Substituted aminopyridines as potent and selective phosphodiesterase-4 inhibitors

The synthesis and the biological evaluation of new potent phosphodiesterase type 4 (PDE4) inhibitors are presented. This new series was elaborated by replacement of the metabolically resistant phenyl hexafluorocarbinol of L-791,943 (1) by a substituted aminopyridine residue. The structure-activity relationship of N-substitution on 3 led to the identification of (-)-3n which exhibited a good PDE4 inhibitor activity (HWB-TNFalpha=0.12 microM) and an improved pharmacokinetic profile over L-791,943 (rat t(1/2)=2 h). (-)-3n was well tolerated in ferret with an emetic threshold of 30 mg/kg (po) and was found to be active in the ovalbumin-induced bronchoconstriction model in guinea pig (54%, 0.1 mg/kg, ip) as well as the ascaris-induced bronchoconstriction model in sheep (64%/97%, early/late, 0.5 mg/kg, iv).     

Discovery of L-791,943: a potent,selective, non emetic and orally active phosphodiesterase-4 inhibitor

Structure-activity relationship studies directed toward improving the potency and metabolic stability of CDP-840 (3) resulted in the discovery of L-791,943 (11n) as a potent (HWB TNF-alpha = 0.67 microM) and orally active phosphodiesterase type 4 (PDE4) inhibitor. This compound, which bears a stable bis-difluoromethoxy catechol and a pendant hexafluorocarbinol, exhibited a long half-life in rat and in squirrel monkey. It is well tolerated in ferret with an emetic threshold greater than 30 mg/kg (po) and was found to be active in the ovalbumin-induced bronchoconstriction model in guinea pig and in the ascaris-induced bronchoconstriction models in sheep and squirrel monkey.     

L-648,051, sodium 4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)- propylsulfonyl]-gamma-oxo-benzenebutanoate: a leukotriene D4 receptor antagonist

L-648,051, sodium 4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy) propylsulfonyl]-gamma-oxo-benzenebutanoate is a selective and competitive inhibitor of [3H]leukotriene D4 (KB value of 4.0 microM) and to a lesser extent [3H]leukotriene C4 (Ki value of 36.7 microM) binding in guinea pig lung homogenates. Functionally, it selectively antagonized contractions of guinea pig trachea induced by leukotrienes C4, D4, E4, and F4 in concentrations that did not antagonize contractions induced by acetylcholine, histamine, serotonin, prostaglandin F2 alpha, or U-44069 (endoperoxide analogue). Schild plot analysis indicated that L-648,051 competitively antagonized contractions of guinea pig ileum induced by leukotriene D4 (pA2 7.7) and contractions of trachea induced by leukotrienes D4, E4, and F4 (pA2 7.3, 7.4, and 7.5, respectively). Contractions of guinea pig trachea induced by leukotriene C4 were inhibited in a noncompetitive fashion (Schild plot slope, 0.45). Developed contractions of trachea induced by the leukotrienes were rapidly reversed by L-648,051 greater than FPL-55712 greater than L-649,923. Intravenous L-648,051 selectively blocked bronchoconstriction induced in anaesthetized guinea pigs by intravenous leukotrienes C4, D4, and E4 but not that induced by arachidonic acid, serotonin, U-44069, or acetylcholine. The compound displayed poor activity following intraduodenal administration. The profile of activity for L-648,051 indicates that it may be a useful topical agent for studying the role of leukotrienes in diseases such as bronchial asthma.     

Soybean Lipoxygenase L-4, a Component of the 94-kilodalton Storage Protein in Vegetative Tissues: Expression and Accumulation in Leaves Induced by Pod Removal and by Methyl Jasmonate

