Resolution and synthesis of optically active alcohols with immobilized ovalbumin and pea protein as new bio-catalysts

It was found that ovalbumin stereoselectively oxidized one of the enantiomers of p-substituted racemic alcohols, thereby providing optically active alcohols with high optical purities. It was found out that, when used appropriately in combination with immobilized pea protein, immobilized ovalbumin made it possible to resolve and synthesize racemic 1-(2-naphthyl)ethanol, 1-phenylethanol, and 1-phenyl-1-propanol. Immobilized ovalbumin could be continuously recycled at least three times without lowering the yield and purity of the products. These results suggested that cereals, beans, and ovalbumin might have additional fourth function among conventional foods. Namely, there might contain nutritional, sensory, biologically regulatory and bio-catalytic functions in conventional foods.     

Enzymatic resolution of racemic amines in a continuous reactor in organic solvents

An enzymatic process has been developed for the continuous production of the pharmaceutically important intermediate (R)-1-aminoindan and of the chiral resolving agent (R)-1-(1-naphthyl)ethylamine. The process consists of the subtilisin catalyzed stereoselective aminolysis of the racemic primary amine with an active ester in organic solvent. The competing nonenzymatic reaction has been suppressed by appropriate choice of solvent and reactant's concentration and by minimizing the time of contact between the amine and the active ester. Subtilisin was immobilized on glass beads and the reaction carried out in a continuous-flow column bioreactor. By using a 450-mL column bioreactor containing 5.7 g of subtilisin immobilized on 570 g of glass beads, 1.6 kg of racemic 1-(1-naphthyl)ethylamine was resolved after 320 h of continuous operation with only a slight loss of the enzymatic activity. During the whole process, the optical purity of the chiral amine eluting from the column was higher than 90%. A facile procedure was developed for separating the unreacted (R)-amine from the (S)-amide and for the recycling of the solvent 3-methyl-3-pentanol and the active ester 2,2,2-trifluoroethyl butyrate. (c) 1992 John Wiley & Sons, Inc.     

Resolution and Synthesis of (S)-1-(2-Naphthyl)ethanol with Immobilized Pea Protein: As a New Biocatalyst

(S)-1-(2-Naphthyl)ethanol was yielded by immobilized pea (Pisum sativum L.) protein (IPP) from (R, S) 2-naphthyl ethanol (>99% ee, yield; about 50%), in which the (R)-enantiomer was selectively oxidized to 2-acetonaphthone. IPP could be reused consecutively at least three times without any decrease of yield and optical purity.     

MalphaNP acid, a powerful chiral molecular tool for preparation of enantiopure alcohols by resolution and determination of their absolute configurations by the (1)H NMR anisotropy method

A novel methodology using a chiral molecular tool of MalphaNP acid (1), 2-methoxy-2-(1-naphthyl)propionic acid, useful for preparation of enantiopure secondary alcohols and determination of their absolute configurations by the (1)H NMR anisotropy method was developed; racemic MalphaNP acid (1) was enantioresolved with (-)-menthol, and the enantiopure MalphaNP acid (S)-(+)-(1) obtained was allowed to react with racemic alcohol, yielding a mixture of diastereomeric esters, which was clearly separated by HPLC on silica gel. By applying the sector rule of (1)H NMR anisotropy effect, the absolute configuration of the first-eluted MalphaNP ester was unambiguously determined. Solvolysis or reduction of the first-eluted MalphaNP esters yielded enantiopure alcohols.     

Resolution and synthesis of optically active alcohols with immobilized water-soluble proteins from green pea, soybean and buckwheat as new bio-catalysts

Kinetic resolution of racemic alcohols, (+/-)-1-(4-substituted phenyl)ethanol and (+/-)-1-(2-naphthyl)ethanol, was done with immobilized green pea, soybean, or buckwheat proteins. The resolution was done stereoselectively by oxidizing only one enantiomer of a racemic alcohol to leave an optically active alcohol with a high purity. In addition, each protein could be reused consecutively at least three times without any decrease of yield or optical purity.     

