Nucleic acid components and their analogs: Design and synthesis of novel cytosine thioglycoside analogs

The synthesis of a new category of novel cytosine 4-thioglycoside analogs has been first accomplished. The main step of this strategy is the synthesis of sodium pyrimidine-4-thiolate through the condensation of 2-cyano-N-arylacetamides with sodium cyanocarbonimidodithioate, followed by coupling with α-bromo-sugars to afford the corresponding cytosine 4-thioglycoside analogs. The free thioglycosides were also prepared. Subsequent studies on the application of this strategy for the preparation of other potent pyrimidine thioglycosides are reported.     

A First Microwave-Assisted Synthesis of a New Class of Purine and Guanine Thioglycoside Analogs

A first microwave-assisted synthesis of a new class of novel purine thioglycoside analogs from readily available starting materials has been described. The key step of this protocol is the formation of sodium pyrazolo[1,5-a]pyrimidine-7-thiolate and 7-mercaptopyrazolo[1,5-a]pyrimidine derivatives via condensation of 5-amino-1H-pyrazoles with sodium 2,2-dicyanoethene-1,1-bis(thiolate) salts or 2-(dimercaptomethylene)malononitrile, respectively, under microwave irradiation, followed by coupling with halo sugars to give the corresponding purine thioglycoside analogs. The obtained purines and purines thioglycosides derivatives were evaluated in vitro against lung (A549), colon (HCT116), liver (HEPG2), and prostate (PC3) cancer cell lines. Some of these compounds (5b, 5d, 5f, and 9a–d) exhibited little potency toward the four cell lines. On the other hand, compound 5a elicited higher cytotoxicity on both prostate (PC3) and colon (HCT116), respectively, while it was found moderate on lung (A549), and inactive on liver (HEPG2). Moreover, compound 5c was found moderate with LC₅₀ values 52.0–88.9 μM for almost all the cell lines.     

A facile synthesis of novel pyrazolopyrimidine thioglycosides as purine thioglycoside analogues

The easy, convenient and high yielding preparation of new thioglycosides incorporating mercaptopyrazolo[1,5-a]pyrimidine moieties from readily accessible starting materials has been reported. The main step of this protocol is the formation of 7-mercaptopyrazolo[1,5-a]pyrimidine-6-carbonitrile derivatives 4a-d by condensation of sodium 2-cyano-3-ethoxy-3-oxoprop-1-ene-1,1-bis(thiolate) 1 with 4-(aryldiazenyl)-1H-pyrazole-3,5-diamines 3a-d to form target compounds 4a-d, which coupled with tetra-O-acetyl-α-D-glycopyranosyl bromides 5a,b in the presence of basic medium to provide the corresponding product purine thioglycoside analogs 6a-h. Ammonolysis of the latter compounds 6a-d at ambient temperature for 10 minutes, led to the free glycoside derivatives 7a-h, which were obtained in approximately quantitative yields. Their structures were created based on the spectroscopic and elemental data.     

Design,synthesis, and in vitro anti-hepatocellular carcinoma of novel thymine thioglycoside analogs as new antimetabolic agents

A first reported direct method for preparation of thymine thioglycoside analogs utilizing novel pyrimidine-2(1H)-thiones and α-bromoglucose or α-bromogalactose tetraacetate as starting components is described. The synthetic potential of the method is demonstrated. The evaluation of antiproliferative activity against HepG-2 cell lines (Liver carcinoma cell lines) shows that most of the compounds have high antitumor activities especially 6b, 6e, 11b, and 12b. Moreover, molecular modelings of these compounds reveal that they have high binding affinity through hydrogen bond interaction with the binding pocket of thymidylate synthase dihydrofolate reductase (TS-DHFR).     

Transient Expression of Drosophila melanogaster Deoxynucleoside Kinase Gene Enhances Cytotoxicity of Nucleoside Analogs

The Drosophila melanogaster deoxynucleoside kinase gene was introduced into HeLa cells with cationic lipids to allow its transient expression, and cytotoxic effects of several nucleoside analogs in the transfected cells were examined. Of the analogs tested, cytotoxicities of 1-β-D-arabinofuranosylcytosine (araC), 5-fluorodeoxyuridine (FUdR), and 1-(2-deoxy-2-methylene-β-D-erythro-pentofuranosyl)cytosine (DMDC) were increased by the deoxynucleoside kinase gene. These results suggest that the combination of the transient expression of the Drosophila deoxynucleoside kinase gene and these nucleoside analogs is a candidate for the suicide gene therapy.     

