Incorporation of the carbocyclic analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate into a synthetic DNA

The carbocyclic analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine, C-BVDU, is a very potent and selective anti-herpes-virus compound. In order to synthesize and study the properties of a DNA that contains C-BVDU, the 5'-triphosphate, C-BVDUTP was prepared and evaluated as a potential substrate of the E. coli Klenow DNA polymerase enzyme. Although C-BVDUTP proved to be a very poor substrate also of this enzyme, it could be incorporated up to 3.6% into the synthetic DNA, poly(dA-dT, C-BVDU). This level of substitution decreased significantly the template activity for DNA and RNA polymerases, as compared to that of poly(dA-dT).     

Double-stranded oligonucleotides containing 5-aminomethyl-2'-deoxyuridine form thermostable anti-parallel triplexes with single-stranded DNA or RNA

This Letter describes the synthesis and properties of double-stranded antisense oligonucleotides connected with a pentaerythritol linker. We found that double-stranded antisense oligonucleotides with aminomethyl residues have high affinity for single-stranded DNA or RNA in buffer solutions with and without MgCl(2). Thus, these oligonucleotides would be useful as antisense oligonucleotides for targeting single-stranded RNA through triplex formation.     

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.     

Synthesis of 5-(pyridinyl and pyridiniumyl)-2'-deoxyuridine nucleosides: reversible electron traps for DNA

The desire to produce reversible electron traps for direct, room temperature studies of excess electron transport in DNA duplexes and hairpins motivated our efforts first to link pyridines to 2'-deoxyuridine (pyridinyl-dU) and then to convert these new conjugates into pyridiniumyl-dU nucleosides. Base sensitivity studies presented here rule out general use of bipyridinediiumyl compounds, but show that pyridiniumyl compounds are suitable for use under the strand cleavage and base deprotection procedures required for automated solid-phase oligonucleotide synthesis. This paper presents the synthesis of four 5'-O-DMT-protected 5-(N-methylpyridiniumyl)-dU conjugates using either ethynyl or ethylenyl linkers to join the pyridiniumyl and dU subunits.     

Potent inhibitory effects of the 5'-triphosphates of (E)-5-(2-bromovinyl)-2'-deoxyuridine and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil on DNA polymerase gamma

(E)-5-(2-Bromovinyl)-2'-deoxyuridine 5'-triphosphate (BrVdUTP) and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil 5'-triphosphate (BrVarafUTP), which are known as specific inhibitors of herpes simplex viral (type 1 and 2) DNA polymerase, were found to be strong inhibitors of DNA polymerase gamma from human KB and murine myeloma cells. In fact BrVdUTP and BrVarafUTP were found to be stronger inhibitors of DNA polymerase gamma than of other DNA polymerases having viral (herpes simplex virus or retrovirus) origin or cellular (eukaryotic alpha and beta, or prokaryotic) origin. The mode of inhibition of DNA polymerase gamma by BrVdUTP and BrVarafUTP was competitive with respect to dTTP, the normal substrate. Whereas BrVdUTP was an efficient substrate for DNA polymerase gamma and other DNA polymerases that were examined, BrVarafUTP failed to serve as a substrate for DNA synthesis. Ki values for BrVdUTP (40 nM) and BrVarafUTP (7 nM) with DNA polymerase gamma, as determined with (rA)n.(dT) as the template.primer, were much smaller than the Km values for dTTP (0.16 microM and 0.71 microM for murine and human DNA polymerase gamma, respectively). Thus, the affinity of BrVdUTP or BrVarafUTP for DNA polymerase gamma was much stronger than that of dTTP.     

Effect of the 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole residue on the stability of DNA duplexes and triplexes.

3-Nitropyrrole (M) was introduced as a non-discriminating 'universal' base in nucleic acid duplexes by virtue of small size and a presumed tendency to stack but not hydrogen bond with canonical bases. However, the absence of thermally-induced hyperchromic changes by single-stranded deoxyoligomers in which M alternates with A or C residues shows that M does not stack strongly with A or C nearest neighbors. Yet, the insertion of a centrally located M opposite any canonical base in a duplex is sometimes even less destabilizing than that of some mismatches, and the variation in duplex stability is small. In triplexes, on the other hand, an M residue centrally located in the third strand reduces triplex stability drastically even when the X.Y target base pair is A.T or G. C in a homopurine. homopyrimidine segment. But, when the target duplex opposition is M-T and the third strand residue is T, the presence of M in the test triplet has little effect on triplex stability. Therefore, a lack of hydrogen bonding in an otherwise helix-compatible test triplet cannot be responsible for triplex destabilization when M is the third strand residue. Thus, M is non-discriminating and none-too-destabilizing in a duplex, but in a triplex it is extremely destabilizing when in the third strand.     

