New Synthesis of 5-Carboxy-2′-deoxyuridine and Its Incorporation into Synthetic Oligonucleotides

Abstract

5-Carboxy-2′-deoxyuridine is a methyl oxidation product of thymidine. It can be formed by the menadione-mediated photosensitization of thymidine in aerated aqueous solution. Here in we present a new four-step synthesis of the 5-carboxy-2′-deoxyuridine phosphoramidite building block based on the alkaline hydrolysis of 5-trifluoromethyl-2′-deoxyuridine. The phosphoramidite derivative has been incorporated at defined sites into oligonucleotides using the solid phase synthesis approach.     

Incorporation of 2'-deoxy-5-(trifluoromethyl)uridine and 5-cyano-2'-deoxyuridine into DNA.

In an attempt to synthesize DNA containing 2'-deoxy-5-(trifluoromethyl)uridine (1) using previously published protocols, we found that the trifluoromethyl group converted into a cyano group, resulting in DNA containing 5-cyano-2'-deoxyuridine (3). We show that nucleoside 1 can be incorporated into DNA using phosphoramidite 2 in combination with acetyl-protected deoxycytidine and phenoxyacetyl-protected purine phosphoramidites. Replacing thymidine in DNA with 1 caused a slight decrease in DNA duplex stability at pH 6.9.     

Synthesis of N-acetyl-D-galactosamine and folic acid conjugated ribozymes

To evaluate potential improvement in tissue specific targeting and cellular uptake of therapeutic ribozymes, we have developed three new phosphoramidite reagents. These reagents can be used in automated solid-phase synthesis to produce oligonucleotide conjugates containing N-acetyl-D-galactosamine (targeting hepatocytes) and folic acid (targeting tumor). N-Acetyl-D-galactosamine was attached through a linker to both 2'-amino-2'-deoxyuridine and D-threoninol scaffolds, and these conjugates were converted to phosphoramidite building blocks. Incorporation of a D-threoninol-based monomer into ribozymes provided multiply labeled ribozyme conjugates. Attachment of the fully protected pteroic acid to the D-threoninol-6-aminocaproyl-L-glutamic acid construct afforded the folic acid conjugate, which was converted into the phosphoramidite and incorporated onto the 5'-end of the ribozyme.     

5-(phenylthiomethyl)-2'-deoxyuridine as an efficient photoreactive precursor to generate single and multiple lesions within DNA fragments

5-(Phenylthiomethyl)-2'-deoxyuridine was successfully incorporated into DNA oligomers by automated DNA synthesis using phosphoramidite chemistry. UV exposure of the latter thionucleoside containing oligonucleotides under anaerobic and aerobic conditions gives rise to specific base lesions. The photoproducts have been isolated and further characterized on the basis of NMR and mass spectrometric analyses.     

Induction and inhibition of Friend erythroleukemia cell differentiation by pyrimidine analogs: analysis of the requirement for intracellular accumulation and incorporation into DNA.

Alkyldeoxyuridines which differ from thymidine by a C5 substitution of straight or branched alkyl chains of two to six carbon atoms have been tested for their ability to be taken up, phosphorylated, and incorporated into DNA. Analysis of the uptake of 5-ethyl-2'-deoxyuridine and 5-propyl-2'-deoxyuridine (n-PrdU)--similar to both thymidine and 5-bromo-2'-deoxyuridine--indicates that transport is dependent upon a functional cellular thymidine kinase. All of the aforementioned pyrimidines with the exception of n-PrdU are phosphorylated to the triphosphate and incorporated into DNA. The homologs 5-iso-propyl-2'-deoxyuridine (iso-PrdU) and 5-hexyl-2'-deoxyuridine are neither transported into the cell, phosphorylated, nor incorporated into DNA. These analogs were tested (i) for their ability to induce in the absence of dimethyl sulfoxide and (ii) to determine whether they enhance or inhibit dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells. Inhibition of erythroid differentiation appears to require the incorporation of thymidine analogs into DNA, and thus only 5-ethyl-2'-deoxyuridine and 5-bromo-2'-deoxyuridine were effective in inhibiting dimethyl sulfoxide-induced differentiation. The observation that iso-PrdU, and to a lesser extent n-PrdU and 5-propyldeoxyuridine monophosphate, induce differentiation under conditions in which they are not detectable intracellularly is strong evidence that this class of inducer acts at the cell membrane.     

