Partially purified Carica papaya lipase: a versatile biocatalyst for the hydrolytic resolution of (R,S)-2-arylpropionic thioesters in water-saturated organic solvents

With the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl thioesters in water-saturated isooctane as a model system, improvements of the specific lipase activity and thermal stability were found when a crude Carica papaya lipase (CPL) was partially purified and employed as the biocatalyst. The partially purified Carica papaya lipase (PCPL) was furthermore explored as an effective enantioselective biocatalyst for the hydrolytic resolution of (R,S)-profen thioesters in water-saturated organic solvents. The kinetic analysis in water-saturated isooctane indicated that both acyl donor and acyl acceptor have profound influences on the lipase activity, E-value, and enantioselectivity. Inversion of the enantioselectivity from (S)- to (R)-thioester was found for (R,S)-fenoprofen and (R,S)-ketoprofen thioesters that contained a bulky substituent at the meta-position of 2-phenyl moiety of the acyl part. Kinetic constants for the acylation step were furthermore estimated for elucidating the kinetic data and postulating an active site model. The thermodynamic analysis indicated that the enantiomer discrimination was driven by the difference of activation enthalpy (DeltaDeltaH) and that of activation entropy (DeltaDeltaS), yet the latter was dominated for most of the reacting systems. The postulated active site model was supported from the variation of DeltaDeltaH and DeltaDeltaS with the acyl moiety, in which a good linear enthalpy-entropy compensation relationship was also illustrated. A comparison of the performances between Candida rugosa lipase (CRL) and PCPL indicated that PCPL was superior to CRL in terms of the better thermal stability, similar or better lipase activity for the fast-reacting substrate, time-course-stability, and lower enzyme cost.     

Lipase-catalyzed enantioselective esterification of (S)-naproxen hydroxyalkyl ester in organic media

A lipase-catalyzed, enantioselective esterification process in organic solvents was developed for the synthesis of (S)-naproxen hydroxyalkyl ester. With the selection of lipase (Candida rugosa lipase) and reaction medium (isooctane and cyclohexane), a high enantiomeric ratio of <100 for the enzyme was obtained. 1,4-Butanediol was the best acyl acceptor. The carbon chain length of the alcohol had a major effect on the enzyme activity and enantioselectivity of lipase-catalyzed esterification.     

Resolution of racemic ketoprofen in organic solvents by lipase from Burkholderia cepacia G63

A lipase from the Burkholderia cepacia strain G63 immobilized on resin was used for the resolution of ketoprofen. To study its catalytic properties in enantioselective esterication, different alcohols and solvents were tested to select the most suitable acyl acceptor and reaction medium. Compared with the low activity of the free lipase, the enzyme activity and E value of the immobilized lipase were significantly enhanced. The enantioselectivity of the immobilized lipase could also be markedly improved by adding a small amount of 18-crown-6. RSM was employed to optimize the reaction parameters. The optimal reaction conditions were: reaction time 22.50 h, additives dosage 0.4322 g (0.33 mmol/mL), and substrate molar ratio 54.11:1. Under optimal conditions, the maximal E value was up to 10.01, which exhibited a better enantioselectivity than some commercial lipases, such as Novozym 435, Lipozyme RM IM and LipozymeTL IM.     

Enantioselective hydrolysis of (R,S)-naproxen 2,2,2-trifluoroethyl ester in water-saturated solvents via lipases from Carica pentagona Heilborn and Carica papaya

A crude lipase prepared from Carica pentagona Heilborn latex was explored as an effective enantioselective biocatalyst for the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl ester in water-saturated organic solvents. Comparisons of the enzyme performance with that from Carica papaya lipase indicated that both lipases showed low tolerance to the hydrophilic solvent and were inhibited by (S)-naproxen and 2,2,2-trifluoroethanol. Improvements on the enzyme activity and enantioselectivty were demonstrated when both lipases in partially purified forms were employed. By using the thermodynamic analysis, the enantiomeric discrimination was mainly driven by the difference of activation enthalpy for all reaction systems except for employing Carica papaya lipase as the biocatalyst for (R,S)-fenoprofen 2,2,2-trifluoroethyl thioester.     

