Studies on the hydrolysis of 3-methyl-2′-deoxycytidine in aqueous solution A synthesis of 3-methyl-2′-deoxyuridine

The hydrolysis of 3-methyl-2′-deoxycytidine in aqueous solution has been investigated. Varying proportions of 3-methylcytosine, 3-methyluracil and 3-methyl-2′-deoxyuridine are formed depending upon conditions of pH and temperature. All three hydrolytic products are formed at pH 6.8 and 90°C. At pH 2, depyrimidination of 3-methylcytosine occurs as the only hydrolysis product. When the pH is increased to 12, 3-methyl-2′-deoxycytidine on heating at 90°C is completely deaminated to 3-methyl-2′-deoxyuridine with few side products formed. This reaction serves as the basis for a convenient synthesis of 3-methyl-2′-deoxyuridine. The 300 MHz spectra of 3-methyl-2′-deoxycytidine and 3-methyl-2′-deoxyuridine indicate that the sugar ring in these compounds is predominantly in ²E conformation.     

Effect of 3′-methyl-4-dimethylaminoazobenzene in the induction of malignant transformation and of 8-azaguanine-resistant mutations and chromosomal aberrations in a diploid clone derived from normal rat liver cells in culture

Summary The effect of 3′methyl-4-dimethylaminoazobenzene (3′-Me-DAB) in the induction of malignant transformation and of 8-azaguanine-resistant mutations and chromosomal aberrations was studied in a diploid strain derived from normal rat liver cells. The cells were malignantly transformed by treatment with 3′-Me-DAB 1.7 μg/ml for 130 to 221 d or 1.7 μg/ml for 53 d followed by 24.9 μg/ml for 27 to 77 d. The untreated control cells did not transform spontaneously until the 232nd d in culture. Some properties of the 3′-Me-DAB-treated cells were compared to those of untreated control cells but no reliable marker for predicting the tumorigenic potential of the cells was found. The single addition of 3′-Me-DAB caused little induction of 8-azaguanine-resistant mutations and chromosomal aberrations to the cells. However, mutations and chromosomal aberrations were significantly induced byN-acetoxy-4-methylaminoazobenzene, an active metabolite of 4-dimethylaminoazobenzene or 3′-Me-DAB in the presence of liver microsomes. This study was supported by a grant for cancer research from the Japanese Ministry of Education.     

Bronchial mucosal and kidney cortex affinity of 4- and 4,4′-substituted sulphur-containing derivatives of 2,2′-5,5′-tetrachlorobiphenyl in mice

In order to evaluate further the structural requirements previously proposed for accumulation of polychlorinated biphenyls (PCB) and their sulphur-containing metabolites in the respiratory tract of mice, 4-methylthio-, 4-methylsulphonyl and 4,4′-bis(methylthio)-2,2′,5,5′-[¹⁴C]tetrachlorobiphenyl were studied by whole body autoradiography. All the compounds gave rise to a strong accumulation of radioactivity in the mucosa of the bronchi, trachea and larynx. The first two substances were also concentrated in the mucosa of the nasal cavities. At the longer post-injection times all the compounds studied were localized in distinct sites of the kidney cortex. However, while the uptake of the monosubstituted sulphur-containing tetrachlorobiphenyl metabolites there was comparatively weak, the bis(methylthio) derivative showed a remarkable accumulation and retention in the kidney cortex. The study makes it possible to formulate the structural requirements for bronchial accumulation on the basis of the structure of the compounds that are accumulated rather than on the structure of the unmetabolized polychlorobiphenyls. Also with regard to the uptake in the kidney cortex a specific structure-dependency seems to exist.     