Antisense oligodeoxynucleotides: synthesis, biophysical and biological evaluation of oligodeoxynucleotides containing modified pyrimidines.

6-Azathymidine, 6-aza-2'-deoxycytidine, 6-methyl-2'-deoxyuridine, and 5,6-dimethyl-2'-deoxyuridine nucleosides have been converted to phosphoramidite synthons and incorporated into oligodeoxynucleotides (ODNs). ODNs containing from 1 to 5 of these modified pyrimidines were compared with known 2'-deoxyuridine, 5-iodo-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, 5-fluoro-2'-deoxyuridine, 5-bromo-2'-deoxycytidine, and 5-methyl-2'-deoxycytidine nucleoside modifications. Stability in 10% heat inactivated fetal calf serum, binding affinities to RNA and DNA complements, and ability to support RNase H degradation of targeted RNA in DNA-RNA heteroduplexes were measured to determine structure-activity relationships. 6-Azathymidine capped ODNs show an enhanced stability in serum (7- to 12-fold increase over unmodified ODN) while maintaining hybridization properties similar to the unmodified ODNs. A 22-mer ODN having its eight thymine bases replaced by eight 6-azathymines or 5-bromouracils hybridized to a target RNA and did not inhibit RNase H mediated degradation.     

Purification of thymidine phosphorylase from Escherichia coli and its photoinactivation in the presence of thymine, thymidine, and some halogenated analogs.

Isoelectric focusing was used as the final step in the isolation of thymidine phosphorylase which was found to have an isoelectric point of 4.1. Analytical acrylamide gel electrophoresis showed the purified enzyme preparation contained one major protein band which stained for thymidine phosphorylase activity and usually a minor, faster migrating band devoid of activity. Inactivation of thymidine phosphorylase alone or in the presence of sensitizers by ultraviolet light, primarily at 253.7 nm, followed first order inactivation kinetics. The rate of inactivation of the enzyme was the same at pH 5 and 7.4 and the addition of various pyrimidine bases and nucleosides enhanced the inactivation rate at both pH values, but to a greater extent at pH 5. Linear plots of inactivation rates versus concentrations of thymidine or thymine were the same. At 7.8 mM thymidine or thymine, 11- and 4.4-fold increases in photoinactivation of thymidine phosphorylase were observed at pH 5 AND 7.4 RESPECTIVELY. Parabolic curves were obtained with increasing concentrations of either 5-iodo-2'-deoxyuridine or 5-iodouracil. 5-Iodouracil at 5.2 mM caused 212- (pH 5) and 100- (pH 7.4) FOLD INCREASES IN THE RATES OF PHOTOINACTIVATION OF THYMIDINE PHOSPHORYLASE. However, 5-iodo-2'-deoxyuridine at 5.0mM only enhanced the photoinactivation of enzyme by factors of 83 (pH 5) and 21 (pH 7.4). Neither 5-bromo-2'-deoxyuridine or 5-bromo-uracil was as potent in sensitizing the enzyme as the iodo analogs. Combinations of 5-iodouracil or 5-iodo-2'-deoxyuridine with thymine resulted in higher inactivation rates than the additive inactivation rates of individual compounds, whereas combinations of either iodo analog with thymidine resulted in lower inactivation rates. Increasing concentrations of phosphate or NaCl lessened the photoinactivation rate of thymidine phosphorylase alone and protected the enzyme from the sensitization caused by the different bases and nucleosides. No quantitative changes in the number of primary amino groups in thymidine phosphorylase was evident as a result of irradiation in the presence or absence of 5-iodouracil or 5-iodo-2'-deoxyuridine. Examination of the irradiated enzyme on Sephadex G-150 indicated that a larger protein species is formed and that 5-iodouracil promotes this process.     

