Biocatalyst engineering exerts a dramatic effect on selectivity of hydrolysis catalyzed by immobilized lipases in aqueous medium

It has been found that enantioselectivity of lipases is strongly modified when their immobilization is performed by involving different areas of the enzyme surface, by promoting a different degree of multipoint covalent immobilization or by creating different environments surrounding different enzyme areas. Moreover, selectivity of some immobilized enzyme molecules was much more modulated by the experimental conditions than other derivatives. Thus, some immobilized derivatives of Candida rugosa (CRL) and C. antarctica-B (CABL) lipases are hardly enantioselective in the hydrolysis of chiral esters of (R,S)-mandelic acid under standard conditions (pH 7.0 and 25°C) (E<2). However, other derivatives of the same enzymes exhibited a very good enantioselectivity under nonstandard conditions. For example, CRL adsorbed on PEI-coated supports showed a very high enantio-preference towards S-isomer (E=200) at pH 5. On the other hand, CABL adsorbed on octyl-agarose showed an interesting enantio-preference towards the R-isomer (E=25) at pH 5 and 4°C. These biotransformations are catalyzed by isolated lipase molecules acting on fully soluble substrates and in the absence of interfacial activation against external hydrophobic interfaces. Under these conditions, lipase catalysis may be associated to important conformational changes that can be strongly modulated via biocatalyst and biotransformation engineering. In this way, selective biotransformations catalyzed by immobilized lipases in macro-aqueous systems can be easily modulated by designing different immobilized derivatives and reaction conditions.     

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.     

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.     

Lipase-catalyzed enantioselective hydrolysis of methyl 2-fluoro-2-arylpropionates in water-saturated isooctane

Lipases from Candida rugosa, Candida antartica B and Carica papaya are employed as the biocatalyst for the hydrolytic resolution of methyl 2-fluoro-2-arylpropionates in water-saturated isooctane, in which excellent to good enantioselectivity without the formation of byproducts is obtained for the papaya lipase when using (R,S)-2-fluoronaproxen methyl ester (1) and methyl (R,S)-2-fluoro-2-(4-methoxyphenyl)propionate (2), but not methyl (R,S)-2-fluoro-2-(naphth-1-yl)propionate (3) as the substrates. The thermodynamic analysis indicates that the enantiomer discrimination for the papaya lipase is driven by the difference in activation enthalpy for compound 1, 2 or (R,S)-naproxen methyl ester (4). The kinetic analysis also demonstrates that in comparison with (S)-4, the insertion of the 2-fluorine moiety in (R)-1 has increased k₂, but not K_m, and consequently the lipase activity.     

Bioreduction of aromatic ketones: preparation of chiral benzyl alcohols in both enantiomeric forms

Substituted phenacyl chlorides are reduced with whole-cell biocatalysts to give (R)- or (S)-chlorohydrines in high yields and to make them good for high enantiomeric excess. Yields and enantiomeric purity of the S-enantiomer could be increased by performing bioreduction in the presence of polymeric absorbing resins. With this methodology, 2-chloro-1(S)-(3,4-dichloro-phenyl)-ethanol of 98% e.e. and 2-(R)-(4-nitro-phenyl)-ethanol of 92% e.e. have been prepared and used respectively as precursors in the synthesis of (+)-cis-1(S),4(S)-sertraline and of the β-blocker (R)-nifenalol®.     

Modulation of Mucor miehei lipase properties via directed immobilization on different hetero-functional epoxy resins: Hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid

Purified lipase from Mucor miehei (MML) has been covalently immobilized on different epoxy resins (standard hydrophobic epoxy resins, epoxy-ethylenediamine, epoxy-iminodiacetic acid, epoxy-copper chelates) and adsorbed via interfacial activation on octadecyl-Sepabeads support (fully coated with very hydrophobic octadecyl groups). These immobilized enzyme preparations were used under slightly different conditions (temperature ranging from 4 to 25 °C and pH values from 5 to 7) in the hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid.

Different catalytic properties (activity, specificity, enantioselectivity) were found depending on the particular support used. For example, the epoxy-iminodiacetic acid-Sepabeads gave the most active preparation at pH 7 while, at pH 5, the ethylenediamine-Sepabeads was superior.

More interestingly, the enantiomeric ratio (E) also depends strongly on the immobilized preparation and the conditions employed. Thus, the octadecyl-MML preparation was the only immobilized enzyme derivative which exhibited enantioselectivity towards R isomer (with E values ranging from 5 at 4 °C and pH 7 to 1.2 at pH 5 and 25 °C).

