Microbiological synthesis of 5-ethyl- and (E)-5-(2-bromovinyl)-2′-deoxyuridine

Summary The title compounds were prepared by an enzymatic transdeoxyribosylation from 2 dGuo or 2 dThd to the respective heterocyclic bases, 5-ethyluracil and (E)-5-(2-bromovinyl)uracil, using the whole bacterial cells ofEscherichia coli as a biocatalyst.     

Synthesis and Properties of 2′4′- and 3′-5′-Linked Ribodinucleoside Monophosphates Containing 2-Aminoadenosine and Uridine

Abstract

Self complementary diribonucleoside monophosphates containing 2-aminoadenosine (n²A) and uridine (U) residues, (2′-5′) n²ApU (1), (3′-5′) n²ApU (2), (2′-5′) Upn²A (3) and (3′-5′) Upn²A (4), were synthesized by condensation of suitably protected nucleoside and nucleotide units using dicyclohexylcarbodiimide (DCC). The dimers, (3) and (41, were also obtained from uridine 2′,3′-cyclic phosphate and unprotected 2-aminoadenosine using 2,4,6-triisopropylbenzenesulfonyl chloride (TPS-Cl) as the condensing agent. The conformational properties of these dimers were examined by UV, CD and NMR spectroscopy. The results reveal that the 2′-5′ isomers take a stacked conformation, which contains a larger base-base overlap and is more stable against thermal perturbation with respect to the 3′-5′ isomers. The n²ApU isomers have more stacked structure than the Upn²A isomers.     

Conformation and Antiherpes Activity of 3′- and 5′-Azido and Amino Analogs of 5-Methoxymethyl-2′-Deoxyuridine

Abstract

The molecular conformations of 3′- and 5′-azido and amino derivatives of 5-methoxymethyl-2′-deoxyuridine, 1, were investigated by nmr. The glycosidic conformation of 5-methoxymethyl-5′-amino-2′,5′-dideoxy-uridine, 5 had a considerable population of the syn form. The 5′-derivatives show a preference for the S conformation of the furanose ring as in 1. In contrast, the 3′-derivatives show preference for the N conformation. For 5-methoxymethyl-3′-amino-2′,3′-dideoxyuridine, 3, the shift towards the N state is pH dependent. The preferred conformation for the exocyclic (C4′,C5′) side chain is g⁺ for all compounds except 5 which has a strong preference for the t rotamer (79%). Compounds 1, 3 and 5 inhibited growth of HSV-1 by 50% at 2, 18 and 70 μg/ml respectively, whereas 2 and 4 were not active up to 256 μg/ml (highest concentration tested). The compounds were not cytotoxic up to 3,000 μM.     

Effect of 2-Amino Substitution on the Antiviral Effects of 5-Ethyl-2′-Deoxyuridine and (E)-5-(2-bromovinyl)-2′-Deoxyuridine and Their Incorporation into DNA

Abstract

The 2-amino derivatives of 5-ethyl-2′-deoxyuridine (EDU) and (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) have been synthesized and evaluated for anti-herpesvirus activity. They were at least 1000-fold less effective against herpes simplex virus replication than the parent compounds EDU and BVDU. The 5′-triphosphates of the 2-amino substituted EDU, BVDU and thymidine derivatives were also synthesized and examined on their substrate/inhibitor properties against different DNA polymerases. None of the compounds proved markedly inhibitory to HSV-1 DNA polymerase or cellular DNA polymerase a. Nor were they incorporated into the growing DNA chain.     

Linkage mapping of α-2 adrenergic receptor genes to mouse Chromosomes 2 and 5

-2 adrenergic receptors can be subdivided into three related subtypes which are conserved in humans, rats, and mice. In the mouse, these receptors are encoded by three genes (Adra-2a, Adra-2b, Adra-2c). To gain insight into the evolution of this multigene family and to investigate whether these genes are candidates for previously identified mouse mutations, we have determined the map positions of the Adra-2b and Adra-2c genes. The Adra-2a gene has been previously mapped to mouse Chromosome (Chr) 19 (Oakey et al. Genomics 10, 338–334, 1991). Using segregation among recombinant inbred strains of a single-stranded conformational polymorphism specific for alleles of Adra-2b and Adra-2c, we present map positions for these genes on mouse Chrs 2 and 5, respectively. In the case of Adra-2b, these results have been confirmed by an analysis of somatic cell hybrids. In addition, we generate AKXD recombinant inbred strain distribution patterns for 11 previously defined SSLP microsatellite markers, further refining the haplotype maps for these chromosomes. Finally, several candidate mouse mutations that map close to Adra-2b and Adra-2c are discussed.     

