P using polynucleotide kinase and [gamma32P]ATP to yield [32P]MRS2500. [32P]MRS2500 bound selectively to Sf9 insect cell membranes expressing the human P2Y1 receptor (Sf9-P2Y1), but did not detectably bind membranes expressing other P2Y receptors. P2Y1 receptor binding to [32P]MRS2500 was saturable with a KD of 1.2 nM. Agonists and antagonists of the P2Y1 receptor inhibited [32P]MRS2500 binding in Sf9-P2Y1 membranes with values in agreement with those observed in functional assays of the P2Y1 receptor. A high-affinity binding site for [32P]MRS2500 (KD=0.33 nM) was identified in rat brain, which exhibited the pharmacological selectivity of the P2Y1 receptor. Distribution of this binding site varied among rat tissues, with the highest amount of binding appearing in lung, liver, and brain. Among brain regions, distribution of the [32P]MRS2500 binding site varied by six-fold, with the highest and lowest amounts of sites detected in cerebellum and cortex, respectively. Taken together, these data illustrate the synthesis and characterization of a novel P2Y1 receptor radioligand and its utility for examining P2Y1 receptor expression in native mammalian tissues.

Regioselective syntheses of 7-nitro-7-deazapurine nucleosides and nucleotides for efficient PCR incorporation.

Substitution at the C(7) position of purine nucleotides by a potent electron-withdrawing nitro group facilitates the cleavage of glycosidic bonds under alkaline conditions. This property is useful for sequence-specific cleavage of DNA containing these analogues. Here we describe the preparation of 7-deaza-7-NO(2)-dA and 7-deaza-7-NO(2)-dG using two different approaches, starting from 2'-deoxy-adenosine and 6-chloro-7-deaza-guanine, respectively. These modified nucleosides were converted to nucleotide triphosphates, each of which can replace the corresponding, naturally occurring triphosphate to support PCR amplification. [structure: see text]

Deoxyribosyl analogues of methionyl and isoleucyl sulfamate adenylates as inhibitors of methionyl-tRNA and isoleucyl-tRNA synthetases.

2'-Deoxy, 3'-deoxy, and 2',3'-dideoxyribosyl surrogates of isoleucyl and methionyl sulfamate adenylates have been investigated to identify the pharmacophoric importance of the ribose group for the inhibition of Escherichia coli methionyl-tRNA (MRS) and isoleucyl-tRNA (IRS) synthetases. Molecular modeling of 2',3'-dideoxyribosyl Met-NHSO2-AMP (9) with the crystal structure of E. coli MRS revealed that the lack of the two hydroxyl groups on ribose was compensated by the formation of an extra hydrogen bond between the ring oxygen and His24, resulting in a small activity reduction.