Investigation of the Impact of Water on the Enantioselectivity Displayed by CALB in the Kinetic Resolution of δ‐Functionalized Alkan‐2‐ol Derivatives

It is shown that the low enantioselectivity of Candida antarctica lipase B (CALB)‐catalyzed transesterification of a δ‐functionalized alkan‐2‐ol to its acetate does not correlate at all with the high enantioselectivity of the CALB‐catalyzed hydrolysis of the corresponding acetate in water. This lack of correlation is unusual and for unfunctionalized alkan‐2‐ol derivatives there is a very good correlation between the enantioselectivity of transesterification of the alcohol and hydrolysis of the corresponding acetate (E>200 in both cases). The results confirm previous predictions from molecular modeling. The water effect was mimicked by CALB variant Ala281Ser, which showed an enhanced enantioselectivity in transesterification of δ‐functionalized alkan‐2‐ols compared to wild‐type CALB.     

Esterification of (RS)-Ibuprofen by native and commercial lipases in a two-phase system containing ionic liquids

Four commercially available lipases and two native lipases from Aspergillus niger AC-54 and Aspergillus terreus AC-430 were used for the resolution of (RS)-Ibuprofen in systems containing the ionic liquids [BMIM][PF₆] and [BMIM][BF₄]. The lipases showed higher conversion in a two-phase system using [BMIM][PF₆] and isooctane compared to that in pure isooctane. Although the best enzyme was a commercially available lipase from Candida rugosa (E = 8.5), another native lipase, produced in our laboratory, from A. niger gave better enantioselectivity (E = 4.6) than the other lipases tested (E = 1.9–3.3.). After thorough optimization of several reaction conditions (type and ratios of isooctane/ionic liquid, amount of enzyme, and reaction time), the E-value of A. niger lipase (15% w/v) could be duplicated (E = 9.2) in a solvent system composed of [BMIM][PF₆] and isooctane (1:1) after 96 h of reaction.     

Chemo‐ and Enzyme‐Catalyzed Reactions Revealing a Common Temperature‐Dependent Dynamic Solvent Effect on Enantioselectivity

The enantiomeric ratio E of enzyme‐catalyzed (Candida antarctica lipase and lipase PS) and chemo‐catalyzed (L ‐proline‐based diamines) acylation reactions of 1‐(naphthalen‐2‐yl)ethanol, 2‐phenylpropanol, and 2‐benzylpropane‐1,3‐diol is dependent on solvent and temperature. Plots of ln E vs. 1/T showed the presence of inversion temperatures (T_inv). The T_inv values for the bio‐catalyzed and the chemo‐catalyzed reactions are fairly in agreement, and correspond as well to the T_NMR values obtained by variable‐temperature ¹³C‐NMR experiments on the substrates in the same solvent of the resolution. This result demonstrates that clustering effects in the substrate solvation manage the chemical and the enzymatic enantioselectivity, and, moreover, that the solute? solvent cluster is always the real reacting species in solution for chemical as well as for enzymatic reactions.     

Screening and Immobilization <Emphasis Type="Italic">Burkholderia</Emphasis> sp. GXU56 Lipase for Enantioselective Resolution of (<Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">S</Emphasis>)-Methyl Mandelate

Microorganisms producing lipase were isolated from soil and sewage samples and screened for enantioselective resolution of (R,S)-methyl mandelate to (R)-mandelic acid. A strain designated as GXU56 was obtained and identified as Burkholderia sp. Preparing immobilized GXU56 lipase by simple adsorption on octyl sepharose CL-4B, the optimum temperature was shifted from 40 °C (free lipase) to 50 °C (immobilized lipase), and the optimum pH was shifted from 8.0 (free lipase) to 7.2 (immobilized lipase). The immobilized enzyme displayed excellent stability in the pH range of 5.0–8.0, at the temperatures below 50 °C and in organic solvents compared with free enzyme. Enantioselectivity ratio for (R)-mandelic acid (E) was dramatically improved from 29.2 to more than 300 by applying immobilized lipase in the resolution of (R,S)-methyl mandelate. After five cycles of use of immobilized lipase, conversion and enantiomeric excess of (R)-mandelic acid were 34.5% and 98.5%, respectively, with enantioselectivity ratio for (R)-mandelic acid (E) of 230. Thus, octyl-sepharose-immobilized GXU56 lipase can be used as a bio-resolution reagent for producing (R)-mandelic acid.     

