Regioselective enzymatic aminoacylation of lobucavir to give an intermediate for lobucavir prodrug

Synthesis of lobucavir prodrug, L-valine, [(1S,2R,3R)-3-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (BMS 233866), requires regioselective coupling of one of the two hydroxyl groups of lobucavir (BMS 180194) with valine. Either hydroxyl group of lobucavir could be selectively aminoacylated with valine by using enzymatic reactions. N-[(Phenylmethoxy)carbonyl]-L-valine, [(1R,2R,4S)-2-(2-amino-6-oxo-1H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester (3, 82.5% yield), was obtained by selective hydrolysis of N,N′-bis[(phenylmethoxy)carbonyl]bis[L-valine], O,O′-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester (1) with lipase M, and L-valine, [(1R,2R,4S)-2-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (4, 87% yield) was obtained by hydrolysis of bis[L-valine], O,O′-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester, dihydrochloride (2), with lipase from Candida cylindracea. The final intermediate for lobucavir prodrug, N-[(phenylmethoxy)carbonyl]-L-valine, [(1S,2R,4R)-3-(2-amino-6-oxo-1H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester (5), could be obtained by transesterification of lobucavir using ChiroCLEC™ BL (61% yield), or more selectively by using immobilized lipase from Pseudomonas cepacia (84% yield).     

Biophysical characterization of double-stranded oligonucleotides using ETBR and isothermal fluorescence spectroscopy: implication for SNP genotyping

The UV-absorption, fluorescence and CD spectra of aps 23 bp oligoduplexes were performed for potential diagnostic purpose. These oligonucleotide sequences were mimicked from natural mutations (mitochondrial genome) of human population (unpublished). This work was designed on the basis of hybridization of non-self complementary oligoduplexes (aps) containing no mismatch, one-mismatch and two-mismatches. Since melting temperature™ is dependent on concentration of the oligoduplex, various concentrations were used in this study protocol. The thermal spectra profiles (UV absorbance and fluorescence) of these oligoduplexes (aps) are different for a particular concentration, and can be implicated for mutations. − dF/dT (or dA/dT) vs T, lnK (or RlnK) vs T_M, ΔG vs T_M, ΔS vs T_M and ΔH vs T_M are also variable for those sequences. All these thermodynamic data were calculated from absorbance (at 260 nm) data. On the contrary to the 23 bp oligoduplexes (aps), the PCR products of 97 bp and 256 bp length were genotyped with ETBR (excitation 530 nm, emission 600 nm) fluorimetrically. But our attempts to genotype these PCR sequences with isothermal UV absorbance spectroscopy were unsuccessful. Isothermal UV absorbance spectra has a limitation of sequence length. However, the structural conformation (all B-type) of the oligoduplexes (aps) was determined using CD. The minor discrepancy in CD spectra of these oligoduplexes are not significant for mutational analysis. 97 bp nested PCR product was an amplicon having either GcT or AcC mutation of mitochondria of normal human population, whereas 256 bp PCR product was an amplicon of human BRCA2 gene (NCBI Accession No. AY151039) of chromosome 13 having either A or G mutation at position − 26.     

遮荫对七子花幼苗光合特性和非结构性碳水化合物含量的影响

为探讨不同光环境下濒危植物七子花(Heptacodium miconioides)的生长及适应规律,以2年生七子花幼苗为研究对象,设置全光照、60%遮荫和90%遮荫3种光照强度,研究不同光环境对七子花幼苗的光合特性和非结构性碳水化合物(NSC)含量的影响。结果表明：(1)3种光照处理下,七子花幼苗的净光合速率(P_n)日变化均呈“单峰型”曲线,P_n日均值为全光照>60%遮荫>90%遮荫,90%遮荫下水分利用效率(W_UE)日均值显著低于全光照和60%遮荫。(2)与全光照相比,60%遮荫下七子花幼苗的光补偿点(L_CP)和暗呼吸速率(R_d)显著下降,而光饱和点(L_SP)、最大净光合速率(P_nmax)、最大羧化速率(V_cmax)、最大电子传递速率(J_max)和磷酸丙糖利用率(T_PU)无显著差异;重度遮荫(90%遮荫)导致植株的P_nmax、L     

