Application of mixtures of tartaric acid derivatives in resolution via supercritical fluid extraction

Racemic N-methylamphetamine (rac-MA) was resolved with 2R,3R-tartaric acid (TA) and its derivatives (O,O'-dibenzoyl-(2R,3R)-tartaric acid monohydrate (DBTA) and O,O'-di-p-toluoyl-(2R,3R)-tartaric acid (DPTTA)), individually and using them in different combinations. After partial diastereomeric salt formation, the free enantiomers were extracted by supercritical fluid extraction using carbon dioxide as solvent. DBTA and DPTTA are efficient resolving agents for rac-MA, the best chiral separation being obtained at a molar ratio of 0.25 resolving agent to racemic compound for both resolving agents (ee(E) = 82.5% and ee(E) = 57.9%, respectively). Compared with the two other acids, TA is practically unsuitable for enantiomer separation (ee(E) < 5%). Applying a mixture of one individually active and one ineffective acid in half the equivalent molar ratio, when the acids are in 1:1 ratio in the mixture, the resolution efficiency values obtained exceeded those obtained by using the components individually. Decreasing the molar ratio of resolving agent mixture to 0.25, at which the individual resolving agents give the best chiral separation, the obtained resolution efficiency values did not differ significantly from those expected. The outcome of the resolution process depended only on the amount of the individually active resolving agents in the mixture.     

Optical resolution of 6-fluoro-2-methyl-1,2,3,4-tetrahydroquinoline by supercritical fluid extraction

6-Fluoro-2-methyl-1,2,3,4-tetrahydroquinoline (FTHQ) enantiomers were separated by supercritical fluid extraction using carbon dioxide. Diastereoisomeric salts were formed from the racemic base with less than one equivalent of O,O'-di-(4-toluoyl)-(2R,3R)-tartaric acid (DPTTA). Further purification was achieved by partial salt formation of the enantiomeric mixture with an achiral acid (HCl) followed by the supercritical fluid extraction of the free enantiomers.     

Cyclic resolution of racemic ibuprofen via coupled efficient lipase and acid-base catalysis

Extracellular lipase LIP prepared in our lab from the yeast Yarrowia lipolytica was used for the resolution of racemic ibuprofen. The (S)-enantiomer was preferred by lipase LIP, and the unreacted (R)-enantiomer was extracted and racemized in basic solvent-water medium to be re-resolved. Solvent, content of solvent, base concentration, and temperature have a strong effect on racemization. The (S)-ester was separated and hydrolyzed to (S)-ibuprofen in acidic dimethyl sulfoxide-water mixture containing 70% dimethyl sulfoxide. The high purity (S)-ibuprofen (ee = 0.98) was obtained using lipase LIP to catalyze hydrolysis of (S)-ester in 0.1 M phosphate buffer (pH = 8).     

Biodegradation of the chlorophenoxy herbicide (R)-(+)-mecoprop by Alcaligenes denitrificans

An Alcaligenes denitrificans strain capable of utilizing theherbicide (R)-(+)-2(2-methyl-4-chlorophenoxy)propionicacid (mecoprop) as a sole carbon source was isolated fromsoil and cultured in liquid medium. Crude cell extracts of thebacterium were utilized in spectrophotometric assays toelucidate a biochemical pathway for degradation ofmecoprop. Results indicated a reaction sequence analogousto the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D).GC-MS analysis provided direct evidence for thebiotransformation of mecoprop to the transient metabolite4-chloro-2-methylphenol (MCP). No NADPH-dependentactivity was observed during this reaction. Pyruvate wasverified as the second product derived from the aliphatic sidechain of mecoprop. MCP was subsequently transformed to asubstituted catechol by an NADPH-dependentmonooxygenase. When grown on mecoprop, A.denitrificans was adapted to oxidize catechol and its 4- and3-methylated derivatives indicating the broad substratespecificity of catechol dioxygenase. The microorganism wasdemonstrated to adopt the ortho mechanism of aromaticcleavage which resulted in the formation of2-methyl-4-carboxymethylene but-2-en-4-olide, a reactionintermediate of the -ketoadipate pathway.     

