Synthesis of 2'-C,4'-C-Methyleneoxy-Bridged Thymidine Derivatives and Properties of Modified Oligonucleotides.

2',4'-Bridged nucleic acid (2',4'-BNA) analogues are used for therapeutic oligonucleotides, owing to their excellent hybridizing ability with complementary RNA and high resistance toward enzymatic degradation. We developed 2',4'-BNA analogues with oxygen atoms at 6'-positions (e.g., EoNA and EoDNAs) and demonstrated that the presence of 6'-oxygen atoms in the bridge structure could show positive effect on the properties of the modified oligonucleotides. Herein, we designed and synthesized 7'-methyl derivatives of methyleneoxy-bridged 2'-deoxyribonucleic acid (MoDNA), possessing a five-membered bridge with 6'-oxygen atom via radical cyclization for the bridge construction. The synthesized monomers were incorporated into the oligonucleotides by solid-phase oligonucleotide synthesis. The MoDNA-modified oligonucleotides showed high affinity toward single-stranded RNA and double-stranded DNA, as well as excellent resistance toward nuclease compared with the corresponding natural oligonucleotide.

Synthesis and anticancer activity of 3'-[4-fluoroaryl-(1,2,3-triazol-1-yl)]-3'-deoxythymidine analogs and their phosphoramidates.

A series of novel 4-chlorophenyl N-alkyl phosphoramidates of 3'-[4-fluoroaryl-(1,2,3-triazol-1-yl)]-3'-deoxythymidines (20-49) was synthesized by means of phosphorylation of 3'-[4-aryl-(1,2,3-triazol-1-yl)]-3'-deoxythymidines (7-11) with 4-chlorophenyl phosphoroditriazolide (14), followed by a reaction with the appropriate amine. The synthesized compounds 7-11 and 20-49 were evaluated along with four known anticancer compounds for their cytotoxic activity in human cancer cell lines: cervical (HeLa), nasopharyngeal (KB), breast (MCF-7), osteosarcoma (143B) (only selected compounds 20, 24, 28, 32-36, 38, 40, 46) and normal human dermal fibroblast cell line (HDF) using the sulforhodamine B (SRB) assay. Among 3'-[4-aryl-(1,2,3-triazol-1-yl)]-3'-deoxythymidines (7-11) the highest activity in all the investigated cancer cells was displayed by 3'-[4-(3-fluorophenyl)-(1,2,3-triazol-1-yl)]-3'-deoxythymidine (9) (IC50 in the range of 2.58-3.61 µM) and its activity was higher than that of cytarabine. Among phosphoramidates 20-49 the highest activity was demonstrated by N-n-propyl phosphoramidate of 3'-[4-(3-fluorophenyl)-(1,2,3-triazol-1-yl)]-3'-deoxythymidine (35) in all the cancer cells (IC50 in the range of 0.97-1.94 µM). Also N-ethyl phosphoramidate of 3'-[4-(3-fluorophenyl)-(1,2,3-triazol-1-yl)]-3'-deoxythymidine (33) exhibited good activity in all the used cell lines (IC50 in the range of 4.79-4.96 µM).

Synthesis and in vitro Biological Evaluation of Novel Thymidine Analogs Containing 1H-1,2,3-Triazolyl, 1H-Tetrazolyl, and 2H-Tetrazolyl Fragments.