A lipoxygenase L-4 gene was isolated from a soybean genomiclibrary. The amino acid sequence of lipoxygenase L-4 is highlyhomologous with the partial amino acid sequence of the 94-kDavegetative storage protein, vsp94, found in paraveinal mesophyllcells in the leaves of depodded soybean plants. No L-4 expressionwas observed in maturing seeds. The L-4 gene is highly expressedin the vegetative tissues of young seedlings, including cotyledons,hypocotyls, roots and primary leaves. L-4 expression followedthe same pattern as lipoxygenase activity in cotyledons peaking3 to 5 days after germination, and returning to a basal levelby 9 days after germination. L-4 gene expression was low inthe roots, stems and leaves of 10-week-old plants. Exposureof 4-week-old plants to atmospheric methyl jasmonate increasedL-4 mRNA in leaves. Continuous pod removal from 7-week-old plantsover a 2 week period resulted in dramatic accumulation of L-4mRNA in leaves. Accumulation of the L-4 protein and three otherlipoxygenase fractions in the leaves of depodded plants wasdemonstrated by ion exchange chromatography. These results indicatethat lipoxygenase L-4 is a component of vsp94. (Received May 31, 1993; Accepted August 9, 1993)     

Apolipoprotein A-I mimetic peptide L-4F prevents myocardial and coronary dysfunction in diabetic mice

Diabetes is a major health problem associated with adverse cardiovascular outcomes. The apolipoprotein A-I mimetic peptide L-4F is a putative anti-diabetic drug, has antioxidant and anti-inflammatory proprieties and improves endothelial function. In obese mice L-4F increases adiponectin levels, improving insulin sensitivity, and reducing visceral adiposity. We hypothesized that the pleiotropic actions of L-4F can prevent heart and coronary dysfunction in a mouse model of genetically induced Type II diabetes. We treated db/db mice with either L-4F or vehicle for 8 weeks. Trans-thoracic echocardiography was performed; thereafter, isolated hearts were subjected to ischemia/reperfusion (IR). Glucose, insulin, adiponectin, and pro-inflammatory cytokines (IL-1β, TNF-α, MCP-1) were measured in plasma and HO-1, pAMPK, peNOS, iNOS, adiponectin, and superoxide in cardiac tissue. In db/db mice L-4F decreased accumulation of subcutaneous and total fat, and increased insulin sensitivity and adiponectin levels while lowering inflammatory cytokines (P < 0.05). L-4F normalized in vivo left ventricular (LV) function of db/db mice, increasing (P < 0.05) fractional shortening and decreasing (P < 0.05) LV dimensions. In I/R experiments, L-4F prevented coronary microvascular resistance from increasing and LV function from deteriorating in the db/db mice. These changes were associated with increased cardiac expression of HO-1, pAMPK, peNOS, and adiponectin and decreased levels of superoxide and iNOS (P < 0.01). In the present study we showed that L-4F prevented myocardial and coronary functional abnormalities in db/db mice. These effects were associated with stimulation of HO-1 resulting in increased levels of anti-inflammatory, anti-oxidative, and vasodilatatory action through a mechanism involving increased levels of adiponectin, pAMPK, and peNOS.     

Leukotriene D4 and platelet-activating factor-acether antagonists on allergic and arachidonic acid-induced reactions in guinea pig airways

Arachidonic acid (AA) and ovalbumin (OA) were used to induce contractions of sensitized guinea pig tracheal spiral (indomethacin-pretreated) and lung parenchymal strip preparations. This model was used to examine the properties of three leukotriene (LT) D4 antagonists and a platelet-activating factor (PAF)-acether receptor antagonist. The three LTD4 antagonists, L-649,923, FPL 57231, and LY163443, inhibited AA-induced contractions of indomethacin-pretreated tracheal spirals selectively. The PAF-acether antagonist, L-652,731, did not inhibit AA-induced contractions of either trachea or parenchyma. This confirmed that AA-induced contractions of trachea involved release and activity of LTD4. The LTD4 antagonists and L-652,731 partially inhibited OA-induced contractions of both trachea and parenchyma. When L-649,923 and L-652,731 or FPL 57231 and L-652,731 were combined, an additive inhibitory effect on OA-induced contractions was observed. When LY163443 and L-652,731 were combined, the inhibitory effect was synergistic. This may be due to the additional effect of LY163443 to inhibit phosphodiesterase. Total inhibition of OA-induced contractions was obtainable with relatively low concentrations when a LTD4 and PAF-acether antagonist were combined. These results suggested that LTD4 and PAF-acether may be the two major mediators in our model of allergic bronchospasm. The LTD4 and PAF-acether antagonists had the capacity to decrease baseline tone, even on tissues that were already relaxed with indomethacin, suggesting that LTD4 and PAF-acether may contribute to intrinsic tone in airway smooth muscle.     