5-Bromovinyl 2'-deoxyuridine phosphorylation by mitochondrial and cytosolic thymidine kinase (TK2 and TK1) and its use in selective measurement of TK2 activity in crude extracts

Mitochondrial thymidine kinase (TK2) is responsible for phosphorylation of thymidine and deoxycytidine and plays a crucial role in mitochondrial DNA precursor synthesis. TK2 is expressed in all tissues at low levels complicating accurate determinations, especially in tissues with high cytosolic thymidine kinase (TK1) activity. Recently, 5-bromovinyl 2 '-deoxyuridine (BvdU) at 0.2 micro M was used to measure TK2 activity selectively. BvdU phosphorylation by pure human TK2 and TK1 was tested here, and the ratio of BvdU phosphorylation by TK2/TK1 was 91 at 0.2 micro M but was 500 at 2.5 micro M. Therefore, for reliable measurement of TK2 activity higher BvdU concentration should be used.     

Synthesis and radioiodination of a stannyl oligodeoxyribonucleotide.

Synthesis and radioiodination of a stannyl oligodeoxyribonucleotide were undertaken to evaluate a gamma ray emitting ODN ligand for thrombus imaging in vivo . Synthesis of the ODN was based on modified automatedbeta-cyanoethyl phosphoramidite chemistry with an organotin nucleoside (dU*) coupled to a thrombin binding aptamer sequence to give d(U*GGTTGGTGTGGTTGG). The synthesis accommodated dU*, which is destannylated by iodine or acids. Fourteen standard synthesis cycles were followed by one 'stannyl synthesis cycle', distinguished by Fmoc protection, omission of capping, oxidation by an organic peroxide and cleavage by ammonium hydroxide. The organotin nucleoside phosphoramidite ¿5'-[fluorenylmethoxycarbonyl]-5-(E)-[2-tri-n -butylstannylvinyl]-2'-deoxyuridine-3'-(2-cyanoethyl N,N-diisopropyl phosphoramidite)¿ was prepared from 5-iodo-2'-deoxyuridine. A customized mild rapid workup included deprotection with methylamine, and reverse phase HPLC with CH3CN/triethylammonium bicarbonate. Pure stannyl ODN was highly retained by reverse phase HPLC. Radioiodination of stannyl ODN (100 microg) provided 123I-labeling yields up to 97%. Five alternative oxidants were effective. High specific activity [123I]- ODN (15 000 Ci/mmol) was recovered, separated from unlabeled isomers. Excellent reverse phase HPLC resolution of ODN isomers (alternatively I, Cl, H or Br in vinyl deoxyuridine) was essential. The affinity of the iodovinyl aptamer analog (Kd = 36 nM) for human alpha-thrombin was similar to the native aptamer (Kd = 45 nM).     

Thymidylate nucleotide supply for mitochondrial DNA synthesis in mouse L-cells. Effect of 5-fluorodeoxyuridine and methotrexate in thymidine kinase plus and thymidine kinase minus cells.

