Phosphorothioate Diester Research Articles

Synthesis of potential UDP‐glucuronosyltransferase inhibitors containing a diphosphate function

AbstractThe synthesis of potential inhibitors of UDP‐glucuronosyltransferase, in which the β‐phosphate moiety of uridine 5′‐diphosphate is linked to phenolic or alcoholic hydroxyl groups, is described. Key intermediates in the formation of the diphosphate function are S‐(4‐methylphenyl) 2‐cyanoethyl phosphorothioate triesters which, after conversion into the corresponding S‐(4‐methylphenyl) phosphorothioate diesters, react with phosphate monoesters, in the presence of iodine, to give the target molecules.

ChemInform Abstract: Reaction of Internucleotidic Phosphorothioate Diesters with Fluorescent Reporter Groups

ChemInformVolume 21, Issue 49 Reviews ChemInform Abstract: Reaction of Internucleotidic Phosphorothioate Diesters with Fluorescent Reporter Groups N. E. CONWAY, N. E. CONWAY Dep. Chem., Boston Coll., Chestnut Hill, MA 02167, USASearch for more papers by this authorJ. A. FIDANZA, J. A. FIDANZA Dep. Chem., Boston Coll., Chestnut Hill, MA 02167, USASearch for more papers by this authorL. W. MCLAUGHLIN, L. W. MCLAUGHLIN Dep. Chem., Boston Coll., Chestnut Hill, MA 02167, USASearch for more papers by this author N. E. CONWAY, N. E. CONWAY Dep. Chem., Boston Coll., Chestnut Hill, MA 02167, USASearch for more papers by this authorJ. A. FIDANZA, J. A. FIDANZA Dep. Chem., Boston Coll., Chestnut Hill, MA 02167, USASearch for more papers by this authorL. W. MCLAUGHLIN, L. W. MCLAUGHLIN Dep. Chem., Boston Coll., Chestnut Hill, MA 02167, USASearch for more papers by this author First published: December 4, 1990 https://doi.org/10.1002/chin.199049352Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume21, Issue49December 4, 1990 RelatedInformation

Reaction of Internucleotidic Phosphorothioate Diesters with Fluorescent Reporter groups

Abstract The substitution of a non-bridging oxygen by sulfur in a DNA intemucleotidic phosphorus residue creates a nucleophilic site which is amenable to labeling by reporter groups such as fluorophores or spin labels. Placement of a single phosphorothioate diester at a selected position allows site-specific attachment of the reporter group. Incorporating a phosphorothioate at each and every intemucleotidic phosphorous linkage allows the incorporation of multiple reporter groups, ideally one for each nucleoside residue.

Identification of a non-junction phosphodiester that influences an autolytic processing reaction of RNA

Oligoribonucleotides with specific sequences derived from the satellite RNA of tobacco ringspot virus undergo autolytic cleavage at the CpA phosphodiester that is the junction between unit sequences of multimeric satellite RNA. Buzayan et al. (Nucleic Acids Res., 16, 4009-4023 (1988)) showed that an oligoribonucleotide with 97 satellite RNA-derived nucleotide residues self-cleaved with greatly reduced efficiency when it was synthesized in vitro from adenosine-5'-O-(1-thiotriphosphate) (abbreviated rATP alpha S) and three rNTPs. No other substitution of one rNTP by the corresponding rNTP alpha S had this effect, suggesting that a phosphorothioate CpA junction inhibits self-cleavage. Here, we replaced the usual CpA junction of a small self-cleaving oligoribonucleotide with a CpU junction. Self-cleavage of this molecule was reduced not only by rUTP alpha S-substitution, as expected, but also by partial and complete rATP alpha S-substitution. By analysis of the locations of rAMPS residues in cleavage products derived from partially rATP alpha S-substituted oligoribonucleotides, we identified A26 as the residue contributing the non-junction phosphorothioate diester that most strongly inhibited self-cleavage. Manganese ions strongly stimulated the self-cleavage of the rATP alpha S-substituted, CpU-junction oligoribonucleotide but was less effective when the junction was CpA.