Thioethyl-, Vinyl-, Ethyl Octanoate Esters and Octanoic Acid as Acyl Donors in Lipase Catalysed Acyl Transfer Reactions

S-Ethyl thiooctanoate, vinyl octanoate, ethyl octanoate and octanoic acid were studied as acyl donors in a lipase catalysed acyl transfer reaction with 2-octanol as acyl acceptor. The reaction conditions had a pronounced effect on the equilibrium displacement and the apparent enantioselectivity. The thioethyl and vinyl esters proved to be efficient acyl donors under atmospheric pressure and 39 d`C, affording a high apparent enantiomeric ratio. Under these reaction conditions the apparent enantioselectivity seemed to be enhanced by water, which was present in the reaction system and caused the production of octanoic acid, by hydrolysis of me acyl enzyme.     

Lipase-catalysed kinetic resolution of secondary alcohols with improved enantioselectivity in propylene carbonate

The lipase-catalysed kinetic resolution of secondary alcohols was studied using vinyl acetate as acyl donor in propylene carbonate. Propylene carbonate offers an environmentally friendly alternative in contrast to conventional solvents. Several different lipases were investigated, and Candida antarctica lipase B (CALB) exhibited better results for all the substrates. It was shown that the addition of non-reactive base triethylamine and silver oxide to the reaction mixture enhanced the reaction rate and enantioselectivity. With propylene carbonate as solvent, CALB could be recycled without significant activity or enantioselectivity losses.     

Enantioselective esterification of ibuprofen by a novel thermophilic Biocatalyst: APE1547

The enantioselective esterification of ibuprofen catalyzed by a novel thermophilic esterase (APE1547) from the archaeon Aeropyrum pernix K1 was successfully conducted in organic solvents. The effects of acyl acceptor, substrate ratio, organic solvent, temperature, and water activity were investigated. Under optimum conditions, the highest enantioselectivity (E = 38.1) was obtained with a higher enzyme activity (216.5 μmol/g/h). Celites were added into the reaction mixture to remove the water produced in the esterification. The reaction achieved its equilibrium in approximately 96 h with a conversion of 57 and 99% (ee) of the un-reacted (S)-ibuprofen obtained.     

Solvent effects on the enantioselectivity of the thermophilic lipase QLM in the resolution of (R,S)-2-octanol and (R,S)-2-pentanol

The thermophilic lipase QLM-catalyzed resolution of (R, S)-2-octanol and (R, S)-2-pentanol via transesterification was carried out in various organic solvents, and the solvent effects on the enzyme's enantioselectivity were investigated. A significant negative correlation between the enantiomeric ratio, E, and the size of the solvent molecules was observed. The highest E value, 21, was obtained in the small molecular-sized solvent dichloromethane when (R, S)-2-octanol was resolved with vinyl acetate as acyl donor. Thermodynamic analysis indicated that the difference in activation free energy between the two enantiomers was about 25.5% lower in dichloromethane than in the solvent-free system, and the change in the difference in activation entropy between the enantiomers was the main contributor to the changes in E values with the molecular size of solvents.     

Resolution of 2-phenoxy-1-propanols through the enantioselective acylation mediated by Achromobacter sp. lipase as biocatalyst

Enantioselective acylation employing vinyl alkanoates as acyl donors was exploited for the resolution of 2-(substituted phenoxy)-1-propanols carrying different substituents on the benzene ring using Achromobacter sp. lipase. These primary alcohols with an oxygen atom at the stereocenter, were resolved with moderate to good enantioselectivity, based on the enantiomeric ratio E (up to 27), through acylation with vinyl butanoate in diisopropyl ether, after the examination of potential factors affecting the reaction such as organic solvents and acyl donors. Using this procedure, enantiomerically enriched (R)-2-(4-chlorophenoxy)-1-propanol was prepared in 97% e.e. and 33% yield in a gram-scale reaction.     

