Synthesis of 3′-Thioribouridine, 3′-Thioribocytidine,and Their Phosphoramidites

Abstract

3′-Thio-3′-deoxyribonucleosides (U and C) have been synthesized via Vorbruggen-type glycosylation with 3-S-benzoyl-5-O-toluoyl-1,2-O-diacetylfuranose, which was obtained from 1,2-O-isopropylidene-5-O-toluoyl-3-O-trifluoromethanesulfonyl-α-D-xylofuranose. 3′-Thio-3′-deoxyuridine has been converted to its phosphoramidite.     

Transport of Eicosapentaenoic Acid-Derived PGE3, PGF3α, and TXB3 by ABCC4

Background

Eicosapentaenoic acid-derived prostaglandin (PG) E₃, PGF_3α, and thromboxane (TX) B₃ are bioactive lipid mediators which have anti-cancer and anti-inflammatory effects. To exert their effects, PGE₃, PGF_3α, and TXB₃ must be released to the extracellular space from cells, but the release mechanism has been unclear. We therefore investigated the contribution of ATP-binding cassette transporter C4 (ABCC4), which has been known as a prostanoids efflux transporter, to the release of PGE₃, PGF_3α, and TXB₃.

Materials and Methods

ATP-dependent transport of PGE₃, PGF_3α, and TXB₃ via ABCC4 was investigated by using inside-out membrane vesicles prepared from ABCC4-overexpressing HEK293 cells. To evaluate the contribution of ABCC4 to the release of PGE₃, PGF_3α, and TXB₃, we measured the extracellular and intracellular levels of PGE₃, PGF_3α, and TXB₃ in A549 cells when we used ABCC4 inhibitors (dipyridamole, MK571, and probenecid) or ABCC4 siRNAs. The quantification of PGE₃, PGF_3α, and TXB₃ was performed by using liquid chromatography-tandem mass spectrometry.

Results

The apparent K_m values for ABCC4-mediated transport were 2.9±0.1 µM for PGE₃, 12.1±1.3 µM for PGF_3α, and 11.9±1.4 µM for TXB₃ and the ATP-dependent accumulation of PGE₃, PGF_3α, and TXB₃ into vesicles was decreased by using typical substrates and inhibitors of ABCC4. ABCC4 inhibitors and ABCC4 knockdown showed the reduction of extracellular/intracellular ratio of PGE₃ (40–60% of control) and PGF_3α (60–80% of control) in A549 cells.

Conclusions

Our results suggest that PGE₃, PGF_3α, and TXB₃ are substrates of ABCC4 and ABCC4 partially contributes to the release of PGE₃ and PGF_3α.     

Design and Synthesis of A3 Adenosine Receptor Ligands, 3′-Fluoro Analogues of Cl-IB-MECA

Abstract

Synthesis of 3′-deoxy-3′-fluoro-N ⁶-substituted adenosines as bioisosteres of Cl-IB-MECA and their binding affinities to A₃ adenosine receptor are described.     

Studies on Glycosides of 3, 4, 6-Trisubstituted Pyrazolo-[3, 4-D]Pyrimidines. Synthesis of 2′-Deoxyribonucleosides

Abstract

A synthesis of 4,6-dimethylthio-1-(2-deoxy-β-D-erythro-pentofuranosyl)pyrazolo[3,4-d]pyrimidine-3-carbonitrile (4) is described using the stereospecific sodium salt glycosylation procedure. Condensation of the sodium salt of 4,6-dimethylthiopyrazolo[3,4-d]pyrimidine-3-carbonitrile (1) with 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-α-D-eythro-pentofuranose (2) gave exclusively the corresponding blocked nucleoside (3) with β-anomeric configuration, which on deprotection provided 2′-deoxyriboside 4. Aglycone functional groups transformations of 4 led to related 3,4,6-trisubstituted pyrazolo[3,4-d]pyrimidine-2′-deoxynucleosides. These compounds are devoid of any significant cytotoxic activity in vitro.     

Heat-Melt of β-1, 3-d-Glucan Gel

Six new products of oxidation of indolyl-3-acetic add catalyzed by horseradish peroxidase were isolated, along with four known ones, 3-hydroxymethyloxindole (1), 3-methyleneoxindole (2), indolyl-3-aldehyde (4), and 3,3-diindolylmethane (10). Based on spectroscopic and chemical evidence, the new products were identified as 3-acetoxyindole (3), 3-(indol-3-ylmethyl)oxindole (6), 3-[(2-mdol-3-ylmethyl)indol-3-ylmethyl]oxindole (9), the 3-hydroxymethyl compounds of 6 and 9 (5 and 7), and 2-(indol-3-ylmethyl)indolyl-3-acetic acid (8), respectively.     