Site specific functionalization of oligonucleotides for attaching two different reporter groups

The synthesis of an oligonucleotide functionalized to attach two different reporter groups at specific internucleotide linkages is described. To incorporate the amine specific reporter group the internucleotide linkage is modified to phosphoramidate (N-1-aminoalkyl) and for a thiol specific reporter group the internucleotide linkage is modified to a phosphorothioate diester. The synthetic cycle for introducing the modified internucleotide linkages at specific sites can be carried out using an automated DNA synthesizer. Combination of reporter groups have been attached successfully.

Two autolytic processing reactions of a satellite RNA proceed with inversion of configuration

Both polarities of the satellite RNA of tobacco ringspot virus occur in infected cells in multimeric forms which are capable of autolytic processing, using different sequences and structures [Feldstein, P.A., et al., Proc. Nat. Acad. Sci. USA (1990) 87 (in press)]. These transesterification reactions generate a 2',3'-cyclophosphate and a 5'-hydroxyl as the two new end groups. Cleavage is at a CpA for the (+) polarity RNA and at an ApG for the (-) polarity RNA. We enzymically synthesized oligoribonucleotides with processing capability and with specific 35S-labeled phosphorothioate diesters in the Rp configuration. After processing had occurred, the terminal nucleoside-2',3'-cyclophosphorothioate diester residues were recovered from the appropriate product by digestion with nuclease and phosphatase. Comparisons with specially prepared endo- and exoisomer reference compounds by thin layer chromatography and autoradiography revealed that the [35S]cytidine- and [35S]adenosine-2',3'-cyclophosphorothioate both were endo-isomers. The results are consistent with transesterification occurring by an inline SN2(P) attack of the 2'-hydroxyl group in the autolytic processing reactions of both polarities of the satellite RNA.

Reaction of Internucleotidic Phosphorothioate Diesters with Fluorescent Reporter Groups

Reaction of Internucleotidic Phosphorothioate Diesters with Fluorescent Reporter Groups

Introduction of reporter groups at specific sites in DNA containing phosphorothioate diesters

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTIntroduction of reporter groups at specific sites in DNA containing phosphorothioate diestersJacqueline A. Fidanza and Larry W. McLaughlinCite this: J. Am. Chem. Soc. 1989, 111, 25, 9117–9119Publication Date (Print):December 1, 1989Publication History Published online1 May 2002Published inissue 1 December 1989https://doi.org/10.1021/ja00207a028RIGHTS & PERMISSIONSArticle Views377Altmetric-Citations48LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (396 KB) Get e-Alerts Get e-Alerts

"Post-assay" covalent labeling of phosphorothioate-containing nucleic acids with multiple fluorescent markers.

A simple protocol has been developed which allows the covalent introduction of multiple fluorescent markers into DNA fragments after gel electrophoresis techniques, that is, while the nucleic acid is imbedded in the polyacrylamide gel matrix. "Post-assay" fluorescent labeling in this manner employs DNA fragments containing phosphorothioate diesters, which can be easily incorporated during chemical and enzymatic synthesis procedures, and can be alkylated with the fluorescent marker monobromobimane. Labeling the internucleotidic phosphorus residue in this manner allows the introduction of a fluorescent marker for each nucleotide residue present. Roughly a linear increase in emitted fluorescence, thus detection sensitivity, is observed with an increasing number of bimane markers. With this technique, oligodeoxynucleotides and DNA fragments can be observed in the gel matrix, without sophisticated electronic detection devices in the low femtomolar (10(-15) mol) range.

An efficient approach toward the synthesis of phosphorothioate diesters via the schönberg reaction

Easily accessible phenacetyl or benzoyl disulfide proved to be very convenient reagents for a rapid P-sulfurization of phosphite-triesters and H-phosphonate diesters, respectively.

RNA mediated formation of a phosphorothioate diester bond

Previous results showed that multimeric, tandemly sequence-repeated forms of satellite tobacco ringspot virus RNA of the encapsidated polarity (STobRV (+)RNA) autolytically process at a specific phosphodiester bond, the junction. Substituting a phosphorothioate diester bond for the STobRV (+)RNA junction drastically slowed autolytic processing. Here we show that for the complementary STobRV (-)RNA, in contrast, replacing sets of phosphodiester bonds with phosphorothioate diester bonds, even at the junction, did not greatly slow autolytic processing or spontaneous ligation, the usual reactions of the unmodified RNA. In the ligation reaction STobRV (-)RNA directed the formation of an ApG phosphorothioate diester bond.

