Synthesis,NMR characterization and divergent biological actions of 2′-hydroxy-ceramide/dihydroceramide stereoisomers in MCF7 cells

A straightforward method for the simultaneous preparation of (2S,3R,2′R)- and (2S,3R,2′S)-2′-hydroxy-ceramides (2′-OHCer) from (2S,3R)-sphingosine acetonide precursors and racemic mixtures of 2-hydroxy fatty acids (2-OHFAs) is described. The obtained 2′-OH-C4-, -C6-, -C12-, -C16-Cer and 2′-OH-C6-dhCer pairs of diastereoisomers were characterized thoroughly by TLC, MS, NMR, and optical rotation. Dynamic and multidimensional NMR studies provided evidence that polar interfaces of 2′-OHCers are extended and more rigid than observed for the corresponding non-hydroxylated analogs. Stereospecific profile on growth suppression of MCF7 cells was observed for (2′R)- and (2′S)-2′-OH-C6-Cers and their dihydro analogs. The (2′R)-isomers were more active than the (2′S)-isomers (IC₅₀ ～3 μM/8 μM and IC₅₀ ～8 μM/12 μM, respectively), surpassing activity of the ordinary C6-Cer (IC₅₀ ～12 μM) and C6-dhCer (IC₅₀ ～38 μM). Neither isomer of 2′-OH-C6-Cers and 2′-OH-C6-dhCers was metabolized to their cellular long chain 2′-OH-homologs. Surprisingly, the most active (2′R)-isomers did not influence the levels of the cellular Cers nor dhCers. Contrary to this, the (2′S)-isomers generated cellular Cers and dhCers efficiently. In comparison, the ordinary C6-Cer and C6-dhCer also significantly increased the levels of their cellular long chain homologs. These peculiar anabolic responses and SAR data suggest that (2′R)-2′-OHCers/dhCers may interact with some distinct cellular regulatory targets in a specific and more effective manner than their non-hydroxylated analogs. Thus, stereoisomers of 2′-OHCers can be potentially utilized as novel molecular tools to study lipid–protein interactions, cell signaling phenomena and to understand the role of hydroxylated sphingolipids in cancer biology, pathogenesis and therapy.     

Distribution of α-Tocopherol Stereoisomers in Rats

The distribution of α-tocopherol (α-Toc) stereoisomers in the tissues of rats fed on a diet containing all-rac-α-tocopheryl acetate was investigated by a newly revised HPLC. The concentrations of 2R-isomers of α-Toc in blood and tissues of the rats were significantly higher than those of 2S-isomers. In most tissues, the levels of 2S-isomers were in order SRS> (SSS +SSR)/2 > SRR.     

Synthesis and evaluation of the inhibitory activity of the four stereoisomers of the potent and selective human γ-glutamyl transpeptidase inhibitor GGsTop

2-Amino-4-{[3-(carboxymethyl)phenoxy](methoxy)phosphoryl}butanoic acid (GGsTop) is a potent, highly selective, nontoxic, and irreversible inhibitor of γ-glutamyl transpeptidase (GGT). GGsTop has been widely used in academic and medicinal research, and also as an active ingredient (Nahlsgen) in commercial anti-aging cosmetics. GGsTop consists of four stereoisomers due to the presence of two stereogenic centers, i.e., the α-carbon atom of the glutamate mimic (l/d) and the phosphorus atom (R_P/S_P). In this study, each stereoisomer of GGsTop was synthesized stereoselectively and their inhibitory activity against human GGT was evaluated. The l- and d-configurations of each stereoisomer were determined by a combination of a chiral pool synthesis and chiral HPLC analysis. The synthesis of the four stereoisomers of GGsTop used chiral synthetic precursors that were separated by chiral HPLC on a preparative scale. With respect to the configuration of the α-carbon atom of the glutamate mimic, the l-isomer (k_on = 174 M^?1 s^?1) was ca. 8-fold more potent than the d-isomer (k_on = 21.5 M^?1 s^?1). In contrast, the configuration of the phosphorus atom is critical for GGT inhibitory activity. Based on a molecular modeling approach, the absolute configuration of the phosphorus atom of the active GGsTop isomers was postulated to be S_P. The S_P-isomers inhibited human GGT (k_on = 21.5–174 M^?1 s^?1), while the R_P-isomers were inactive even at concentrations of 0.1 mM.     

