Lipase mediated resolution of γ-azidoalcohols in aqueous and organic media: Synthesis of (R)- and (S)-fluoxetine and duloxetine

A simple and efficient method for the synthesis of optically active γ-azidoalcohols is described. The lipase catalyzed kinetic resolutions of acetates of γ-azidoalcohols in aqueous as well as organic media have been studied. The enantiomerically pure γ-azidoalcohols obtained by the kinetic resolution in high enantiopurity have been utilized towards the synthesis of enantiomeric pairs of anti-depressant drugs, fluoxetine and duloxetine.     

Synthesis of (S)-2-fluoro- -daunosamine and (S)-2-fluoro- -ristosamine

Derivatives of (S)-2-fluoro- -daunosamine and (S)-2-fluoro- -ristosamine were synthesized, starting ultimately from 2-amino-2-deoxy- -glucose which was converted, according to the literature, into methyl 2-benzamido-4,6-O-benzylidene-2-deoxy-3-O-(methylsulfonyl)-α- -glucopyranoside (2). Treatment of 2 with tetrabutylammonium fluoride gave a 63% yield of (known) methyl 3-benzamido-4,6-O-benzylidene-2,3-dideoxy-2-fluoro-α- -altropyranoside (4), together with a 6% yield of its 2-benzamido-2,3-dideoxy-3-fluoro-α- -gluco isomer. From 4, the corresponding 6-bromo-2,3,6-trideoxyglycoside 4-benzoate (6) was obtained by Hanessian-Hullar reaction. Dehydrobromination of 6, followed by catalytic hydrogenation of the resulting 5-enoside, and subsequent debenzoylation and N-trifluoroacetylation, afforded the fluorodaunosaminide, methyl 2,3,6-trideoxy-2-fluoro-3-trifluoroacetamido-β- -galactopyranoside. Reductive debromination of 6, followed by debenzoylation and N-trifluoroacetylation, gave the fluororistosaminide, methyl 2,3,6-trideoxy-2-fluoro-3-trifluoroacetamido-α- -altropyranoside. The ¹H-n.m.r. spectra of the new aminofluoro sugars are discussed with respect to the effects of neighboring amino and acylamido substituents on geminal and vicinal ¹H–¹⁹F coupling constants, in comparison with the reported effects of oxyge substituents.     

Assessing the absolute configuration of (7S,8R)-(−)-epoxyjasmone

The absolute configuration of cis-epoxyjasmone (−)-2, isolated from Trichosporum cutaneum CCT 1903 whole cells, has been unambiguously established as (7S,8R), [α]_D²⁰ −29.0° (c 1.3, CHCl₃), by a new two step method, using a regioselective epoxide opening as the key step followed by Mosher acid derivatization.     

(3S,4S,5R)-3,4,5-trihydroxy-1-cyclohexene-carboxylic acid from Sequoiadendron giganteum

From the leafy lateral branchlets of Sequoiadendron giganteum, (3S,4S,5R)-3,4,5-trihydroxy-1-cyclohexenecarboxylic acid has been isolated. Its structure was proved spectroscopically.     

Enantioselective analysis of (R)- and (S)-atenolol in urine samples by a high-performance liquid chromatography column-switching setup

An HPLC column-switching method for the enantioselective determination of (R,S)-atenolol in human urine was developed and validated. Diluted urine samples were injected onto a LiChrospher ADS restricted access column and atenolol was separated from most of the matrix components using 0.01 M Tris buffer. The atenolol peak was sharpened by a step gradient of 30% acetonitrile and the atenolol-containing fraction was switched onto an enantioselective column. Separation of the atenolol enantiomers was carried out on a Chirobiotic T (Teicoplanin) column using acetonitrile–methanol–acetic acid–triethylamine (55:45:0.3:0.2, v/v/v/v) as eluent. Detection of the effluent was performed by fluorescence measurement. Several experiments were carried out to suppress the high blank reading, which was efficiently achieved using Tris buffer in the first dimension. For the enantioselective analysis of (R)- and (S)-atenolol in plasma under the same conditions the sample capacity of the ADS column is considerably lower.     