Enhancement by LDL of transfer of L-4F and oxidized lipids to HDL in C57BL/6J mice and human plasma

The apoA-I mimetic peptide L-4F [(Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2) synthesized from all L-amino acids] has shown potential for the treatment of a variety of diseases. Here, we demonstrate that LDL promotes association between L-4F and HDL. A 2- to 3-fold greater association of L-4F with human HDL was observed in the presence of human LDL as compared with HDL by itself. This association further increased when LDL was supplemented with the oxidized lipid 15S-hydroxy-5Z, 8Z, 11Z, 13E-eicosatetraenoic acid (15HETE). Additionally, L-4F significantly (P = 0.02) promoted the transfer of 15HETE from LDL to HDL. The transfer of L-4F from LDL to HDL was demonstrated both in vitro and in C57BL/6J mice. L-4F, injected into C57BL/6J mice, associated rapidly with HDL and was then cleared quickly from the circulation. Similarly, L-4F loaded onto human HDL and injected into C57BL/6J mice was cleared quickly with T(1/2) = 23.6 min. This was accompanied by a decline in human apoA-I with little or no effect on the mouse apoA-I. Based on these results, we propose that i) LDL promotes the association of L-4F with HDL and ii) in the presence of L-4F, oxidized lipids in LDL are rapidly transferred to HDL allowing these oxidized lipids to be acted upon by HDL-associated enzymes and/or cleared from the circulation.     

Pharmacology of L-660,711 (MK-571): a novel potent and selective leukotriene D4 receptor antagonist

L-660,711 (3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl) ((3-dimethyl amino-3-oxo propyl)thio)methyl)thio)propanoic acid is a potent and selective competitive inhibitor of [3H]leukotriene D4 binding in guinea pig (Ki value, 0.22 nM) and human (Ki value, 2.1 nM) lung membranes but is essentially inactive versus [3H]leukotriene C4 binding (IC50 value in guinea pig lung, 23 microM). Functionally it competitively antagonized contractions of guinea pig trachea and ileum induced by leukotriene (LT) D4 (respective pA2 values, 9.4 and 10.5) and LTE4 (respective pA2 values, 9.1 and 10.4) and contractions of human trachea induced by LTD4 (pA2 value, 8.5). L-660,711 (5.8 x 10(-8)M) antagonized contractions of guinea pig trachea induced by LTC4 in the absence (dose ratio = 28) but not in the presence of 45 mM L-serine borate (dose ratio less than 2). L-660,711 (1.9 x 10(-5)M) did not block contractions of guinea pig trachea induced by histamine, acetylcholine, 5-hydroxytryptamine, PGF2 alpha, U-44069, or PGD2. In the presence of atropine, mepyramine, and indomethacin, L-660,711 (1.9 x 10(-5)M) inhibited a small component of the response to antigen on guinea pig trachea but completely blocked anti-IgE-induced contractions of human trachea. L-660,711 (i.v.) antagonized bronchoconstriction induced in anesthetized guinea pigs by i.v. LTC4, LTD4, and LTE4 but did not block bronchoconstriction to arachidonic acid, U-44069, 5-hydroxytryptamine, histamine, or acetylcholine. Intraduodenal L-660,711 antagonized LTD4 (0.2-12.8 micrograms/kg)-induced bronchoconstriction in guinea pigs, and p.o. L-660,711 blocked LTD4- and Ascaris-induced bronchoconstriction in conscious squirrel monkeys and ovalbumin-induced bronchoconstriction in conscious sensitized rats treated with methysergide (3 micrograms/kg). The pharmacological profile of L-660,711 indicates that it is a potent, selective, orally active leukotriene receptor antagonist which is well suited to determine the role played by LTD4 and LTE4 in asthma and other pathophysiologic conditions.     

Topical anti-androgenicity of a new 4-azasteroid in the hamster.