Diterpenoid and other components of Cistus laurifolius

From the ethanol extract of C. laurifolius was isolated a new diterpene, 6β,8-dihydroxy-ent-13E-labden-15-oic acid, which we called laurifolic acid. Four glucosides were separated in the form of their acetyl derivatives: β-D-glucopyranosiloxyethane, 4-β-D-glucopyranosiloxyacetophenone, roseoside and 1,3-dihydroxy-5-β-D-glucopyranosiloxybenzene. An inositol, 1-O-methyl-epi-inositol, was also identified.     

The naphthylamine palladium(II) template induced E-Z isomerism of exocyclic vinyl group in five-membered P-N chelates: detailed spectroscopic studies

The exocyclic CC bond E-Z isomerism of chelating Ph₂PC(CHPh)-CHNAr in organopalladium complexes containing orthometallated [(S)-1-(dimethylamino)ethyl]naphthalene is reported. In dilute solutions of non-coordinating CH₂Cl₂ or CHCl₃, all the original E-isomers, in which the CHPh phenyl rings are located trans to PPh₂ moieties were partly converted to their Z-isomers. The isomerism was found to be dependent on temperature, concentration and solvent. At higher temperature, the Z-isomers were transformed completely back to their original E-isomers. Removal of the chiral auxiliaries of the E-Z mixtures by concentrated HCl, gave only the dichloro complexes of the E-isomers. The E-Z isomerization processes were well established by detailed spectroscopic studies, including ³¹P NMR, ¹H NMR and 2D ¹H-¹H ROESY NMR studies. It is noteworthy that the dichloro complexes and free P-N ligands did not show such isomerization processes, indicating that the isomerization processes were triggered by the orthopalladated naphthylamine moiety.     

Resolution of the stereoisomers of leucovorin and 5-methyltetrahydrofolate by chiral high-performance liquid chromatography

Leucovorin (5-formyltetrahydrofolate, LV) is a reduced folate that has been in clinical use for many years as a rescue agent following methotrexate (MTX) therapy. Commercially available LV is a 1:1 mixture of [6R]-and [6S]-isomers. Due to the lack of a specific method for directly separating and quantitating the stereoisomers of LV, it has been difficult to precisely define the pharmacokinetic and biological characteristics of each stereoisomer. We have now developed a novel HPLC method to completely separate [6S]-LV and [6S]-5-methyltetrahydrofolate (MeTHF) from their respective [6R]-isomers using bovine serum albumin (BSA)-bonded silica as the chiral stationary phase. Baseline separation was achieved using 5 and 25 mM sodium phosphate buffers (pH 7.4) as the mobile phase with resolution factors of 1.65 for LV and 2.31 for MeTHF, respectively. The purity of each isomer prepared by this HPLC method is greater than 99%. The stereoisomers were identified by examining their ability to protect CEM cells from MTX (0.04 microM)-induced inhibition of growth. In the LV chromatogram, the first eluted peak provided complete protection from MTX growth inhibition when LV concentrations of 0.1 microM and above were used, whereas the last eluted peak failed to reverse MTX toxicity at concentrations up to 1.0 microM. Chemically pure synthetic [6R]-and [6S]-LV standards confirmed that the first eluted, biologically active peak is the [6S]-isomer. For MeTHF, only the last eluted peak effectively protects cells from MTX growth inhibition and is therefore believed to be the [6S]-isomer. This new HPLC method will serve as a useful tool to elucidate the clinical and cellular pharmacology of the stereoisomers of LV and MeTHF.     

Simultaneous determination of the stereoisomers of guggulsterone in serum by high-performance liquid chromatography

Simultaneous separation of E- and Z-guggulsterone, which is the main ingredient of ‘Guggulip', an ayurvedic drug, was accomplished by HPLC on a C₁₈ column using methanol, acetonitrile, buffer and tetrahydrofuran as a mobile phase. The compounds were monitored at 248 nm on a photodiode array detector. The assay method was used for the simultaneous determination of stereoisomers (E and Z) of guggulsterone in spiked serum and dosed (50 mg/kg, p.o.) rats. The recoveries of E- and Z-isomers from serum samples were always greater than 90%. The calibration graph was linear over the range of 25–2500 ng/ml for Z- and E-isomers. Lowest quantitation limit of Z- and E-guggulsterones was 25 ng/ml.     

