Simple synthesis of 5-vinyl- and5-ethynyl-2′-deoxyuridine-5′-triphosphates
TL;DR: In this article, the acid-sensitive nucleoside 5′-O-triphosphates can be obtained in good yield directly from the unprotected nucleosides in the presence of a proton sponge.
About: This article is published in Tetrahedron Letters.The article was published on 1988-01-01. It has received 143 citations till now. The article focuses on the topics: Deoxyuridine & Nucleoside.
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TL;DR: In this article, a method for purification and synthesis of RNA molecules and enzymatic RNA molecules in enzymatically active form is presented, and the method is used to synthesize RNA molecules.
Abstract: Method for purification and synthesis of RNA molecules and enzymatic RNA molecules in enzymatically active form.
599 citations
Patent•
12 Nov 2004TL;DR: In this article, a polynucleotide sequence is sequenced by stopping an extension cycle in a sequence by synthesis reaction before the reaction has run to near or full completion, which is called stopping the cycle.
Abstract: The invention provides methods for sequencing a polynucleotide comprising stopping an extension cycle in a sequence by synthesis reaction before the reaction has run to near or full completion.
415 citations
TL;DR: The synthesis of the 5'-triphosphate derivative of 1 and the study of its ability to be inserted into replicating DNA strands by the Klenow fragment of Escherichia coli DNA polymerase I find that this nucleotide derivative is a surprisingly good substrate for KF.
Abstract: Compound 1 (F), a nonpolar nucleoside analog that is isosteric with thymidine, has been proposed as a probe for the importance of hydrogen bonds in biological systems. Consistent with its lack of strong H-bond donors or acceptors, F is shown here by thermal denaturation studies to pair very poorly and with no significant selectivity among natural bases in DNA oligonucleotides. We report the synthesis of the 5′-triphosphate derivative of 1 and the study of its ability to be inserted into replicating DNA strands by the Klenow fragment (KF, exo− mutant) of Escherichia coli DNA polymerase I. We find that this nucleotide derivative (dFTP) is a surprisingly good substrate for KF; steady-state measurements indicate it is inserted into a template opposite adenine with efficiency (Vmax/Km) only 40-fold lower than dTTP. Moreover, it is inserted opposite A (relative to C, G, or T) with selectivity nearly as high as that observed for dTTP. Elongation of the strand past F in an F–A pair is associated with a brief pause, whereas that beyond A in the inverted A–F pair is not. Combined with data from studies with F in the template strand, the results show that KF can efficiently replicate a base pair (A–F/F–A) that is inherently very unstable, and the replication occurs with very high fidelity despite a lack of inherent base-pairing selectivity. The results suggest that hydrogen bonds may be less important in the fidelity of replication than commonly believed and that nucleotide/template shape complementarity may play a more important role than previously believed.
312 citations
Patent•
18 Jan 2011TL;DR: In this paper, a real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer is presented.
Abstract: The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions.
258 citations
TL;DR: Kevin Burgess is a professor at Texas A&M University, where he has been since September 1992, and his research interest focuses on ways in which synthetic organic chemists can help to solve problems in medicinal chemistry and biotechnology.
Abstract: * To whom correspondence should be addressed. † Texas A&M University. Telephone: 979-845-4345. Fax: 979-8458839. E-mail: burgess@tamu.edu. ‡ ISIS Pharmaceuticals. Fax: 760-931-9200. E-mail: dcook@ isiph.com. Dan Cook, a keep-fit fanatic, was born in 1944 in the farm community of Clovis, NM. On his way to obtaining his doctorate in organic chemistry, under the direction of Raymond N. Castle, he attended the New Mexico Military Institute, Eastern New Mexico University, University of New Mexico, and Brigham Young University. He completed an industrial postdoctoral fellowship with Roland K. Robins at ICN Pharmaceutical in Costa Mesa, CA, in 1970−72, spent 11 years as a medicinal chemist at Warner-Lambert/ Parke-Davis Pharmaceutical Research Division in Ann Arbor, MI, and one year at Eastman Kodak Research Laboratories in Rochester, NY. After that he moved to Malvern, PA, with the Eastman Pharmaceutical Division/ Sterling Research Group. In 1989, he joined Isis Pharmaceuticals in Carlsbad, CA, where he is now Vice President, Research Chemistry. His research interests include heterocycles, nucleosides, nucleotides, and oligonucleotides as they relate to medicinal applications. Figure 1 Kevin Burgess is a professor at Texas A&M University, where he has been since September 1992. His research interest focuses on ways in which synthetic organic chemists can help to solve problems in medicinal chemistry and biotechnology. Specific research projects in The Burgess Group include peptidomimetics of protein−protein interactions, combinatorial chemistry, libraries of asymmetric catalysis, and methodology for high-throughput DNA sequencing. It is interest in the latter project that provided motivation to write this review. 2047 Chem. Rev. 2000, 100, 2047−2059
208 citations
References
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TL;DR: During an investigation of the factors affecting the reaction of 2′,3′-O-isopropylidene nucleosides with phosphoryl chloride, trialkyl phosphates were found to be powerful accelerators and useful solvents for the phosphorylation.
