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.     

Biocatalytic synthesis of optically active tertiary alcohols

The enzymatic preparation of optically pure tertiary alcohols under sustainable conditions has received much attention. The conventional chemical synthesis of these valuable building blocks is still hampered by the use of harmful reagents such as heavy metal catalysts. Successful examples in biocatalysis used esterases, lipases, epoxide hydrolases, halohydrin dehalogenases, thiamine diphosphate-dependent enzymes, terpene cyclases, -acetylases, and -dehydratases. This mini-review provides an overview on recent developments in the discovery of new enzymes, their functional improvement by protein engineering, the design of chemoenzymatic routes leading to tertiary alcohols, and the discovery of entirely new biotransformations.     

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.     

Synthesis of optically active aminooxy alcohols

Racemic secondary alcohols with an N-protected oxyamino function in the β-position were prepared by a base-catalyzed epoxide ring opening with N-hydroxyphthalimide or acetone oxime. The enantiomers were separated with a good selectivity by a lipase-catalyzed acetylation of the racemates with vinyl acetate. The protecting group of the aminooxy alcohol was split off by a hydrochloric acid hydrolysis to yield the hydrochloride of one of the enantiomeric forms of the title compounds.     

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.     

Preparation of optically active β-Ketoesters via PLE-catalyzed Resolution

Substituted cyclopentane β-ketoesters can be prepared in enantiomerically pure form by PLE catalyzed hydrolysis of their racemic precursors. Possessing an asymmetric quaternary centre, these molecules represent usefull intermediates in natural product synthesis.     

A new approach to the synthesis of optically active alkylated adenine derivatives

A new synthesis of chiral acyclic nucleoside and nucleotide analogues starting from d(-)- or l(+)-riboses was proposed. Antiviral properties of the synthesized compounds towards the pox virus family were evaluated.     

Highly active mutants of carbonyl reductase S1 with inverted coenzyme specificity and production of optically active alcohols

A wild type NADPH-dependent carbonyl reductase from Candida magnoliae (reductase S1) has been found not to utilize NADH as a coenzyme. A mutation to exchange the coenzyme specificity in reductase S1 has been designed by computer-aided methods, including three-dimensional structure modeling and in silico screening of enzyme mutants. Site-directed mutagenesis has been used to introduce systematic substitutions of seven or eight amino acid residues onto the adenosine-binding pocket of the enzyme according to rational computational design. The resulting S1 mutants show NADH-dependency and have lost their ability to utilize NADPH as a coenzyme, but retain those catalytic activities. Kinetic parameter V(max) and K(m) values of those mutants for NADH are 1/3- to 1/10-fold those of the wild type enzyme for NADPH. As a model system for industrial production of optically active alcohols, the S1 mutants can be applied to an asymmetric reduction of ketones, cooperating with a coenzyme-regeneration system that uses an NAD-dependent formate dehydrogenase.     

Highly selective anti-Prelog synthesis of optically active aryl alcohols by recombinant Escherichia coli expressing stereospecific alcohol dehydrogenase

Biocatalytic asymmetric synthesis has been widely used for preparation of optically active chiral alcohols as the important intermediates and precursors of active pharmaceutical ingredients. However, the available whole-cell system involving anti-Prelog specific alcohol dehydrogenase is yet limited. A recombinant Escherichia coli system expressing anti-Prelog stereospecific alcohol dehydrogenase from Candida parapsilosis was established as a whole-cell system for catalyzing asymmetric reduction of aryl ketones to anti-Prelog configured alcohols. Using 2-hydroxyacetophenone as the substrate, reaction factors including pH, cell status, and substrate concentration had obvious impacts on the outcome of whole-cell biocatalysis, and xylose was found to be an available auxiliary substrate for intracellular cofactor regeneration, by which (S)-1-phenyl-1,2-ethanediol was achieved with an optical purity of 97%e.e. and yield of 89% under the substrate concentration of 5 g/L. Additionally, the feasibility of the recombinant cells toward different aryl ketones was investigated, and most of the corresponding chiral alcohol products were obtained with an optical purity over 95%e.e. Therefore, the whole-cell system involving recombinant stereospecific alcohol dehydrogenase was constructed as an efficient biocatalyst for highly enantioselective anti-Prelog synthesis of optically active aryl alcohols and would be promising in the pharmaceutical industry.     

Regulation of protein synthesis in hen's oviducts. II. Extracellular ovalbumin as an inhibitor of ovalbumin synthesis in the oviducts.

Washed, preincubated minced hen's oviducts, which contained low levels of extracellular and intracellular proteins, synthesized egg-white proteins actively. The addition of ovalbumin to the incubation medium resulted in inhibition of the synthesis of egg-white proteins by the washed, preincubated oviduct cells, while the addition of bovine serum albumin seemed to stimulate protein synthesis and hen's egg-white lysozyme had no effect. The inhibitory or stimulatory effect on protein synthesis was proportional to the amount of protein added to the medium. The inhibitory effect of added ovalbumin was shown not to be due to the incorporation of ovalbumin into the oviduct cells from the incubation medium. Egg-white proteins added to the medium also inhibited protein synthesis inside the cells and the extent of the inhibition appeared to correspond to the amount of ovalbumin present in egg-white.     

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%.     