Attraction of newly hatched codling moth larvae (Laspeyresia pomonella) to synthetic stereo-isomers of farnesene

Newly hatched Laspeyresia pomonella larvae were attracted to only two of six synthetic stereo-isomers of the acyclic sesquiterpene farnesene. These were (E,E)-α-farnesene and (Z,E)-α-farnesene which together comprise a natural attractant for the insect. Two other α-isomers and two β-isomers had no influence on larval behaviour. The activity of isomers is correlated with their molecular shape.     

Syntheses of 6-(2-hydroxy-6-phenylhexyl)-5,6-dihydro-2H-pyran-2-one derivatives and their cytotoxicities

The cytotoxicities against cancer cells (HL-60, HeLa) and insect cells (Sf9) of four stereoisomers of 6-(2-hydroxy-6-phenylhexyl)− 5,6-dihydro-2H-pyran-2-one (1) were evaluated, and then their structure-activity relationships examined. The 2′-dehydroxy derivative 5 of (6 R,2′R)- and (6 R,2′S)-1 showed the highest activity against HeLa cells (IC₅₀ = 1.4 μM). To evaluate the effect of the 2′-hydroxy group of 1, 6R-and 6S-oxetane derivatives were also synthesized and their activities examined. Against HeLa and HL-60 cells, the activities of the less potent stereoisomers were enhanced 3–4-fold by the introduction of the oxetane moieties at the 2′-position. Against the insect cell line (Sf9), phenyl derivative 7 showed the highest activity with an IC₅₀ value of 8.0 μM.     

Efficient and environmentally friendly method for carotenoid extraction from Paracoccus carotinifaciens utilizing naturally occurring Z-isomerization-accelerating catalysts

This study aimed to improve the efficiency of Paracoccus carotinifaciens-derived carotenoid (astaxanthin, adonirubin, adonixanthin) extraction using environmentally friendly Z-isomerization-accelerating catalysts. Adding naturally occurring catalysts such as isothiocyanates and polysulfides to the extraction solvent significantly improved the efficiency of carotenoid extraction, likely because of enhanced solubility of carotenoid Z-isomers compared with all-E-isomers. Indeed, addition of the catalysts markedly increased the content of carotenoid Z-isomers in the resulting extract. The use of a catalyst allyl isothiocyanate, which is abundantly included in Brassicaceae plant family, at high extraction temperature and long extraction time led to increased carotenoid recovery and Z-isomer content. These findings will enhance the efficiency of organic solvent-based extraction of carotenoids from carotenoid-rich sources. Numerous studies have reported that the Z-isomers of carotenoids exhibit greater bioavailability and antioxidant capacity than the all-E-isomers. Hence, the method proposed here utilizing Z-isomerization-accelerating catalysts could enhance both the extraction efficiency and beneficial health effects of carotenoids.     

cis-Isomers of Cytokinins Predominate in Chickpea Seeds throughout Their Development

Trans-isomers of cytokinins (CK) are thought to predominate and have greater biological activity than corresponding cis-isomers in higher plants. However, this study demonstrates a system within which the predominant CK are cis-isomers. CK were measured at four developmental stages in developing chickpea (Cicer arietinum L. cultivar Kaniva) seeds by gas chromatography-mass spectrometry. Concentrations were highest at an early endospermic fluid stage and fell considerably when the cotyledons expanded. The cis-isomers of zeatin nucleotide ([9R-MP]Z), zeatin riboside ([9R]Z), and zeatin (Z) were present in greater concentrations than those of corresponding trans-isomers: (trans)[9R-MP]Z, (trans)[9R]Z, (trans)Z, or dihydrozeatin riboside. Dihydrozeatin, dihydrozeatin nucleotide, and the isopentenyl-type CK concentrations were either low or not detectable. Root xylem exudates also contained predominantly cis-isomers of [9R-MP]Z and [9R]Z. Identities of (cis)[9R]Z and (cis)Z were confirmed by comparison of ion ratios and retention indices, and a full spectrum was obtained for (cis)[9R]Z. Tissues were extracted under conditions that minimized the possibility of RNase hydrolysis of tRNA following tissue disruption, being a significant source of the cis-CK. Since no isomerization of (trans)[²H]CK internal standards occurred, it is unlikely that the cis-CK resulted from enzymic or nonenzymic isomerization during extraction. Although quantities of total CK varied, similar CK profiles were found among three different chickpea cultivars and between adequately watered and water-stressed plants. Developing chickpea seeds will be a useful system for investigating the activity of cis-CK or determining the origin and metabolism of free CK.     

