Synthesis of 8-bromo- and 8-azido-2'-deoxyadenosine-5'-O-(1-thiotriphosphate)

LABEIT S., LEHRACH H., GOODY R.S.

A new procedure for determining DNA nucleotide sequences is reported. In the first step of the method, four DNAs, each separately substituted with a different deoxynucleoside phosphorothioate in place of the corresponding monophosphate, are prepared by template-directed polymerization catalyzed by DNA polymerase. In the second step, these DNAs are subjected to stringent exonuclease III treatment, which produces only fragments terminating with a phosphorothioate internucleotide linkage. These can then be separated by standard gel electrophoresis techniques and the sequence can be read directly as in presently used sequencing methods.

Fidanza J.A., Ozaki H., McLaughlin L.W.

A phosphorothioate diester can be incorporated site-specifically into DNA sequences to provide a nucleophilic site that is amenable to alkylation by labels containing haloacetamide, aziridine sulfonamide, or γ-bromo-α,β-unsaturated carbonyl functionalities. Labeling reactions proceed most efficiently at 50 o C in the pH range 5.0-8.0 and require a number of hours for completion. HPLC techniques employing reversed-phase columns can be used to rapidly purify the labeled materials, and a variety of examples are shown including the purification of a site-specifically labeled 30-mer

Catalano C.E., Allen D.J., Benkovic S.J.

The synthesis of an azidoDNA duplex and its use to photolabel DNA polymerases have been previously described (Gibson & Benkovic, 1987). We now present detailed experiments utilizing this azidoDNA photoprobe as a substrate for Escherichia coli DNA polymerase I (Klenow fragment) and the photoaffinity labeling of the protein. The azidoDNA duplex is an efficient substrate for both the polymerase and 3'----5' exonuclease activities of the enzyme. However, the hydrolytic degradation of the azido-bearing base is dramatically impaired. On the basis of the ability of these duplexes to photolabel the enzyme, we have determined that the protein contacts between five and seven bases of duplex DNA. Incubation of azidoDNA with the Klenow fragment in the presence of magnesium results in the in situ formation of a template-primer with the azido-bearing base bound at the polymerase catalytic site of the enzyme. Photolysis of this complex followed by proteolytic digestion and isolation of DNA-labeled peptides results in the identification of a single residue modified by the photoreactive DNA substrate. We identify Tyr766 as the modified amino acid and thus localize the catalytic site for polymerization in the protein. A mansyl-labeled DNA duplex has been prepared as a fluorescent probe of protein structure. This has been utilized to determine the location of the primer terminus when bound to the Klenow fragment. When the duplex contains five unpaired bases in the primer strand of the duplex, the primer terminus resides predominantly at the exonuclease catalytic site of the enzyme. Removal of the mismatched bases by the exonuclease activity of the enzyme yields a binary complex with the primer terminus now bound predominantly at the polymerase active site. Data are presented which suggest that the rate-limiting step in the exonuclease activity of the enzyme is translocation of the primer terminus from polymerase to exonuclease catalytic sites.

Meffert R., Rathgeber G., Schäfer H., Dose K.

The synthesis of 8-azido-2'-deoxyadenosine-5'-triphosphate is described. The photoreactive dATP analog was characterized by thin layer chromatography, proton resonance spectroscopy, infrared spectroscopy and UV spectroscopy. Its photolysis upon UV irradiation was studied. After incorporation of this dATP analog into DNA containing the tet operator sequence the investigation of the interactions between tet operator DNA and Tet repressor protein by UV photocross-linking becomes possible. Photocross-linking of protein to DNA was demonstrated by the reduced migration of the DNA in SDS polyacrylamide gel electrophoresis. Addition of the inducer tetracycline prior to UV irradiation significantly reduces the DNA-protein cross-linking rate. The long wave UV light applied here does not significantly alter the DNA or the protein under the photocross-linking conditions.

Ruffner D.E., Uhlenbeck O.C.

A hammerhead domain of less than 50 nucleotides is responsible for a self-cleavage reaction in the replication of plant RNA pathogens. The hammerhead is composed of three helices joining at a central conserved core of 11 single stranded nucleotides. The core is believed to fold into a tertiary structure that provides functional groups for catalysis and to coordinate one or more divalent metal ions. In this study we use a phosphorothioate substitution interference assay to identify four phosphates in the conserved core which also play a role in the self-cleavage reaction.

Milligan J.F., Uhlenbeck O.C.

The binding of the bacteriophage R17 coat protein to its RNA binding site is an example of a specific RNA-protein interaction. Extensive analysis has revealed that the binding is dependent upon a unique hairpin structure that contains four essential single-stranded nucleotides. Additional specificity is thought to be due to four or five ionic contacts between the protein and phosphates on the RNA. Transcription of synthetic DNA with T7 RNA polymerase, using one of the nucleoside 5'-O-(1-thiotriphosphates) [NTP(alpha S)s], allows the synthesis of RNAs specifically substituted with thiophosphates. Eleven sequence variants of the R17 coat protein binding site were synthesized with different NTP(alpha S)s and tested for coat protein binding to deduce positions of thiophosphates that alter the binding affinity. Of the twenty-one phosphate positions in the molecule, two were found to decrease the Ka 3-fold when substituted with a thiophosphate, one position decreased the Ka 10-fold, and one position increased the Ka 10-fold. Substitution of any of the other 17 positions with thiophosphates does not alter the Ka. The four positions that alter the Ka are located in a uniquely structured region of the RNA, and it is postulated that these thiophosphates affect binding because they contact coat protein directly.