Liquid chromatographic determination of total celiprolol or (S)-celiprolol and (R)-celiprolol simultaneously in human plasma

A method has been developed for the determination of total celiprolol (sum of enantiomers) or the enantiomers (R)-celiprolol and (S)-celiprolol in plasma by high-performance liquid chromatography with UV and fluorescence detection. After extraction from alkalinized plasma with methyl-tert-butyl ether and back-extraction into 0.01 M HCl (for total celiprolol determination) or after evaporation of the organic phase and derivatisation with R(−)-1-(1-naphthyl)ethyl isocyanate (enantiomer determination), total celiprolol or its diastereomeric derivatives were chromatographed on a reversed-phase HPLC column with a mixture of acetonitrile and phosphate buffer pH 3.5 (+0.05% triethylamine). Acebutolol was used as internal standard. Linearity was obtained in the range of 5 to 2000 ng/ml for total and 2.5 to 500 ng/ml for enantiomer determination. Intra-day and inter-day variation was lower than 10%. The method can be applied for analysis of plasma samples obtained from patients treated with oral racemic celiprolol doses.     

Molecular pharmacology of the AMPA agonist, (S)-2-amino-3-(3-hydroxy-5-phenyl-4-isoxazolyl)propionic acid [(S)-APPA] and the AMPA antagonist, (R)-APPA

The heterocyclic analogue of (S)-glutamic acid, (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid [(S)-AMPA] is a potent and selective AMPA receptor agonist, whereas the enantiomeric compound, (R)-AMPA, is virtually inactive. We have previously characterized (RS)-2-amino-3-(3-hydroxy-5-phenyl-4-isoxazolyl)propionic acid [(RS)-APPA] as a partial AMPA receptor agonist showing about 60% of the efficacy of (RS)-AMPA. This partial agonism produced by (RS)-APPA is, however, only apparent, since resolution of (RS)-APPA has now been shown to provide the full AMPA receptor agonist, (S)-APPA, whereas (R)-APPA is a acid (non-NMDA) receptor antagonist showing preferential AMPA blocking effects. In agreement with classical theories for competitive interaction between agonists and antagonists, the efficacy of depolarizations produced by (S)-APPA in the rat cortical wedge preparation was shown to be progressively reduced with increasing molar ratios of (R)-APPA/(S)-APPA. These compounds and the competitive antagonists (RS)-2-amino-3-(3-carboxymethoxy-5-methyl-4-isoxazolyl)propionic acid [(RS)-AMOA], 6-cyano-7-nitroquinoxalin-2,3-dione (CNQX) and 6-nitro-7-sulfamoylbenzo(f)quinoxalin-2,3-dione (NBQX) were also tested in [³H]AMPA and [³H]CNQX binding systems, the latter ligand being used in the absence or presence of thiocyanate ions. On the basis of these studies it is suggested that (RS)-AMPA and the AMPA agonist (S)-APPA interact with a high-affinity receptor conformation, whereas the competitive antagonists (RS)-AMOA and (R)-APPA, derived from these agonists, preferentially bind to a low-affinity AMPA receptor conformation. The competitive antagonists, CNQX and NBQX which are structurally unrelated to (RS)-AMPA or (RS)-APPA, do not seem to discriminate between these two AMPA receptor conformations. The modified [³H]CNQX binding assay containing thiocyanate ions was shown to provide receptor affinity data for AMPA receptor agonists as well as antagonists, which correlate with the potencies of these compounds in the cortical wedge preparation. Using autoradiographic techniques, (S)- and (R)-APPA were shown to exhibit significantly different absolute potencies as inhibitors of [³H]AMPA binding in a number of regions of the rat brain.     

Stereoselective total synthesis of a potent natural antifungal compound (6S)-5,6,dihydro-6-[(2R)-2-hydroxy-6-phenyl hexyl]-2H-pyran-2-one

A practical stereoselective synthesis of (6S)-5,6,dihydro-6-[(2R)-2-hydroxy-6-phenyl hexyl]-2H-pyran-2-one (1), a potent natural antifungal compound, is described. The sequence involves diastereoselective iodine-induced electrophilic cyclization, epoxide ring opening with a vinyl Grignard reagent and ring closing metathesis (RCM) as the key steps.     