The other immobilized preparations, in contrast, were S selective. Immobilization on iminodiacetic acid-Sepabeads afforded the catalyst with the highest enantioselectivity (E=59 under optimum conditions).     

酵母脂肪酶分子进化及催化合成(S)-1,4-苯并二口恶烷*

Objective: 1,4-Benzodioxane is an important chiral intermediate for antihypertensive (Proroxan and Doxazosin), antidepressant (MCK-242) and other drugs, and it displays a broad spectrum of applications in the pharmaceutical field. Currently, in spite of high-yield advantage of chemical synthesis, there are some problems of environmental pollution and low production safety. Using lipase to catalyze synthesis of 1,4-benzodioxane provides a new pathway of green synthesis of 1,4-benzodioxane. However, natural enzymes face the dilemma of poor enantioselectivity. Therefore, molecular evolution was performed on Candida antarctica lipase B, and a technical route for the catalytic synthesis of 1,4-benzodioxane was established. Methods: Firstly, the key amino acid residues involved in substrate binding and conversion in the active center of Candida antarctica lipase B were analyzed, and saturation mutagenesis libraries on the interaction sites were constructed. Improved mutants with high efficiency and high enantioselectivity were then obtained using HPLC detection. Furthermore, catalytic synthesis conditions of mutant D223N/A225K were systematically optimized. Results: The results indicated that the mutants mainly derived from the pairwise site D223/A225 (such as D223N/A225K and D223G/A225W) were biased towards the synthesis of (S)-isoforms, while most of the mutants derived from the pairwise site E188/I189 (such as E188D/I189M) showed a bias for the synthesis of (R)-isoforms. Compared with WT, the ee_s value of the best mutant D223N/A225K to synthesize (S)-1,4-benzodioxane was increased from 11.9% to 29.3%. After systematic optimization of the reaction conditions, an ee_s value of (93.9±0.16)% and a conversion rate of (47.5±2.33)% were achieved using mutant D223N/A225K to catalyze kinetic resolution of methyl (R,S)-2,3-dihydro-1,4-benzodioxin-2-carboxylate in n-butanol/phosphate buffered saline (20∶80, V/V) biphasic solvent at 37℃ for 50 min. Conclusion: An efficient kinetic resolution of methyl (R,S)-2,3-dihydro-1,4-benzodioxin-2-carboxylate was successfully achieved by molecular evolution and optimization of conditions, which provides a new example for the creation of new enzymes by protein engineering technology, and also provides a theoretical and technical foundation for the efficient synthesis of (S)-1,4-benzodioxane molecules by enzymatic methods.     

Novel microbial epoxide hydrolases for biohydrolysis of glycidyl derivatives

Microbial isolates from biofilters and petroleum-polluted bioremediation sites were screened for the presence of enantioselective epoxide hydrolases active towards tert-butyl glycidyl ether, benzyl glycidyl ether, and allyl glycidyl ether. Out of 270 isolated strains, which comprised bacteria, yeasts, and filamentous fungi, four were selected based on the enantioselectivities of their epoxide hydrolases determined in biotransformation reactions. The enzyme of Aspergillus niger M200 preferentially hydrolyses (S)-tert-butyl glycidyl ether to (S)-3-tert-butoxy-1,2-propanediol with a relatively high enantioselectivity (the enantiomeric ratio E is about 30 at a reaction temperature of 28 °C). Epoxide hydrolases of Rhodotorula mucilaginosa M002 and Rhodococcus fascians M022 hydrolyse benzyl glycidyl ether with relatively low enantioselectivities, the former reacting predominantly with the (S)-enantiomer, the latter preferring the (R)-enantiomer. Enzymatic hydrolysis of allyl glycidyl ether by Cryptococcus laurentii M001 proceeds with low enantioselectivity (E = 3). (R)-tert-Butyl glycidyl ether with an enantiomeric excess (ee) of over 99%, and (S)-3-tert-butoxy-1,2-propanediol with an ee-value of 86% have been prepared on a gram-scale using whole cells of A. niger M200. An enantiomeric ratio of approximately 100 has been determined under optimised biotransformation conditions with the partially purified epoxide hydrolase from A. niger M200. The regioselectivity of this enzyme was determined to be total for both (S)-tert-butyl glycidyl ether and (R)-tert-butyl glycidyl ether.     