Use of molecular modeling, docking, and 3D-QSAR studies for the determination of the binding mode of benzofuran-3-yl-(indol-3-yl)maleimides as GSK-3β inhibitors

Molecular modeling and docking studies along with three-dimensional quantitative structure relationships (3D-QSAR) studies have been used to determine the correct binding mode of glycogen synthase kinase 3β (GSK-3β) inhibitors. The approaches of comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) are used for the 3D-QSAR of 51 substituted benzofuran-3-yl-(indol-3-yl)maleimides as GSK-3β inhibitors. Two binding modes of the inhibitors to the binding site of GSK-3β are investigated. The binding mode 1 yielded better 3D-QSAR correlations using both CoMFA and CoMSIA methodologies. The three-component CoMFA model from the steric and electrostatic fields for the experimentally determined pIC₅₀ values has the following statistics: R²(cv) = 0.386 nd SE(cv) = 0.854 for the cross-validation, and R² = 0.811 and SE = 0.474 for the fitted correlation. F (3,47) = 67.034, and probability of R² = 0 (3,47) = 0.000. The binding mode suggested by the results of this study is consistent with the preliminary results of X-ray crystal structures of inhibitor-bound GSK-3β. The 3D-QSAR models were used for the estimation of the inhibitory potency of two additional compounds.     

Synthesis of 3′-deoxy-3′-carboxymethylnucleosides,precursors of oligonucleotides with an amide internucleoside bond

An improved method for the synthesis of 3-deoxy-3-carboxymethyl nucleosides was suggested. Oxidation of 5-O-benzoyl-1,2-O-isopropylidene-α-D-xylofuranose resulted in the 3-keto derivative, which was treated with triethylphosphonoacetate in the presence of sodium hydride to obtain the 3-deoxy-3-ethoxycarbonylmethylene derivative. Hydrogenation of the unsaturated compound proceeded strictly stereospecifically and gave the product with the ribo-configuration. Acetolysis of the resulting compound with AcOH-Ac₂O-CH₃SO₃H led to 1,2-di-O-acetyl-5-O-benzoyl-3-deoxy-3-ethoxycarbonylmethyl-D-ribofuranose, whose interaction with persilylated nucleic bases gave 3-deoxy-3-ethoxycarbonylmethylnucleosides in a total yield of 42–49% from the starting compound.     

Production,Purification and Crystallization of 3α-Hydroxysteroid Dehydrogenase of Pseudomonas putida

The ability of formation of 3α-hydroxysteroid dehydrogenase was studied in bacteria and actinomycetes. The enzyme activity was found in several bacteria belonging to the genera Pseudomonas, Bacillus and Corynebacterium, when they were grown on cholic acid as a sole source of carbon. Of these bacteria, Pseudomonas putida NRRL B–11064 isolated from soil, showed the highest activity of 3α-hydroxysteroid dehydrogenase. The enzyme was purified from the cell-free extract by procedure including fractionation with ammonium sulfate and column chromatographies on DEAE-celluIose, Sephadex G–100 and hydroxylapatite. Crystals of the enzyme were obtained by the addition of ammonium sulfate to the purified enzyme in the presence of glycerol or polyethylene glycol. The overall purification was about 550-fold with an yield of 18.5%. The crystalline enzyme was homogeneous on polyacrylamide disc electrophoresis and analytical ultracentrifugation (s_20,w=3.2).     