Optimization of (R,S)-1-phenylethanol kinetic resolution over Candida antarctica lipase B in ionic liquids

The kinetic resolution of racemates constitutes one major route to manufacture optically pure compounds. The enzymatic kinetic resolution of (R,S)-1-phenylethanol over Candida antarctica lipase B (CALB) by using vinyl acetate as the acyl donor in the acylation reaction was chosen as model reaction. A systematic screening and optimization of the reaction parameters, such as enzyme, ionic liquid and substrates concentrations with respect to the final product concentration, were performed. The enantioselectivity of immobilized CALB commercial preparation, Novozym 435, was assayed in several ionic liquids as reaction media. In particular, three different ionic liquids: (i) 1-butyl-3-methylimidazolium hexafluorophosphate [bmim][PF₆], (ii) 1-butyl-3-methylimidazolium tetrafluoroborate [bmim][BF₄] and (iii) 1-ethyl-3-methylimidazolium triflimide [emim][NTf₂] were tested. At 6.6% (w/w) of Novozym 435, dispersed in 9.520 M of [bmim][PF₆] at 313.15 K, using an equimolar ratio of vinyl acetate/(R,S)-1-phenylethanol after 3 h of bioconversion, the highest possible conversion (50%) was reached with enantiomeric excess for substrate higher than 99%.     

Kinetic modelling of aldolase-catalyzed addition between dihydroxyacetone phosphate and (S)-alaninal

This paper is focused on the development of a kinetic model for an aldolase-catalyzed reaction. The aldol addition between dihydroxyacetone phosphate (DHAP) and (S)-benzyloxycarbonyl-alaninal ((S)-Cbz-alaninal) catalyzed by the four DHAP-dependent aldolases is a promising way for the synthesis of four complementary diastereoisomers with potential biological activity. The reaction catalyzed by fuculose-1-phosphate aldolase (FucA) conducts to a synthesis product with a 100% diastereomeric excess. A kinetic model has been proposed including both the synthesis and a parallel non-desired secondary reaction. The model involved an ordered two-substrate mechanism for the synthesis and non-competitive inhibition by (S)-Cbz-alaninal and competitive inhibition by methylglyoxal byproduct in both reactions. The values of the model kinetic parameters were determined and the model validated in batch and fed-batch synthesis reactions. The obtained model could be extended to explain the behavior of other class II DHAP-dependent aldolases and exploited in simulation for reactor design purposes.     

Biotransformation of 3-substituted methyl (R,S)-4-cyanobutanoates with nitrile- and amide-converting biocatalysts

Whole cells of Rhodococcus equi A4 chemoselectively hydrolyzed methyl (R,S)-3-benzoyloxy-4-cyanobutanoate and methyl (R,S)-3-benzyloxy-4-cyanobutanoate into monomethyl (R,S)-3-benzoyloxyglutarate and monomethyl (R,S)-3-benzyloxyglutarate, respectively. The intermediates of the biotransformations were the corresponding amides which were also obtained using the purified nitrile hydratase from the same microorganism.     

Pharmacokinetic behaviour of R-(+)- and S-(−)-amlodipine after single enantiomer administration

Amlodipine, 3-ethyl 5-methyl-2-[(2-aminoethoxymethyl]-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedicarboxylate, is a chiral calcium antagonist, currently on the market and in therapeutic use as a racemate. The pharmacokinetic behaviour of R-(+)- and S-(−)-amlodipine after single enantiomer administration to healthy male human volunteers together with comparative administration of the racemic mixture of both enantiomers were studied. Plasma levels were studied as a function of time and assayed using an enantioselective chromatographic method (coupled chiral and achiral HPLC) with on-line solid-phase extraction and UV absorbance detection. The method was validated separately for the R-(+)- and S-(−)-enantiomer, respectively. Results of the study indicate that the pharmacokinetic behaviour of R-(+)- and S-(−)-amlodipine after single enantiomer administration is comparable to that of each enantiomer after administration of the racemate. No racemization occurs in vivo in human plasma after single enantiomer administration.     

Occurrence of 13(S)-Hydroxyoctadecadienoic Acid in Biological Samples

The oxygenated metabolite of linoleic acid, 13(S)-hydroxyoctadecadienoic acid has recently been shown to play a role in cellular regulation. To detect this molecule in biological systems, we recently developed a specific polyclonal antibody. Using this antibody, we report the presence of 13(S)-hydroxyoctadecadienoic acid in human urine, cell culture media, and untreated goat serum for the first time by a specific, sensitive, and rapid enzyme immunoassay. Furthermore, the enzyme linked immunosorbent assay data are verified by gas chromatography/mass spectrometry analysis of the same samples.     