The effects of 5-fluorodeoxyuridine and methotrexate on [3H]thymidine and 32P labeling of mtDNA were studied in two lines of mouse L-cells. LMTK- cells, which lack the major cellular thymidine kinase (EC 2.7.1.21) but contain a genetically distinct mitochondrial enzyme, were compared to LA9 cells, which contain both thymidine kinase activities. LMTK- cells were resistant to 5-flurodeoxyuridine by a factor of 200 in comparison to LA9 cells. In both cells lines appropriate drug treatment increased utilization of exogenous thymidine for mtDNA synthesis. The maximum enhancement was 10- to 12-fold for LA9 cells and approximately 20-fold for LMTK- cells when treated with 10 muM methotrexate. The rates of mtDNA and nuclear DNA synthesis during drug treatment were analyzed with 32P labeling and 5-bromo-2'-deoxyuridine density labeling experiments. Synthesis of both mtDNA and nuclear DNA were strongly inhibited by drug treatment of either LA9 or LMTK- cells in the absence of exogenous thymidine. The rate of mtDNA synthesis substantially exceeded that of nuclear DNA in LA9 cells treated with 4 muM 5-fluorodeoxyuridine and less than 5 muM thymidine. Both synthetic rates approached those of untreated LA9 control cultures if 20 muM thymidine was present during 5-fluorodeoxyuridine treatment. In contrast, in LMTK- cells treated with 10 muM methotrexate and 20 muM thymidine, mtDNA synthesis continued at 50 to 60% of the control rate for at least 10 hours while nuclear DNA synthesis was 96% inhibited. Synthesis of mtDNA mass-labeled in both strands with 5-bromouracil occurred when LMTK- cells were incubated for 30 hours with 10 muM methotrexate and 20 muM 5-bromodeoxyuridine. These results indicate that mtDNA synthesis is resistant to a limitation of the thymidine triphosphate supply and is not strictly dependent upon concomitant nuclear DNA synthesis in these cells.     

DNA and LNA oligonucleotides containing N2'-functionalised derivatives of 2'-amino-2'-deoxyuridine

Synthesis of various N-acylated derivatives of 2'-amino-2'-deoxyuridine is described together with their incorporation into DNA and LNA oligonucleotides using the phosphoramidite approach on an automated DNA synthesizer. The thermal stabilities of duplexes formed by these 2'-amino-DNA-modified DNA or LNA/DNA chimeric strands and complementary DNA or RNA strands have been studied. Introduction of LNA monomers around the functionalised 2'-amino-DNA modifications results in reversal of the affinity-decreasing effect of the latter. This represents a novel general approach for design and synthesis of high-affinity functionalised oligonucleotides for biotechnological or medicinal applications.     

Microwave-assisted synthesis of O'-adamantylated uracil-derived nucleosides

Microwave-induced synthesis of O'-adamantyl derivatives of AZT, thymidine, 2'-deoxyuridine and uridine was investigated. Contrary to heterocyclus adamantylation of uracil and uridine in trifluoroacetic acid, the microwave-induced reaction provided sugar-substituted compounds.     

Selective inhibition of the proliferation of herpes simplex virus type 1 thymidine kinase gene-transformed murine mammary FM3A carcinoma cells by (E)-5-(2-bromovinyl)-2'-deoxyuridine and related compounds

Mouse mammary carcinoma FM3A cells deficient in thymidine kinase were transformed by a cloned gene for herpes simplex virus type 1 thymidine kinase. Among several anti-herpetic nucleoside analogues, (E)-5-(2-bromovinyl)-2'-deoxyuridine, (E)-5-(2-iodovinyl)-2'-deoxyuridine and (E)-5-(2-bromovinyl)-2'-deoxycytidine inhibited the growth of the transformed cells at concentrations 5000- to 20000-fold lower than those required to inhibit the growth of the corresponding wild-type cells. The selective inhibitory action of these compounds was due to a specific phosphorylation by the viral thymidine kinase. From the transformed cells, thymidine-auxotrophic mutants that are deficient in thymidylate synthase were isolated. These mutant cell lines should prove useful in elucidating the mechanism of action of the antiherpetic nucleoside analogues.     

Detection of S-phase cell cycle progression using 5-ethynyl-2'-deoxyuridine incorporation with click chemistry, an alternative to using 5-bromo-2'-deoxyuridine antibodies

The 5-bromo-2'-deoxyuridine (BrdU) labeling of cells followed by antibody staining has been the standard method for direct measurement of cells in the S-phase. Described is an improved method for the detection of S-phase cell cycle progression based upon the application of click chemistry, the copper(I)-catalyzed variant of the Huisgen [3+2] cycloaddition between a terminal alkyne and an azide. 5-ethynyl-2'-deoxyuridine (EdU) is a nucleoside analog of thymidine that is incorporated into DNA during active DNA synthesis, just like BrdU. While the BrdU assay requires harsh chemical or enzymatic disruption of helical DNA structure to allow for direct measurement of cells in the S-phase by the anti-BrdU antibody, the EdU method does not. Elimination of this requirement results in the preservation of helical DNA structure and other cell surface epitopes, decreased assay time, and increased reproducibility.     