Metabolism of the carbocyclic analogue of (E)-5-(2-iodovinyl)-2'-deoxyuridine in herpes simplex virus-infected cells. Incorporation of C-IVDU into DNA

The carbocyclic analogues of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU), in which the sugar moiety is replaced by a cyclopentane ring and which have been designated as C-BVDU and C-IVDU, respectively, are, like their parent compounds BVDU and IVDU, potent and selective inhibitors of herpes simplex virus type 1 (HSV-1) and, to a lesser extent, herpes simplex virus type 2 (HSV-2) replication. We have now synthesized the radiolabeled C-IVDU analogue, C-[125I]IVDU, and determined its metabolism by HSV-infected and mock-infected Vero cells. C-[125I]IVDU was effectively phosphorylated by HSV-1-infected cells and, to a lesser extent, HSV-2-infected cells. C-[125I]IVDU was not phosphorylated to an appreciable extent by either mock-infected cells or cells that had been infected with a thymidine kinase-deficient mutant of HSV-1. Furthermore, C-[125I]IVDU was incorporated into both viral and cellular DNA of HSV-1-infected Vero cells. This finding represents the first demonstration of the incorporation of a cyclopentylpyrimidine into DNA.     

Induction of gene mutations by 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), an antiviral pyrimidine nucleoside analogue

5-(2-chloroethyl)-2'-deoxyuridine (CEDU) had been developed for the treatment of herpes simplex infections. In the Salmonella reverse mutation test, the compound was found to be mutagenic in strains TA1535 and TA102 at very high concentrations (> or =2500 micro g/plate), both with and without S9-mix. The mutagenic potential of CEDU was further investigated in vivo and in vitro. It did not induce DNA repair in rat hepatocyte primary cultures, and was negative in the micronucleus test in V79 cells and in the comet assay in human leukocytes. In vivo, CEDU was negative in the bone marrow micronucleus test in CD1 mice. The mouse spot test provided a clearly positive result. Treatment of mice on day 9 of pregnancy with 2000 mg/kg resulted in 5.9% of the F1 animals having genetically relevant spots, whereas the corresponding vehicle control group had a spot rate of 1.9%. Since these data clearly identified CEDU as an inducer of gene mutations in vivo, this potential was further investigated in lacZ transgenic Muta Mouse. Six female animals were treated daily on five consecutive days with 2000 mg/kg/day and sacrificed, after a treatment-free sampling time, 14 days later. The data showed a clear increase in the mutant frequency in the bone marrow, the lung and in the spleen. CEDU is an exception in the group of nucleoside analogues, because it was found to be a strong gene mutagen and, in contrast to the other compounds of this group investigated so far, had no considerable clastogenic effects.     

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, protonation behavior, conformational analysis, and regioselective enzymatic acylation of the novel diamino analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU)

(E)-3',5'-Diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. The protonation behavior of 5 has been studied by means of pH-metric measurements and NMR spectroscopy. This study allows the determination of the basicity constants and the stepwise protonation sites. Thus, the main species at physiological pH is the monoprotonated form. The conformational analysis of this nucleoside analogue was also carried out through 1H NMR spectroscopy. In addition, a convenient synthesis of N-3' and N-5' acylated derivatives was developed by regioselective enzymatic acylation. Thus, Candida antarctica lipase B (CAL-B) selectively acylated the 5'-amino group, thus furnishing nucleosides 8. On the other hand, immobilized Pseudomonas cepacia lipase (PSL-C) exhibited the opposite selectivity, conferring acylation at the 3'-amino group, thus affording derivatives 9.     

A convenient synthesis of a novel nucleoside analogue: 4-(alpha-diformyl-methyl)-1-(beta-D-ribofuranosyl)-2-pyrimidinone

The nucleoside analogue 4-(alpha-diformyl-methyl)-1-(beta-D-ribofuranosyl)-2-pyrimidinone (5) was prepared from the corresponding 4-methyl pyrimidinone nucleoside by means of the Vilsmeier reaction. The unprotected nucleoside can be phosphorylated directly with phosphorus oxychloride in triethyl phosphate.     

Inhibition of terminal deoxynucleotidyltransferase by (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate

(E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate (BVdUTP), known as a specific inhibitor of herpes simplex virus (type 1)-DNA polymerase, was found to be a potent inhibitor of the activity of terminal deoxynucleotidyltransferase (TdT) from calf thymus. BVdUTP was not an efficient substrate of TdT, but it inhibited the incorporation of normal deoxynucleotide substrates in competitive fashion at the nucleotide binding site of TdT molecule. The Ki value for BVdUTP (5 microM) was much less than the Km value for dGTP (83 microM), indicating stronger affinity of the inhibitor to TdT than that of the substrate. These results indicate the usefulness of BVdUTP as a potent inhibitor of TdT for elucidation of the reaction mechanism of this enzyme.     

The relationship between incorporation of E-5-(2-Bromovinyl)-2'-deoxyuridine into herpes simplex virus type 1 DNA with virus infectivity and DNA integrity

E-5-(2-Bromovinyl)-2'-deoxyuridine (BrvdUrd) produced a dose-dependent shift in the density of herpes simplex virus type 1 (HSV-1) DNA at concentrations which yielded potent inhibition of virus replication in cultured Vero cells. Although the density of cellular DNA was not altered by these concentrations of BrvdUrd, incorporation of this analogue into cellular DNA of HSV-1-infected cells has been previously observed in this laboratory. The degree of inhibition correlated with the amount of BrvdUrd substituted for thymidine in HSV-1 DNA. BrvdUrd-substituted DNA was more labile as determined by a dose-dependent increase in single strand breaks when examined by centrifugation in alkaline sucrose gradients. Thus, the potent antiviral action of BrvdUrd observed in cell culture correlates not only with its incorporation into HSV-1 DNA but also with an altered stability of this DNA.     

