Pharmacokinetics of theanine enantiomers in rats

Theanine, first discovered in tea, is a chiral nonproteinic amino acid that has been reported to have cardiovascular, neurological, and oncological effects. It is being considered as a therapeutic/medicinal agent and additive in consumer products. The present study evaluated the pharmacokinetics of D-theanine, L-theanine, and D,L-theanine in plasma and urine using LC-ESI/MS in rats after oral (p.o.) and intraperitoneal (i.p.) administration. Oral administration data indicated that gut absorption of d-theanine was far less than that of L-theanine. However, after i.p. administration, plasma theanine concentrations of L- and D-theanine were similar. This indicated that D- and L-theanine may exhibit a competitive effect with respect to intestinal absorption. Regardless of the route of administration, p.o. or i.p., the presence of the other enantiomer always decreased theanine plasma concentrations, indicating D,L-theanine competition with respect to urinary reabsorption. Data on urinary concentrations of D-theanine suggested that the D-isomer may be eliminated with minimal metabolism. L-Theanine appeared to be preferentially reabsorbed and metabolized by the kidney while D-theanine was preferentially excreted. Clearly, the bioequivalencies of D,L-theanine and its enantiomers were found to be quite different from one another. Consequently, the efficacy of commercial theanine products containing D-theanine, L-theanine, or D,L-theanine may be quite different.     

Elucidation of the absolute configuration of JNJ-27553292, a CCR2 receptor antagonist, by vibrational circular dichroism analysis of two precursors

The absolute configurations of two precursors, that is, 1-(3',4'-dichlorophenyl)-propanol and 1-(3',4'-dichlorophenyl)-propanamine, of a potent 2-mercapto-imidazole CCR-2 receptor antagonist, JNJ-27553292, were determined using vibrational circular dichroism. As a consequence, the absolute configuration of the antagonist itself was also determined. The two precursor compounds were subjected to a thorough conformational analysis and rotational strengths were calculated at the B3LYP/cc-pVTZ level of theory. Based on these data, vibrational circular dichroism spectra were simulated, which in turn were compared with experimental spectra. Agreement between the spectra allowed the assignment of the absolute configuration, which is in agreement with the proposed configuration based on stereospecific reactions on similar compounds.     

Semipreparative enantioseparation of Tröger base derivatives by HPLC

We describe the enantioseparation of functionalized derivatives of the Tr?ger base by HPLC on commercially available chiral stationary phases. Cellulose-derived Chiralcel OJ and brush-type Whelk O1 are demonstrated to be complementary to each other in their scope. On the basis of the results obtained, the separation of selected compounds was successfully transferred onto semipreparative columns. We believe that the availability of enantiopure functionalized derivatives of the Tr?ger base will stimulate the further use of this interesting molecular scaffold in molecular recognition, asymmetric catalysis, and related areas of research and technology.     

Synthesis of new enantiomerically pure macrocycles containing phenanthroline subunits

New enantiomerically pure macrocycles have been prepared by combining 1,10-phenanthroline 2,9-dicarboxylic acid and two alpha-amino-acids linked through spacers. Different diamine linkers have been employed in order to modify the dimensions and the properties of the macrocycles whose structures have been studied by 1H and 13C NMR spectroscopy. The ability of the (L)-valine containing macrocycles to bind metal ions and phenolic molecules has been investigated by 1H NMR experiments and Molecular Mechanics calculations.     

Synthesis, chiral HPLC resolution and configuration assignment of 1-phenylglyceryl trinitrate stereomers

As an introductory study of in vitro vasodilating activity, the access to the four stereomers of 1-phenylglyceryl trinitrate is described using achiral and chiral chromatography. For semi-preparative separation of the enantiomers, a Chiralcel OD (250 x 10 mm, 10 microm) was used. Catalytic reduction leading to the corresponding stereomers of 1-phenylglycerol allowed absolute configuration assignments. The same methods were used for the separation and configuration assignment of the enantiomers of 3-phenylpropane-1,2-diyl dinitrate.     

Lipoxygenases during Brassica napus seed germination

The peroxidation of polyunsaturated fatty acids is mostly catalyzed by members of the lipoxygenase enzyme family. Lipoxygenase products can be metabolized further in the oxylipin pathway and are known as signalling substances that play a role in plant development as well as in plant responses to wounding and pathogen attack. Apart from accumulating data in model plants like Arabidopsis, information on the relevance of lipid peroxide metabolism in the crop plant oilseed rape is scarce. Thus we aimed to analyze lipoxygenases and oxylipin patterns in seedlings of oilseed rape. RNA isolated from 3 day etiolated seedlings contains mRNAs for at least two different lipoxygenases. These have been cloned as cDNAs and named Bn-Lox-1fl and Bn-Lox-2fl. The protein encoded by Bn-Lox-2fl was identified as a 13-lipoxygenase by expression in Escherichia coli. The Bn-Lox-1fl yielded an inactive protein when expressed in E. coli. Based on Bn-Lox-1fl active site determinants and on sequence homology the Bn-Lox-1fl is most likely a 9-lipoxygenase. Both genes are expressed in light-grown and etiolated cotyledons as well as in leaves. Bn-Lox-2fl protein is more abundant in cotyledons of etiolated seedlings than in cotyledons of green seedlings. Both 13- and 9-lipoxygenase-derived hydroperoxides can be detected during germination. Etiolated seedlings contain more lipoxygenase-derived hydroperoxides in non esterified fatty acids than green seedlings. The 13-lipoxygenase derivatives are 6-8-fold more abundant than the 9-derivatives. Lipoxygenase-derived hydroperoxides in esterified lipids are almost not present during germination. These results suggest that 13-lipoxygenases acting on free fatty acids dominate during B. napus seed germination.     