DNA and RNA Sequence Determination Based on Phosphorothioate Chemistry

The difference in reactivity between phosphate and phosphorothioate diesters is the basis of a chemical degradation scheme for the sequencing of DNA and RNA. The phosphorothioate groups are incorporated into the nucleic acid in four separate enzymatic reactions, with three of the natural nucleoside triphosphates and one alpha-thiotriphosphate in each reaction. Selective strand cleavage is achieved through alkylation to form the hydrolytically labile phosphorothioate triester. As an example, the sequence analysis is presented of M13 phage DNA and of RNA prepared by transcription with SP6 RNA polymerase.

Accuratein vitrocleavage by RNase in of phosphorothioate-substituted RNA processing signals in bacteriophage T7 early mRNA

To test the ability of an RNA processing enzyme to cleave chemically-modified RNA substrates, RNA transcripts containing RNase III cleavage sites were enzymatically synthesized in vitro to contain specific phosphorothioate diester internucleotide linkages. One transcript (R1.1 RNA) was generated using phage T7 RNA polymerase and a cloned segment of phage T7 DNA containing the R1.1 RNase III processing site. The second transcript was the phage T7 polycistronic early mRNA precursor, which was synthesized using E. coli RNA polymerase and T7 genomic DNA. The RNA transcripts contained phosphorothioate diester groups at positions including the scissile bonds. The modified RNAs were stable to incubation in Mg2+-containing buffer, and were specifically cleaved by RNase III. RNA oligonucleotide sequence analysis showed that the modified R1.1 RNA processing site was the same as the canonical site and contained a phosphorothioate bond. Furthermore, RNase III cleaved the phosphorothioate internucleotide bond with 5' polarity. RNase III cleavage of phosphorothioate substituted T7 polycistronic early mRNA precursor produced the same gel electrophoretic pattern as that obtained with the control transcript. Thus, RNase III cleavage specificity is not altered by phosphorothioate internucleotide linkages.

Autolytic processing of a phosphorothioate diester bond

A small satellite RNA of tobacco ringspot virus replicates in tissues infected with tobacco ringspot virus and accumulates in virus capsids, forming virus-like particles. Previous research showed that multimeric forms of this satellite RNA have tandem repeats of the "monomeric" satellite RNA sequence of 359 or 360 nucleotide residues. The multimeric RNAs undergo autolytic processing at a specific CpA phosphodiester bond, the junction, to generate the monomeric RNA. We substituted phosphorothioate diester bonds for various sets of phosphodiester bonds, in dimeric and truncated forms of the satellite RNA. The degree of reduction in autolytic cleavage varied both with the sites of substitution and the size of the RNA molecules. Analyses of a product of the autolysis reaction suggest that one phosphorothioate diester bond most strongly interferes with processing, the one introduced at the CpA junction during its synthesis from adenosine-5'-0-(1-thiotriphosphate). However, extensive introduction of phosphorothioate diester bonds elsewhere in the molecule also decreased processing, possibly by altering conformation.

Stereochemical course of hydrolysis of DNA by exonuclease I from Escherichia coli.

Exonuclease I has been purified from an overproducing strain of Escherichia coli K12 [Prasher, D. C., Conarro, L., & Kushner, S. R. (1983) J. Biol. Chem. 258, 6340-6343]. The enzyme hydrolyzes deoxyribonucleic acids that contain chiral phosphorothioate diester linkages, and the stereochemical course of the reaction is inversion of configuration at phosphorus. This result is most consistent with hydrolysis occurring via the direct attack of water on a phosphorothioate diester rather than through the intermediacy of a covalent nucleotidyl-enzyme intermediate. This finding represents the first example of a processive exonuclease whose stereochemical pathway has been determined.