The topical anti-androgenic activity of L-651,580 (methyl 3-oxo-4-methyl-4-aza-5 alpha-androst-1-ene-17 beta-carboxylate) was established in a series of for experiments using castrated male hamsters. During each 21-day experiment, the animals received a daily subcutaneous injection of 40 micrograms testosterone propionate or 20 micrograms dihydrotestosterone propionate. Test compound in 25 microliters of gel was applied daily to the left flank organ. Compounds assayed included L-651,580, WIN 17,665 (17 alpha-propyltestosterone), and SH-434 (17 beta-hydroxy-1 alpha-methyl-17 alpha-propyl-5 alpha-androstan-3-one). Endpoints were flank organ area, sebaceous gland area, and prostate weight. Very similar results were obtained with L-651,580 and WIN 17,665. Daily doses of 0.25 mg or more of either compound usually produced a significant reduction in the areas of treated flank organs and sebaceous glands underlying treated flank organs. Neither compound caused significant changes in the area of the contralateral flank organs and sebaceous glands, which indicated they possess little or no systemic activity at topically effective treatment levels. In direct comparisons, SH-434 was less anti-androgenic than L-651,580 or WIN 17,665, although in one experiment, 0.5 mg/d of SH-434 significantly reduced the area of treated flank organs and sebaceous glands. Neither WIN 17,665 nor SH-434 caused a change in prostate weight; however, in one of four tests, a significant decrease was induced by the 0.5 mg/d level of L-651,580. The results of these experiments show that the topical anti-androgenicity of L-651,580 compares very favorably with that of WIN 17,665 and SH-434. They also indicate that the topical administration of effective dosage levels of L-651,580 causes few, if any, systemic effects.     

Photosynthetic Characteristics of Segregates from Hybrids between Flaveria brownii (C4 Like) and Flaveria linearis (C3-C4)

Characteristics related to C4 photosynthesis were studied in reciprocal F1 hybrids and F2 plants from Flaveria brownii (C4 like) and Flaveria linearis (C3-C4). The reciprocal F1 plants differed in 13C/12C ratios of leaves and the percentage of 14C initially incorporated into C4 acids, being more like the pollen parents in these traits. They did not differ in apparent photosynthesis or in O2 inhibition of apparent photosynthesis and differed only slightly in CO2 compensation concentration at 175 [mu]mol quanta m-2 s-1 and 400 mL L-1 O2. The 13C/12C ratios of 78 F2 progeny from the two F1 plants exhibited a normal distribution centered between those of the parents, with a few values slightly higher and lower than the parents. Apparent photosynthesis at 130 [mu]L L-1 CO2 and inhibition of photosynthesis by O2 was nearly normally distributed in the F2 population, but no values for F2 plants approached those for F. brownii (15.4 [mu]mol m-2 s-1 and 7.8%, respectively). Distribution of the CO2 compensation concentration measured at 1000 [mu]mol quanta m-2 s-1 and 400 mL L-1 of O2 in the F2 population was skewed toward F. brownii with 72% of the progeny having values <9 [mu]L of CO2 L-1 compared to 1.5 and 27.2 [mu]L L-1 for F. brownii and F. linearis, respectively. Correlations among traits of F2 plants were low (coefficients of 0.30 to -0.49), indicating that the C4- related traits are not closely linked in segregating populations. Plants in the F2 population selected for high or low apparent photosynthesis at 130 [mu]L of CO2 L-1 (six each) did not rank consistently high or low for 13C/12C ratios, O2 inhibition of apparent photosynthesis, CO2 compensation concentration, or activities of phosphoenolpyruvate carboxylase or NADP-malic enzyme. This study confirms results of earlier work that indicates independent segregation of C4 traits and also shows that the C4-like parental type can be recovered, at least for some characteristics (13C/12C ratio), in segregating populations. Recovery of fully functional C4 plants awaits further experimentation with C4 x C3 or C4 x C3-C4 hybrid plants that produce fertile progeny.     