酵母脂肪酶分子进化及催化合成(S)-1,4-苯并二口恶烷*

Objective: 1,4-Benzodioxane is an important chiral intermediate for antihypertensive (Proroxan and Doxazosin), antidepressant (MCK-242) and other drugs, and it displays a broad spectrum of applications in the pharmaceutical field. Currently, in spite of high-yield advantage of chemical synthesis, there are some problems of environmental pollution and low production safety. Using lipase to catalyze synthesis of 1,4-benzodioxane provides a new pathway of green synthesis of 1,4-benzodioxane. However, natural enzymes face the dilemma of poor enantioselectivity. Therefore, molecular evolution was performed on Candida antarctica lipase B, and a technical route for the catalytic synthesis of 1,4-benzodioxane was established. Methods: Firstly, the key amino acid residues involved in substrate binding and conversion in the active center of Candida antarctica lipase B were analyzed, and saturation mutagenesis libraries on the interaction sites were constructed. Improved mutants with high efficiency and high enantioselectivity were then obtained using HPLC detection. Furthermore, catalytic synthesis conditions of mutant D223N/A225K were systematically optimized. Results: The results indicated that the mutants mainly derived from the pairwise site D223/A225 (such as D223N/A225K and D223G/A225W) were biased towards the synthesis of (S)-isoforms, while most of the mutants derived from the pairwise site E188/I189 (such as E188D/I189M) showed a bias for the synthesis of (R)-isoforms. Compared with WT, the ee_s value of the best mutant D223N/A225K to synthesize (S)-1,4-benzodioxane was increased from 11.9% to 29.3%. After systematic optimization of the reaction conditions, an ee_s value of (93.9±0.16)% and a conversion rate of (47.5±2.33)% were achieved using mutant D223N/A225K to catalyze kinetic resolution of methyl (R,S)-2,3-dihydro-1,4-benzodioxin-2-carboxylate in n-butanol/phosphate buffered saline (20∶80, V/V) biphasic solvent at 37℃ for 50 min. Conclusion: An efficient kinetic resolution of methyl (R,S)-2,3-dihydro-1,4-benzodioxin-2-carboxylate was successfully achieved by molecular evolution and optimization of conditions, which provides a new example for the creation of new enzymes by protein engineering technology, and also provides a theoretical and technical foundation for the efficient synthesis of (S)-1,4-benzodioxane molecules by enzymatic methods.     

Characterization of the biological conversion of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene in direct micellar systems

The whole cell biological conversion of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene by the E. coli JM109(pPS1778) recombinant strain carrying the naphthalene dioxygenase and regulatory genes cloned from Pseudomonas fluorescens N3 in micellar systems has been investigated using biochemical and chemico-physical techniques. Reverse and direct micellar systems have been tested. Non-ionic surfactants (Tween and Triton X series) were found not to inhibit either the growth of the bacteria and the expression of the hydroxylating dioxygenase enzyme in such systems and were utilized in order to speed up the naphthalene conversion by increasing its solubility and also its bioavailability. The phase behavior of the direct micellar system was characterized through light scattering and other chemico-physical techniques. Further addition of isopropyl-palmitate 1–2% v/v to the micellar systems resulted in an increase of the apparent substrate concentration in solution and particularly its bioavailability thus allowing faster catalytic conversions resulting in an increase in productivity for the process. Since the cis-dihydrodiols are acquiring considerable potential as chiral pool synthons in asymmetric synthesis for a variety of industrial processes, possible applications for efficient small and large-scale production of such compounds are discussed.     

Effect of (6R)- and (6S)-tetrahydrobiopterin on L-3,4-dihydroxyphenylalanine (DOPA) formation in NRK fibroblasts transfected with human tyrosine hydroxylase type 2 cDNA