Evidence for sequence-specific conformational changes in DNA from the melting temperatures of DNA phosphorothioate derivatives.

Analogs of alternating purine-pyrimidine DNA polymers such as poly(dA-dT)-poly(dA-dT) can be made with phosphorothioate groups in the DNA backbone. A phosphorothioate diester at the 5'-purine-pyrimidine-3' step causes a significant lowering of the polymer's melting temperature compared to a phosphorothioate diester at the 5'-pyrimidine-purine-3' step. This may occur because sulfur substitution increases anionic charge density in the DNA minor groove and 5'-purine-pyrimidine-3' steps narrow the minor groove. The ability to modulate charge density in the DNA backbone via sulfur substitution should prove useful in studies of sequence-dependent conformational changes in DNA.

Stereochemical evidence for a phosphorylpyridinium intermediate in the iodine-mediated desulphurisation of a phosphorothioate diester

Desulphurisation of (Sp)-5′-O-(2′-deoxyadenosyl)-3′-O-thymidyl phosphorothioate to 5′-O-(2′-deoxyadenosyl)-3′-O-thymidyl [18O]phosphate proceeds with epimerisation at phosphorus using iodine in aqueous pyridine but with predominant inversion of configuration in aqueous 2,6-lutidine.

Stereochemistry of internucleotide bond formation by polynucleotide phosphorylase from Micrococcus luteus

Polynucleotide phosphorylase catalyzes the formation of polynucleotides from the Sp diastereomer of adenosine 5'-O-(l-thiodiphosphate) ADPalphaS), whereas the Rp diastereomer is a competitive inhibitor. The absolute configuration of the phosphorothioate diester bond in the polymer was determined by copolymerizing ADPalpha S, Sp isomer with UDP and degrading the resulting copolymer with R Nase A and spleen phosphodiesterase to give, inter alia, uridine 2',-3'-cyclic phosphorothioate. The latter product was shown to be the endo isomer by high-performance liquid chromatography. No evidence for the presence of the exo isomer was obtained. It can thus be concluded that the Sp diastereomer of ADPalphaS polymerizes with inversion of configuration at phosphorus without racemization to give a phosphorothioate diester bond with the Rp configuration.

Stereochemistry of internucleotidic bond formation by tRNA nucleotidyltransferase from bakers' yeast

Isomer A of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) is a substrate for tRNA nucleotidyltransferase from baker's yeast, whereas isomer B is a competitive inhibitor. The tRNA resulting from this reaction has a phosphorothioate instead of a phosphate diester linkage at the last internucleotidic linkage between cytidine and adenosine. On limited digestion of this tRNA with RNase A, one can isolate cytidine 2',3'-cyclic phosphorothioate which can be deaminated to uridine 2',3'-cyclic phosphorothioate. It can be shown that this compound is the endo isomer and that, therefore, the phosphorothioate diester bond in the tRNA must have had the R configuration. This result indicates that no racemization during the condensation of ATP alpha S, isomer A, onto the tRNA had occurred. Whether inversion or retention of configuration had taken place awaits elucidation of the absolute configuration of isomer A of ATP alpha S.

Stereochemistry of polymerization by DNA-dependent RNA-polymerase from Escherichia coli: an investigation with a diastereomeric ATP-analogue.

The phosphodiester bond formation by DNA-dependent RNA-polymerase (RNA nucleotidyltransferase, nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) can in principle result in retention, inversion, or racemization of configuration at the alpha-phosphorus of the nucleoside 5'-triphosphate being polymerized. As a first step in elucidating the stereochemistry of this reaction, one diastereomer (A) of adenosine 5'-O-(1-thiotriphosphate) (ATPalphaS) was polymerized with UTP in the presence of poly(dA-dT)-poly(dA-dT). The resulting polymer was enzymatically cleaved to uridine 2',3'-cyclic phosphorothioate which was determined to be the endo-isomer by comparison with an authentic sample. This shows that no reacemization had occurred and that isomer A of ATPalphaS gives a phosphorothioate diester bond with the R-configuration. Whether this represents inversion of retention of configuration awaits elucidation of the absolute configuration of isomer A for ATPalphaS.