Selective separation of cis-trans geometrical isomers of beta-carotene via CO2 supercritical fluid extraction

We investigated a novel method for the selective separation of beta-carotene isomers from a freeze-dried powder of the algae Dunaliella bardawil using supercritical fluid extraction. The separation method relies on the different dissolution rate of the 9Z and all-E isomers of beta-carotene in SC-CO(2). At first, the equilibrium solubility of the two isomers in SC-CO(2) was determined at the extraction conditions of 44.8 MPa and 40 degrees C. The solubility of the 9Z isomer was found to be nearly 4 times higher than that of the all-E isomer (1.92 x 10(-5) g all-E isomer/g CO(2) compared to 7.64 x 10(-5) g 9Z isomer/g CO(2)). When supercritical fluid extraction was applied to a carotenoid concentrate from the algae (29 wt% beta-carotene) or a freeze-dried powder of the algae (3.1% beta-carotene), a selective separation of the 9Z/all-E isomers of beta-carotene was obtained. Thirty-nine percent recovery of beta-carotene with 80% purity of 9Z isomer was achieved at the initial stages of extraction (40 mL CO(2)). The extraction rate of beta-carotene from the freeze-dried algae powder was slower than that from the carotenoid concentrate, resulting in a reduction in the recovery and purity of the 9Z isomer. This indicates that even at the initial stage of the extraction the internal mass resistance is significant. Isomer purity and recovery could be enhanced upon grinding of the algae powder.     

Chiral resolution of some antimalarial agents by sub- and supercritical fluid chromatography on an (S)-naphthylurea stationary phase

The behavior of mefloquine, halofantrine, enpiroline, quinine, quinidine, chloroquine and primaquine is studied by subcritical fluid chromatography on a (S)-naphthylurea column (250 mm × 4.6 mm ID) with a subcritical mobile phase composed of carbon dioxide, methanol and triethylamine (flow rate of 3 ml/min). Except for primaquine and chloroquine, each enantiomer was separated at a temperature between 40 and 60°C, and at a pressure below 15 MPa. A 98/2, v/v CO₂/methanol 0.1% triethylamine mixture allowed the separation of halofantrine enantiomers while the enantiomers of the more polar metabolite (N-desbutylhalofantrine) were separated with a 80–20 v/v mixture as used for mefloquine, enpiroline, quinine and quinidine. The influence of temperature, pressure and of the nature of the mobile phase is discussed. © 1993 Wiley-Liss, Inc.     

Resolution of 2-chloromandelic acid with (R)-(+)-N-benzyl-1-phenylethylamine: chiral discrimination mechanism

During the resolution of 2-chloromandelic acid with (R)-(+)-N-benzyl-1-phenylethylamine, the crystals of the less soluble salt were grown, and their structure were determined and presented. The chiral discrimination mechanism was investigated by examining the weak intermolecular interactions (such as hydrogen bond, CH/π, and van der Waals interactions) and molecular packing mode in crystal structure of the less soluble diastereomeric salt. A one-dimensional double-chain hydrogen-bonding network and a "lock-and-key" supramolecular packing mode are disclosed. The investigation demonstrates that hydrophobic layers with corrugated surfaces can fit into the grooves of one another to realize a compact packing, when the molecular structure of resolving agent is much larger than that of the racemate. This "lock-and-key" assembly is recognized to be another characteristic of molecular packing contributing to the chiral discrimination, in addition to the well-known sandwich-like packing by hydrophobic layers with planar boundary surfaces.     