Mechanism-based inactivation of cytochrome P450 3A4 by L-754,394

Mechanism-based inactivation of human liver P450 3A4 by L-754,394, a Merck compound synthesized as a potential HIV protease inhibitor, was investigated using recombinant P450 3A4. Enzyme inactivation was characterized by a small partition ratio (3.4 or 4.3 +/- 0.4), i.e., the total number of metabolic events undergone by the inhibitor divided by the number of enzyme inactivating events, lack of reversibility upon extensive dialysis, no decrease in the characteristic 450-nm species relative to control, and covalent modification of the apoprotein. The major and minor products formed during the inactivation of P450 3A4 were the monohydroxylated and the dihydrodiol metabolites of L-754,394, respectively. L-754,394 that had been adducted to P450 3A4 was hydrolyzed under the conditions used for SDS-PAGE, Ni(2+) affinity chromatography, and proteolytic digestion. In addition, the modification was not stable to the acidic conditions of HPLC separation and CNBr digestion. The labile nature of the peptide adduct and the nonstoichiometric binding of the inactivating species to P450 3A4 precluded the direct identification of a covalently modified amino acid residue or the peptide to which it was attached. However, Tricine SDS-PAGE in combination with MALDI-TOF-MS and homology modeling, allowed I257-M317 to be tentatively identified as an active site peptide, while prior knowledge of the stability of N-, O-, and S-linked conjugates of activated furans implicates Glu307 as the active site amino acid that is labeled by L-754, 394.     

L-4F treatment reduces adiposity, increases adiponectin levels, and improves insulin sensitivity in obese mice

We hypothesized that the apolipoprotein mimetic peptide L-4F, which induces arterial anti-oxidative enzymes and is vasoprotective in a rat model of diabetes, would ameliorate insulin resistance and diabetes in obese mice. L-4F (2 mg/kg/d) administered to ob/ob mice for 6 weeks limited weight gain without altering food intake, decreased visceral (P < 0.02) and subcutaneous (P < 0.045) fat content, decreased plasma IL-1beta and IL-6 levels (P < 0.05) and increased insulin sensitivity, resulting in decreased glucose (P < 0.001) and insulin (P < 0.036) levels. In addition, L-4F treatment increased aortic and bone marrow heme oxygenase (HO) activity and decreased aortic and bone marrow superoxide production (P < 0.001). L-4F treatment increased serum adiponectin levels (P < 0.037) and decreased adipogenesis in mouse bone marrow (P < 0.039) and in cultures of human bone marrow-derived mesenchymal stem cells (P < 0.022). This was manifested by reduced adiposity, improved insulin sensitivity, improved glucose tolerance, increased plasma adiponectin levels, and reduced IL-1beta and IL-6 levels in obese mice. This study highlights the existence of a temporal relationship between HO-1 and adiponectin that is positively affected by L-4F in the ob/ob mouse model of diabetes, resulting in the amelioration of the deleterious effects of diabetes.     

Effect of L-2-oxothiazolidine-4-carboxylate administration on glutathione and cysteine concentrations in guinea pig liver and kidney

Effect of L-2-oxothiazolidine-4-carboxylate administration on the glutathione and cysteine concentrations in liver and kidney was studied in the guinea pig. Liver glutathione concentration increased significantly by 21 to 29% at one to three hours after the intraperitoneal injection of 5 mmol of sodium L-2-oxothiazolidine-4-carboxylate per kg of body weight. Cysteine concentration did not change significantly. In contrast to the liver, a significant increase in cysteine and also a significant decrease in glutathione concentrations were observed in the kidney. Incubation of L-2-oxothiazolidine-4-carboxylate with liver and kidney homogenates resulted in cysteine formation of 1.21 and 0.56 mumol per g of fresh tissue per 30 min, respectively. These results seem to indicate that, in the liver, L-2-oxothiazolidine-4-carboxylate administration resulted in the formation of cysteine, which was utilized for glutathione synthesis. In the kidney, it seems to be suggested that the administration of this compound accelerated glutathione turnover.     