Synthesis of DNA oligonucleotides containing 5-(mercaptomethyl)-2'-deoxyuridine moieties

Recently thiolated oligonucleotides have attracted significant interest due to their ability to efficiently undergo stable bond formation with gold nanoparticles and surfaces to form DNA conjugates. In this respect we became interested in the synthesis of oligonucleotides that bear short thioalkyl functions located at the nucleobase. Here we present a strategy for the synthesis of DNA oligonucleotides that bear 5-(mercaptomethyl)-2'-deoxyuridine moieties. The building blocks were synthesized in a straightforward manner from thymidine. Only moderate changes of standard protocols for automated DNA synthesis are required for the generation of modified oligonucleotides containing the thiolated building blocks.     

Lymphoblasts already in the DNA synthesis phase of the cell cycle can be reversibly arrested at the R/G transition

Early and late S-phase of the cell cycle are separated by the R-band/G-band (R/G) transition. This corresponds to the time at which R-band synthesis has been completed while G-band synthesis has yet to begin. The aim of this work was to study cell cycle kinetics during S-phase using different blocking agents: mimosine, methotrexate, 5-fluorouracil, 5-fluoro-2'-deoxyuridine and an excess of thymidine. The stage at which these blocking agents arrest the cell cycle and their efficiency at blocking Epstein-Barr virus transformed lymphoblasts at the R/G transition were evaluated using flow cytometric techniques. Mimosine blocked 90% of the cells near the G1/S-phase boundary. Methotrexate, 5-fluoro-2'-deoxyuridine and 5-fluorouracil, and particularly thymidine, let a significant proportion of cells enter S-phase. The cells were released from the arrest state and their progression through early S-phase was monitored by flow cytometry. Before the cells reached the R/G transition, a second agent was added to inhibit cell cycle progression. For example, the use of mimosine followed by thymidine allowed up to 60% of the cells to be blocked at the R/G transition. The arrest of DNA replication at the R/G transition was confirmed by a marked decrease of 5-bromo-2'-deoxyuridine (BrdUrd) incorporation, revealed by using bivariate flow cytometric analysis. The blocking agent was then removed and the cell cohort was released in the presence of BrdUrd so that replication banding analysis could be performed on the harvested mitotic cells. This yielded a mitotic index of approximately 10% and chromosomes showing replication bands. Flow cytometric analysis combined with cytogenetic banding analysis suggested that the R/G transition is an arrest point within the S-phase of the cell cycle and allowed us to conclude that only cells that have already initiated S-phase are blocked at this point. It corresponds to a susceptible site where S-phase can be arrested easily. The R/G transition could also be a regulatory checkpoint within S-phase, a checkpoint that could respond to imbalance in deoxyribonucleotide pools.     

Synthesis of 5-(2,2'-bipyridinyl and 2,2'-bipyridinediiumyl)-2'-deoxyuridine nucleosides: precursors to metallo-DNA conjugates

The synthesis of 2,2'-bipyridinyl-2'-deoxyuridine metal-chelator nucleosides (Bipy-dU) with either ethynyl or ethylenyl linkers was now been accomplished. These new nucleosides will permit the construction of a number of corresponding metallo-DNA conjugates where many types of metals can be complexed to the 2,2'-bipyridinyl chelator group and the resulting metallo-dU conjugates incorporated into DNA oligonucleotides. Additionally this paper also reports the synthesis of a di-N-alkylated bipyridinediiumyl-2'-deoxyuridine nucleoside (Bipy(2+)-dU) with an ethylenyl linker. The Bipy(2+)-dU nucleoside was found to decompose under basic conditions precluding its use in standard automated DNA-synthesis by the phosphoramidite method. No such restrictions apply to the two Bipy-dU nucleosides reported here for use as metal chelators.     