Resolution of 2-phenoxy-1-propanols through the enantioselective acylation mediated by Achromobacter sp. lipase as biocatalyst

Enantioselective acylation employing vinyl alkanoates as acyl donors was exploited for the resolution of 2-(substituted phenoxy)-1-propanols carrying different substituents on the benzene ring using Achromobacter sp. lipase. These primary alcohols with an oxygen atom at the stereocenter, were resolved with moderate to good enantioselectivity, based on the enantiomeric ratio E (up to 27), through acylation with vinyl butanoate in diisopropyl ether, after the examination of potential factors affecting the reaction such as organic solvents and acyl donors. Using this procedure, enantiomerically enriched (R)-2-(4-chlorophenoxy)-1-propanol was prepared in 97% e.e. and 33% yield in a gram-scale reaction.     

(R,S)-2-chlorophenoxyl pyrazolides as novel substrates for improving lipase-catalyzed hydrolytic resolution

The best reaction condition of Candida antartica lipase B as biocatalyst, 3-(2-pyridyl)pyrazole as leaving azole, and water-saturated methyl t-butyl ether as reaction medium at 45°C were first selected for performing the hydrolytic resolution of (R,S)-2-(4-chlorophenoxyl) azolides (1-4). In comparison with the kinetic resolution of (R,S)-2-phenylpropionyl 3-(2-pyridyl)pyrazolide or (R,S)-α-methoxyphenylacetyl 3-(2-pyridyl)pyrazolide at the same reaction condition, excellent enantioselectivity with more than two order-of-magnitudes higher activity for each enantiomer was obtained. The resolution was then extended to other (R,S)-3-(2-pyridyl)pyrazolides (5-7) containing 2-chloro, 3-chloro, or 2,4-dichloro substituent, giving good (E > 48) to excellent (E > 100) enantioselectivity. The thermodynamic analysis for 1, 2, and 4-7 demonstrates profound effects of the acyl or leaving moiety on varying enthalpic and entropic contributions to the difference of Gibbs free energies. A thorough kinetic analysis further indicates that on the basis of 6, the excellent enantiomeric ratio for 4 and 7 is due to the higher reactivity of (S)-4 and lower reactivity of (R)-7, respectively.     

Enantioselective properties of Fusarium solani pisi cutinase on transesterification of acyclic diols: activity and stability evaluation

The regio and enantioselectivity of a recombinant cutinase from Fusarium solani pisi was tested on three racemic and one prochiral phenylalkanediols via irreversible transesterification with vinyl acetate. The optimization of the reaction conditions involved the screening of different organic solvents as well as the variation of the substrate concentrations. Thus, the enzymatic activity was checked by measuring initial reaction rates, overall yields, and enantiomeric excess of the reaction products. Only the smaller molecules were recognized by the enzyme, and a denaturing effect of the acyl donor was observed. Nevertheless, a stabilising effect on the enzyme caused by a pre-incubation with the diol was also noted.     

Lipase resolution of new (±)-3-aryloxy-1-halogenopropan-2-ols: Versatile building blocks for β-adrenergic receptor antagonists

Using two commercial immobilized lipases Lipozyme^® TL and Novozym^® 435 effective kinetic resolution of several novel 3-aryloxy-1-halogenopropan-2-ols was achieved by acyl transfer reaction in organic solvents, yielding both enantiomers with 89–99% ee. In preparative resolutions carried out in tert-butyl methyl ether at 25 °C with vinyl acetate as acyl donor enantioselectivity ratio E was from 64 to 99. The resolved enantiomers were successfully used as chiral building blocks in the synthesis of new 1-alkylamino-3-aryloxypropan-2-ols, by nucleophilic halogen substitution with isopropylamine and tert-butylamine. The obtained products will be evaluated in vitro as potential new β-adrenergic receptors antagonists.     