Enzymatic synthesis of 5-bromo-2’-deoxyuridine-2-14C and of 5-iodo-2’-deoxyuridine-2-14C and their incorporation into deoxyribonucleic acid (Allium cepa)

5-Bromo-2’-deoxyuridine-2-¹⁴C was prepared from 5-bromouracil-2-¹⁴C and 2’-de-oxyguanosine using trans-N-deoxyribosylase fromLactobacillus helveticus and incorporated into DNA ofAllium cepa roots. After isolating the DNA and hydrolyzing it enzymatically to deoxynucleoside-5’-phosphates a radioactive nucleotide was detected which yielded 5-bromo-2’-deoxyuridine-2-¹⁴C on enzymatic dephosphorylation. The incorporation of 5-iodo-2’-deoxy-uridine-2-¹⁴C was followed only by microautoradiography.     

Interaction of (2′ -5′) and (3′ -5′) Linked 2-Aminoadenylyl-3-aminoadenosines with Polyuridylic Acid

Abstract

2′-5′ and 3′-5′ linked 2-aminoadenylyl-2-aminoadenosines [(2′-5′)n²Apn²A (1) and (3′-5′)n²Apn²A (2)] were synthesized by condensation of 5′-O-monomethoxytrityl-N ² N ⁶-dibenzoyl-2-aminoadenosine and N ²,N ⁶,2′,3′-O-tetrabenzoyl-2-aminoadenosine 5′-phosphate using dicyclohexylcarbodiimide (DCC). The conformational properties of these dimers 1 and 2 were examined by UV, NMR and CD spectroscopy. The results reveal that the 2′-5′-isomer 1 takes a stacked conformation, which contains a larger base-base overlap and is more stable against thermal perturbation with respect to the 3′-5′-isomer 2. Interactions of 1 and 2 with polyuridylic acid (Poly (U)) were also examined by Tm, mixing curves, UV and CD spectra. Both the dinucleoside isomers 1 and 2 formed a complex of 1 : 2 stoichiometry with poly(U), which was much more stable than that of the corresponding ApA isomer     

Synthesis and Antiviral Activity Assay of Novel (E)-3′,5′-Diamino-5-(2-bromovinyl)-2′,3′,5′-trideoxyuridine

Abstract

(E)-3′,5′-diamino-5-(2-bromovinyl)-2′,3′,5′-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. In contrast with BVDU, compound 5 did not show activity against herpes simplex virus or varicella-zoster virus.     

Studies Towards the Large Scale Chemical Synthesis of the Precursors of Ribonucleosides-3′,4′,5′,5″- 2H 4 and -2′,3′,4′,5′,5″- 2H 5

Abstract

A summary delineating the large scale synthetic studies to prepare labeled precursors of ribonucleosides-3′,4′,5′,5″- ²H ₄ and -2′,3′,4′,5′,5″- ²H ₅ from D-glucose is presented. The recycling of deuterium-labeled by-products has been devised to give a high overall yield of the intermediates and an expedient protocol has been elaborated for the conversion of 3-O-benzyl-α,β-D-allofuranose-3,4-d ₂ 6 to 1-O-methyl-3-O-benzyl-2-O-t-butyldimethylsilyl-α,β-D-ribofuranose-3,4,5,5′-d ₄ 16 (precursor of ribonucleosides-3′,4′,5′,5″- ²H ₄ ) or to 1-O-methyl-3,5-di-O-benzyl-α,β-D-ribofuranose-3,4,5,5′-d ₄ 18 (precursor of ribonucleosides-3′,4′,5′,5″- ²H ₄ ).     

5-(2-Pyrrolidinyl)oxazolidinones and 2-(2-pyrrolidinyl)benzodioxanes: Synthesis of all the stereoisomers and α4β2 nicotinic affinity

The four stereoisomers of 2-oxazolidinone 5-substituted with 1-methyl-2-pyrrolidinyl (1), of 1,4-benzodioxane 2-substituted with the same residue (2) and of the nor-methyl analogue of this latter (2a) were synthesized as candidate nicotinoids. Of the 12 compounds, two N-methylated pyrrolidinyl-benzodioxane stereoisomers, namely those with the same relative configuration at the pyrrolidine stereocentre as (S)-nicotine, bind at α4β2 nicotinic acetylcholine receptor with submicromolar affinity. Consistently with the biological data, docking analysis enlightens significant differences in binding site interactions not only between 1 and 2, but also between 2 and 2a and between the stereoisomers of 2 accounting for the critical role played, in the case of the pyrrolidinyl-benzodioxanes, by the chirality of both the stereolabile and stereostable stereogenic atoms, namely the protonated tertiary nitrogen and the two asymmetric carbons.     