A novel resolution of a pharmaceutically important bridged bicyclic ketone intermediate via selective enzymatic reduction with a commercially available ketoreductase

An efficient route to the pharmaceutically important (6S,9R)-11-oxo-5,6,7,8,9,10-hexahydro-6,9-methanobenzocyclooctene intermediate has been demonstrated via kinetic resolution of 11-oxo-5,6,7,8,9,10-hexahydro-6,9-methanobenzocyclooctene using a commercially available ketoreductase. Biocatalytics KRED 101 has been shown to selectively reduce the (6R,9S) enantiomer leaving behind the desired (6S,9R) enantiomer. This novel reaction is the first demonstration of a high yielding (44% versus 50% maximum theoretical yield) highly stereoselective (>99% ee) resolution of a bicyclic ketone, via enzymatic reduction using a commercially available ketoreductase, where the stereochemistry of the substrate is determined by a bridged ring system. Several challenges were overcome, including enhancing the selectivity of the enzyme by controlling temperature and increasing substrate solubility by employing a combination of cyclodextrin and organic co-solvent in the aqueous reaction system.     

下调Fsp27基因表达联合杨梅素干预对3T3-L1细胞脂解的影响

目的:下调脂肪特异性蛋白27(Fsp27)基因表达联合杨梅素干预,观察对3T3-L1细胞中脂质代谢的影响,并探究脂滴发生、发展变化的调控机制。方法:常规培养3T3-L1前脂肪细胞,采用"鸡尾酒"法诱导其分化为成熟脂肪细胞。脂质体法转染sh-Fsp27干扰载体,以杨梅素浓度为100μmol/L的完全培养基干预成熟脂肪细胞72h。油红O染色,观察脂滴形态及大小的变化;酶法测定细胞内甘油及甘油三酯的含量,观察细胞脂质代谢的变化。Western blot检测Fsp27、激素敏感性甘油三酯脂肪酶(HSL)、甘油三酯脂肪酶(ATGL)以及丝裂原活化蛋白激酶(MAPK)信号通路蛋白的表达。结果:1. 3T3-L1细胞诱导分化后,形态由纤维样变成圆形,并伴随有细胞体积的增大。2.与对照组相比,杨梅素组和转染组细胞中甘油三酯含量下降,甘油含量升高(P 0. 05)。与其他三组相比,联合干预组细胞中甘油三酯含量减少,甘油含量增加(P 0. 05)。3.与对照组相比,其余三组细胞内Fsp27蛋白的表达量均降低,ATGL和PPARγ的表达量升高(P 0. 05)。另外,联合干预组和杨梅素组细胞内HSL的表达量和p-p38MAPK/p38MAPK的比值均大于sh-Fsp27组和对照组(P 0. 05)。结论:1. Fsp27基因沉默与杨梅素联合干预可以更大程度地促进脂肪分解代谢。2.杨梅素可通过激活MAPK信号通路,上调HSL和ATGL的蛋白表达来发挥其促脂解的作用; sh-Fsp27干扰载体通过调节PPARγ和Fsp27蛋白的表达,增加ATGL含量来加速脂肪分解。     

Hormone-sensitive lipase: control of intracellular tri-(di-)acylglycerol and cholesteryl ester hydrolysis