Identification of CD3ε, CD4, CD8β splice variants of Atlantic salmon

In vertebrates, CD3 complex and CD4 and CD8 co-receptors are essential for signal transduction during T cell activation. In the present study, we report the mRNA spliced variants of the Atlantic salmon CD3ε, CD4 and CD8β and the effect of pathogen encounter on the expression of these variants. CD3ε is alternatively spliced in thymus, head kidney, spleen and gills to give rise to the complete mRNA sequence and to an alternative product that lacks the transmembrane exon. CD4 is also alternatively spliced in the thymus, head kidney, spleen and gills to form two variants, although the alternative product is barely detectable. The alternative product lacks the exon 1B encoding the D1 domain, which is essential for binding to MHC class II proteins. Two amplicons were also found for the CD8β gene; sequencing analysis revealed that the main PCR product corresponds to the previously reported CD8β sequence, whereas the variant sequence encodes a potential protein that lacks the Ig-like domain. The expression of CD3, CD4, CD8β genes also analyzed in head kidney of LPS-treated and IPNV infected salmon and different patterns of expression were observed. The presence and balance of the different variants of T cell co-receptors could be related to the ability of fish to induce a particular type of immune response, as well as, the ability of the pathogen to modify the fish immune response.     

Identification of Novel Regulatory Cholesterol Metabolite, 5-Cholesten, 3β,25-Diol,Disulfate

Oxysterol sulfation plays an important role in regulation of lipid metabolism and inflammatory responses. In the present study, we report the discovery of a novel regulatory sulfated oxysterol in nuclei of primary rat hepatocytes after overexpression of the gene encoding mitochondrial cholesterol delivery protein (StarD1). Forty-eight hours after infection of the hepatocytes with recombinant StarD1 adenovirus, a water-soluble oxysterol product was isolated and purified by chemical extraction and reverse-phase HPLC. Tandem mass spectrometry analysis identified the oxysterol as 5-cholesten-3β, 25-diol, disulfate (25HCDS), and confirmed the structure by comparing with a chemically synthesized compound. Administration of 25HCDS to human THP-1-derived macrophages or HepG2 cells significantly inhibited cholesterol synthesis and markedly decreased lipid levels in vivo in NAFLD mouse models. RT-PCR showed that 25HCDS significantly decreased SREBP-1/2 activities by suppressing expression of their responding genes, including ACC, FAS, and HMG-CoA reductase. Analysis of lipid profiles in the liver tissues showed that administration of 25HCDS significantly decreased cholesterol, free fatty acids, and triglycerides by 30, 25, and 20%, respectively. The results suggest that 25HCDS inhibits lipid biosynthesis via blocking SREBP signaling. We conclude that 25HCDS is a potent regulator of lipid metabolism and propose its biosynthetic pathway.     

Preparation,Hydrolysis and Intramolecular Transesterification of 3′-Deoxy-3′-thioinosine 3′-S-Dimethylphosphorothiolate

Abstract

The hydrolytic reactions of the dimethyl ester of 3′-deoxy-3′-thioinosine 3′-S-phosphorothiolate have been followed over a wide aciditty range by HPLC. At pH > 3, only hydroxide ion catalyzed isomerization to the 2′-dimethylphosphate takes place, whereas under more acidic conditions hydrolysis to the 2′-monomethylphosphate and 3′-S-monomethylphosphorothiolate competes. The latter is the only product accumulating in very acidic solutions (1 M hydrochloric acid). Mechanisms of the reactions are discussed.     

Non-Enzymatic Oligomerization of 3’, 5’ Cyclic AMP

Recent studies illustrate that short oligonucleotide sequences can be easily produced from nucleotide precursors in a template-free non-enzymatic way under dehydrating conditions, i.e. using essentially dry materials. Here we report that 3’,5’ cyclic AMP may also serve as a substrate of the reaction, which proceeds under moderate conditions yet with a lower efficiency than the previously reported oligomerization of 3’,5’ cyclic GMP. Optimally the oligomerization requires (i) a temperature of 80°C, (ii) a neutral to alkaline environment and (iii) a time on the order of weeks. Differences in the yield and required reaction conditions of the oligomerizations utilizing 3’,5’ cGMP and cAMP are discussed in terms of the crystal structures of the compounds. Polymerization of 3’,5’ cyclic nucleotides, whose paramount relevance in a prebiotic chemistry context has been widely accepted for decades, supports the possibility that the origin of extant genetic materials might have followed a direct uninterrupted path since its very beginning, starting from non-elaborately pre-activated monomer compounds and simple reactions.     