Utility of ionic liquid for Geotrichum candidum-catalyzed synthesis of optically active alcohols

Ionic liquids have recognized as a solvent for Geotrichum candidum-catalyzed optical resolution and/or deracemization of racemic secondary alcohols, giving optically active alcohols. The immobilized Geotrichum candidum proceeded the enantioselective oxidation of alcohols, producing chiral alcohols in an ionic liquid. Further, deracemization of racemic alcohols was proceeded to give the corresponding chiral alcohols in high yield with excellent stereoselectivity by the Geotrichum candidum–NaBH₄ system in the mixture of MES buffer solution and ionic liquid.     

Convenient method for determining the absolute configuration of chiral alcohols with racemic 1H NMR anisotropy reagent,M(alpha)NP acid: use of HPLC-CD detector

A convenient method for determining the absolute configuration of chiral secondary alcohols using the racemic NMR anisotropy reagent, (+/-)-2-methoxy-2-(1-naphthyl)propionic acid [(+/-)-M(alpha)NP acid], and an HPLC-CD detector was developed. The method was successfully applied to some chiral alcohols derived from (-)-alpha-santonin.     

Easily recyclable chiral polymeric Mn(III) salen complexes for oxidative kinetic resolution of racemic secondary alcohols

Chiral polymeric Mn(III) salen complexes were used efficiently for oxidative kinetic resolution of racemic secondary alcohols at room temperature. High chiral purity (ee; >99%) was achieved for the oxidative kinetic resolution of racemic secondary alcohols with 0.6 mol % catalyst loading in 60 min. The catalyst was easily recycled for five successive catalytic experiments.     

Advances in asymmetric oxidative kinetic resolution of racemic secondary alcohols catalyzed by chiral Mn(III) salen complexes

Enantiomerically pure secondary alcohols are essential compounds in organic synthesis and are used as chiral auxiliaries and synthetic intermediates in the pharmaceutical, agrochemical, and fine chemical industries. One of the attractive and practical approaches to achieving optically pure secondary alcohols is oxidative kinetic resolution of racemic secondary alcohols using chiral Mn(III) salen complexes. In the last decade, several chiral Mn(III) salen complexes have been reported with excellent enantioselectivity and activity in the homogeneous and heterogeneous catalysis of the oxidative kinetic resolution of racemic secondary alcohols. This review article is an overview of the literature on the recent development of chiral Mn(III) salen complexes for oxidative kinetic resolution of racemic secondary alcohols. The catalytic activity of monomeric, dimeric, macrocyclic, polymeric, and silica/resin supported chiral Mn(III) salen complexes is discussed in detail.     

Enhancing cofactor recycling in the bioconversion of racemic alcohols to chiral amines with alcohol dehydrogenase and amine dehydrogenase by coupling cells and cell-free system

Alcohol dehydrogenase (ADH) and amine dehydrogenase (AmDH)-catalyzed one-pot cascade conversion of an alcohol to an amine provides a simple preparation of chiral amines. To enhance the cofactor recycling in this reaction, we report a new concept of coupling whole-cells with the cell-free system to enable separated intracellular and extracellular cofactor regeneration and recycling. This was demonstrated by the respective biotransformation of racemic 4-phenyl-2-butanol 1a and 1-phenyl-2-propanol 1b to (R)-4-phenylbutan-2-amine 3a and (R)-1-phenylpropan-2-amine 3b . Escherichia coli cells expressing S-enantioselective CpsADH, R-enantioselective PfODH, and NADH oxidase (NOX) was developed to oxidize racemic alcohols 1a–b to ketones 2a–b with full conversion via intracellular NAD⁺ recycling. AmDH and glucose dehydrogenase (GDH) were used to convert ketones 2a–b to amines (R)- 3a–b with 89–94% conversion and 891–943 times recycling of NADH. Combining the cells and enzymes for the cascade transformation of racemic alcohols 1a–b gave 70% and 48% conversion to the amines (R)- 3a and (R)-3 b in 99% ee, with a total turnover number (TTN) of 350 and 240 for NADH recycling, respectively. Improved results were obtained by using the E. coli cells with immobilized AmDH and GDH: (R)- 3a was produced in 99% ee with 71–84% conversion and a TTN of 1410-1260 for NADH recycling, the highest value so far for the ADH–AmDH-catalyzed cascade conversion of alcohols to amines. The concept might be generally applicable to this type of reactions.     