Crystal structure of varicella zoster virus thymidine kinase

Herpes virus thymidine kinases are responsible for the activation of nucleoside antiviral drugs including (E)-5-(2-bromovinyl)-2'-deoxyuridine. Such viral thymidine kinases (tk), beside having a broader substrate specificity compared with host cell enzymes, also show significant variation in nucleoside phosphorylation among themselves. We have determined the crystal structure of Varicella zoster virus (VZV, human herpes virus 3) thymidine kinase complexed with (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-monophosphate and ADP. Differences in the conformation of a loop region (residues 55-61) and the position of two alpha-helices at the subunit interface of VZV-tk compared with the herpes simplex virus type 1 (human herpes virus 1) enzyme give rise to changes in the positioning of residues such as tyrosine 66 and glutamine 90, which hydrogen bond to the substrate in the active site. Such changes in combination with the substitution in VZV-tk of two phenylalanine residues (in place of a tyrosine and methionine), which sandwich the substrate pyrimidine ring, cause an alteration in the positioning of the base. The interaction of the (E)-5-(2-bromovinyl)-2'-deoxyuridine deoxyribose ring with the protein is altered by substitution of tyrosine 21 and phenylalanine 139 (analagous to herpes simplex virus type 1 histidine 58 and tyrosine 172), which may explain some of the differences in nucleoside sugar selectivity between both enzymes. The altered active site architecture may also account for the differences in the substrate activity of ganciclovir for the two thymidine kinases. These data should be of use in the design of novel antiherpes and antitumor drugs.     

Monitoring DNA replication in fission yeast by incorporation of 5-ethynyl-2'-deoxyuridine

We report procedures to allow incorporation and detection of 5-ethynyl-2'-deoxyuridine (EdU) in fission yeast, a thymidine analogue which has some technical advantages over use of bromodeoxyuridine. Low concentrations of EdU (1 μM) are sufficient to allow detection of incorporation in cells expressing thymidine kinase and human equilibrative nucleoside transporter 1 (hENT1). However EdU is toxic and activates the rad3-dependent checkpoint, resulting in cell cycle arrest, potentially limiting its applications for procedures which require labelling over more than one cell cycle. Limited DNA synthesis, when elongation is largely blocked by hydroxyurea, can be readily detected by EdU incorporation using fluorescence microscopy. Thus EdU should be useful for detecting early stages of S phase, or DNA synthesis associated with DNA repair and recombination.     

Cooperative effects between two acyclovir resistance loci in herpes simplex virus.

The acyclovir-resistant mutant SC16 R9C2 (H.J. Field, G. Darby, and P. Wildy , J. Gen. Virol. 49:115-124, 1980) has been shown to contain two resistance loci which segregate independently on recombination with wild-type virus. One locus is in thymidine kinase, and the other is in DNA polymerase. Both induced enzymes have altered properties, thymidine kinase showing a low affinity for acyclovir and low activity, and DNA polymerase showing a low affinity for acyclovir triphosphate. Other properties of both enzymes are described which distinguish them from their wild-type counterparts. Recombinants containing either mutant thymidine kinase ( RSC -11) or mutant DNA polymerase ( RSC -26), but not both, have been used to investigate the relative contribution of each lesion to resistance and pathogenicity. Although SC16 R9C2 and both recombinants grow as well as does wild-type virus in tissue culture, they are considerably attenuated in vivo, the greatest attenuation of virulence being seen with SC16 R9C2 and RSC -26. With respect to both acyclovir resistance and in vivo growth, the lesions appear to behave synergistically. Cross resistance studies have shown the recombinant RSC -26, which contains mutant DNA polymerase but which evidently expresses wild-type thymidine kinase, to be cross resistant to both 5-iodo-2'-deoxyuridine and 5-trifluoromethyl-2'-deoxyuridine but not to (E)-5-(2-bromovinyl)-2'-deoxyuridine or 9-beta-D-arabinofuranosyladenine.