Effect of organic solvents on enantioselectivity of protease catalysis

The protease-catalyzed transesterifications between N-trifluoroacetyl-DL-phenylalanine 2,2,2-trifluoroethyl ester and 1-propanol were studied in a variety of anhydrous organic solvents at 30 degrees C. The protease preparations lyophilized from phosphate buffer solutions (pH 8.0) were used as catalysts. The organic solvent affected both rate of reaction and enantioselectivity differently. Proteases such as Aspergillus oryzae protease, subtilisin Carlsberg, and subtilisin BPN' always preferred the L-enantiomer in both hydrophilic and hydrophobic solvents, indicating no inversion of the L-specificity in hydrophobic solvents such as toluene. However, enantioselectivity was rather poor, with E (enantiomeric ratio) values not exceeding even one order of magnitude except for acetonitrile. There was a weak inverse correlation between E values of subtilisin Carlsberg and solvent hydrophobicity (logP). Acetonitrile was a preferable solvent in terms of both rate of reaction and enantioselectivity (E= 15 to 25) for processing L-amino acid derivatives in organic media. Organic solvents generally have potential advantages of processing D-amino acid derivatives. (c) 1997 John Wiley & Sons, Inc.     

Intrinsic effects of solvent polarity on enzymic activation energies

The effect of organic solvents on subtilisin Carlsberg catalysis has been investigated with the aid of a thermodynamic analysis. Saturation solubility experiments were performed to provide a quantitative measure of substrate desolvation from the reaction medium. This enabled calculation of the intrinsic enzymic activation energy and resulted in a linear free energy relationship with respect to solvent polarity. The results indicate that the intrinsic activation energy of subtilisin catalysis is lowest in polar organic solvents, which may be due to transition state stabilization of the enzyme's polar transition state for transesterification.     

Hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl thioester by Carica papaya lipase in water-saturated organic solvents

For the first time, the Carica papaya lipase (CPL) stored in crude papain is explored as a potential enantioselective biocatalyst for obtaining chiral acids from their racemic thioesters. Hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl thioester in water-saturated organic solvents is employed as a model system for studying the effects of temperature and solvents on lipase activity and enantioselectivity. An optimal temperature of 60 degrees C, based on the initial rate of (S)-thioester and a high enantiomeric ratio (i.e., E-value defined as the ratio of initial rates for both substrates) of >100 at 45 degrees C in isooctane, is obtained. Kinetic analysis, considering product inhibition and enzyme deactivation, is also performed, showing agreement between the experimental and best-fit conversions for (S)-thioester. A comparison of the kinetic and thermodynamic behaviors of CPL and Candida rugosa lipase (CRL) in isooctane and cyclohexane indicates that both lipases are very similar in terms of thermodynamic parameters DeltaDeltaH and DeltaDeltaS, initial rate of (S)-substrate, and E-value when (R,S)-naproxen 2,2,2-trifluoroethyl thioester or ester is employed as substrate.     

Surfactant-protease complex as a novel biocatalyst for peptide synthesis in hydrophilic organic solvents*

The peptide synthesis from N-acetyl-L-phenylalanine ethyl ester with alaninamide catalyzed by a surfactant-protease complex has been performed in anhydrous hydrophilic organic solvents. Proteases derived from various sources were converted to surfactant-coated complexes with a nonionic surfactant. The surfactant-subtilisin Carlsberg (STC) complex had a higher enzymatic activity than the other protease complexes and the initial reaction rate in tert-amyl alcohol was 26-fold that of STC lyophilized from an optimum aqueous buffer solution. Native STC hardly catalyzed the same reaction. The addition of water to the reaction medium activated the lyophilized STC, however, the reaction rate was much lower than that of the STC complex, and a hydrolysis reaction preferentially proceeded. The STC complex exhibited a high catalytic activity in hydrophilic organic solvents (e.g. tertiary alcohol). The addition of dimethylformamide as a cosolvent improved the solubility of amino acid amides and further activated the STC complex due to the water mimicking effect. When hydrophilic amino acid amides were employed as an acyl acceptor, the peptide formation proceeded efficiently compared to that using hydrophobic substrates. The surfactant-STC complex is a powerful biocatalyst for peptide synthesis because the STC complexes display a high catalytic activity in anhydrous hydrophilic organic solvents and did not require the excess amount of water. Thus the side (hydrolysis) reaction is effectively suppressed and the yield in the dipeptide formation is considerably high.     