3′-3′,3′-5′ and 5′-5′ TpT-Amides: Synthesis,Characterization and Alkaline Hydrolysis

Abstract

A simple procedure is described for the preparation of the title compounds 1, 8 and 9. 3′-3′ or 3′-5′ or 5′-5′ TpT was reacted with a twofold molar excess of TPS in anhydrous DMF, at room temperature, for 5 min, followed by a 1 min in situ treatment of the reaction mixture with excess 7.0 N NH₄OH, at 0°C. The alkaline hydrolysis of 1, 8 and 9 proceeds without the assistance of 3′- and 5′-hydroxyl groups resulting in equimolar mixtures of thymidine (4) and thymidine 3′-phosphoramidate (6) (for the 3′-3′ isomer) or thymidine 5′-phosphoramidate (7) (for the 5′-5′ isomer) or 6 and 7 in equal quantities (for the 3′-5′ isomer).     

Synthesis and fluorescent properties of 5-(1-pyrenylethynyl)-2′-deoxyuridine-containing oligodeoxynucleotides

Novel reagents for the fluorescent labeling of oligo- and polynucleotides have been prepared: 5-(1-pyrenylethynyl)-2′-deoxyuridine 3′-phosphoramidite and a solid support carrying this nucleoside. Oligo-nucleotides containing one or several modified units have been synthesized, and the fluorescence of these probes has been shown to change upon hybridization with the complementary sequence. Fluorescent Nucleosides. III. The previous communications, see [1, 2]. Prefix “d” in the oligodeoxynucleotide designations is omitted.     

Photobromination of 1,5-Anhydro-2,3-O-isopropylidene-β-d-ribofuranose and Synthesis of (5R) and (5S)-(5-2H1)-d-Riboses

The photobromination of 1,5-anhydro-2,3-O-isopropylidene-β-d-ribofuranose gave the corresponding (5S)-5-bromo compound. The reduction of the bromide with triphenyltindeuteride gave (5S)-(5-²H₁)-1,5-anhydro-2,3-O-isopropylidene-β-d-ribofuranose, with a chiral purity of 76% at C-5, which was converted to (5R)- and (5S)-(5-²H₁)-d-riboses and other ribofuranose derivatives.     

Structure Sensitivity of Amino Proton Exchange in 2′ - and 5′ - Guanosine Monophosphate Dianions

Abstract

Proton NMR line broadening methods were used to determine the rates of amino proton exchange for disordered 2′ - and 5′ - GMP dianions in aqueous solutions containing tetramethylammonium (TMA⁺) cations. Replacing TMA⁺ with Na⁺ does not substantially alter the exchange rates, provided that H-bonded, Na⁺-directed tetramer structures are absent. Activation enthalpies (kcal/mol) and entropies (eu) for 2′ - GMP are: ΔH^# = 18.5 ± 1.3, ΔS^# = 9.6 ± 4.2 for theTMA⁺ salt atpH 8.10, and ΔH^# = 14.7 ± 2.6, ΔS^# = -3.7 ± 8.0 for the Na⁺ salt at pH 8.11. Extrapolated values of pseudo first-order rate constants at 25° Care in the range of k = 1–10 sec^?1. At suitable concentrations and temperatures, the Na⁺ salts of both 2′ - and 5′ - GMP formed stacked and unstacked tetramer units. Relative to the exchange kinetics observed for the disordered nucleotide, the exchange process in the tetramer units was catalyzed in half the amino protons and inhibited in the other half. The catalytic process (k < 10³ sec^?3) has been attributed to amino protons not involved in interbase H-bonding, where as the inhibited process (k > 10^?1 sec^?1) was assigned to those protons which do form such bonds. The structure-catalyzed process in both the stacked and unstacked tetramers was manifested by a loss of NMR amino proton intensity due to weighted time-averaging with the resonance for bulk water. A bridging water molecule between an amino proton and a phosphate on an adjacent nucleotide in the tetramer unit may provide a mechanistic pathway for the structure-catalyzed process.     