Lilac alcohol epoxide: a linalool derivative in Actinidia arguta flowers

Lilac alcohol epoxide (2-(5-methyl-5-(oxiran-2-yl)-tetrahydrofuran-2-yl)propan-1-ol), a previously unreported monoterpene, was identified in the solvent extract of the flowers of seven Actinidia arguta genotypes. The diastereomeric lilac alcohol epoxides co-occurred with the lilac aldehydes and alcohols. Another compound, the lilac diol (2-(5-(1-hydroxyethyl)-5-methyl-tetrahydrofuran-2-yl)propan-1-ol) was synthesised as part of our efforts to identify the lilac alcohol epoxide.     

Practical enantioresolution of alcohols with 2-methoxy-2-(1-naphthyl)propionic acid and determination of their absolute configurations by the (1)H NMR anisotropy method.

The enantioresolution of racemic alcohols as esters of 2-methoxy-2-(1-naphthyl)propionic acid (MalphaNP acid 1) and the determination of their absolute configurations on the basis of (1)H NMR anisotropy effect are described. The enantiopure MalphaNP acid (S)-(+)-1 was allowed to react with racemic 2-alkanols and 1-octyn-3-ol, yielding diastereomeric mixtures of esters, which were easily separated by HPLC on silica gel. To determine the absolute configurations of the first-eluted diastereomeric esters by the (1)H NMR anisotropy method, the general scheme was proposed. Separated esters were reduced with LiAlH(4) or hydrolyzed with KOH/EtOH to recover enantiopure alcohols.     

Experimental studies of competition between enantiomers in chiral liquid chromatography through their system peaks

Competition between the (+)- and (?) enantiomers of 2,2,2-trifluoro-1-(9-anthryl) ethanol as mobile phase additives was indicated by the chromatographic behavior of their system peaks. Two types of chiral stationary phases were used, one based on dinitrobenzoylphenylglycine and the other on dinitrobenzylphenylethylamine plus tartaric acid. The racemic mixture was used as the mobile phase additive and k′ of their system peaks was studied as a function of the mixture concentration in the mobile phase in both cases. A shift in k′ of the two system peaks was observed and considered as an indication that competition occurred. The areas of the two system peaks were also studied as a function of the concentration of the enantiomers in the samples, using two different compositions of the mobile phase. The dependency of system peaks' area on the sample composition indicated whether competition between the enantiomers occurred. One mobile phase contained 0.1 mM of the racemic mixture, where the area of the two retained system peaks behaved independently, i.e., only the peak corresponding to the enantiomer was affected by its presence in the sample. The other mobile phase contained 0.75 mM of the racemic mixture, and both peaks were affected by the injection of any one of the enantiomers. The interdependency of the system peaks' area on both the enantiomers indicated that their distribution in the chiral system was interrelated due to mutual interactions. A quantitative treatment of the interdependency and competition was excluded, due to the irreversible adsorption of the two enantiomers on the chiral stationary phase after using overloading concentrations. This irreversible adsorption was visualized by the appearance of two retained system peaks of the two residual enantiomers. These system peaks were detected only when the sample contained pure enantiomers due to competition between the enantiomer in the sample with the residual enantiomers in the stationary phase. © 1994 Wiley-Liss, Inc.     

Separation and quantitation of (R)- and (S)-atenolol in human plasma and urine using an alpha 1-AGP column

A method for the determination of (R)- and (S)-atenolol in human plasma and urine is described. The enantiomers of atenolol are extracted into dichloromethane containing 3% heptafluorobutanol followed by acetylation with acetic anhydride at 60 degrees C for 2 h. The acetylated enantiomers were separated on a chiral alpha 1-AGP column. Quantitation was performed using fluorescence detection. A phosphate buffer pH 7.1 (0.01 M phosphate) containing 0.25% (v/v) acetonitrile was used as mobile phase. The described procedure allows the detection of less than 6 ng of each enantiomer in 1 ml plasma. The relative standard deviation is 4.4% at 30 ng/ml of each enantiomer in plasma. The plasma concentration of (R)- and (S)-atenolol did not differ significantly in two subjects who received a single tablet of racemic atenolol. The R/S ratio of atenolol in urine was approximately 1.     