Analysis of developmental switching in transgenic mice with 5′ and 3′ deletions in the human β globin gene

Our interest in thecis-acting elements that promote the up-regulation of the globin gene has led to a systematic deletion analysis of portions of the globin gene in the context of the HS2 and globin gene using transgenic mice. In constructs that delete the 5 region to only 265 bp, high-level erythroid-specific expression was observed. Further deletion to 122 bp, however, results in significantly reduced expression levels A substitution of a minilocus control region for the single HS2 site was also produced, resulting in increased globin expression over that seen with the HS2 alone. These results are consistent with the presence of an enhancer-like element between –122 and –265. In addition, a construct in which the entire globin gene promoter was replaced by a thymidine kinase promoter was tested. Interestingly, no expression was detected in these transgenic mice. This may indicate the requirement for an erythroid-specific promoter to drive this gene. Finally, the 3 region of the globin gene was deleted in order to examine the effect of a previously defined 3 enhancer region. With deletion of this region, the expression of the human globin gene in transgenic mice is unchanged relative to the parental constructs.     

Rhodium catalyzed stereospecific reductive carbocyclization of 1,6-enynes and synthesis of 4′-methyl-6′-substituted aristeromycins

The need of long-term treatment for chronic HBV, emergence of drug-resistant viruses and inefficiency of currently approved therapies to eliminate covalently closed circular DNA (cccDNA), mandates identification of potent and selective inhibitors of HBV replication with novel mechanisms of action. Entecavir, a carbocyclic guanosine nucleoside analog, is the most potent inhibitor of HBV replication on the market. Moreover, the naturally occurring carbocyclic nucleosides aristeromycin are known for their wide range of antiviral activities.

In this research, we have utilized BINAP directed rhodium catalyzed reductive carbocyclization of 1,6-enynes (8a–b) through asymmetric hydrogenation which is an approach, not yet explored in carbocyclic sugar synthesis. Interestingly, we obtained exclusive anti-(9a) and Z-anti (9b) carbocyclic sugars. The new aristeromycin analogs (10a–b) with scaffold combination of entecavir and aristeromycin were then synthesized using the Mitsunobu reaction followed by deprotection.     

Temperature Dependency of the Mutagenic Action of N-Methyl-N′-nitro-N-nitrosoguanidine in Streptomyces cacaoi

The effectiveness of N-methyl-N′-nitro-N-nitrosoguanidine (NG) in inducing mutations in Streptomyces cacaoi was demonstrated with two systems: reversion to prototrophy of an isoleucine-requiring mutant and the induction of forward mutation of a prototrophic strain. An optimal condition for yielding a high frequency mutation was set as follows: Treatment of spores with 1 mg/ml of NG in 0.016 m phosphate buffer, pH 6, at 42°C for 60 min. In Streptmyces cacaoi, the mutagenesis depended highly on the temperature of NG treatment; elevation of the treatment temperature resulted higher frequency of mutation. Only a little dependency on temperature was shown in Escherichia coli, but the dependency was also observed when the Streptomyces was treated with ethyl methanesulfonate. The significance of these results for considering mechanism of NG action was discussed.     

Deuteration and Tritiation of 2′-Deoxyribonucleosides in the 5′ and 4′ Positions

Abstract

The deuterations of 2′-deoxyguanosine in the 4′ and 5′ positions have been described elsewhere (1). The starting material is the 5′-aldehyde formed by mild oxidation with N,N-dicyclohexyl carbodiimide in dimethyl sulphoxide of the fully protected nucleoside with free 5′-alcoholic function. The 5′4euteration was achieved by reduction with deuterated sodium borohydride. Incorporation of deuterium in the 4′-position was achieved v i a an enhanced keto-enol tautomerim by heating the aldehyde in 50/50 D20/pyridine, with subsequent reduction of the aldehyde with NaBH4. The 6-furanoid form was isolated from the I-lyxo by-product by reverse phase HPLC. Applied to pyrimidine 2′-deoxyribonucleosides, this method was shown to give deuterated 2′-deoxycytidine and thymidine in good yield.     