The Functional Roles of the MDM2 Splice Variants P2-MDM2-10 and MDM2-∆5 in Breast Cancer Cells

BACKGROUND: MDM2 is a negative regulator of p53 and is upregulated in numerous human cancers. While different MDM2 splice variants have been observed in both normal tissues and malignant cells, their functions are poorly understood. METHODS: We evaluated the effect of MDM2 splice variants by overexpression in MCF-7 cells and analyses of expression of downstream genes (qPCR and Western blot), subcellular localization (immunofluorescence), cell cycle assays (Nucleocounter3000), apoptosis analysis (Annexin V detection), and induction of senescence (β-galactosidase analysis). RESULTS: In a screen for MDM2 splice variants in MCF-7 breast cancer cells, extended with data from healthy leukocytes, we found P2-MDM2-10 and MDM2-Δ5 to be the splice variants expressed at highest levels. Contrasting MDM2 full-length protein, we found normal tissue expression levels of P2-MDM2-10 and MDM2-Δ5 to be highest in individuals harboring the promoter SNP309TT genotype. While we detected no protein product coded for by MDM2-Δ5, the P2-MDM2-10 variant generated a protein markedly more stable than MDM2-FL. Both splice variants were significantly upregulated in stressed cells (P = 4.3 × 10^?4 and P = 7.1 × 10^?4, respectively). Notably, chemotherapy treatment and overexpression of P2-MDM2-10 or MDM2-Δ5 both lead to increased mRNA levels of the endogenous MDM2-FL (P = .039 and P = .070, respectively) but also the proapoptotic gene PUMA (P = .010 and P = .033, respectively), accompanied by induction of apoptosis and repression of senescence. CONCLUSION: We found P2-MDM2-10 and MDM2-Δ5 to have distinct biological functions in breast cancer cells. GENERAL SIGNIFICANCE: Alternative splicing may influence the oncogenic effects of the MDM2 gene.     

A Direct and Efficient Synthesis of 5′-Deoxy-2′, 3′-

Abstract

A direct and efficient synthesis of 5′-deoxy-2′,3′-O-isopropylideneinosine, 7, from readily available inosine is described. An example of a potentially general synthesis of N -substituted-5′-deoxyadenosines from 7 is also described.     

Increased Δ5- and Δ6-desaturase, cyclooxygenase-2, and lipoxygenase-5 expression and activity are associated with fatty acid and eicosanoid changes in cystic fibrosis

Patients with cystic fibrosis consistently demonstrate selective abnormalities in essential fatty acid concentrations, including decreased linoleate (LA) and docosahexaenoate (DHA), with variably increased arachidonate (AA). These changes appear important for the pathophysiology of the disease. However, the mechanisms of these changes are not clearly understood. The current study demonstrates that metabolism of LA and alpha linolenate (LNA) to AA and eicosapentaenoate (EPA), respectively, are significantly increased in two different cell culture models of cystic fibrosis. These changes correlated with increased expression of fatty acid Δ5- and Δ6-desaturases, key enzymes in this metabolic pathway. In contrast, cystic fibrosis cells showed decreased metabolism of AA and EPA to docosapentaenoate (DPA) and docosahexaenoate (DHA), respectively, although metabolism of 22:5n-3 to DHA was relatively unchanged. In addition, the expression and activity of both cyclooxygenase-2 and lipoxygenase-5 was markedly increased in these cells. Taken together, these findings are consistent with the conclusion that the diminished LA and increased AA in cystic fibrosis result from increased metabolism of LA, while the observed decrease in DHA is at least partly due to decreased elongation and desaturation beyond EPA.     

5-溴甲基-2(5H)-呋喃酮的生理活性初探

本文报道了原白头翁素衍生物5-溴甲基-2(5n)-呋喃酮的合成和抗癌活性及其抑制农作物病原菌活性的探讨结果,表明在5～20μg/mL范围内,5-溴甲基-2(5H)-呋喃酮对人肺癌A549细胞株有明显的抑制生长的作用;在50～200μg/mL范围内,其对玉米大斑病菌等的生长抑制率可达到95%以上.     

Absorption,Translocation and Metabolism of 2-tert-Butyl-4-(2,4-dichloro-5-isopropoxyphenyl)-Δ2-1,3,4-oxadiazolin-5-one (Oxadiazon) in Rice Plants

Absorption, translocation and metabolism of 2-tret-butyl-4-(2,4-dichloro-5-isopro-poxyphenyl)-Δ²-1,3,4-oxadiazolin-5-one (oxadiazon) in rice plants were investigated. Three types of ¹⁴C-labeled oxadiazon were used in this study. ¹⁴C-Labeled oxadiazon was applied to soil under submerged conditions and plants were taken for the preparations at various stages of growth and development. Oxadiazon was translocated remarkably to shoots and accumulated in the lower leaves and stems. A small portion of oxadiazon intaken in plants was translocated to head parts. Oxadiazon was chemically transformed in plants to produce dealkylated compounds, oxidized alcohol and carboxylic acid as metabolites. In addition, two unidentified metabolites were detected, one of which was translocated to head parts from leaves and stems. Translocation and accumulation of oxadiazon and its metabolites were discussed in relation to physiological conditions of rice plants.