Synthesis and absolute configuration of the stereoisomers of [2-(1-diethylaminopropyl)] 1-hydroxy-1,1′-bicyclohexyl-2-carboxylate,a muscarinic antagonist

The compound [2-(1-diethylaminopropyl)] 1-hydroxy-1,1′-bicyclohexyl-2-carboxylate 1 is a muscarinic antagonist characterized by the presence of three chiral atoms and eight possible stereoisomers. The binding affinities to the five cloned m₁–m₅ muscarinic receptors of the stereoisomers of 1 were previously investigated and proved to be related to the chirality of the molecules. The eight isomers are prepared through the synthesis of their racemates followed by chemical resolution as (+) and (−) tartrate or (+) and (−) dibenzoyltartrate salts. The isomers with cis-configuration of OH and COOH substituents of the cyclohexane are also obtained by coupling optically active (1S, 2S) or (1R,2R)-1-hydroxy-[1,1′-bicyclohexyl]-2-carboxylic acid with (S)- or (R)-1-diethylamino-2-propanol. Chiral GC and HPLC methods are used to determine their optical purity. The absolute configurations of the four cis- and four trans-isomers are established by stereospecific synthesis and X-ray crystallographic data. Chirality 9:713–721, 1997. © 1997 Wiley-Liss, Inc.     

Biosynthesis of pisatin: Experiments with enantiomeric precursors

Feeding experiments in cupric chloride-treated Pisum sativum pods and seedlings have demonstrated the preferential incorporation of (+)-(6aS,11aS)-[³H]maackiain over (?)-(6aR, 11aR)-[¹⁴C]maackiain into (+)-(6aR, 11aR)-pisatin, establishing that the 6a-hydroxylation of pterocarpans proceeds with retention of configuration. (+)- (6aR,11aR)-6a-hydroxymaackiain was similarly incorporated much better than (?)-(6aS,11aS)-6a- hydroxymaackiain. Where (?)-isomers were incorporated, optical activity measurements on the pisatin produced indicated significant synthesis of (?)-pisatin as well as the normal (+)-pisatin. 7,2′-Dihydroxy-4′,5′- methylenedioxyisoflav-3-ene and both enantiomers of 7,2′-dihydroxy-4′,5′-methylenedioxyisoflavan were poor precursors of pisatin.     

Stereospecificity of 6′-C-Neplanocin A Analogues as Inhibitors of S-Adenosylhomocysteine Hydrolase Activity and Human Immunodeficiency Virus Replication†

Abstract

The R- and S-isomers of 6′-C-neplanocin A analogues, which are all known as inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase, were studied for their inhibitory effects on Human Immunodeficiency Virus type 1 (HIV-1) replication and HIV-1 Tat-mediated transactivation. The R-isomers showed much greater activity against AdoHcy hydrolase than the S-isomers. The same differential activity was observed against the HIV-1 replication and the Tat transactivation.

     

Increased ��-tocotrienol content in seeds of transgenic rice overexpressing Arabidopsis ��-tocopherol methyltransferase

Vitamin E comprises a group of eight lipid soluble antioxidant compounds that are an essential part of the human diet. The ??-isomers of both tocopherol and tocotrienol are generally considered to have the highest antioxidant activities. ??-tocopherol methyltransferase (??-TMT) catalyzes the final step in vitamin E biosynthesis, the methylation of ??- and ??-isomers to ??- and ??-isomers. In present study, the Arabidopsis ??-TMT (AtTMT) cDNA was overexpressed constitutively or in the endosperm of the elite japonica rice cultivar Wuyujing 3 (WY3) by Agrobacterium-mediated transformation. HPLC analysis showed that, in brown rice of the wild type or transgenic controls with empty vector, the ??-/??-tocotrienol ratio was only 0.7, much lower than that for tocopherol (~19.0). In transgenic rice overexpressing AtTMT driven by the constitutive Ubi promoter, most of the ??-isomers were converted to ??-isomers, especially the ??- and ??-tocotrienol levels were dramatically decreased. As a result, the ??-tocotrienol content was greatly increased in the transgenic seeds. Similarly, over-expression of AtTMT in the endosperm also resulted in an increase in the ??-tocotrienol content. The results showed that the ??-/??-tocopherol ratio also increased in the transgenic seeds, but there was no significant effect on ??-tocopherol level, which may reflect the fact that ??-tocopherol is present in very small amounts in wild type rice seeds. AtTMT overexpression had no effect on the absolute total content of either tocopherols or tocotrienols. Taken together, these results are the first demonstration that the overexpression of a foreign ??-TMT significantly shift the tocotrienol synthesis in rice, which is one of the world??s most important food crops.     