Anti-AIDS agents 84. Synthesis and anti-human immunodeficiency virus (HIV) activity of 2′-monomethyl-4-methyl- and 1′-thia-4-methyl-(3′R,4′R)-3′,4′-di-O-(S)-camphanoyl-(+)-cis-khellactone (DCK) analogs

In a continuing investigation into the pharmacophores and structure–activity relationship (SAR) of (3′R,4′R)-3′,4′-di-O-(S)-camphanoyl-(+)-cis-khellactone (DCK) as a potent anti-HIV agent, 2′-monomethyl substituted 1′-oxa, 1′-thia, 1′-sulfoxide, and 1′-sulfone analogs were synthesized and evaluated for inhibition of HIV-1 replication in H9 lymphocytes. Among them, 2′S-monomethyl-4-methyl DCK (5a)³ and 2′S-monomethyl-1′-thia-4-methyl DCK (7a) exhibited potent anti-HIV activity with EC₅₀ values of 40.2 and 39.1 nM and remarkable therapeutic indexes of 705 and 1000, respectively, which were better than those of the lead compound DCK in the same assay. In contrast, the corresponding isomeric 2′R-monomethyl-4-methyl DCK (6) and 2′R-monomethyl-1′-thia-4-methyl DCK (8) showed much weaker inhibitory activity against HIV-1 replication. Therefore, the bioassay results suggest that the spatial orientation of the 2′-methyl group in DCK analogs can have important effects on anti-HIV activity of this compound class.     

The effect of ethanol on the formation of N 2-ethylidene-dG adducts in mice: implications for alcohol-related carcinogenicity of the oral cavity and esophagus

The present study aimed to experimentally confirm that long-term alcohol drinking causes a high risk of oral and esophageal cancer in aldehyde dehydrogenase 2 (ALDH2)-deficient individuals. Aldh2 knockout mice, an animal model of ALDH2-deficiency, were treated with 8% ethanol for 14 months. Levels of acetaldehyde-derived DNA adducts were increased in esophagus, tongue and submandibular gland. Our finding that a lack of Aldh2 leads to more DNA damage after chronic ethanol treatment in mice supports epidemiological findings on the carcinogenicity of alcohol in ALDH2-deficient individuals who drink chronically.     

Effects of adenosine 3′ : 5′-monophosphate and guanosine 3′ : 5′-monophosphate on calcium uptake and phosphorylation in membrane fractions of vascular smooth muscle

The effects of adenosine 3′ : 5′-monophosphate (cyclic AMP), guanosine 3′ : 5′-monophosphate (cyclic GMP) and exogenous protein kinase on Ca uptake and membrane phosphorylation were studied in subcellular fractions of vascular smooth muscle from rabbit aorta. Two functionally distinct fractions were separated on a continuous sucrose gradient: a light fraction enriched in endoplasmic reticulum (fraction E) and a heavier fraction containing mainly plasma membranes (fraction P).While cyclic AMP and cyclic GMP had no effect on Ca uptake in the absence of oxalate, both cyclic nucleotides inhibited the rate of oxalate-activated Ca uptake when used at concentrations higher than 10^?5 M. The addition of bovine heart protein kinase to either fraction produced an increase in the rate of oxalate-activated Ca uptake which was further augmented by cyclic AMP. Cyclic GMP caused smaller stimulations of protein kinase-catalyzed Ca uptake than cyclic AMP.Mg-dependent phosphorylation, attributable to endogenous protein kinase(s), was inhibited in fraction E by low concentrations (10^?8 M) of both cyclic AMP and cyclic GMP. In fraction P, an inhibition by cyclic AMP occurred also at a concentration of 10^?8 M, while with cyclic AMP a concentration of 10^?5 M was required for a similar inhibition. Bovine heart protein kinase stimulated the phosphorylation of the membrane fractions much more than Ca uptake. In fraction E, in the presence of bovine protein kinase, both cyclic AMP and cyclic GMP stimulated phosphorylation up to 200%. Under these conditions, no stimulation was observed in fraction P.These results are compatible with the hypothesis that in vascular smooth muscle soluble rather than particulate protein kinases are involved in the regulation of intracellular Ca concentration.     