Lipase‐catalyzed enantioselective transesterification of prochiral 1‐((1,3‐dihydroxypropan‐2‐yloxy)methyl)‐5,6,7,8‐tetrahydroquinazoline‐2,4(1H,3H)‐dione in ionic liquids

The application of ionic liquids as solvents for transesterification of prochiral pirymidine acyclonucleoside using lipase (EC 3.1.1.3) Amano PS from Burkholderia cepacia (BCL) is reported. The effect of using medium reaction, acyl group donor, and temperature on the activity and enantioselectivity of BCL was studied. From the investigated ionic solvents, the hydrophobic ionic liquid [BMIM]PF₆] was the preferred medium for enzymatic reactions. However, the best result was obtained in the mixture [BMIM][PF₆]:TBME (1:1 v/v) at 50°C. Enzyme activity and selectivity in [BMIM][PF₆]:TBME (1:1 v/v) was slightly higher in than in conventional organic solvents (for example, TBME), and in this condition, good activity and enantioselectivity were associated with unique properties of ionic liquid such as hydrophobicity and high polarity. Independently of solvents, monester of (R)‐configuration was obtained in excess. Under optimal conditions, desymmetrization of the prochiral compound using different acyl donors was performed. If vinyl butyrate was used as the acylating agent, BCL completely selectively acylated enantiotopic hydroxyl groups.     

Efficient kinetic resolution of (R,S)-1-trimethylsilylethanol via lipase-mediated enantioselective acylation in ionic liquids

Efficient enantioselective acylation of (R,S)-1-trimethylsilylethanol {(R,S)-1-TMSE} with vinyl acetate catalyzed by immobilized lipase from Candida antarctica B (i.e., Novozym 435) was successfully conducted in ionic liquids (ILs). A remarkable enhancement in the initial rate and the enantioselectivity of the acylation was observed by using ILs as the reaction media when compared to the organic solvents tested. Also, the activity, enantioselectivity, and thermostability of Novozym 435 increased with increasing hydrophobicity of ILs. Of the six ILs examined, the IL C4MIm.PF6 gave the fastest initial rate and the highest enantioselectivity, and was consequently chosen as the favorable medium for the reaction. The optimal molar ratio of vinyl acetate to (R,S)-1-TMSE, water activity, and reaction temperature range were 4:1, 0.75, and 40 -50 degrees C, respectively, under which the initial rate and the enantioselectivity (E value) were 27.6 mM/h and 149, respectively. After a reaction time of 6 h, the ee of the remaining (S)-1-TMSE reached 97.1% at the substrate conversion of 50.7%. Additionally, Novozym 435 was effectively recycled and reused in C4MIm.PF6 for five consecutive runs without substantial lose in activity and enantioselectivity. The preparative scale kinetic resolution of (R,S)-1-TMSE in C4MIm.PF6 is shown to be very promising and useful for the industrial production of enantiopure (S)-1-TMSE.     

Enantioselective lipase-catalyzed kinetic resolution of N-(2-ethyl-6-methylphenyl)alanine

The enzyme (BSL2), a highly active lipase expressed from newly constructed strain of Bacillus subtilis BSL2, is used in the kinetic resolution of N-(2-ethyl-6-methylphenyl)alanine from the corresponding racemic methyl ester. Reaction conditions are optimized to enhance the enantioselectivity. The effects of various racemic alkyl esters, substrate concentration, operating temperature, pH of the aqueous medium and organic solvents on activity and enantioselectivity of BSL2 for kinetic resolution are also studied. A high enantiomeric ratio (E = 60.7) is reached in diisopropyl ether/water (10%, v/v) and the enantioselectivity is about 22-fold higher than that in pure buffered aqueous solution. The results show that the reaction medium greatly influences BSL2 reaction and its enantioselectivity in the hydrolysis of racemic methyl ester.