Evaluation of urinary 1-hydroxypyrene,S-phenylmercapturic acid,trans,trans-muconic acid, 3-methyladenine, 3-ethyladenine, 8-hydroxy-2′-deoxyguanosine and thioethers as biomarkers of exposure to cigarette smoke

The objective was to evaluate the utility of urinary 1-hydroxypyrene (1-OHP), S-phenylmercapturic acid (S-PMA), trans,trans-muconic acid (t,t-MA), 3-methyladenine (3-MeAd), 3-ethyladenine (3-EtAd), 8-hydroxy-2′-deoxyguanosine (8-OHdG) and thioethers as biomarkers for assessing the exposure in adult smokers who switched from smoking conventional cigarettes to candidate potential reduced exposure products (PREP) or who stopped smoking. Two electrically heated smoking systems (EHCSS) were used as prototype cigarettes that have significant reductions in a number of mainstream smoke constituents as measured by smoking machines relative to those from conventional cigarettes. Urine samples were collected from a randomized, controlled, forced-switching study in which 110 adult smokers of a conventional cigarette brand (CC1) were randomly assigned to five study groups. The groups included the CC1 smoking group, a lower-tar conventional cigarette (CC2) smoking group, EHCSS1 group, EHCSS2 group and a no smoking group that were monitored for 8 days. Biomarkers were measured at baseline and day 8. The daily excretion levels of these biomarkers were compared among the groups before and after switching, and the relationships between the daily excretion levels of these biomarkers and cigarette smoking-related exposure were investigated using Pearson product-moment correlation and multiple regression analyses. It was concluded that under controlled study conditions: (1) 1-OHP, S-PMA and t,t-MA are useful biomarkers that could differentiate exposure between smoking conventional and EHCSS cigarettes or between smoking conventional cigarettes and no smoking; between S-PMA and t,t-MA, the former appeared to be more sensitive; (2) 3-MeAd could only differentiate between smoking conventional cigarettes and no smoking; the results for 3-EtAd were not conclusive because contradictory results were observed; (3) 8-OHdG had a questionable association with smoking and therefore the utility of this biomarker for smoking-related exposure could not be established; and (4) urinary excretion of thioethers as a biomarker lacked sensitivity to demonstrate a clear dose–response relationship in conventional cigarette smokers, although it could differentiate the excretion levels between those subjects who smoked a conventional cigarette and those who stopped smoking.     

Comparison of pharmacokinetic properties,physicochemical stability,and pump compatibility of 3 rapid-acting insulin analogues— aspart,lispro, and glulisine

ObjectiveTo compare how the rapid-acting insulin analogues (RAIAs) aspart, lispro, and glulisine perform in continuous subcutaneous insulin infusion (CSII) therapy regarding (1) pharmacokinetic properties, (2) chemical and physical stability, and (3) pump compatibility.MethodsPubMed was searched for articles pertaining to the use of RAIAs in CSII, without a restriction on the time period.ResultsThese RAIAs have pharmacokinetic profiles that more closely mimic endogenous insulin in comparison with regular human insulin and tend to produce less hypoglycemia. Among these RAIAs, the rates of absorption and clinical efficacy in terms of glycemic control were similar. Although glulisine showed a faster onset of action in some studies with aspart and lispro, this advantage lasted only for a maximum of 1 hour, after which results were similar for glulisine and aspart or lispro. Each RAIA is created by making minor amino acid substitutions to the regular human insulin molecule and adding a stabilizer to help prevent fibrillation. A series of chemical and covalent changes affecting the primary structure of an insulin preparation, however, may cause decomposition during storage, handling, and use, diminishing the potency of the insulin molecule while contained in an insulin pump. Precipitation, fibrillation, and occlusion may ensue, undermining compatibility for CSII pump use. Aspart has demonstrated the greatest chemical and physical stability in the insulin pump, with the lowest rates of overall occlusion in comparison with lispro and glulisine (aspart 9.2%, lispro 15.7%, and glulisine 40.9%; P < .01).ConclusionAspart is the most compatible of the 3 RAIAs for pump use. (Endocr Pract. 2011;17:271-280)     

Localization of a Class II Phosphatidylinositol 3-Kinase,PI3KC2α, to Clathrin-Coated Vesicles