A differential effect of 3-(3′4′ dichlorophenyl)-1,1 dimethyl urea and atrazine on fluorescence kinetics in chloroplasts

It was found that DCMU had a differential effect at two concentration ranges on variable fluorescence kinetics in isolated chloroplasts. The increase in fluorescence rate at low concentrations of DCMU was abolished by preincubation of chloroplasts with ferricyanide or formate, treatments which were shown to convert Fe in the PS II reaction center (i.e., the FeQ_A complex) into a non-oxidizable form, but it was not affected by Tris treatment. Increase in fluorescence kinetics (at the initial linear rate) at high concentrations of DCMU was found to be abolished by Tris treatment but it was only marginally affected by ferricyanide or formate treatments. The effect of Tris could be abolished by addition of hydroquinone-ascorbate, which restored electron flow to the pool of secondary acceptors.Contrary to the effect of DCMU, no such differential concentration dependence of the variable fluorescence kinetics was found for atrazine.The increase in fluorescence kinetics (at the initial linear rate) at a low concentration rate of DCMU is presumably restricted to units which contain an oxidizable Fe in the FeQ_A complex. Increase in fluorescence kinetics (at the initial linear rate) at high DCMU concentration is probably related to the effect of DCMU at the Q_B site.Abbreviations DCMU 3-(34 dichlorophenyl)-1,1 dimethyl urea - PS II Photosystem II - Tris tris (hydroxymethyl) aminomethane     

Mutagenic specificity of N′-methyl-N′-nitro-N-nitrosoguanidine in the gpt gene on a chromosome of Chinese hamster ovary cells and of Escherichia coli cells

Summary DNA base sequence changes induced by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis have been determined for the Escherichia coli gpt gene stably incorporated in a chromosome of Chinese hamster ovary cells and in the chromosome of both growing and starving E. coli cells, instead of on a plasmid as in most previous studies. In the three cases, nearly all mutations were G: C to A: T transitions, with a 2-to 4-fold higher mutation rate, compared to other sites, at guanines flanked on the 5 side by another guanine. Mutagenic hot spots in these experiments were less prominent than in published results for MNNG mutagenesis of gpt and of other genes. A suggested explanation involves repair of O⁶meG. At low levels of mutagenic products, most are repaired and even small differences in the repair rates leads to large differences in the relative amounts of residual O⁶meG at various sites; in contrast, at high levels of mutagenic products there is little effect of repair on the distribution.Abbreviations MNNG N-methyl-N-nitro-N-nitrosoguanidine - MNU N-methyl-N-nitrosourea - O⁶meG O⁶-methylguanine - N7meG N7-methylguanine - CHO Chinese hamster ovary     

A review of MHC‐based mate preferences and fostering experiments in two congenic strains of mice

We review our studies of mate choice with two MHC‐congenic strains of mice. This work was stimulated by findings from Yamazaki and colleagues showing that male mice exhibited mate preferences for females whose MHC‐haplotype was different from their own, while female mice exhibited either no preference or a weak preference for males of a particular MHC‐haplotype (see Beauchamp et al., 1988). Since these findings were unexpected (mate choice theory predicts that females will be more selective than males), we studied the preferences of mice from two additional MHC‐congenic strains to assess the generality of the previous findings. Specifically, the goals of our research were: (1) to determine the mate preferences of congenic mice with MHC‐haplotypes derived from wild populations, (2) to compare the mate preferences of male and female mice in a test situation where each sex has a clear opportunity to make a choice, and (3) to estimate effects of cross‐fostering on each sex. This revised version was published online in July 2006 with corrections to the Cover Date.     