Purine and Guanine Thioglycoside Analogs: Novel Synthesis of a New Class of Pyrazolo[1,5-a][1,3,5]Triazine-4-Thioglycoside Derivatives under Microwave Activation

A first microwave-assisted synthesis of a new class of novel purine thioglycoside analogues from readily available starting materials has been described. The key step of this protocol is the formation of sodium pyrazolo[1,5-a][1,3,5]triazine-4-thiolates via condensation of 5-amino-1H-pyrazoles with sodium cyanocarbonimidodithioate salt under microwave irradiation, followed by coupling with halo sugars to give the corresponding purine thioglycoside analogues. Further studies on the application of this method for the synthesis of other highly functionalized biologically active glycosides are underway.     

Syntheses of methyl (4,6-dideoxy-alpha-L-lyxo-hexopyranosyl)-(1-->3)- and (4-deoxy-4-fluoro-alpha-L-rhamnopyranosyl)-(1-->3)- 2-acetamido-2-deoxy-alpha-D-glucopyranosides, analogs of the mycobacterial arabinogalactan linkage disaccharide

We have made thioglycoside donors for the 4,6-dideoxy-L-lyxo-hexopyranosyl ('4-deoxy-L-rhamnosyl') and 4-deoxy-4-fluoro-L-rhamnosyl monosaccharide residues. The preparation of the deoxyfluororhamnose was not straightforward, and revealed some unexpected behavior of the diethylaminosulfur trifluoride (DAST) reagent. The new glycosyl donors were used to synthesize two analogs of the mycobacterial arabinogalactan linkage disaccharide -->4)-alpha-L-Rha-(1-->3)-alpha-D-GlcNAc. These analogs are prototypes for a family of potential inhibitors of the enzymes involved in the early stages of cell-wall construction in mycobacteria.     

Physiological and biochemical contributions to the taxonomy of the genus Prototheca

16 strains of the genus Prototheca do not produce extracellular amylolytic enzymes. The base composition of their DNA shows rather continuous values from 62% to 78% GC (guanine + cytosine). Their assignment to four species and their possible relationship with Chlorella protothecoides are discussed.Abbreviations Used GC guanine + cytosine - SSC saline sodium citrate     

Molecular-Mechanical studies of the mismatched base analogs of d(CGCGAATTCGCG)2:d(CGTGAATTCGCG)2, d(CGAGAATTCGCG)2, d(CGCGAATTCACG)2, d(CGCGAATTCTCG)2, and d(CGCAGAATTCGCG)·d(CGCGAATTCGCG)

Molecular-mechanical simulations have been carried out on “mismatched base” analogs of the DNA double-helical structure d(CGCGAATTCGCG)₂, in which the base pairs CG at the 3 and 10 positions have been replaced by CA, AG, TC, and TG base pairs, as well as an insertion analog in which an extra adenine has been incorporated into one strand of the above structure between bases 3 and 4. The results of these simulations (calculated relative stabilities, structures, and nmr ring-current shifts) have been compared with calorimetric and nmr data. The calculated relative stabilities of the double-helical parent dodecamer and the various “wobble” base pairs qualitatively correlate with the experimental melting temperatures. The base-pairing structure for the GT wobble pair is in agreement with that previously determined from nmr experiments. For the GA base pair, the structure with both bases anti has a slightly more favorable energy from base pairing and stacking than a structure with non-Watson-Crick H-bonding with adenine syn, in agreement with nmr experiments. The CA wobble base is calculated to favor an adenine 6NH₂ …? cytosine N3 H-bond over cytosine 4NH₂ …? adenine N1, again, in agreement with nmr experiments. There is no definitive experimental data on the TC base pair, but the existence of (somewhat long and weak) H-bonds involving cytosine 4NH₂ …? thymine 4CO and cytosine N3 …? thymine HN3 seems reasonable. We find a structure in which the extra adenine base of the insertion analogs sits “inside” the double helix.     