Hormone-sensitive lipase (HSL) is an intracellular neutral lipase that is capable of hydrolyzing triacylglycerols, diacylglycerols, monoacylglycerols, and cholesteryl esters, as well as other lipid and water soluble substrates. HSL activity is regulated post-translationally by phosphorylation and also by pretranslational mechanisms. The enzyme is highly expressed in adipose tissue and steroidogenic tissues, with lower amounts expressed in cardiac and skeletal muscle, macrophages, and islets. Studies of the structure of HSL have identified several amino acids and regions of the molecule that are critical for enzymatic activity and regulation of HSL. This has led to important insights into its function, including the interaction of HSL with other intracellular proteins, such as adipocyte lipid binding protein. Accumulating evidence has defined important functions for HSL in normal physiology, affecting adipocyte lipolysis, steroidogenesis, spermatogenesis, and perhaps insulin secretion and insulin action; however, direct links between abnormal expression or genetic variations of HSL and human disorders, such as obesity, insulin resistance, type 2 diabetes, and hyperlipidemia, await further clarification. The published reports examining the regulation, and function of HSL in normal physiology and disease are reviewed in this paper.     

Catalytic properties of cytochrome P-450scc from bovine and porcine adrenocortical mitochondria: effect of Tween20 concentration

Cholesterol side-chain cleavage activities of cytochrome P-450ssc purified from bovine adrenocortical mitochondria were measured for various substrates, including cholesterol, 20[S]-hydroxycholesterol, 22[R]-hydroxycholesterol and 20[R], [R]-dihydroxycholesterol, in the reconstituted enzyme system at various Tween20 concentrations. The side-chain cleavage activity for cholesterol showed more than 10-fold enhancement upon addition of 0.1% Tween20, compared with that without the detergent. Addition of Tween20 did not cause any enhancement of the side-chain cleavage activities for 20[S]-hydroxycholesterol and 22[R]-hydroxycholesterol; rather, it resulted in an inhibition of the activities. The side-chain cleavage activity for 20[R],22[R]-dihydroxycholesterol showed a very high value even without the detergent. As the stimulatory effect of Tween20 was only specific for cholesterol, Tween20 seemed to enhance the rate of access of cholesterol to cytochrome P-450scc. These results are consistent with the suggestion that a transfer of substrate, cholesterol, in mitochondrial inner membrane, to the substrate-binding site of cytochrome P-450scc is the rate-limiting step in the cholesterol side-chain cleavage reaction.     

Synthesis and HMG CoA reductase inhibition of 4-thiophenyl quinolines as potential hypocholesterolemic agents

A series of novel 4-thiophenyl quinoline-based mevalonolactone derivatives were synthesized from ethyl 6,7,8-trisubstituted-4-chloro-quinoline-3-carboxylates by several reactions and evaluated for their ability to inhibit the rat HMG CoA reductase in vitro. It was found that substitution with a variety of thiophenyl groups at position 4 in quinoline resulted in retention or enhancement of the inhibition and the preferable groups were 4-isopropyl-thiophenyl and 3-methoxy-thiophenyl. (4R,6S)-6-[(E)-2-(6,7,8-trifluoro-4-isopropylthiophenyl-quinoline-3-yl)-ethenyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (A16) and (4R, 6S)-6-[(E)-2-(6-fluoro-4,7-di-(3-methoxy-thiophenyl)-quinoline-3-yl)-ethenyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (A23) were approximately three times more potent than rosuvastatin or pitavastatin in inhibiting HMG CoA reductase and selected as the hypocholesterolemic candidates for further evaluation.     

Novel antioxidant agents deriving from molecular combinations of vitamins C and E analogues: 3,4-dihydroxy-5(R)

Molecular combinations of two antioxidants (i.e., ascorbic acid and the pharmacophore of -tocopherol), namely the 2,3-dihydroxy-2,3-enono-1,4-lactone and the chromane residues, have been designed and tested for their radical scavenging activities. When evaluated for their capability to inhibit malondialdehyde (MDA) production in rat liver microsomal membranes, the 3,4-dihydroxy-5R-2(R,S)-(6-hydroxy-2,5,7,8-tetramethylchroman-2(R,S)yl-methyl)-1,3]dioxolan-4S-yl]-5H-furan-2-one (11a–d), exhibited an interesting activity. In particular the 5R,2R,2R,4S and 5R,2R,2S,4S isomers (11c,d) displayed a potent antioxidant effect compared to the respective synthetic -tocopherol analogue (5) and natural -tocopherol or ascorbic acid, used alone or in combination. Moreover, the mixture of stereoisomers 11a–d also proved to be effective in preventing damage induced by reperfusion on isolated rabbit heart, in particular at the higher concentration of 300 μM. In view of these results our study represents a new approach to potential therapeutic agents for applications in pathological events in which a free radical damage is involved. Design, synthesis and preliminary biological activity are discussed.     