Synthesis and polymerization of benzyl (3R,4R)-3-methylmalolactonate via enzymatic preparation of the chiral precursor

β-methylaspartate ammonia-lyase, EC 4.3.1.2, (β-methylaspartase) from Clostridium tetanomorphum was used to produce a 40/60 molar ratio of (2S,3R) and (2S,3S)-3-methylaspartic acids, 2a and 2b , respectively, from mesaconic acid 1 as substrate, on a large scale. To prepare (3R,4R)-3-methyl-4-(benzyloxycarbonyl)-2-oxetanone (benzyl 3-methylmalolactonate) 6, 2a and 2b were transformed, in the first step, into 2-bromo-3-methylsuccinic acids 3a and 3b and separated. After three further steps, (2S,3S)- 3a yielded the α,β-substituted β-lactone (3R,4R) 6 with a very high diastereoisomeric excess (>95% by chiral gas chromatography). The corresponding crystalline polymer, poly[benzyl β-(2R,3S)-3-methylmalate] 8 , prepared by an anionic ring opening polymerization, was highly isotactic as determined by ¹³C NMR. Catalytic hydrogenolysis of lactone 6 yielded (3R,4R)-3-methyl-4-carboxy-2-oxetanone (3-methylmalolactonic acid) 7 , to which reactive, chiral, or bioactive molecules can be attached through ester bonds leading to polymers with possible therapeutic applications. Because of the ability of β-methylaspartase to catalyse both syn- and anti-elimination of ammonia from (2S,3RS)-3-methylaspartic acid 2ab at different rates, the (2S,3R)-stereoisomer 2a was retained and isolated for further reactions. These results permit the use of the chemoenzymatic route for the preparation of both optically active and racemic polymers of 3-methylmalic acid with well-defined enantiomeric and diastereoisomeric compositions. Chirality 10:727–733, 1998. © 1998 Wiley-Liss, Inc.     

Optical resolution of 1-(1-naphthyl)ethylamine by its dicarboxylic acid derivatives: Structural features of the oxalic acid derivative diastereomeric salt pair

Optical resolution methods were established for racemic 1-(1-naphthyl) ethylamine. The resolving agents were synthesized by N-derivatizing (R)-1-(1-naphthyl) ethylamine with dicarboxylic acids. Oxalic, malonic, and succinic acid derivatives were found to be suitable resolving agents. These resolutions are parallel to a series of optical resolutions of 1-phenylethylamine which had been previously performed by our research group using similar derivative resolving agents (Balint et al., Tetrahedron: Asymmetry 2001;12:1511-1518.) The comparison of the results of the enantiomer separations is performed. The diastereomeric salts formed with (R)-N-[1-(1-naphthyl)ethyl]oxalamic acid were investigated by single crystal X-ray diffraction. The crystal structures were compared with the previously published structures of the diastereomers of the phenyl-substituted analogue, namely (R)- and (S)-1-phenylethylammonium (R)-N-(1-phenylethyl)oxalamates (Balint et al., Tetrahedron: Asymmetry 2001;12:1511-1518).     

Comparative toxicity of (+)-(R)- and (−)-(S)-1,2-dibromo-3-chloropropane

The haloalkane 1,2-dibromo-3-chloropropane (DBCP), an environmental pollutant that was widely used as a soil fumigant, is a carcinogen and a mutagen and displays target-organ toxicity to the testes and the kidneys. Because little is known about effects of stereochemistry on the metabolism and toxicity of halogenated alkyl compounds and because DBCP, which has a chiral center at C-2, may show enantioselectivity in its metabolism and/or toxicities, the optically pure enantiomers of DBCP were tested in vivo in rats for organ toxicity as well as for bacterial mutagenicity. Organ toxicity studies showed that (S)-DBCP was slightly more renal toxic than (R)-DBCP but was not significantly more toxic than the racemate, and that no significant differences were observed in the extents of testicular necrosis and atrophy caused by either enantiomer or the racemate. In contrast, (R)-DBCP was more mutagenic than either (S)-DBCP or the racemate to Salmonella typhimurium (S. typhimurium) strains TA 100 and TA104. However, there was little or no enantioselectivity in glutathione S-transferase (GST)-catalyzed conjugation reactions of glutathione with DBCP based on the lack of selectivity in the rates of disappearance of the enantiomers of DBCP in the presence of glutathione (GSH) and GSTs as monitored by chiral gas chromatography (GC). © 1995 Wiley-Liss, Inc.     