S‐endo‐2‐Norbornyl‐N‐n‐butylcarbamate as a Potential Pseudomonas Lipase Inhibitor to Probe the Enantioselectivity of the Enzyme by Kinetic and Molecular Docking Evaluation

Obesity is a complex health issue and it can cause many health and social problems. Previous studies reported that lipase is a main target for obesity treatment. We synthesized R‐exo‐2‐norbornyl‐N‐n‐butylcarbamate and S‐exo‐2‐norbornyl‐N‐n‐butylcarbamate as potential pseudomonas lipase inhibitors to probe the enantioselectivity of the enzyme and demonstrated that R‐exo‐2‐norbornyl‐N‐n‐butylcarbamate had better enzyme enantioselectivity, ki and the docking model with Pseudomonas species lipase in our previous studies. In this article, we reported the property of the Pseudomonas species lipase inhibitors, R‐and S‐endo‐2‐norbornyl‐N‐n‐butylcarbamate and compared the docking models of these two compounds with R‐ and S‐exo‐2‐norbornyl‐N‐n‐butylcarbamates by AutoDock. We found that S‐endo‐2‐norbornyl‐N‐n‐butylcarbamate has the best enantioselectivity, ki and docking model and this study could provide useful information about enzyme enantioselectivity for the development of Pseudomonas species lipase inhibitors for obesity treatment.     

A Novel 1‐Glycosyl‐1H‐triazole‐Based P,N Ligand for Rhodium‐Catalyzed Asymmetric Hydrosilylation of Ketones

A novel class of chiral glycosyl‐triazole‐based P,N ligands were synthesized by ‘click chemistry’ and were applied to the Rh‐catalyzed asymmetric hydrosilylation of a range of substituted acetophenones. Enantioselective hydrosilylation of acetophenone gave (S)‐1‐phenylethanol in moderate enantioselectivity (72% ee) and in good conversion (93%).     

Enzyme‐ and Ruthenium‐Catalyzed Enantioselective Transformation of α‐Allenic Alcohols into 2,3‐Dihydrofurans

An efficient one‐pot method for the enzyme‐ and ruthenium‐catalyzed enantioselective transformation of α‐allenic alcohols into 2,3‐dihydrofurans has been developed. The method involves an enzymatic kinetic resolution and a subsequent ruthenium‐catalyzed cycloisomerization, which provides 2,3‐dihydrofurans with excellent enantioselectivity (up to >99 % ee). A ruthenium carbene species was proposed as a key intermediate in the cycloisomerization.     

Purification and Application of a Lipase from <Emphasis Type="Italic">Penicillium expansum</Emphasis> PED-03

An extracellular lipase was purified from the fermentation broth of Penicillium expansum PED-03 by DEAE-Sepharose chromatography, followed by sephacryl S-200 chromatography. The enzyme was purified 81.8-fold with 19.8% recovery and a specific activity of 85.94 U/mg. The molecular weight of the homogeneous enzyme was about 28 kDa, determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The enzymatic resolution of racemic ibuprofen was carried out by the lipase from P. expansum PED-03, and the conversion reached 46% with excellent enantioselectivity(E > 200 ), which showed a good application potential in the production of optically pure ibuprofen.     

Free radical and nitroxide mediated polymerization of hydroxy–functional acrylates prepared via lipase–catalyzed transacylation reactions

3‐Hydroxypropyl acrylate, 4‐hydroxybutyl acrylate, 2‐methyl‐3‐hydroxypropyl acrylate, 2‐hydroxypropyl acrylate, neopentyl glycol acrylate, glyceryl acrylate, and dihydroxyhexyl acrylate were prepared via transacylation reaction of methyl acrylate with diols and triols catalyzed by Candida antarctica lipase B. After removal of the enzyme by filtration and the methyl acrylate by distillation, the monomers were polymerized via free radical polymerization (FRP) with azobisisobutyronitrile as initiator and nitroxide mediated polymerization (NMP) employing Blocbuilder? alkoxyamine initiator and SG‐1 free nitroxide resulting in hydroxy functional poly(acrylates). The NMP kinetics are discussed in detail. In addition, the polymers obtained by FRP and NMP are compared and the results are related to the amount of bisacrylates that are present in the initial monomer mixtures resulting from the transacylation reactions. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 2610–2621, 2010     

Immobilization does not influence the enantioselectivity of CAL-B catalyzed kinetic resolution of secondary alcohols

Decreasing enantioselectivity (E-value) by increasing conversion has been observed in transesterification reactions of secondary alcohols catalyzed by a pure protein formulation of lipase B from Candida antarctica (Novozym 525 F). Addition of a range of enantiopure alcohols caused a temporary increase in selectivity of the transesterification reaction of 3-chloro-1-phenoxy-2-propanol with vinyl butanoate. The corresponding immobilized lipase B, (Novozym 435) showed a similar relationship between the E-value and degree of conversion.     