Oxidation of 1,4-alkanediols into γ-lactones via γ-lactols using Rhodococcus erythropolis as biocatalyst

Whole cells of Rhodococcus erythropolis DSM 44534 grown on ethanol, (R)- and (S)-1,2-propanediol were used for biotransformation of racemic 1,4-alkanediols into γ-lactones. The cells oxidized 1,4-decanediol (1a) and 1,4-nonanediol (2a) into the corresponding γ-lactones 5-hexyl-dihydro-2(3H)-furanone (γ-decalactone, 1c) and 5-pentyl-dihydro-2(3H)-furanone (γ-nonalactone, 2c), respectively, with an EE(R) of 40–75%. The transient formation of the γ-lactols 5-hexyl-tetrahydro-2-furanol (γ-decalactol, 1b) and 5-pentyl-tetrahydro-2-furanol (γ-nonalactol, 2b) as intermediates was observed by GC–MS. 1,4-Pentanediol (3a) was transformed into 5-methyl-dihydro-2(3H)-furanone (γ-valerolactone, 3c) whereas (R)- and (S)-2-methyl-1,4-butanediol (4a) was converted to the methyl-substituted γ-butyrolactones 4-methyl-dihydro-2(3H)-furanone (4c₁) and 3-methyl-dihydro-2(3H)-furanone (4c₂) in a ratio of 80:20 with a yield of 55%. Also cis-2-buten-1,4-diol (5a) was transformed resulting in the formation of 2(5H)-furanone (γ-crotonolactone, 5c). At the higher pH values of 8.8 the yield of lactone formed was improved; however, the enatiomeric excesses were slightly higher at the lower pH of 5.2.     

Kinetic resolution of a diltiazem intermediate by lipase-catalyzed enantioselective alcoholysis

A novel enzymatic resolution of an important alcohol intermediate in the Diltiazem process was developed. The enzymatic reaction involved alcoholysis of the alcohol acetate with butanol, thus obtaining the (R,R)-alcohol and the remaining (S,S)-acetoxy-alcohol in >95% enantiomeric excess. This resolution may serve as the key step in a possible recycling procedure for the waste streams of the Diltiazem process, which will allow a significant increase in the overall process yield.     

Direct synthesis of 6-oxabicyclo[3.2.1]octane derivatives from deoxyinososes

In the presence of bases, even those (for example, pyridine) normally used for acylation reactions, 2 -(2,4,5/3)-2,3,4,-tribenzoyloxy-5-hydroxycyclohexanone (3) readily gives (2 -(2,4/3)-2,3,4-tribenzoyloxycyclohex-5-enone or aromatic products. Under acid conditions, efficient O-acylation and tetrahydropyranylation can be effected. The main product (60% isolated) formed on treatment of 3 with diazomethane is (1R,2S,3R,4S,5S)-2,3,4-tribenzoyloxy-1-hydroxy-6-oxabicyclo [3.2.1]octane (15); small proportions of the epimeric spiro-epoxides and 1-acetyl-6-benzoyloxy-7-methoxy-1H-indazole are also formed. On photobromination, the acetate (17) of 15 undergoes substitution at C-7 and, from the product, C-formyl-deoxyinositol derivatives are produced.     