Experiment and simulation on kinetic resolution of (R,S)-2-chloromandelic acid by enzymatic transesterification

Optically pure 2-chloromandelic acid (ClMA) is a very important chiral drug intermediate for synthesis of (S)-clopidogrel, belonging to the platelet aggregation inhibitor. Enantioselective resolution of (R,S)-2-chloromandelic acid was carried out in organic solvent through irreversible transesterification catalyzed by lipase AK with vinyl acetate acting as the acyl donor. Effects of various conditions on enantioselectivity and activity of lipase were investigated, including organic solvents, temperature, water content, substrate ratio, enzyme loading, and reaction time. Based on homogeneous reaction and Ping-Pong bi-bi mechanism, a quantitative model was constructed to simulate and optimize the reaction process. Under the optimal conditions, excellent results were obtained with high conversion of (R)-2-ClMA (c _R, ≥98.85%) and large enantiomeric excess of substrate (ee _s, ≥98.15%). There is a good agreement between predicted values and experiment data, which indicates that the established method is a powerful tool for optimization of the enantioselective transesterification process for enantiomers separation.     

Effects of pressure and temperature of supercritical carbon dioxide on the extraction of triacylglycerols from plant tissue

The rate of extraction of triacylglycerols with supercritical carbon dioxide can be greatly enhanced by raising the pressure of the fluid to 600 bar, or higher, and its temperature to 60°C, or higher. Both the amount of carbon dioxide and the time required for complete extraction are reduced at such high pressures and temperatures.     

Identification and Characterization of a Mycobacterial (2R,3R)-2,3-Butanediol Dehydrogenase

Bacterial strain B-009, capable of using racemic 1,2-propanediol (PD), was identified as a rapid-growing member of the genus Mycobacterium. The strain is phylogenetically related to M. gilvum, but has slightly different physiological characteristics. An NAD⁺-dependent enantioselective alcohol dehydrogenase, which acts on R-PD, was purified from the strain. The enzyme was a homodimer of a peptide coded by a 1047-bp gene (mbd1). A highly conserved sequence for medium-chain dehydrogenase/reductases with a preference for secondary alcohols was found in the gene. Hydroxyacetone was produced from R-PD by an enzymatic reaction, indicating that position 2 of the substrate was oxidized. The enzyme activity was highest for (2R,3R)-2,3-butanediol (R,R-BD), enabling the enzyme to be identified as (2R,3R)-2,3-butanediol dehydrogenase (R,R-BD-DH). A homology search revealed M. gilvum, M. vanbaalenii, and M. semegmatis to have ORFs similar to mbd1, suggesting the widespread distribution of genes encoding R,R-BD-DH among mycobacterial strains.     

Application of chiralcel OJ in supercritical fluid chromatography for the resolution of different groups of frequently used drug racemates

Supercritical fluid chromatography was applied to evaluate the direct chiral discriminative properties of Chiralcel OJ, a tris(4-methylbenzoate) cellulose column towards frequently administered drugs. Two groups of acidic drugs, profen and barbiturate derivatives, and a few basic drugs of the benzodiazepine type were analysed. The effect on enantioselectivity of carbon dioxide when using methanol are acetonitrile as primary modifier was studied. Acetonitrile proved to be a good alternative for methanol, especially for the profen compounds that were not well resolved using methanol. The results were compared to normal phase chromatographic applications on the same column. SFC was not necessarily superior to high-performance liquid chromatography. Chirality 9:126–132, 1997. © 1997 Wiley-Liss, Inc.     

Pharmacokinetic data of propranolol enantiomers in a comparative human study with (S)- and (R,S)-propranolol

The pharmacokinetics of (S)-propranolol were compared after the oral administration of a 40 mg dose of the pure enantiomer and an 80 mg dose of a racemic mixture of (R,S)-propranolol. The results of this study indicate that the bioavailability of (S)-propranolol, as expressed by the mean area under the concentration-time curve (AUC) and maximum serum concentration, is lower after 40 mg of the optically pure drug than after the racemic drug.     