Thioglycosides as glycosylating agents in oligosaccharide synthesis

A review is given of the use of thioglycosides as glycosyl donors in oligosaccharide synthesis. Both indirect use, by conversion of the thioglycoside into a glycosyl halide and direct use, by electrophilic activation of the thioglycoside, are discussed.Abbreviations DMTST dimethyl(methylthio)sulfonium trifluoromethanesulfonate - Bz benzoyl - Bn benzyl - pNBz p-nitrobenzoyl - Phth phthallyl - Ph phenyl     

Synthesis of deoxy and methoxy analogs of octyl alpha-D-mannopyranosyl-(1-->6)-alpha-D-mannopyranoside as probes for mycobacterial lipoarabinomannan biosynthesis

A panel of analogs of the disaccharide alpha-D-Manp-(1-->6)-alpha-D-Manp-OOctyl, a known acceptor substrate for a polyprenol monophosphomannose-dependent alpha-(1-->6)-mannosyltransferase involved in the assembly of the alpha-(1-->6)-linked mannan core of mycobacterial lipoarabinomannan, has been synthesized. Described are synthetic routes to the target deoxy and methoxy analogs in which one of the hydroxyl groups of the parent disaccharide has been modified. All glycosylation reactions involved the use of octyl glycoside acceptors and thioglycoside donors using iodonium-ion activation, and the stereochemistry of the mannopyranoside bond formed was established by measurement of the 1J(C-1,H-1). Depending on the target, the key methylation or deoxygenation reactions were carried out on either mono- or disaccharide substrates. This series of analogs will be useful for probing the substrate specificity of the enzyme, in particular, its steric and hydrogen-bonding requirements.     

Binding of the EcoRII methylase to azacytosine-containing DNA.

Binding of DNA(cytosine-5)methyltransferases to azacytosine containing DNA is stimulated by the presence of S-adenosyl-methionine or its analogs sinefungin or S-adenosyl-L-homocysteine. Methylation of the DNA is therefore not necessary for binding to occur. There is no relationship between the affinity of the analog for the EcoRII enzyme and its ability to stimulate binding. The DNA-enzyme complex partially dissociates on incubation in 0.1% sodium dodecyl sulfate and 0.5 M ammonium acetate. Some of this DNA could again form a tight complex with enzyme, indicating that DNA-enzyme complex formation is reversible. Binding occurs when the second cytosine in the sequence CCAGG is substituted by azacytosine. This is the cytosine that would normally be methylated by the enzyme. The binding is therefore due to specific interaction of the methylase with azacytosine at the site it would normally methylate.     

DNA base compositions of halophilic and nonhalophilicBacillus firmus strains of marine origin

The mineral salt requirements of 27 strains ofBacillus firmus were determined. Twenty-six of these strains were of marine origin and one terrestrial strain was used as a reference. Three strains demonstrated strictly halophilic behavior, i.e., they showed no growth in media prepared without sodium chloride. Seven strains were nonhalophilic. The growth of 17 strains was stimulated by the addition of sodium chloride, but the cells were able to grow without it. These results were compared with the DNA base compositions of the strains. In contrast to literature data, relationships between the DNA base ratios and the halophilic or nonhalophilic behavior of the cells could not be detected. But strains with guanine plus cytosine values above 41 mol% did grow well at 44°C, and those strains showing poor growth at this temperature had lower guanine plus cytosine percentages.     

Induction of Systemic Acquired Resistance by Heat Shock Treatment in Arabidopsis

A 3,6-di-O-benzylated demethylallosamizoline derivative was glycosylated at the 4-position with an N, N′-diphthaloylchitobiosyl moiety by using the thioglycoside method. After de-protections, the resulting demethylallosamidin-like pseudotrisaccharide was evaluated as an inhibitor against a couple of chitinases.     

A survey of cytosine and cytidine salvage in fungi

Twenty-five fungi, including representatives from each of the four major classes, were surveyed with regard to their ability to utilize exogenous cytosine and cytidine. Two assay methods were employed: incorporation of radiolabeled precursors and sensitivity to 5-fluorinated structural analogs. The following taxonomic correlations were found: (i) the Ascomycetes utilize exogenous cytidine and/or cytosine; (ii) the lower fungi utilize cytidine but not cytosine; (iii) the Basidiomycetes utilize neither; and (iv) the Dueteromycetes, with some exceptions, closely resemble the Ascomycetes. Additionally, natural resistance to 5-fluorocytosine via both defective transport and pyrimidine excretion was observed.     