Reactive oxygen species facilitate translocation of hormone sensitive lipase to the lipid droplet during lipolysis in human differentiated adipocytes

In obesity, there is an increase in reactive oxygen species (ROS) within adipose tissue caused by increases in inflammation and overnutrition. Hormone sensitive lipase (HSL) is part of the canonical lipolytic pathway and critical for complete lipolysis. This study hypothesizes that ROS is a signal that integrates regulation of lipolysis by targeting HSL. Experiments were performed with human differentiated adipocytes from the subcutaneous depot. Antioxidants were employed as a tool to decrease ROS, and it was found that scavenging ROS with diphenyliodonium, N-acetyl cysteine, or resveratrol decreased lipolysis in adipocytes. HSL phosphorylation of a key serine residue, Ser552, as well as translocation of this enzyme from the cytosol to the lipid droplet upon lipolytic stimulation were both abrogated by scavenging ROS. The phosphorylation status of other serine residues on HSL were not affected. These findings are significant because they document that ROS contributes to the physiological regulation of lipolysis via an effect on translocation. Such regulation could be useful in developing new obesity therapies.     

Stereoselective synthesis of opine-type secondary amine car☐ylic acids by a new enzyme opine dehydrogenase use of recombinant enzymes

The substrate specificity of the recently discovered enzyme, opine dehydrogenase (ODH) fromArthrobacter sp. strain 1C for amino donors in the reaction that forms secondary amines using pyruvate as a fixed amino acceptor is examined. The enzyme was active toward short-chain aliphatic (S)-amino acids and those substituted with acyloxy, phosphonooxy, and halogen groups. The enzyme was named N-[1-(R)-(car☐yl)ethyl]-(S)-norvaline: NAD⁺ oxidoreductase (L-norvaline forming). Other substrates for the enzyme were 3-aminobutyric acid and (S)-phenylalaninol. Optically pure opine-type secondary amine car☐ylic acids were synthesized from amino acids and their analogs such as (S)-methionine, (S)-isoleucine, (S)-leucine, (S)-valine, (S)-phenylalanine, (S)-alanine, (S)-threonine, (S)-serine, and (S)-phenylalaninol, and -keto acids such as glyoxylate, pyruvate, and 2-oxobutyrate using the enzyme, with regeneration of NADH by formate dehydrogenase (FDH) fromMoraxella sp. C-1. The absolute configuration of the nascent asymmetric center of the opines was of the (R) stereochemistry with > 99.9% e.e. One-pot synthesis of N-[1-(R)-(car☐yl)ethyl]-(S)-phenylalanine from phenylpyruvate and pyruvate by using ODH, FDH, and phenylalanine dehydrogenase (PheDH) fromBacillus sphaericus, is also described.     

Comparative studies of the role of hormone-sensitive lipase and adipose triglyceride lipase in human fat cell lipolysis

Hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) regulate adipocyte lipolysis in rodents. The purpose of this study was to compare the roles of these lipases for lipolysis in human adipocytes. Subcutaneous adipose tissue was investigated. HSL and ATGL protein expression were related to lipolysis in isolated mature fat cells. ATGL or HSL were knocked down by RNA interference (RNAi) or selectively inhibited, and effects on lipolysis were studied in differentiated preadipocytes or adipocytes derived from human mesenchymal stem cells (hMSC). Subjects were all women. There were 12 lean controls, 8 lean with polycystic ovary syndrome (PCOS), and 27 otherwise healthy obese subjects. We found that norepinephrine-induced lipolysis was positively correlated with HSL protein levels (P < 0.0001) but not with ATGL protein. Women with PCOS or obesity had significantly decreased norepinephrine-induced lipolysis and HSL protein expression but no change in ATGL protein expression. HSL knock down by RNAi reduced basal and catecholamine-induced lipolysis. Knock down of ATGL decreased basal lipolysis but did not change catecholamine-stimulated lipolysis. Treatment of hMSC with a selective HSL inhibitor during and/or after differentiation in adipocytes reduced basal lipolysis by 50%, but stimulated lipolysis was inhibited completely. In contrast to findings in rodents, ATGL is of less importance than HSL in regulating catecholamine-induced lipolysis and cannot replace HSL when this enzyme is continuously inhibited. However, both lipases regulate basal lipolysis in human adipocytes. ATGL expression, unlike HSL, is not influenced by obesity or PCOS.     