Biotransformation of the fungistatic compound (R)-(+)-1-(4′-chlorophenyl)propan-1-ol by Botrytis cinerea

The biotransformation of the fungistatic agent (R)-(+)-1-(4′-chlorophenyl)propan-1-ol (1) by the phytopathogen Botrytis cinerea has been studied. The main reaction pathways involved hydroxylations on several positions as well as condensations with secondary metabolites of the fungus. The antifungal activity of compound 1 against B. cinerea has also been determined.     

猴头菌子实体超临界流体技术萃取活性物质初探

以猴头菌子实体为原料,萃取物得率作为指标,采用CO₂超临界流体萃取技术,以萃取压力和CO₂流量等参数为考察因素,结合正交试验获得优化的萃取工艺：萃取压力为30MPa,温度为45℃,时间为1.5h,CO₂流量为20g/min,夹带剂（乙醇）与猴头菌子实体的物料比为5:1（mL:g）,在此条件下,萃取物得率为2.78%。与猴头菌子实体的醇提物相比,猴头菌子实体超临界萃取物具有更好的体外抗氧化能力和抗肿瘤活性,本研究结果为合理地开发和利用猴头菌子实体超临界萃取物产品提供科学的数据。     

Synthesis of (R)-2-trimethylsilyl-2-hydroxyl-propionitrile catalyzed by (R)-hydroxynitrile lyase from Prunus japonica seed meal

Synthesis of (R)-2-trimethylsilyl-2-hydroxyl-propionitrile via asymmetric transcyanation of acetyltrimethylsilane with acetone cyanohydrin in an aqueous/organic biphasic system catalyzed by (R)-hydroxynitrile lyase from Prunus japonica seed meal was successfully carried out for the first time. The optimal volume ratio of aqueous to organic phase, buffer pH value and reaction temperature were 15% (v/v), 5.0 and 30°C, respectively, under which both substrate conversion and product enantiomeric excess (ee) were 99%. Silicon atom in the substrate showed great effect on the reaction. Acetyltrimethylsilane was a much better substrate for (R)-hydroxynitrile lyase from Prunus japonica than its carbon analogue.     

Synthesis of (R)-2-trimethylsilyl-2-hydroxyl-propionitrile catalyzed by (R)-hydroxynitrile lyase from Prunus japonica seed meal

Synthesis of (R)-2-trimethylsilyl-2-hydroxyl-propionitrile via asymmetric transcyanation of acetyltrimethylsilane with acetone cyanohydrin in an aqueous/organic biphasic system catalyzed by (R)-hydroxynitrile lyase from Prunus japonica seed meal was successfully carried out for the first time. The optimal volume ratio of aqueous to organic phase, buffer pH value and reaction temperature were 15% (v/v), 5.0 and 30°C, respectively, under which both substrate conversion and product enantiomeric excess (ee) were 99%. Silicon atom in the substrate showed great effect on the reaction. Acetyltrimethylsilane was a much better substrate for (R)-hydroxynitrile lyase from Prunus japonica than its carbon analogue.     

羰基还原酶产生菌Candida ontarioensis制备(R)-2-氯-1-(3-氯苯基)乙醇

从实验室保藏的菌株中筛选获得Candida sp.PT2A,并通过18S rRNA鉴定为安大略假单胞菌Candida on-tarioensis。对C.ontarioensis不对称还原合成(R)-2-氯-1-(3-氯苯基)乙醇的发酵产酶条件和转化条件进行优化,确定了最适的发酵产酶条件和转化条件:温度30℃,初始pH 6.5,摇床转速180 r/min,菌体质量浓度200 g/L。采用2-氯-1-(3-氯苯基)乙酮质量浓度为10 g/L时,还原反应72 h,(R)-2-氯-1-(3-氯苯基)乙醇的e.e.值为99.9%,产率为99%;底物质量浓度提高至30 g/L时,产率下降为84.3%。采用十六烷基三甲基溴化铵(CTAB)对C.ontarioensis细胞进行通透性处理(CTAB g/L,4℃下处理20 min),在30 g/L底物下反应24 h,产物的e.e.和产率分别达到99.9%和97.5%。