Study of the enantioselectivity of the CAL-B-catalysed transesterification of α-substituted α-propylmethanols and α-substituted benzyl alcohols

A study of the enantioselectivity exhibited by the lipase B from Candida antarctica in the transesterification of different α-substituted α-propylmethanols with vinyl acetate is shown. The best results are obtained when the large-sized (L) substituent of the alcohol is either a phenyl group or more especially a cyclohexyl group, although the reaction rates are lower than when linear or slightly branched groups are present. It is also found that ramification at the β-position of the L substituent has a deleterious effect on both lipase activity and enantioselectivity. Moreover, some α-substituted benzyl alcohols bearing medium-sized (M) substituents larger than an ethyl and smaller than a propyl group are resolved by means of this methodology with moderate-good enantioselectivities (E=46–57) and similar reaction rates.     

Preparation and Evaluation of a Novel Cellulose Tris(N‐3,5‐dimethylphenylcarbamate) Chiral Stationary Phase

A novel cellulose tris(N‐3,5‐dimethylphenylcarbamate) (CDMPC) chiral stationary phase (CSP) was prepared by coating CDMPC on TiO₂/SiO₂, which was prepared by coating titania nanoparticles on silica through a self‐assemble technique. At first, 2‐hydroxyl‐phenyl acetonitrile and α‐phenylethanol were separated on this new CSP to evaluate the chiral separation ability. Then, two pesticides, matalaxyl and diclofop‐methyl were separated. The influence of the mobile phase composition on the enantioselectivity was discussed, and the repeatability and stability of the CSP were studied too.     

Dynamic Kinetic Resolution of Homoallylic Alcohols: Application to the Synthesis of Enantiomerically Pure 5,6‐Dihydropyran‐2‐ones and δ‐Lactones

Dynamic kinetic resolution of various homoallylic alcohols with the use of Candida antarctica lipase B and ruthenium catalyst 2 afforded homoallylic acetates in high yields and with high enantioselectivity. These enantiopure acetates were further transformed into homoallylic acrylates after hydrolysis of the ester function and subsequent DMAP‐catalyzed esterification with acryloyl chloride. After ring‐closing metathesis 5,6‐dihydropyran‐2‐ones were obtained in good yields. Selective hydrogenation of the carbon? carbon double bond afforded the corresponding δ‐lactones without loss of chiral information.     

Selective Activation of C−H Bonds in a Cascade Process Combining Photochemistry and Biocatalysis

Selective oxyfunctionalizations of inert C−H bonds can be achieved under mild conditions by using peroxygenases. This approach, however, suffers from the poor robustness of these enzymes in the presence of hydrogen peroxide as the stoichiometric oxidant. Herein, we demonstrate that inorganic photocatalysts such as gold–titanium dioxide efficiently provide H₂O₂ through the methanol‐driven reductive activation of ambient oxygen in amounts that ensure that the enzyme remains highly active and stable. Using this approach, the stereoselective hydroxylation of ethylbenzene to (R )‐1‐phenylethanol was achieved with high enantioselectivity (>98 % ee ) and excellent turnover numbers for the biocatalyst (>71 000).     

A parametric study on biphasic medium conditions for the enantioselective production of naproxen by <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase

A parametric study to increase the enantioselectivity of Candida rugosa lipase (CRL) toward S-Naproxen production by the hydrolysis of racemic Naproxen methyl ester in an aqueous-organic biphasic batch system was carried out. Effects of organic solvent type, aqueous phase/organic solvent volume ratio, agitation rate, concentrations of the substrate and the enzyme, pH of the aqueous phase, and temperature on the en antiomeric excess for the product (ee_p), on the enantiomeric ratio (E) and on the conversion (x) were evaluated. Employing isooctane as the solvent resulted in higher ee_p, E, and x than those obtained in hexane, cyclohexane, and toluene. The higher the volume ratio of aqueous phase/organic solvent employed, the higher the conversion and enantioselectivity achieved. The increase in agitation rate increased the hydrolysis rate. Higher concentration of racemic Naproxen methyl ester than 10 mg/mL decreased both the conversion and enantioselectivity. The increase in crude CRL concentration resulted in enhancement of x, but the decrease of fee_p and E. Acidic pH led to higher conversion and enantioselectivity than the medium and alkaline pH values. A further increase in temperature to over 45°C decreased the conversion and enantioselectivity. The highest enantiomeric ratio achieved in the S-Naproxen production was E=171.1, with x=49.8% and ee_p=95.7%.     