Ludwig J., Eckstein F.

Gish G., Eckstein F.

Evans R.K., Haley B.E.

A photoactive nucleotide analogue of dUTP, 5-azido-2'-deoxyuridine 5'-triphosphate (5-N3dUTP), was synthesized from dUMP in five steps. The key reaction in the synthesis of 5-N3dUTP is the nitration of dUMP in 98% yield in 5 min at 25 degrees C using an excess of nitrosonium tetrafluoroborate in anhydrous dimethylformamide. Reduction of the resulting 5-nitro compound with zinc and 20 mM HCl gave 5-aminodeoxyuridine monophosphate (5-NH2dUMP). Diazotization of 5-NH2dUMP with HNO2 followed by the addition of NaN3 to the acidic diazonium salt solution gave a photoactive nucleotide derivative in 80-90% yield. The monophosphate product was identified as 5-N3dUMP by proton NMR, UV, IR, and chromatographic analysis as well as by the mode of synthesis and its photosensitivity. After formation of 5-N3dUTP through a chemical coupling of pyrophosphate to 5-N3dUMP, the triphosphate form of the nucleotide was found to support DNA synthesis by Escherichia coli DNA polymerase I at a rate indistinguishable from that supported by dTTP. When UMP was used as the starting compound, 5-N3UTP was formed in an analogous fashion with similar yields and produced a photoactive nucleotide which is a substrate for E. coli RNA polymerase. To prepare [gamma-32P]-5-N3dUTP for use as an active-site-directed photoaffinity labeling reagent, a simple method of preparing gamma-32P-labeled pyrimidine nucleotides was developed. [gamma-32P]-5-N3dUTP is an effective photoaffinity labeling reagent for DNA polymerase I and was found to bind to the active site with a 2-fold higher affinity than dTTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Gibson K.J., Benkovic S.J.

To investigate protein-DNA interactions, we have synthesized a versatile phthalimide-protected 5-(3-aminopropyl)-2'-deoxyuridine nucleoside probe. The modified residue was incorporated into deoxyoligonucleotides by automated synthesis. The standard oligonucleotide workup also exposed the pendent amino group, which was found to react with either fluorescent labelling agents or, as detailed below, a photoactivatable cross-linking agent. In the dark, a strand with a photolabile group adjacent to the 3' end served as a primer for synthetic template-directed DNA synthesis by the Klenow fragment of E. coli DNA polymerase I, by bacteriophage T4 DNA polymerase, and by avian myeloblastosis virus (AMV) reverse transcriptase. Brief illumination with 302 nm light afforded covalent complexes between DNA and the polymerases; labelling of AMV reverse transcriptase was predominantly in the beta subunit.

Evans R.K., Johnson J.D., Haley B.E.

We have synthesized the photoactive deoxyuridine nucleotide 5-azido-2'-deoxyuridine 5'-triphosphate (5-N3dUTP) and used it to synthesize light-sensitive DNA by enzymatic incorporation. In the absence of ultraviolet light, 5-N3dUTP is a substrate for Escherichia coli DNA polymerase I. In in vitro DNA synthesis reactions using bacteriophage M13 single-stranded DNA as the template and 5-N3dUTP in place of dTTP, a photoactive complementary strand was synthesized by DNA polymerase I. The complementary strand was not synthesized when the 5-N3dUTP was substituted for dCTP or when it was exposed to ultraviolet light prior to the addition of DNA polymerase I. Using a synthetic lac operator template of 26 bases and a 15-base primer, we generated a photoactive 26-base-pair lac operator by enzymatically incorporating 5-N3dUMP with DNA polymerase I. Crosslinking of this photoactive DNA fragment to lac repressor was totally dependent on the presence of UV light and was reduced 78% by 150 microM isopropyl beta-D-thiogalactoside. Under the same conditions no crosslinking to lac repressor was observed using a nonphotoactive 26-base-pair lac operator. Photoactivatable deoxyuridine analogs have potential application as reagents to crosslink DNA binding proteins to 5-azidouracil-containing DNA and as active-site-directed photoaffinity labelling reagents.

Goody R.S., Isakov M.

Nucleoside 5′-0-(1-thiodi-) and triphosphates can be obtained in yields of up to 45% directly from the nucleosides. Their diastereomers can be separated by preparative reversed phase chromatography.

Eckstein F.

Potter B.V., Eckstein F., Uznański B.

Fully protected diastereoisomers of deoxyguanylyl (3' leads to 5') deoxyadenosine stereospecifically labelled on phosphorus with oxygen-18 have been synthesized by oxidation of phosphite triester intermediates in the presence of 18O-labelled water. The diastereoisomers have been chromatographically separated and their absolute configuration at phosphorus determined. (Rp)-[18O]deoxyguanylyl (3' leads to 5')deoxyadenosine has been prepared by complete deprotection of the parent diastereoisomer of the Sp configuration. Methylation of the former compound permits assignment of the absolute configurations of the methyl esters of N1-methyldeoxyguanylyl (3' leads to 5') N1-methyldeoxyadenosine.