Cross-linked crystals of hydroxynitrile lyase as catalyst for the synthesis of optically active cyanohydrins

Purified hydroxynitrile lyase (HNL) from Manihot esculenta was crystallized by the sitting-drop vapour-diffusion method. The bipyramidal crystals formed (10–20 μm) were cross-linked with different amounts of glutaraldehyde and used as biocatalyst for the synthesis of optically active cyanohydrins. The cross-linked crystals were more stable than Celite-immobilized enzymes when incubated in organic solvents, especially in polar solvents. After six consecutive batch reactions in dibutylether, the remaining activity of the cross-linked crystals was more than 70 times higher than for the immobilized enzymes. Nevertheless, the specific activity of the cross-linked crystals (per milligram protein) was reduced compared to the activity of immobilized enzymes. The product enantiopurity was independent of the type of enzyme preparation used.     

Cloning and characterization of three ketoreductases from soil metagenome for preparing optically active alcohols

Objectives

To discover novel ketoreductases (KRED) from soil metagenome preparation of chiral alcohols.

Results

Three putative KRED were cloned, heterologously expressed in Eschericha coli and characterized based on the sequence analysis of soil metagenome. All the three enzymes (KRED424, KRED432, and KRED433) had maximum activity at 55 °C and pH 7. KRED424 had a broader substrate spectrum compared with the other two. Three prochiral carbonyl compounds were used to evaluate the abilities of enantioselective reductions of the KRED. For N-Boc-3-pyrrolidone, all enzymes produced an (S)-type alcohol in enantiomeric excess (>99 % ee). For ethyl 2-oxo-4-phenylbutyrate, KRED424 showed a higher conversion (91.5 %) and enantioselectivity (S-type, >99 % ee) than KRED432 and KRED433. For ethyl 4-chloroacetoacetate (COBE), both of KRED424 and KRED433 completely converted 20 mM substrate and KRED433 could obtain an (R)-alcohol with 94 % ee.

Conclusions

The three ketoreductases have potential in the preparation of pharmaceuticals and fine chemicals.

     

Novel synthesis of optically active γ-carboxyglutamic acid and its derivatives

A novel synthesis of optically active L-γ-carboxyglutamic acid (GLA), found in prothrombin and other vitamin K-dependent factors, is described. This method involves the formation of γ-carbanion of the protected glutamic acid esters by means of a non-nucleophilic base, followed by the treatment with benzyl chloroformate or other electrophilic agents, and deprotection by hydrogenation or hydrolysis. This convenient technique is potentially useful for preparation of not only the γ-carboxyglutamic acid itself, but also of the mixed protected ester and amino functions for further peptide syntheses containing this amino acid.     

Monoterpene ketones in the synthesis of optically active insect pheromones

This review considers the synthetic possibilities of monoterpene ketones, such as (R)-(+)- and (S)-(-)-pulegones, (-)-menthone, (R)-(-)- and (S)-(+)-carvones, (2R,5S)-dihydrocarvone, (S)-(+)- and (R)-(-)-camphors, (R)-(-)-nopinone, (R)-(+)- and (S)-(-)-verbenones by the examples of synthesis of optically pure and enantiomerically enriched insect pheromones.     

Microwave-induced synthesis of new optically active and soluble polyamides containing pendent 4-(2-phthalimidiylpropanoylamino)benzoylamino-groups

An aromatic chiral diacid monomer, 5-[4-(2-phthalimidiylpropanoylamino)-benzoylamino]isophthalic acid was synthesized in five steps under conventional heating in high yield and purity. A series of soluble, thermally stable and optically active polyamides (PA)s containing pendent groups made of phthalimide, flexible l-alanine and benzamide sequence have been successfully synthesized under microwave irradiation. Excellent yields and very short reaction time were the main characteristics of this method. The same polymerization reactions were also carried out by conventional thermal heating and the results are compared. The resulting PAs had inherent viscosity in the range of 0.50–0.79 dL g⁻¹. All of the these polymers are readily dissolved in various solvents such as N-methyl-2-pyrrolidinone, N,N-dimethylacetamide and N,N-dimethylformamide and showed glass-transition temperature above 200°C. Thermogravimetric analysis demonstrated that the 10% weight-loss temperatures in nitrogen were 372 and 422°C for selected two PAs. All of these polymers showed optical rotation which is due to successful insertion of l-alanine in the structure of chiral diacid monomer.     

Preparation of optically active D-arylglycines for use as side chains for semisynthetic penicillins and cephalosporins using immobilized subtilisins in two-phase systems

Optically active D-arylglycines, which are of interest for preparation of semisynthetic penicillins and cephalosporins, were isolated from the racemic mixtures of their derivatives using immobilized proteolytic enzyme subtilisin (EC No. 3.4.4. 16). The performance of these reactions in two-phase systems, consisting of water and an immiscible organic solvent, improved the yield, purity, and economics of the process by increasing the substrate solubility and reducing the rate of nonenzymatic hydrolysis. The proportion of the organic phase can be as much as 75% of the overall volume without seriously impairing the enzymatic activity. The optically pure D-and L-arylglycines were liberated from their D- and L-derivatives by acid hydrolysis. The substituent influence of the various arylglycine derivatives on the rate of the enzymatic cleavage reaction was investigated.     

Enzymatic synthesis of electroconductive biocomposites based on DNA and optically active polyaniline

Electroconductive interpolymer polyaniline complexes are synthesized on the DNA matrix, using the method of oxidative polymerization of aniline with two different biocatalyzers: horseradish root peroxidase and micropiroxidase-11 biomimetic. The spectral characteristics and morphology of the acquired biocomposites have been studied. The stereospecificity of the acquired samples of interpolymer complexes is shown, depending on the biocatalyzers used. The results acquired indicate the important role of a biocatalyzer in the formation of the twist direction of an electroconductive polymer spiral on the DNA matrix; i.e., the optical activity of the polymer samples acquired is apparently associated with the biocatalyzer properties.