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.     

Two novel prevalent polymorphisms in the hormone-sensitive lipase gene have no effect on insulin sensitivity of lipolysis and glucose disposal.

Free fatty acids released during triglyceride lipolysis play an important role in obesity-associated insulin resistance of glucose disposal. Individual sensitivity of lipolysis to the suppressive effect of insulin varies greatly among healthy subjects. It is possible that genetic factors contribute to this variation. Among the many proteins involved in the regulation of lipolysis, hormone-sensitive lipase (HSL) represents a prime candidate for genetic variants contributing to the biological variation of insulin sensitivity of lipolysis. We determined the insulin sensitivity of lipolysis (suppression of isotopically [primed-continuous infusion of d5 glycerol] measured glycerol rate of appearance) and of glucose disposal, using a three-step (n = 20) or standard (n = 53) hyperinsulinemic euglycemic clamp in 73 healthy, unrelated subjects. To assess the possible role of genetic polymorphisms, we directly sequenced the coding region of the HSL gene and the noncoding exon B from these subjects. We identified two silent mutations and three amino acid polymorphisms: Arg262Met (prevalence, 5%), Glu620Asp (prevalence, 31%) and Ser681Ile (prevalence, 22%). The latter two are located in the regulatory domain of HSL but neither had a significant impact on insulin sensitivity of lipolysis or glucose disposal (with and without adjustment for obesity and age as covariates; all P values > 0.20). We conclude that a number of genetic polymorphisms in HSL exist, some of which are highly prevalent. Neither of the polymorphisms we identified in the coding region, however, contributed measurably to the biological variation of insulin sensitivity in our lean, healthy population.     

Catalytic performance of a highly enantioselective (R)-ester hydrolase from a new isolate Acinetobacter sp. CGMCC 0789

A highly enantioselective (R)-ester hydrolase was partially purified from a newly isolated bacterium, Acinetobacter sp. CGMCC 0789, whose resting cells exhibited a highly enantioselective activity toward the acetate of (4R)-hydroxy-3-methyl-2-(2-propynyl)- cyclopent-2-enone (R-HMPC). The optimum pH and temperature of the partially purified enzyme were 8.0 and 60 °C, respectively. The enantioselectivity of the crude enzyme was increased by 1.2-fold from 16 to 20 when the reaction temperature was raised from 30 to 60 °C. The activity of the crude enzyme was enhanced by 4.1-fold and the enantioselectivity (E-value) was markedly enhanced by 4.3-fold from 16 to 68 upon addition of a cationic detergent, benzethonium chloride [(diisobutyl phenoxyethoxyethyl) dimethyl benzylammoniom chloride]. The hydrolysis of 52 mM (R,S)-HMPC acetate to (R)-HMPC was completed within 8 h, with optical purity of 91.4% ee_p and conversion of 49%.     

Fed-batch production of (S)-flurbiprofen in lipase-catalyzed dispersed aqueous phase reaction system induced by succinyl β-cyclodextrin and its extractive purification

Optically active (S)-flurbiprofen was produced fed-batch-wisely in a lipase-catalyzed dispersed aqueous phase reaction system induced by succinyl β-cyclodextrin (suβ-CD). A highly concentrated 480 mM (S)-flurbiprofen, corresponding to 117.0 g/l, with an enantiomeric excess of 0.98 and conversion yield of 0.48 was obtained. (S)-Flurbiprofen produced in an inclusion complex form with suβ-CD was extractively purified using three-step procedures: decomplexation of (S)-flurbiprofen and residual (R)-flurbiprofen ethyl ester ((R)-FEE) using the ethyl acetate, dissolution of (S)-flurbiprofen from (R)-FEE using a sodium bicarbonate solution, and selective precipitation of (S)-flurbiprofen using 2-propanol. Consequently, an extremely high concentration of 420 mM (S)-flurbiprofen with an optical purity higher than 98% was recovered after purification.