Chen J., Benkovic S.J.

Treatment of unprotected nucleosides with an excess of phosphorous acid and stoichiometric proportions of N,N'-di-p-tolylcarbodiimide in anhydrous pyridine gives predominantly deoxynucleoside monophosphites and minor amounts of 5' :3'-diphosphites; for deoxyadenosine and deoxyguanosine, the monophosphite products are exclusively 5'-phosphites, whereas for deoxycytidine and thymidine, the yields of the 5'-phosphites are 85% and 92% respectively. Sulfurization of these deoxynucleoside monophosphites with sulfur in the presence of trialkylamines and trimethylsilyl chloride in dry pyridine nearly quantitatively produces deoxynucleoside phosphorothioates. Condensation of these phosphorothioates with pyrophosphate forms diastereomers of the alpha-thio-derivatives of deoxynucleoside triphosphate. The individual diastereomers of each deoxynucleoside 5'-O-(1-thio)triphosphate can be separated, on a preparative scale, by ion exchange chromatography.

Elgemeie G.H., Mohamed-Ezzat R.A.

Purine nucleoside analogs are effective compounds have a significant and pivotal effects in several fields. Novel synthetic strategies for many anticancer purine nucleoside analogs were reported.

Sekiguchi H., Muranaka K., Osada A., Ichikawa S., Matsuda A.

The PU-H58-dimers 13a-15b were efficiently synthesized and their biological properties were evaluated. The copper-catalyzed alkyne azide coupling was effective in simultaneously linking three components via a triazole formation to afford the target dimers. These synthesized dimers exhibited binding affinity to the N-terminal domain of Hsp90, cytotoxicity, and client degradation activity although these activities were comparative or weak comparable with that of the parent compound.

Ustinov A.V., Stepanova I.A., Dubnyakova V.V., Zatsepin T.S., Nozhevnikova E.V., Korshun V.A.

The use of azide and alkyne cycloaddition reaction in the synthesis of conjugates of nucleic acids and oligodeoxyribonucleotides is reviewed. Data on the chemical and enzymatic methods for introducing azides and alkynes into DNA are summarized.

Abou-Elkhair R.A., Netzel T.L.

The challenge in working with anthraquinone-2'-deoxyadenosine (AQ-dA) conjugates is that they are insoluble in water and only sparingly soluble in most organic solvents. However, water-soluble AQ-dA conjugates with short linkers are required for study of their electrochemical and intramolecular electron transfer properties in this solvent prior to their use in laser kinetics investigations of photoinduced hole (cation) transport in DNA. This article first describes the synthesis of a water-soluble, ethynyl-linked AQ-dA conjugate, 8-[(anthraquinone-2-yl)ethynyl]-2'-deoxyadenosine 3'-benzyl hydrogen phosphate, based on initial formation of a 5'-O-(4,4'-dimethoxytrityl) (5'-O-DMTr) intermediate. Because intended H2 over Pd/C reduction of the ethynyl linker in 5'-O-DMTr-protected 2'-deoxyadenosines cleaves the DMTr protecting group and precipitates multiple side products, this work also describes the synthesis of an ethylenyl-linked AQ-dA conjugate, 8-[2-(anthraquinone-2-yl)ethyl]-2'-deoxyadenosine 3'-benzyl hydrogen phosphate, starting with a 5'-O-tert-butyldiphenylsilyl protecting group.

Rose J.D., Parker W.B., Someya H., Shaddix S.C., Montgomery J.A., Secrist J.A.

A common reason for the lack of cytotoxicity of certain nucleosides is thought to be their inability to be initially activated to the monophosphate level by a nucleoside kinase or other activating enzyme. In a search for other nucleosides that might be worthwhile anticancer agents, we have begun to examine the utilization of monophosphate prodrugs in order to explore whether any enhanced cytotoxicity might be found for the prodrugs of candidate nucleosides that have little or no cytotoxicity. To that end, 5'-bis(pivaloyloxymethyl) phosphate prodrugs of two weakly cytotoxic compounds, 8-aza-2'-deoxyadenosine (5) and 8-bromo-2'-deoxyadenosine (9), have been prepared. These prodrugs (8 and 12) were examined for their cytotoxicity in CEM cells and were found to possess significantly enhanced cytotoxicity when compared with the corresponding parent nucleosides. Further cell culture experiments were conducted to gain insight into the mechanisms of cytotoxicity of these two prodrugs, and those data are reported.

CaroliRezende M., SepúlvedaBoza S.

The phosphitylation of 2-α-O-Benzyl-N-acetylneuraminic acid (2) and of the methyl 2-α-O-methyl-N-acetylneuraminate (3) with 2-chloro-4H-1,2,3-benzodioxaphosphorin-4-one (4) is described. The reaction affords a ready access to 9-phosphorylated derivatives of N-acetylneuraminic acid (1).