Treatment of patients with cardiovascular disease with L-4F, an apo-A1 mimetic, did not improve select biomarkers of HDL function

L-4F, an apolipoprotein A-I (apoA-I) mimetic peptide (also known as APL180), was administered daily by either intravenous (IV) infusion for 7 days or by subcutaneous (SC) injection for 28 days in patients with coronary heart disease in two distinct clinical studies. L-4F was well tolerated at all doses tested. Despite achieving plasma levels (mean maximal plasma concentration of 2,907 ng/ml and 395 ng/ml, following IV infusion and SC injection, respectively), that were effective in previously published animal models, treatment with L-4F, as assessed by biomarkers of HDL function such as HDL-inflammatory index (HII), and paraoxonase activity, did not improve. Paradoxically, there was a 49% increase in high-sensitivity C-reactive protein (hs-CRP) levels after seven IV infusions of 30 mg L-4F (P < 0.05; compared with placebo) and a trend for hs-CRP increase in subjects receiving 30 mg SC injection for 28 days. In a subsequent, ex vivo study, addition of L-4F at concentrations of 150, 375, or 1,000 ng/ml to plasma from subjects prior to L-4F treatment resulted in significant dose-dependent HII improvement. In conclusion, in vivo L-4F treatment, delivered by either SC injection or IV infusion, did not improve HDL functional biomarkers despite achieving plasma levels that improved identical biomarkers ex vivo and in animal models.     

L-649,923, sodium (beta S*, gamma R*)-4-(3-(4-acetyl-3-hydroxy-2-propylphenoxy)-propylthio)- gamma-hydroxy-beta-methylbenzenebutanoate, a selective, orally active leukotriene receptor antagonist

L-649,923, Sodium (beta S*, gamma R*)-4-(3-(4-acetyl-3-hydroxy-2-propylphenoxy)propylthio)- gamma- hydroxy-beta-methylbenzenebutanoate is a selective and competitive inhibitor of [3H]leukotriene D4 (Ki value of 400 nM) and to a lesser extent [3H]leukotriene C4 (Ki value of 8.6 microM) binding in guinea-pig lung homogenates. Functionally, it selectively antagonized contractions of guinea pig trachea induced by leukotriene C4, D4, E4, and F4 but not those induced by acetylcholine, histamine, serotonin, prostaglandin F2 alpha, or U-44069 (stable endoperoxide analogue). Schild plot analysis indicated a competitive inhibition of contractions of guinea-pig ileum induced by leukotriene D4 (pA2 8.1) and contractions of guinea-pig trachea induced by leukotrienes E4 and F4 (pA2 7.1 and 6.9, respectively). In contrast, contractions of guinea-pig trachea induced by leukotrienes C4 (pA2 7.2; slope 0.6) and D4 (pA2 7.2; slope 0.7) were inhibited in a noncompetitive fashion. In vivo, intravenously administered L-649,923 selectively blocked bronchoconstriction induced in anesthetized guinea pigs by leukotriene C4 and D4 (ED50 values i.v. 0.38 and 0.26 mg/kg, respectively) but not that induced by histamine, arachidonic acid, serotonin, U-44069, or acetylcholine. Following intraduodenal administration, L-649,923, blocked leukotriene D4 induced bronchoconstriction (5 and 10 mg/kg). The present findings indicate that selective antagonists, such as L-649,923, may be useful for defining the role of leukotrienes in diseases such as bronchial asthma.     

Transport of L-4-azaleucine in Escherichia coli.

The uptake of L-4-azaleucine was examined in Escherichia coli K-12 strains to determine the systems that serve for its accumulation. L-4=Azaleucine in radio-labeled form was synthesized and resolved by the action of hog kidney N-acylamino-acid amidohydrolase (EC 3.5.1.B) on the racemic alpha-N-acetyl derivative of DL-[dimethyl-14C]4-azaleucine. L-4-Azaleucine is taken up in E. coli by energy-dependent processes that are sensitive to changes in the pH and to inhibition by leucine and the aromatic amino acids. Although a single set of kinetic parameters was obtained by kinetic experiments, other evidence indicates that transport systems for both the aromatic and the branched-chain amino acids serve for azaleucine. Azaleucine uptake in strain EO317, with a mutation leading to derepression and constitutive expression of branched-chain amino acid (LIV) transport and binding proteins, was not repressed by growth with leucine as it was in parental strain EO300. Lesions in the aromatic amino acid transport system, aroP, also led to changes in the regulation of azaleucine uptake activity when cells were grown on phenylalanine. Experiments on the specificity of azaleucine uptake and exchange experiments with leucine and phenylalanine support the hypothesis that both LIV and aroP systems transport azaleucine. The ability of external azaleucine to exchange rapidly with intracellular leucine may be an important contributor to azaleucine toxicity. We conclude from these and other studies that at least four other process may affect azaleucine sensitivity: the level of branched-chain amino acid biosynthetic enzymes; the level of leucine, isoleucine, and valine transport systems; the level of the aromatic amino acid, aroP, uptake system; and, possibly, the ability of the cell to racemize D and L amino acids. The relative importance of these processes in azaleucine sensitivity under various conditions is not known precisely.     