Incorporation of two modified nucleosides allows selective platination of an oligonucleotide making it suitable for duplex cross-linking

Platinated oligonucleotides are promising tools for the control of gene expression, since they may target and cross-link nucleic acid chains. Here we describe a method for the preparation of platinated oligonucleotides that has proved able to selectively cross-link complementary sequences, making use of 5-methylcytidine analogs with thioether or imidazole groups attached to the 4-position. These nucleoside analogs were derivatized as phosphoramidites and introduced in oligonucleotide chains using standard phosphite triester chemistry. Different oligonucleotide sequences containing either one or two analogs appending from the 5′-end were synthesized and used in preliminary platination studies. The reaction of transplatin with oligonucleotides containing the thioether-modified nucleobase was fast, but generally afforded unstable adducts and complex reaction mixtures. The imidazole-containing oligonucleotides reacted with transplatin much more slowly, in particular at slightly basic pH, and it was found that the imidazole-modified cytosine was less reactive than the natural nucleobases. In contrast, transplatin selectively reacted with the thioether and imidazole groups of oligonucleotides containing the two cytosine analogs in neighboring positions, even in the presence of the four nucleobases and particularly three guanines, affording platinated oligonucleotides suitable for cross-linking. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synthesis of a Forssman antigen derivative for use in a conjugate vaccine

The total chemical synthesis of a Forssman antigen analog is described. The pentasaccharide contains a functionalized tether which should facilitate future conjugation with immunogenic proteins. We found that the total synthesis can be efficiently achieved by following a convergent 2+3 strategy, and using N-Troc protected GalNAc thioglycoside as a donor.     

Potential of ribozymes against deoxycytidine kinase to confer drug resistance to cytosine nucleoside analogs

Hematopoietic toxicity is the dose-limiting side effect produced in cancer chemotherapy with deoxycytidine nucleoside analogs. Deletion of the deoxycytidine kinase (dCK), results in a drug resistance phenotype to these analogs. An interesting gene therapy strategy to confer drug resistance to cytosine nucleoside analogs would be to specifically inactivate the dCK in normal hematopoietic stem cell. In this study, we designed hammerhead ribozymes that can specifically cut and downregulate the murine dCK mRNA. Three different ribozymes were identified and shown to cleave in vitro the dCK RNA. After introduction of ribozyme cDNA into murine L1210 leukemic cells by retroviral transfer, two of the ribozymes showed some capacity in reducing dCK activity. However, analysis of transduced L1210 clones showed that the significant reduction in the dCK mRNA was not sufficient to confer drug resistance to cytosine arabinoside. Nevertheless, these results provide a new avenue of modulating the dCK enzyme activity and with improved modifications may have the potential for use in gene therapy to confer drug resistance to deoxycytidine analogs.     

Highly efficient incorporation of the fluorescent nucleotide analogs tC and tCO by Klenow fragment

Studies of the mechanisms by which DNA polymerases select the correct nucleotide frequently employ fluorescently labeled DNA to monitor conformational rearrangements of the polymerase–DNA complex in response to incoming nucleotides. For this purpose, fluorescent base analogs play an increasingly important role because they interfere less with the DNA–protein interaction than do tethered fluorophores. Here we report the incorporation of the 5′-triphosphates of two exceptionally bright cytosine analogs, 1,3-diaza-2-oxo-phenothiazine (tC) and its oxo-homolog, 1,3-diaza-2-oxo-phenoxazine (tC^O), into DNA by the Klenow fragment. Both nucleotide analogs are polymerized with slightly higher efficiency opposite guanine than cytosine triphosphate and are shown to bind with nanomolar affinity to the DNA polymerase active site, according to fluorescence anisotropy measurements. Using this method, we perform competitive binding experiments and show that they can be used to determine the dissociation constant of any given natural or unnatural nucleotide. The results demonstrate that the active site of the Klenow fragment is flexible enough to tolerate base pairs that are size-expanded in the major groove. In addition, the possibility to enzymatically polymerize a fluorescent nucleotide with high efficiency complements the tool box of biophysical probes available to study DNA replication.     

EXPANSION OF TRIPLEX RECOGNITION CODES BY THE USE OF NOVEL BICYCLIC NUCLEOSIDE DERIVATIVES (WNA)

Recently, we have developed new base analogs (WNA) and demonstrated that WNA·T with thymine and WNA·C with cytosine stabilize non-natural antiparallel triplexes with a TA or a CG interrupting site, respectively. However, limitations in recognizable sequences with the WNAcontaining TFO were also found. The objective of this study is to search better WNA analogs for expansion of triplex recognition codes to general duplex sequences. In this study, we designed new WNA analogs by systematic modification of the aromatic part and the recognition part. The new WNA analogs with the benzene ring substituted with bromide or cyanide have determined for selective stabilization of triplexes at a TA interrupting site, and general formation of triplexes having a TA interrupting site has been achieved.