Measurement of in vitro leucocyte mitogenesis in fish: ELISA based detection of the thymidine analogue 5'-bromo-2'-deoxyuridine

In this study we present a method for the measurement of in vitro mitogenesis in fish leucocytes that is based on the incorporation of the thymidine analogue 5'-bromo-2'-deoxyuridine (BrdU) into the DNA of replicating cells, followed by ELISA-based detection. This technique, adapted from methods developed for mammalian cells, operates on a similar biological principle to (3)H-thymidine incorporation, but circumvents the logistical and safety issues inherent with the radioactive label. Because it directly measures DNA proliferation, the assay has advantages over other colorimetric methods that may be strongly influenced by leucocyte metabolic status. Using BrdU incorporation followed by ELISA, we evaluate the responsiveness of rainbow trout (Oncorhynchus mykiss [Walbaum]) leucocytes to the mammalian T-cell mitogen Concanavalin A (Con A) as well as the differential response of white perch (Morone americana [Gmelin]) leucocytes to Con A and pokeweed mitogen. Specific considerations intrinsic to the assay system are discussed, including the implications of utilising enzyme-based detection.     

Enantiospecific synthesis of carbocyclic (+)-(E)-5-(2-bromovinyl)-2'-deoxyuridine

(+)-1-[(1R, 3S, 4R)-3-hydroxy-4-hydroxymethylcyclopentyl]-5-[(E)-2- bromovinyl]-1H,3H-pyrimidin-2,4-dione 10 was synthesized starting from (+)-endo-5-norbornen-2-yl acetate. This chiral educt was obtained by enzymatic hydrolysis of racemic esters of endo-5-norbornen-2-ol.     

Mechanism-based inactivation of thymidylate synthase by 5-(3-fluoropropyn-1-yl)-2'-deoxyuridine 5'-phosphate

5-Fluoropropynyl-2'-deoxyuridine 5'-phosphate (3) was designed as a mechanism-based inactivator of thymidylate synthase (TS). The inhibitor was synthesized from 5-iodo-2'-deoxyuridine and propargyl alcohol by palladium-catalyzed coupling, followed by fluorination and selective phosphorylation. Incubation of TS with 3, in the presence or absence of the CH2H4folate cofactor, caused rapid, irreversible inactivation of the enzyme.     

DNA triple helix formation at target sites containing several pyrimidine interruptions: stabilization by protonated cytosine or 5-(1-propargylamino)dU.

DNase I footprinting has been used to study the formation of parallel triplexes at oligopurine target sequences which are interrupted by pyrimidines at regular intervals. TA interruptions are targeted with third strand oligonucleotides containing guanine, generating G x TA triplets, while CG base pairs are targeted with thymine, forming T x CG triplets. We have attempted to optimize the stability of these complexes by varying the base composition and sequence arrangement of the target sites, and by replacing the third strand thymines with the positively charged analogue 5-(1-propargylamino)dU (U(P)). For the target sequence (AAAT)(5)AA, in which pyrimidines are positioned at every fourth residue, triplex formation with TG-containing oligonucleotides is only detected in the presence of a triplex-binding ligand, though stable triplexes were detected at the target site (AAAAAT)(3)AAAA. Triplex stability at targets containing pyrimidines at every fourth residue is increased by introducing guanines into the duplex repeat unit using the targets (AGAT)(5)AA and (ATGA)(5)AA. In contrast, placing C(+) x GC triplets on the 5'-side of G x TA, using the target (AGTA)(5)TT, produces complexes of lower stability. We have attempted further to increase the stability of these complexes by using the positively charged thymine base analogue U(P), and have shown that (TU(P)TG)(5)TT forms a more stable complex with target (AAAT)(5)AA than the unmodified third strand, generating a footprint in the absence of a triplex-binding ligand. Triplex formation at (AGTA)(5)AA is improved by using the modified oligonucleotide (TCGU(P))(5)TT, generating a complex in which the charged triplets C(+) x GC and U(P) x AT alternate with uncharged triplets. In contrast, placing U(P) x AT triplets adjacent to C(+) x GC, using the third strand oligonucleotide (U(P)CGT)(5)TT, reduces triplex formation, while the third strand with both substitutions, (U(P)CGU(P))(5)TT, produces a complex with intermediate stability. It appears that, although adjacent U(P) x AT triplets form stable triplexes, placing U(P) x AT adjacent to C(+) x GC is unfavorable. Similar results were obtained with fragments containing CG inversions within the oligopurine tract, though triplexes at (AAAAAC)(3)AA were only detected in the presence of a triplex-binding ligand. Placing C(+) x GC on the 5'-side of T x CG triplets also reduces triplex formation, while a 3'-C(+) x GC produces complexes with increased stability.