3'-Azidothymidine (AZT) reacts with 1-propargyl-5-R-1H- and 2-propargyl-5-R-2H-tetrazoles (R = H, Me, CH2COOEt, CH2CON(CH3)2, Ph, 2-CH3-C6H4, or 4-NO2-C6H4) via the Cu(I)-catalyzed asymmetric [3 + 2] cycloaddition to give 3'-modified thymidine analogs incorporating 1H-1,2,3-triazolyl, 1H-, and 2H-tetrazolyl fragments in 41-76% yield. The structures of the obtained compounds have been elucidated by means of HRESI+-MS, 1H and 13 C{1H} NMR, and single crystal X-ray diffraction {for 3'-[4-(1H-5-N,N-dimethylaminocarbonylmethyltetrazol-1-yl)-1H-1,2,3-triazol-1-yl]thymidine 10d}. In vitro biological evaluation of the prepared compounds has been performed; they have exhibited low activity against phenotypic HIV-1899A. Moderate anti-influenza activity against influenza virus A/Puerto Rico/8/34 (H1N1) strain has been observed in the cases of 3'-(4-(1H-tetrazol-1-ylmethyl)-1H-1,2,3-triazol-1-yl)thymidine 10a (IC50 39.6 µg/mL), 3'-(4-(2H-5-ethoxycarbonyltetrazol-2-ylmethyl)-1H-1,2,3-triazol-1-yl)thymidine 11c (IC50 31.6 µg/mL), and 3'-(4-(2H-5-(4-nitrophenyl)-tetrazol-2-ylmethyl)-1H-1,2,3-triazol-1-yl)thymidine 11g (IC50 46.4 µg/mL). The tested compounds possess very low cytotoxicity towards MDCK and MT4 cells as well as tumor human cervical carcinoma HeLa and promyelocytic leukemia HL-60 cells.

Separation of Protactinium Employing Sulfur-Based Extraction Chromatographic Resins.

Protactinium-230 ( t1/2 = 17.4 d) is the parent isotope of 230U ( t1/2 = 20.8 d), a radionuclide of interest for targeted alpha therapy (TAT). Column chromatographic methods have been developed to separate no-carrier-added 230Pa from proton irradiated thorium targets and accompanying fission products. Results reported within demonstrate the use of novel sulfur bearing chromatographic extraction resins for the selective separation of protactinium. The recovery yield of 230Pa was 93 ± 4% employing a R3P═S type commercially available resin and 88 ± 4% employing a DGTA (diglycothioamide) containing custom synthesized extraction chromatographic resin. The radiochemical purity of the recovered 230Pa was measured via high purity germanium γ-ray spectroscopy to be >99.5% with the remaining radioactive contaminant being 95Nb due to its similar chemistry to protactinium. Measured equilibrium distribution coefficients for protactinium, thorium, uranium, niobium, radium, and actinium on both the R3P═S type and the DGTA resin in hydrochloric acid media are reported, to the best of our knowledge, for the first time.

An organocatalyzed Stetter reaction as a bio-inspired tool for the synthesis of nucleic acid-based bioconjugates.

An N-Heterocyclic Carbene (NHC) catalyzed biomimetic Stetter reaction was applied for the first time as a bioconjugation reaction to sensitive nucleoside-type biomolecules to provide original pyrrole linked nucleolipids. A versatile approach allowed the functionalization of thymidine at the three reactive positions (O-5', O-3' and N-3) providing a structural diversity oriented synthesis. This strategy was applied to the synthesis of an original glyconucleolipid amphiphile in the hope that the pyrrole aromatic moiety would induce additional self-assembling properties.

Synthesis and cytotoxic activity of novel acyclic nucleoside analogues with functionality in click chemistry.

We describe synthesis of novel acyclic nucleoside analogues which are building blocks for CuAAC reaction and their activity against two types of human cancer cell lines (HeLa, KB). Three of chosen compounds show promising cytotoxic activity. Synthesis pathway starting from simple and easily accessible substrates employing DMT or TBDPS protective groups is described. Adenosine and thymidine analogues containing alkyne moiety and adenosine analogue containing azido group were synthesized. The obtained units showed ability of forming triazole motif under the CuAAC reaction conditions.

Independent Generation and Reactivity of Thymidine Radical Cations.