Abstract: During an investigation of the factors affecting the reaction of 2′,3′-O-isopropylidene nucleosides with phosphoryl chloride, trialkyl phosphates were found to be powerful accelerators and useful solvents for the phosphorylation. The addition of a trialkyl phosphate to a large excess of phosphoryl chloride was markedly effective in promoting the phosphorylation; further, the lower alkyl phosphates, such as trimethyl and triethyl phosphates, were able to replace the excessive amount of phosphoryl chloride as solvents. When unprotected nucleosides were treated with phosphoryl chloride in trimethyl phosphate, the corresponding 5′-phosphates were mainly produced, together with a small amount of highly-phosphorylated products, in good yields. On the contrary, the treatment of 5′-O-acetyl nucleosides in a similar manner gave the corresponding 2′(or 3′)-nucleotides in low yields. The formation of the 2′(or 3′)-phosphate was strongly inhibited by the addition of a small amount of water to the reaction mixture. Th...
335 citations
TL;DR: In this article, a hygroscopic complex (Complex 1), an active phosphorylating agent in the present phosphorylation of nucleosides, was identified as an adduct composed of tetrachloropyrophosphate and pyridinium chloride.
Abstract: 5′-Nucleotides were directly prepared by selective phosphorylation with phosphoryl chloride in the presence of water and pyridine in acetonitrile. The rate of phosphorylation with phosphoryl chloride was markedly improved by the addition of suitable amounts of water and pyridine; the maximal formation of 5′-nucleotides was obtained by mixing these three reactants phosphoryl chloride, water, and pyridine in a molar ratio of 2 : 1 : 2 at a low temperature, followed by adding nucleosides corresponding to a one-fourth or one-fifth of the phosphoryl chloride. By mixing phosphoryl chloride, water, and pyridine in acetonitrile under controlled conditions, a hygroscopic complex (Complex 1), an active phosphorylating agent in the present phosphorylation of nucleosides, was isolated. It was identified as an adduct composed of tetrachloropyrophosphate and pyridinium chloride by means of its infrared spectra and by its chromatographic behavior. One of the proposed structures of the adduct is trichloropyrophosphopyrid...
108 citations
TL;DR: A number of structurally related 5-substituted pyrimidine 2'-deoxyribonucleosides were synthesized and tested for antiviral activity against herpes simplex virus type 1 (HSV-1) in cell culture, and from a comparison of values a number of conclusions were drawn with regard to those molecular features that enhance or reduce antiviral Activity.
Abstract: A number of structurally related 5-substituted pyrimidine 2'-deoxyribonucleosides were synthesized and tested for antiviral activity against herpes simplex virus type 1 (HSV-1) in cell culture. A minimum inhibitory concentration was determined for each compound, and from a comparison of these values a number of conclusions were drawn with regard to those molecular features that enhance or reduce antiviral activity. Optimum inhibition of HSV-1 in cell culture occurred when the 5-substituent was unsaturated and conjugated with the pyrimidine ring, was not longer than four carbon atoms in length, had E stereochemistry, and included a hydrophobic, electronegative function but did not contain a branching point. Such features are contained in (E)-5-(2-bromovinyl)-2'-deoxyuridine, which was the most active of the compounds described.
79 citations
TL;DR: In this article, the nucleoside 5′-0-(1-thiodi-) and triphosphates can be obtained in yields of up to 45% directly from nucleosides.
25 citations
TL;DR: This chapter discusses chemotherapeutic agents for herpesvirus infections and the chemical synthesis of phosphonoformate (PFA) and phosphono compounds in general.
Abstract: Publisher Summary This chapter discusses chemotherapeutic agents for herpesvirus infections. Antiviral chemotherapy has come of age and has been revolutionized by the advent of the second generation of antiherpes drugs, which, in contrast with the first generation antiherpes drugs, are much more selective in their antiherpetic activity. The chemical synthesis of phosphonoformate (PFA) and phosphono compounds in general goes back to the early history of organic chemistry. Much of the methodology was established by the Russian school of phosphorus chemistry in the early years of the 20 th century. The potency of an antiviral agent in vitro is not necessarily predictive of its potency in vivo , because the latter is determined by several factors, such as the intrinsic activity of the compound, its pharmacokinetic profile, route of administration (topical or systemic), absorption from the gut (if administered perorally), penetration through the skin (if administered topically), tissue distribution (i.e., penetration into the brain), presence of competing metabolites (i.e., dThd) in the peripheral tissues, metabolism (i.e., phosphorylation rate) in the peripheral tissues, and clearance from the bloodstream.
20 citations