Kinetics and Protein‐Inhibitor Docking Studies of Enantiomers of exo‐2‐Norbornyl‐N‐n‐butylcarbamates as Pseudomonas Lipase Inhibitors to Probe the Enantioselectivity of the Enzyme

The Pseudomonas species lipase inhibition shows enantioselectivity for R‐enantiomer over S‐enantiomer of exo‐2‐norbornyl‐N‐n‐butylcarbamates. R‐, S‐, and racemic‐exo‐2‐norbornyl‐N‐n‐butylcarbamates are all characterized as pseudo substrate inhibitors of the enzyme. Thus, the mechanism for Pseudomonas species lipase‐catalyzed hydrolysis of the inhibitor is formation of the first enzyme‐inhibitor Michaelis complex via nucleophilic attack of the active site serine to the inhibitor (K_i step) then formation of the butylcarbamyl enzyme intermediate from this complex (k₂ step). Comparison of bimolecular rate constants (k_i = k₂ / K_i) of the inhibitors indicates that R‐enantiomer is 1.8 times more potent than S‐enantiomer. Thus, Pseudomonas species lipase shows enantioselectivity of 1.8 for R‐exo‐2‐norbornyl‐N‐n‐butyl‐carbamate over S‐exo‐2‐norbornyl‐N‐n‐butylcarbamate. Protein‐ligand interaction studies on both enantiomers of exo‐2‐norbornyl‐N‐n‐butylcarbamate as inhibitors of Pseudomonas species lipase using AutoDock suggest that R‐enantiomer binds more tightly into the active site of the enzyme than S‐enantiomer. The norbornyl ring of S‐exo‐2‐norbornyl‐N‐n‐butylcarbamate is repulsive to Ser 82 and His 251 of the catalytic triad as well as to Met 16 of the oxyanion hole. These repulsions may create few unfavorable interactions between S‐exo‐2‐norbornyl‐N‐n‐butylcarbamate and the enzyme and make this inhibitor a less potent one.     

Engineering of P450pyr Hydroxylase for the Highly Regio‐ and Enantioselective Subterminal Hydroxylation of Alkanes

Terminal‐selective cytochrome P450pyr has been successfully engineered through directed evolution for the subterminal hydroxylation of alkanes with excellent regio‐ and enantioselectivity. A sensitive colorimetric high‐throughput screening (HTS) assay was developed for the measurement of both the regioselectivity and the enantioselectivity of a hydroxylation reaction. By using the HTS assay and iterative saturation mutagenesis, sextuple‐mutant P450pyrSM1 was created for the hydroxylation of n‐octane ( 1 ) to give (S)‐2‐octanol ( 2 ) with 98 % ee and >99 % subterminal selectivity. The engineered P450 is the first enzyme for this type of highly selective alkane hydroxylation, being useful for the C? H activation and functionalization of alkanes and the preparation of enantiopure alcohols. Molecular modeling provided structure‐based understanding of the fully altered regioselectivity and the excellent enantioselectivity. Another sextuple‐mutant P450pyrSM2 catalyzed the hydroxylation of propylbenzene ( 3 ) to afford (S)‐1‐phenyl‐2‐propanol ( 4 ) with 95 % ee and 98 % subterminal selectivity.     

Ultrasound Irradiation Promoted Enzymatic Transesterification of (<em>R/S</em>)-1-Chloro-3-(1-naphthyloxy)-2-propanol

(R)-1-Chloro-3-(1-naphthyloxy)-2-propanol (3), which is the key intermediate of (S)-propranolol, was successfully prepared via enantioselective transesterification catalyzed by lipase under ultrasound irradiation. Compared with conventional shaking, the enzyme activity and enantioselectivity were dramatically increased under ultrasound exposure. Effects of various reaction conditions on the synthetic activity of enzyme as well as enantioselectivity, including the type of enzyme, ultrasound power, solvent, acyl donor, temperature and substrate molar ratio, were investigated. Pseudomonas sp. lipase (PSL) showed an excellent catalytic performance under optimum conditions (enzyme activity: 78.3 ± 3.2 μmol·g^-1·min^-1, E value: 98 ± 6).     

Chiral‐Organotin‐Catalyzed Kinetic Resolution of Vicinal Amino Alcohols

A highly efficient kinetic resolution of racemic amino alcohols has been achieved for the first time with a chiral tin catalyst. A chiral organotin compound with 3,4,5‐trifluorophenyl groups at the 3,3′‐positions of the binaphthyl framework enabled this transformation with excellent yield and high enantioselectivity. The process tolerates aryl‐ and alkyl‐substituted amino alcohols and a variety of other substrates, affording the corresponding products in high enantioselectivity and with s factors up to >500.     

Lipase‐catalysed kinetic resolution of hydroxymethylchromanes

An effective kinetic resolution of hydroxymethylchromanes racemic 2a and 3a has been achieved by means of enantioselective transesterification with vinyl acetate in organic solvents. The alcohols (‐)‐R‐ 2a and (‐)‐S‐ 3a were obtained with high optical purities (94 and 98% ee) in 70% and 38% yields, respectively. The influence of the enzyme source and the character of the solvent on the enantioselectivity were studied.