Thymidine radical cation (1) is produced by ionizing radiation and has been invoked as an intermediate in electron transfer in DNA. Previous studies on its structure and reactivity have utilized thymidine as a precursor, which limits quantitative product analysis because thymidine is readily reformed from 1. In this investigation, radical cation 1 is independently generated via ß-heterolysis of a pyrimidine radical generated photochemically from an aryl sulfide. Thymidine is the major product (33%) from 1 at pH 7.2. Diastereomeric mixtures of thymidine glycol and the corresponding 5-hydroxperoxides resulting from water trapping of 1 are formed. Significantly lower yields of products such as 5-formyl-2'-deoxyuridine that are ascribable to deprotonation from the C5-methyl group of 1 are observed. Independent generation of the N3-methyl analogue of 1 (NMe-1) produces considerably higher yields of products derived from water trapping, and these products are formed in much higher yields than those attributable to the C5-methyl group deprotonation in NMe-1. N3-Methyl-thymidine is, however, the major product and is produced in as high as 70% yield when the radical cation is produced in the presence of excess thiol. The effects of exogenous reagents on product distributions are consistent with the formation of diffusively free radical cations (1, NMe-1). This method should be compatible with producing radical cations at defined positions within DNA.

Site-specific covalent capture of human O6-alkylguanine-DNA-alkyltransferase using single-stranded intrastrand cross-linked DNA.

A methodology is reported to conjugate human O6-alkylguanine-DNA-alkyltransferase (hAGT) to the 3'-end of DNA in excellent yields with short reaction times by using intrastrand cross-linked (IaCL) DNA probes. This strategy exploited the substrate specificity of hAGT to generate the desired DNA-protein covalent complex. IaCL DNA linking two thymidine residues, or linking a thymidine residue to a 2'-deoxyguanosine residue (either in a 5'→3' or 3'→5' fashion), lacking a phosphodiester linkage at the cross-linked site, were prepared using a phosphoramidite strategy followed by solid-phase synthesis. All duplexes containing the model IaCL displayed a reduction in thermal stability relative to unmodified control duplexes. The O4-thymidine-alkylene-O4-thymidine and the (5'→3') O6-2'-deoxyguanosine-alkylene-O4-thymidine IaCL DNA adducts were not repaired by any of the AGTs evaluated (human AGT and Escherichia coli homologues, OGT and Ada-C). The (5'→3') O4-thymidine-alkylene-O6-2'-deoxyguanosine IaCL DNA containing a butylene or heptylene tethers were efficiently repaired by the human variant, whereas Ada-C was capable of modestly repairing the heptylene IaCL adduct. The IaCL strategy has expanded the toolbox for hAGT conjugation to DNA strands, without requiring the presence of a complementary DNA sequence. Finally, hAGT was functionalized with a fluorescently-labelled DNA sequence to demonstrate the applicability of this conjugation method.

Reaction of Thymidine with Hypobromous Acid in Phosphate Buffer.

When thymidine was treated with hypobromous acid (HOBr) in 100 mM phosphate buffer at pH 7.2, two major product peaks appeared in the HPLC chromatogram. The products in each peak were identified by NMR and MS as two isomers of 5-hydroxy-5,6-dihydrothymidine-6-phosphate (a novel compound) and two isomers of 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol) with comparable yields. 5-Hydroxy-5,6-dihydrothymidine-6-phosphate was relatively stable, and decomposed with a half-life of 32 h at pH 7.2 and 37°C generating thymidine glycol. The results suggest that 5-hydroxy-5,6-dihydrothymidine-6-phosphate in addition to thymidine glycol may have importance for mutagenesis by the reaction of HOBr with thymine residues in nucleotides and DNA.

Synthesis of new C-5-triazolyl-functionalized thymidine analogs and their ability to engage in aromatic stacking in DNA : DNA and DNA : RNA duplexes.

1-Phenyl-1,2,3-triazole scaffolds on the 5-position of pyrimidine nucleosides have previously shown to enhance nuclease stability and increase the duplex thermal stability (Tm) by engaging in duplex stacking interactions. In this study, we have introduced two new derivatives of this scaffold in DNA : DNA and DNA : RNA duplexes in order to explore the thermal effects of (1) using a 1,5-triazole instead of the usual 1,4-triazole, and (2) replacing the apolar phenyl substituent with a polar uracil-5-yl substituent.

Synthesis of activated 3'-amino-3'-deoxy-2-thio-thymidine, a superior substrate for the nonenzymatic copying of nucleic acid templates.