We have analysed phosphatidylinositol 3-kinase activity associated with subcellular fractions prepared from rat brains. Phosphatidylinositol 3-kinase activity is not markedly enriched with synaptic vesicle purification; whilst the activity associated with the most pure fractions is inhibited at low concentrations of wortmannin (IC₅₀ ∼ 4–5 nM). In contrast, clathrin-coated vesicle (CCV) fractions showed increased enzyme activity compared to light membrane fractions from which they are purified. In addition to a wortmannin-sensitive activity, we also detected an activity that could only be inhibited at higher concentrations of wortmannin (IC₅₀ ∼ 400 nM), characteristic of certain class II enzymes (including phosphatidylinositol 3-kinase C2α) to be highly enriched in CCV fractions. Immunoblotting with an antibody raised against phosphatidylinositol 3-kinase C2α, confirmed that this enzyme is highly enriched in CCVs and displays an enrichment profile during the purification that mirrors enrichment of the low nanomolar wortmannin-insensitive activity. If the CCV purification protocol is adapted to favour nerve terminally derived vesicles, we find reduced levels of the C2α enzyme in the CCV fractions, suggesting that the enzyme may principally reside on vesicles associated with the cell body.     

The effects of testosterone, 5α-dihydrotestosterone, 3α-androstanediol,and 3β-androstanediol on the maturation of rabbit epididymal spermatozoa in organ culture

Summary The fertilizing ability of spermatozoa from epididymal tubules maintained in organ cultures from 1 to 7 days was assessed after artificial insemination into receptive does. It was found that spermatozoa from the distal corpus which were already capable of fertilizing eggs prior to the cultures retain this ability for 1 day without addition of hormone and for 3–4 days when testosterone (0.5 g/ml) or 5-dihydrotestosterone (0.5 g/ml) is added to the culture medium. Spermatozoa from the proximal corpus which were not capable of fertilizing eggs prior to the cultures remain so after 1 day in cultures without addition of hormone. Testosterone, 5-dihydrotestosterone, 3-androstanediol, or 3-androstanediol was added to cultures of proximal corpus at a concentration of 0.5 g/ml. Only with 5-DHT is the mean percentage of fertilization significantly higher than the percentage obtained without addition of hormone. Insulin does not potentiate the effect of 5-DHT on sperm fertilizing ability. Epithelial growth factor is ineffective. Spermatozoa from the caput epididymidis kept in cultures for 1 to 4 days remain infertile. The results are discussed in light of the morphological findings presented in the preceding communication and in relation to the physiological requirement for sperm maturation in the epididymis.     

Ischemic post-conditioning reduces infarct size of the in vivo rat heart: role of PI3-K, mTOR, GSK-3β, and apoptosis

Post-conditioning by repetitive cycles of reperfusion/ischemia after prolonged ischemia protects the heart from infarction. The objectives of this study were: Are kinases (PI3-kinase, mTOR, and GSK-3β) involved in the signaling pathway of post-conditioning? Does post-conditioning result in a diminished necrosis or apoptosis? In open chest rats the infarct size was determined after 30 min of regional ischemia and 30 min of reperfusion using propidium iodide and microspheres. Post-conditioning was performed by three cycles of 30 s reperfusion and reocclusion each, immediately upon reperfusion. PI3-kinase and mTOR were blocked using wortmannin (0.6 mg/kg) or rapamycin (0.25 mg/kg), respectively. The phosphorylation of GSK-3β and p70S6K was determined with phospho-specific antibodies. TUNEL staining and detection of apoptosis-inducing factor (AIF) were used for the determination of apoptosis. Control hearts had an infarct size of 49 ± 3%, while post-conditioning significantly reduced it to 29 ± 3% (P < 0.01). Wortmannin as well as rapamycin completely blocked the infarct size reduction of post-conditioning (51 ± 2% and 54 ± 5%, respectively). Western blot analysis revealed that post-conditioning increased the phosphorylation of GSK-3β by 2.3 times (P < 0.01), and this increase could be blocked by wortmannin, a PI3-kinase inhibitor. Although rapamycin blocked the infarct size reduction, phosphorylation of p70S6K was not increased in post-conditioned hearts. After 2 h of reperfusion, the post-conditioned hearts had significantly fewer TUNEL-positive nuclei (35 %) compared to control hearts (53%; P < 0.001). AIF was equally reduced in post-conditioned rat hearts (P < 0.05 vs. control). Infarct size reduction by ischemic post-conditioning of the in vivo rat heart is PI3-kinase dependent and involves mTOR. Furthermore, GSK-3β, which is thought to be a regulator of the mPTP, is part of the signaling pathway of post-conditioning. Finally, apoptosis was inhibited by post-conditioning, which was shown by two independent methods. The role of apoptosis and/or autophagy in post-conditioning has to be further elucidated to find therapeutic targets to protect the heart from the consequences of acute myocardial infarction.