Synthesis with good enantiomeric excess of both enantiomers of α-ketols and acetolactates by two thiamin diphosphate-dependent decarboxylases

In addition to the decarboxylation of 2-oxo acids, thiamin diphosphate (ThDP)-dependent decarboxylases/dehydrogenases can also carry out so-called carboligation reactions, where the central ThDP-bound enamine intermediate reacts with electrophilic substrates. For example, the enzyme yeast pyruvate decarboxylase (YPDC, from Saccharomyces cerevisiae) or the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1) can produce acetoin and acetolactate, resulting from the reaction of the central thiamin diphosphate-bound enamine with acetaldehyde and pyruvate, respectively. Earlier, we had shown that some active center variants indeed prefer such a carboligase pathway to the usual one [Sergienko, Jordan, Biochemistry 40 (2001) 7369–7381; Nemeria et al., J. Biol. Chem. 280 (2005) 21,473–21,482]. Herein is reported detailed analysis of the stereoselectivity for forming the carboligase products acetoin, acetolactate, and phenylacetylcarbinol by the E477Q and D28A YPDC, and the E636A and E636Q PDHc-E1 active-center variants. Both pyruvate and β-hydroxypyruvate were used as substrates and the enantiomeric excess was analyzed by a combination of NMR, circular dichroism and chiral-column gas chromatographic methods. Remarkably, the two enzymes produced a high enantiomeric excess of the opposite enantiomer of both acetoin-derived and acetolactate-derived products, strongly suggesting that the facial selectivity for the electrophile in the carboligation is different in the two enzymes. The different stereoselectivities exhibited by the two enzymes could be utilized in the chiral synthesis of important intermediates.     

Overcoming the thermodynamic limitation in asymmetric hydrogen transfer reactions catalyzed by whole cells

Whole lyophilized cells of an Escherichia coli overexpressing the alcohol dehydrogenase (ADH-'A') from Rhodococcus ruber DSM 44541 were used for the asymmetric reduction of ketones to secondary alcohols. The recycling of the required nicotinamide cofactor (NADH) was achieved in a coupled-substrate process. In the course of the reaction the ketone is reduced to the alcohol and the hydrogen donor 2-propanol is oxidized to acetone by one enzyme. This leads to a thermodynamic equilibrium between all four components determining the maximum achievable conversion. To overcome this limitation an in situ product removal technique (ISPR) for the application with whole cells was developed. In this method the most volatile compound is separated from the reaction vessel by an air flow resulting in a shift of the equilibrium towards the desired secondary alcohol. The so-called stripping process represents a simple and efficient method to overcome the thermodynamic limitation in biocatalytic reactions. Employing this method, the conversion of selected biotransformations was increased up to completeness.     

槐庶尺蛾性信息素腺体EAG活性成分绝对构型的鉴定(英文)

槐庶尺蛾Semiothisa cinerearia Bremer et Grey （鳞翅目： 尺蛾科）是我国北方国槐Sophora japonica L.上的重要食叶害虫。本研究的主要目的是阐明槐庶尺蛾性信息素成分化学结构的绝对构型, 为在城市地区环境友好地防控槐庶尺蛾的为害提供一种新方法。经与标准品比较气相色谱保留时间和质谱特征离子, 从槐庶尺蛾处女雌蛾（2-3日龄）性信息素腺体溶剂提取物中检测到顺6, 顺9-顺-3, 4-环氧-十七碳二烯烃和顺3, 顺6, 顺9-3, 6, 9-十七碳三烯烃2种成分, 在腺体中以100∶4.8±1.3 （N=12）的比例存在。槐庶尺蛾性信息素腺体提取物进一步经手性毛细管色谱柱（CycloSil-B, 30 m×0.25 mm×0.25 μm 液膜厚）分离, 在优化的程序升温条件下发现腺体成分顺6, 顺9-顺-3, 4-环氧-十七碳二烯烃具有3R, 4S的绝对构型。两种合成的对映异构体混合物顺6, 顺9-3R, 4S-环氧-十七碳二烯烃和顺6, 顺9-3S, 4R-环氧-十七碳二烯烃以1.28∶1的比例加到腺体提取物中, 比例变为1.55∶1。根据这一分析, 腺体成分顺6, 顺9-顺-3, 4-环氧-十七碳二烯烃进一步确认具有3R, 4S的绝对构型。该研究结论将为生产上研发高效的槐庶尺蛾性信息素诱芯奠定坚实的基础。