We present a scalable synthesis of 3'-amino-3'-deoxy-2-thio-thymidine-5'-phosphoro-2-methylimidazolide, an activated monomer that can copy adenosine residues in nucleic acid templates rapidly without a polymerase. The sulfur atom substitution enhances the rate of template copying by 5-fold compared with the 3'-amino-3'-deoxy-T monomer, while the 3'-amino monomers exhibit a 2- to 30-fold enhancement compared with their ribonucleotide counterparts.

Synthesis and base pairing studies of geranylated 2-thiothymidine, a natural variant of thymidine.

The synthesis and base pairing of DNA duplexes containing the geranylated 2-thiothymidine have been investigated. This naturally existing hydrophobic modification could grant better base pairing stability to the T-G pair over normal T-A and other mismatched pairs in the duplex context. This study provides a potential explanation for the different codon recognition preferences of the geranylated tRNAs.

Scalable Synthesis of the Potent HIV Inhibitor BMS-986001 by Non-Enzymatic Dynamic Kinetic Asymmetric Transformation (DYKAT).

Described herein is the synthesis of BMS-986001 by employing two novel organocatalytic transformations: 1) a highly selective pyranose to furanose ring tautomerization to access an advanced intermediate, and 2) an unprecedented small-molecule-mediated dynamic kinetic resolution to access a variety of enantiopure pyranones, one of which served as a versatile building block for the multigram, stereoselective, and chromatography-free synthesis of BMS-986001. The synthesis required five chemical transformations and resulted in a 44% overall yield.

Participation of an additional 4'-hydroxymethyl group in the cleavage and isomerization of ribonucleoside 3'-phosphodiesters.

4'-(Hydroxymethyl)uridylyl-3',5'-thymidine, an RNA model bearing an extra hydroxymethyl group at the 4'-position of the 3'-linked nucleoside, has been prepared and its cleavage and isomerization reactions studied over a wide pH range (from 0 to 12). Overall, the pH-rate profiles of these reactions were very similar to those of uridylyl-3',5'-uridine (UpU) - only a very modest acceleration was observed under acidic and neutral conditions. Evidently, hydrogen bond assistance by the additional hydroxymethyl function does not play a significant role.

Sulfonamide-bridged nucleic acid: synthesis, high RNA selective hybridization, and high nuclease resistance.

2'-N,4'-C-(N-methylamino)sulfonylmethylene-bridged thymidine (SuNA), which has a six-membered linkage including a sulfonamide moiety, was synthesized and introduced into oligonucleotides. The oligonucleotides containing SuNA exhibited excellent nuclease resistance, a high affinity toward single-stranded RNA, and a low affinity toward single-stranded DNA compared to the natural oligonucleotide.

Antiherpesvirus activities of two novel 4'-thiothymidine derivatives, KAY-2-41 and KAH-39-149, are dependent on viral and cellular thymidine kinases.

The emergence of drug-resistant herpesviruses represents a significant problem in clinical practice, primarily in immunocompromised patients. Furthermore, effective antiviral therapies against gammaherpesvirus-associated diseases are lacking. Here, we present two thiothymidine derivatives, KAY-2-41 and KAH-39-149, with different spectra of antiviral activity from those of the reference antiherpetic drugs, showing inhibitory activities against herpes simplex virus, varicella-zoster virus (VZV), and particularly against Epstein-Barr virus, with high selectivity in vitro. While KAY-2-41- and KAH-39-149-resistant herpesviruses were found to harbor mutations in the viral thymidine kinase (TK), these mutations conferred only low levels of resistance to these drugs but high levels to other TK-dependent drugs. Also, antiviral assays in HeLa TK-deficient cells showed a lack of KAY-2-41 and KAH-39-149 activities against herpes simplex virus 1 (HSV-1) and HSV-2 TK-deficient mutants. Furthermore, enzymatic TK assays showed the ability of HSV-1 TK, VZV TK, and cellular TK1 and TK2 to recognize and phosphorylate KAY-2-41 and KAH-39-149. These results demonstrate that the compounds depend on both viral and host TKs to exert antiviral activity. Additionally, the antiviral efficacy of KAH-39-149 proved to be superior to that of KAY-2-41 in a mouse model of gammaherpesvirus infection, highlighting the potential of this class of antiviral agents for further development as selective therapeutics against Epstein-Barr virus.