-erythro-5,6,7,8-Tetrahydrobiopterin (BH₄), which is the cofactor of aromatic amino acid hydroxylases, plays an important role in the biosyntheses of monoamine neurotransmitters. BH₄ exists as natural (6R)- and unnatural (6S)-isomers. In our previous reports, only (6R)-isomer significantly stimulated cofactor activity for tyrosine, tryptophan and phenylalanine hydroxylases (TH, TPH, PAH) in whole animals or in tissue slices. In this study we have compared the in situ cofactor activity on TH between natural (6R)- and unnatural (6S)-isomers in clonal cells. We have transfected human TH type 2 cDNA into the normal rat kidney (NRK) fibroblasts. These cells expressed TH protein, but had neither DOPA decarboxylase (DDC) nor BH₄. Thus, TH activity was observed only in the presence of exogenous BH₄. We compared the difference in in situ DOPA formation by TH activity in the presence of (6R)- or (6S)-BH₄ in the human TH-transfected cells. The effect of exogenous BH₄ was also compared between (6R)- and (6S)-isomers in rat pheochromocytoma PC12h cells, which contained approximately 100 μM endogenous (6R)-BH₄. The rate of uptake of both BH₄ isomers into these cells increased in proportion to the pterin cofactor concentrations in the incubation medium up to 400 μM but was nearly saturated at 1 mM BH₄. TH-transfected NRK fibroblasts formed DOPA only in the presence of exogenously added (6R)- or (6S)-BH₄ dose-dependently and released DOPA into the medium. At a saturating concentration of 1 mM, (6R)-BH₄ was approximately three times as active as (6S)-BH₄. In contrast, in PC12h cells which contained endogenous (6R)-BH₄ (approximately 100 μM), exogenous (6R)-BH₄ activated DOPA formation maximally at 500 μM about 10-fold, while (6S)-BH₄ activated it only slightly, about 2.5-fold. These results suggest that (6S)-isomer has lower cofactor activity with TH in the cells than (6R)-isomer. This TH transfected fibroblasts should be useful to assess cofactor activities of tetrahydropteridines in the cell.     

Chemoenzymatic synthesis of (1S,2R)-1-amino-2-indanol, a key intermediate of HIV protease inhibitor, indinavir

The synthesis of (1S,2R)-1-amino-2-indanol, a key component of HIV protease inhibitor is accomplished in four steps starting from indanone efficiently and with high levels of diastereo- and enantioselectivity. The starting material is converted into 2-acetoxy-1-indanone involving Manganese (III) acetate oxidation . The 2-acetoxyketone is hydrolyzed to 2-hydroxy-1-indanone enantioselectively using Rhizopus oryzae. Selective reduction of 2-hydroxyoxime derivative, derived from the 2-hydroxyketone, gives the amino alcohol up to 98% diastereo- and enantioselectivity.     

金缕梅科红花荷属野生资源及其园林开发潜力评估

为了解我国红花荷属的野生种质资源状况及其观赏价值,笔者对我国红花荷属(Rhodoleia Champ. ex Hook. f.) 6种野生种质资源进行了系统的野外调查,采集种质资源并开展了栽培试验,采用线性加权综合法,建立综合评价指标体系,对野生资源的园林开发潜力进行了评估。结果表明：红花荷、窄瓣红花荷和小花红花荷的综合价值高(Ⅰ级,分值＞2.0);小脉红花荷的综合价值中等(Ⅱ级,分值1.5~2.0);绒毛红花荷和大果红花荷综合价值低(Ⅲ级,分值＜1.5)。红花荷、窄瓣红花荷和小花红花荷作为优良的乡土木本花卉,在园林观赏性、栽培适应性、开发新颖性上的应用潜力非常高,值得进一步开发利用。     

Industrial production of (R)-1,3-butanediol by new biocatalysts

We have developed the economical and convenient biocatalytic process for the preparation of (R)-1,3-butanediol (BDO) by stereo-specific microbial oxido-reduction on an industrial scale. (R)-1,3-BDO is an important chiral synthon for the synthesis of various optically active compounds such as azetidinone derivatives lead to penem and carbapenem antibiotics.

We studied on two approaches to obtain (R)-1,3-BDO. The first approach was based on enzyme-catalyzed asymmetric reduction of 4-hydroxy-2-butanone; the second approach was based on enantio-selective oxidation of the undesired (S)-1,3-BDO in the racemate. As a result of screening for yeasts, fungi and bacteria, the enzymatic resolution of racemic 1,3-BDO by the Candida parapsilosis IFO 1396, which showed differential rates of oxidation for two enantiomers, was found to be the most practical process to produce (R)-1,3-BDO with high enantiomeric excess and yield.

We characterized the (S)-1,3-BDO dehydrogenase purified from a cell-free extract of C. parapsilosis. This enzyme was found to be a novel secondary alcohol dehydrogenase (CpSADH). We have attempted to clone and characterize the gene encoding CpSADH and express it in Escherichia coli. The CpSADH activity of a recombinant E. coli strain was more than two times higher than that of C. parapsilosis. The production yield of (R)-1,3-BDO from the racemate increased by using the recombinant E. coli strain. Interestingly, we found that the recombinant E. coli strain catalyzed the reduction of ethyl 4-chloro-3-oxo-butanoate to ethyl (R)-4-chloro-3-hyroxy-butanoate with high enantiomeric excess.