Chemical constituents from Sarcopyramis nepalensis Wall

One new megastigmane glycoside 1 and two new terpenic glycosides 2 and 3, along with three known compounds, roseoside, pumilaside A, and terminolic acid were isolated from Sarcopyramis nepalensis Wall. The structures of these new compounds were elucidated on the basis of 1D, 2D NMR, MS spectroscopic analysis, and chemical methods.     

Determination of (R)- and (S)-alprenolol and (R)- and (S)-metoprolol as their diastereomeric derivatives in human plasma by reversed-phase liquid chromatography

A high-performance liquid chromatographic method is described for the determination of (R)- and (S)-alprenolol and (R)- and (S)-metoprolol in human plasma. Separation of the enantiomers was accomplished after preparation of diastereomeric derivatives with symmetrical anhydrides of tert.-butoxycarbonyl-l-leucine followed by treatment with trifluoroacetic acetic acid at 0°C to remove the tert.-butoxycarbonyl group. The separations of the diastereomeric derivatives were performed using a reversed-phase system with μBondapak C₁₅ as support and phosphate buffer pH 3.0 with the addition of acetonitrile as the mobile phase. High stability of the chromatographic system was achieved.The reproducibilities in the determination of (R)- and (S)-alprenolol and (R)- and (S)-metoprolol in human plasma were 9.4 and 9.8% (relative standard deviation) for alprenolol and metoprolol, respectively, at drug levels of 0.5 ng/ml.In two subjects who received single oral doses of alprenolol (100-mg tablet) and metoprolol (50-mg tablet) the plasma levels of the (R)-isomers were lower than for the (S)-isomers.     

Enzymic reduction of [6]-gingerol,a major pungent principle of ginger,in the cell-free preparation of rat liver

A reductive metabolism of S-(+)-[6]-gingerol [1-(4'-hydroxy-3'-methoxyphenyl)-5-hydroxydecan-3-one], the major pungent principle of ginger, was investigated in vitro with phenobarbital-induced rat liver 10,000 x g supernatant containing the NADPH-generating system. The ethyl acetate-extractable products were isolated and two metabolites were identified as diastereomers of [6]-gingerdiol by gas chromatrography/mass spectrometry. The ratio of two isomers formed in the above reaction was about 1:5, suggesting the stereospecific reduction of S-(+)-[6]-gingerol by carbonyl reductase activity present in the postmitochondrial supernatant fraction of rat liver. The enzymic reduction of S-(+)-[6]-gingerol thus introduces the second asymmetric carbon center in the molecule with concomitant production of S,S- and R,S-isomers of [6]-gingerdiol in different proportions. This stereospecific reduction of [6]-gingerol may be relevant to the clinical use of the compound.     

Systematic strategy for the synthesis of cyanobacterin and its stereoisomers. 2. Asymmetric total synthesis of the 2- and 3-epimers of dechloro-cyanobacterin

Stereocontrolled total syntheses of the (2S,3R)- and (2R,3S)-isomers of the non-chlorinated analog of cyanobacterin, a potent photosynthesis inhibitor, were achieved. Since both the (2R,3R)- and (2S,3S)-isomers of this compound had been previously synthesized from the same starting material, a systematic strategy for the synthesis of all stereoisomers could be established.     

Pheromones and phylogenetic relations of leafrollers (Lepidoptera,Tortricidae)

Changes in the number of carbon atoms and in the proportion between cis- and trans-isomers of the components of leafroller pheromones were studied. The evolution of pheromone components at the tribe level included the decrease in the number of carbon atoms and the increase in the fraction of trans-isomers. The pattern of changes in the cis- and trans-isomers ratio allows tracing the evolution of species within a single genus.