Kinetic resolution of 1-chloro-3-(1-naphthyloxy)-2-propanol,an intermediate in the synthesis of beta-adrenergic receptor blockers

(R)- and (S)-1-chloro-3-(1-naphthyloxy)-2-propanol are intermediates in the synthesis of β-adrenergic blocking agents and antihypertensive drugs such as propranolol and nadoxolol. Herein, improvement in the preparation of racemic 1-chloro-3-(1-naphthyloxy)-2-propanol generated from 1-naphthol and epichlorohydrin are reported. In addition, kinetic resolution studies have been conducted to obtain both (R) and (S)-1-chloro-3-(1-naphthyloxy)-2-propanol. These compounds were obtained in highly optically pure form by the stereoselective hydrolysis of its acyl derivatives using whole cell preparations containing enzymes from native sources. The results were compared with those obtained using commercial lipases.     

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.     

MiR-448-5p inhibits TGF-β1-induced epithelial-mesenchymal transition and pulmonary fibrosis by targeting Six1 in asthma

MicroRNAs (miRNAs) are small yet versatile gene tuners that regulate a variety of cellular processes, including cell growth and proliferation. The aim of this study was to explore how miR-448-5p affects airway remodeling and transforming growth factor-β1 (TGF-β1)-stimulated epithelial-mesenchymal transition (EMT) by targeting Sine oculis homeobox homolog 1 (Six1) in asthma. Asthmatic mice models with airway remodeling were induced with ovalbumin solution. MiRNA expression was evaluated using quantitative real-time polymerase chain reaction. Transfection studies of bronchial epithelial cells were performed to determine the target genes. A luciferase reporter assay system was applied to identify whether Six1 is a target gene of miR-448-5p. In the current study, we found that miR-448-5p was dramatically decreased in lung tissues of asthmatic mice and TGF-β1-stimulated bronchial epithelial cells. In addition, the decreased level of miR-448-5p was closely associated with the increased expression of Six1. Overexpression of miR-448-5p decreased Six1 expression and, in turn, suppressed TGF-β1-mediated EMT and fibrosis. Next, we predicted that Six1 was a potential target gene of miR-448-5p and demonstrated that miR-448-5p could directly target Six1. An SiRNA targeting Six1 was sufficient to suppress TGF-β1-induced EMT and fibrosis in 16HBE cells. Furthermore, the overexpression of Six1 partially reversed the protective effect of miR-448-5p on TGF-β1-mediated EMT and fibrosis in bronchial epithelial cells. Taken together, the miR-448-5p/TGF-β1/Six1 link may play roles in the progression of EMT and pulmonary fibrosis in asthma.     

Double enantioselective esterification of racemic acids and alcohols by lipase from Candida cylindracea

Summary Doubly enantioselective lipase-catalyzed esterification of racemic acids and alcohols was proceeded in n-hexane. The enantioselectivities of the lipase toward both of racemic substrates were affected reciprocally. It showed that the active site of the lipase was quite flexible.     

Lipase-catalyzed optical resolution of trifluoro(aryl)ethanols

Optical resolutions of racemic 2,2,2-trifluoro-1-(aryl)ethanols — (1-naphthyl), (2-naphthyl), (4-methylnaphthyl), (phenyl), (1-pyrenyl) — were achieved by lipase-catalyzed enantioselective acetylations with vinyl acetate as an acetyl donor in octane, and (S)-acetates and (R)-alcohols were obtained. Among the lipases tested, lipase from Pseudomonas aeruginosa (lipase LIP, Toyobo) showed good enantioselectivity for above ethanols. However, no acetylation occurred with sterically hindered alcohols — (9-phenanthryl), (9-anthryl), (2-methylnaphthyl), (2, 4, 6-trimethylphenyl) — by various lipases. The resolutions of the three alcohols were carried out by the enantioselective alcoholysis or hydrolysis of their chloroacetates by lipase LIP.     

Production of optically active esters and alcohols from racemic alcohols by lipase-catalyzed stereoselective transesterification in non-aqueous reaction system

Microbial lipase-catalyzed transesterification between vinyl acetate and (RS)-2-octanol or (RS)-1-phenylethanol was investigated in a reaction system without addition of aqueous or organic solvents. From a screening test with various lipases, it was found that the enzymes from Pseudomonas species could efficiently catalyze the reaction, and R-enantiomers of the racemic alcohols were preferentially esterified by them. Enantiomeric purities of the optically active alcohols (S) and esters (R) obtained from (RS)-1-phenylethanol by the stereoselective transesterification of these lipases were all more than 95%.