Loss of μ-crystallin causes PPARγ activation and obesity in high-fat diet-fed mice

The thyroid hormone-binding protein μ-crystallin (CRYM) mediates thyroid hormone action by sequestering triiodothyronine in the cytoplasm and regulating the intracellular concentration of thyroid hormone. As thyroid hormone action is closely associated with glycolipid metabolism, it has been proposed that CRYM may contribute to this process by reserving or releasing triiodothyronine in the cytoplasm. We aimed to clarify the relationship between CRYM and glycolipid metabolism by comparing wild-type and CRYM knockout mice fed a high-fat diet. Each group was provided a high-fat diet for 10 weeks, and then their body weight and fasting blood glucose levels were measured. Although no difference in body weight was observed between the two groups with normal diet, the treatment with a high-fat diet was found to induce obesity in the knockout mice. The knockout group displayed increased dietary intake, white adipose tissue, fat cell hypertrophy, and hyperglycemia in the intraperitoneal glucose tolerance test. In CRYM knockout mice, liver fat deposits were more pronounced than in the control group. Enhanced levels of PPARγ, which is known to cause fatty liver, and ACC1, which is a target gene for thyroid hormone and is involved in the fat synthesis, were also detected in the livers of CRYM knockout mice. These observations suggest that CRYM deficiency leads to obesity and lipogenesis, possibly in part through increasing the food intake of mice fed a high-fat diet.     

Genetic and biochemical studies on flavonoid 3′-hydroxylation in flowers of Petunia hybrida

Summary In flower extracts of defined genotypes of Petunia hybrida, an enzyme activity was demonstrated which catalyses the hydroxylation of naringenin and dihydrokaempferol in the 3-position. Similar to the flavonoid 3-hydroxylases of other plants, the enzyme activity was found to be localized in the microsomal fraction and the reaction required NADPH as cofactor. A strict correlation was found between 3-hydroxylase activity and the gene Ht1, which is known to be involved in the hydroxylation of flavonoids in the 3-position in Petunia. Thus, the introduction of the 3-hydroxyl group is clearly achieved by hydroxylation of C₁₅-intermediates, and the concomitant occurrence of the 3,4-hydroxylated flavonoids quercetin and cyanidin (paeonidin) in the presence of the functional allele Ht1 is due to the action of one specific hydroxylase catalysing the hydroxylation of common precursors for both flavonols and anthocyanins.     

The effect of glucose, insulin and noradrenaline on lipolysis and on the concentrations of adenosine 3′:5′-cyclic monophosphate and adenosine 5′-triphosphate in adipose tissue

1. The deoxyfluoro-d-glucopyranose 6-phosphates were prepared from the corresponding deoxyfluoro-d-glucoses and ATP by using hexokinase. 2. 3-Deoxy-3-fluoro- and 4-deoxy-4-fluoro-d-glucose 6-phosphate were substrates for glucose phosphate isomerase, and in addition the products of this reaction, 3-deoxy-3-fluoro- and 4-deoxy-4-fluoro-d-fructose 6-phosphate respectively, were good substrates for phosphofructokinase. 3. Some C-2-substituted derivatives of d-glucose 6-phosphate were found to be competitive inhibitors of glucose phosphate isomerase. 4. The possible role of the hydroxyl groups in the binding of d-glucose 6-phopshate to glucose phosphate isomerase is discussed.     

New syntheses of α-methyl- and α,α′-dimethylspermine

-Methylspermine and ,-dimethylspermine were synthesized in high overall yields starting from N-(benzyloxycarbonyl)-3-aminobutanol in order to study polyamine biochemistry in vitro and in vivo.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 200–205.Original Russian Text Copyright © 2005 by Grigorenko, Vepsalainen, Jarvinen, Keinanen, Alhonen, Janne, Khomutov.     

Convenient Synthesis of 4-C-Branched Lactones and 3′-C-Branched 2′,3′-Dideoxynucleosides

Abstract

The regio- and stereoselective photocatalysed addition of 2-propanol and cyclopentanol to (5S)-hydroxymethylfuran-2(5H)-one (1) gave 4-C-branched lactones 2 and 3 after selective silylations. The lactones 2 and 3 were radically deoxygenated affording lactones 4 and 5, respectively. As an example, compound 2 was transformed without purification of the intermediates into an anomeric mixtures of deprotected 3′-C-branched 2′, 3′-dideoxynucleosides 6 by the following reaction sequence: silylation, reduction, acetylation, coupling with silylated thymine and desilylation.