Synthesis and antiviral activity of L-2'-deoxy-2'-up-fluoro-4'-thionucleosides.

L-2'-Deoxy-2'-up-fluoro-4'-thionucleosides were efficiently synthesized from D-xylose via L-4-thioarabitol derivative as a key intermediate and evaluated for antiviral activities against HIV-1, HSV-1,2 and HBV.     

骆驼刺发根农杆菌转化系的原生质体培养和植株再生

从发根农杆菌A4转化的荒漠植物—璐驼刺毛状根愈伤组织中分离的原生质体培养的结果表明,酶解新转代7～10d的淡黄色松软愈伤组织,可获得大量有活力的原生质体。原生质体在附加有1．5mg．L-1 2,4．D、0．2mg．L-1 6．BA、0．3m01．L-1甘露醇、2％（W/V）蔗糖和500mg·L-1水解酪蛋白的DPD培养基中进行液体浅层培养可持续分裂。培养基的最适渗透压为（450±3）mOsm·kg-1,原生质体的最适植板密度为4×10^5个．mL-1。制备原生质体的愈伤组织以低温（4℃）预处理后,原生质体的产率和分裂频率均提高,分裂频率最高可达50％。原生质体分裂形成的愈伤组织转移在附加1-2mg．L-1 6-BA（或KT）和0．2mg·L-1NAA的MS培养基上培养后,可以分化并获得再生植株。纸电泳检测表明,原生质体再生的愈伤组织和分化植株仍然含有毛状根转化系的特异产物——冠瘿碱。     

Identification,cloning, and sequencing of the genes involved in the conversion of D,L-2-amino-delta2-thiazoline-4-carboxylic acid to L-cysteine in Pseudomonas sp. strain ON-4a

The newly isolated strain Pseudomonas sp. ON-4a converts D,L-2-amino-delta2-thiazoline-4-carboxylic acid to L-cysteine via N-carbamoyl-L-cysteine. A genomic DNA fragment from this strain containing the gene(s) encoding enzymes that convert D,L-2-amino-delta2-thiazoline-4-carboxylic acid into L-cysteine was cloned in Escherichia coli. Transformants expressing cysteine-forming activity were selected by growth of an E. coli mutant defective in the cysB gene. A positive clone, denoted CM1, carrying the plasmid pCM1 with an insert DNA of approximately 3.4 kb was obtained, and the nucleotide sequence of a complementing region was analyzed. Analysis of the sequence found two open reading frames, ORF1 and ORF2, which encoded proteins of 183 and 435 amino acid residues, respectively. E. coli DH5alpha harboring pTrCM1, which was constructed by inserting the subcloned sequence into an expression vector, expressed two proteins of 25 kDa and 45 kDa. From the analyses of crude extracts of E. coli DH5alpha carrying deletion derivatives of pTrCM1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by enzymatic activity, it was found that the 25-kDa protein encoded by ORF1 was the enzyme L-2-amino-delta2-thiazoline-4-carboxylic acid hydrolase, which catalyzes the conversion of L-2-amino-delta2-thiazoline-4-carboxylic acid to N-carbamoyl-L-cysteine, and that the 45-kDa protein encoded by ORF2 was the enzyme N-carbamoyl-L-cysteine amidohydrolase, which catalyzes the conversion of N-carbamoyl-L-cysteine to L-cysteine.