4'-C-[(4-trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl]thymidine as a sensitive (19)F NMR sensor for the detection of oligonucleotide secondary structures.

4'-C-[(4-Trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl]thymidine was synthesized and incorporated as a phosphoramidite into oligonucleotide sequences. Its applicability as a sensor for the (19)F NMR spectroscopic detection of DNA and RNA secondary structures was demonstrated. On DNA, the (19)F NMR measurements were focused on monitoring of duplex-triplex conversion, for which this fluorine-labeled 2'-deoxynucleoside proved to be a powerful sensor. This sensor seemingly favors DNA, but its behavior in the RNA environment also turned out to be informative. As a demonstration, invasion of a 2'-O-methyl oligoribonucleotide into an RNA hairpin model (HIV-1 TAR) was monitored by (19)F NMR spectroscopy. According to the thermal denaturation studies by UV spectroscopy, the effect of the 4'-C-(4-trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl moiety on the stability of these DNA and RNA models was marginal.

Nucleoside-based probes for imaging tumor proliferation using positron emission tomography.

Cancer is one of the leading causes of human death, and early detection can be beneficial for its timely therapy and management. For the early detection of cancer, positron emission tomography (PET) is more accurate and sensitive than other imaging modalities, such as computed tomography and magnetic resonance imaging. [(18) F]-Labeled fluorodeoxyglucose is the most useful PET probe in early detection of cancer; however, its nonspecific accumulation and consequent false-positive findings warrant the identification of other PET probes. Thymidine (TdR) and its analogs have been radiolabeled for PET imaging of cellular proliferation and DNA synthesis. Because of its in vivo instability, radiolabeled TdR has not been successful in PET imaging. However, some of its radiolabeled analogs have been developed for PET imaging of cellular proliferation and DNA synthesis. In this review, the radiochemistry and production of (11) C-TdR and (11) C/(18) F-labeled TdR analogs published to date are presented.

Synthesis of a probe for monitoring HSV1-tk reporter gene expression using chemical exchange saturation transfer MRI.

In experiments involving transgenic animals or animals treated with transgenic cells, it is important to have a method to monitor the expression of the relevant genes longitudinally and noninvasively. An MRI-based reporter gene enables monitoring of gene expression in the deep tissues of living subjects. This information can be co-registered with detailed high-resolution anatomical and functional information. We describe here the synthesis of the reporter probe, 5-methyl-5,6-dihydrothymidine (5-MDHT), which can be used for imaging of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene expression in rodents by MRI. The protocol also includes data acquisition and data processing routines customized for chemical exchange saturation transfer (CEST) contrast mechanisms. The dihydropyrimidine 5-MDHT is synthesized through a catalytic hydrogenation of the 5,6-double bond of thymidine to yield 5,6-dihydrothymidine, which is methylated on the C-5 position of the resulting saturated pyrimidine ring. The synthesis of 5-MDHT can be completed within 5 d, and the compound is stable for more than 1 year.

Disaccharide pyrimidine nucleosides and their derivatives: a novel group of cell-penetrating inhibitors of poly(ADP-ribose) polymerase 1.

Nearly 30 synthetic nucleosides were tested with human recombinant poly(ADP-ribose) polymerase 1 as potential inhibitors of this enzyme. The most active compounds were some disaccharide analogues of thymidine: 3'-O-ß-D-ribofuranosyl-5-iodo-dUrd (2d; IC50 = 45 µM), 3'-O-ß-D-ribofuranosyl-2'-deoxythymidine (2e; IC50 = 38 µM), and 3'-O-ß-D-ribofuranosyl-2'-deoxythymidine oxidized (4; IC50 = 25 µM). These compounds also reduced H2O2-induced synthesis of poly(ADP-ribose) in cultured human ovarian carcinoma (SKOV-3) cells in a dose-dependent manner. Furthermore, compounds 2d or 2e until a concentration of 1 mM did not affect growth of SKOV-3 cells, whereas dialdehyde compound 4, as well as thymidine, exhibited a significant cytotoxicity.