Enantioselective cleavage of activated amino acid esters promoted by chiral palladacycles

The ester cleavage of R- and S-isomers N-CBZ-leucine p-nitrophenyl ester intermolecularly catalyzed by R- (a) and S-stereoisomers (b) of the Pd(II) metallacycle [Pd(C₆H₄C^*HMeNMe₂)Cl(py)] (3) follows the rate expression k_obs = k_o + k_cat [3], where the rate constants k_cat equal 25.8 ± 0.4 and 7.6 ± 0.5 dm³ mol⁻¹ s⁻¹ for the S- and R-ester, respectively, in the case of 3a, but are 5.7 ± 0.6 and 26.7 ± 0.5 dm³ mol⁻¹ s⁻¹ for the S- and R-ester, respectively, in the case of 3b (pH 6.23 and 25°C). Thus, the best catalysis occurs when the asymmetric carbons of the leucine ester and Pd(II) complex are R and S, or S and R configured, respectively. Molecular modeling suggests that the stereoselection results from the spatial interaction between the CH₂CHMe₂ radical of the ester and the -methyl group of 3. A hydrophobic/stacking contact between the leaving 4-nitrophenolate and the coordinated pyridine also seems to play a role. Less efficient intramolecular enantioselection was observed for the hydrolysis of N-t-BOC-S-metthionine p-nitrophenyl ester with R- and S-enantiomers of [Pd(C₆H₄C*HMeNMe₂)Cl] coordinated to sulfur.     

Resolution of 1,3-dioxolane derivatives using the lipase from an Acinetobacter junii SY-01

Acinetobacter junii SY-01 producing a lipase enantioselectively hydrolyzing 1,3-dioxolane derivatives was isolated from water sludge sample and the effect of solvent, acyl donor, vinyl acetate concentration, substrate concentration, operating temperature and immobilization on activity and enantioselectivity was studied for the resolution of 1,3-dioxolane derivatives through transesterification reaction using a lipase from the isolated strain. Best selectivity was obtained at lower substrate concentration (3–5 mM), higher vinyl acetate concentration (500–1000 mM) and lower temperature (30–40 °C) in the reaction mixture. Lipase immobilized onto Accurel MP-1000 (micro-porous polypropylene) gave the best results and the reactivity was about 29-fold higher than the free enzyme without the decrease of enantioselectivity. Resolution of 1,3-dioxolane derivatives was carried out in flask scale containing 100 ml solvents using the lipase immobilized onto Accurel MP-1000. In this reaction, the yield and enantiomeric excess of the remaining (2R, 4S)-alcohol were 31.2% and 98.2%, respectively. This result suggests that it can be used as an alternative method, compared to the present synthetic method, for the production of optically pure (2R, 4S)-itraconazole.     

Covalent immobilization of pure isoenzymes from lipase of Candida rugosa

Covalent immobilization of pure lipases A and B from Candida rugosa on agarose and silica is described. The immobilization increases the half-life of the biocatalysts ( ) with respect to the native pure lipases ( ). The percentage immobilization of lipases A and B is similar in both supports (33–40%). The remaining activity of the biocatalysts immobilized on agarose (70–75%) is greater than that of the enzymatic derivatives immobilized on SiO₂ (40–50%). The surface area and the hydrophobic/hydrophilic properties of the support control the lipase activity of these derivatives. The thermal stability of the immobilized lipase A derivatives is greater than that of lipase B derivatives. The nature of the support influences the thermal deactivation profile of the immobilized derivatives. The immobilization in agarose (hydrophilic support) gives biocatalysts that show a greater initial specific reaction rate than the biocatalysts immobilized in SiO₂ (hydrophobic support) using the hydrolysis of the esters of (R) or (S) 2-chloropropanoic and of (R,S) 2-phenylpropanoic acids as the reaction test. The enzymatic derivatives are active for at least 196 h under hydrolysis conditions. The stereospecificity of the native and the immobilized enzymes is the same.     

Lipase-catalyzed kinetic resolutions of racemic β- and γ-thiolactones

Several racemic β- and γ-thiolactones were synthesized and kinetic resolutions of them were executed using lipases. While a lipase from Pseudomonas cepacia (PCL) showed the highest enantioselectivity for (S)-form (>99% ee_S at 53% conversion, E > 100) in the kinetic resolution of racemic -methyl-β-propiothiolactone (rac-MPTL), it showed no hydrolysis activity in the kinetic resolution of -benzyl--methyl-β-propiothiolactone (rac-BMPTL), suggesting that the changes in the size of alkyl group from rac-MPTL to rac-BMPTL leads to lower hydrolysis activity and enantioselectivity. In contrast, racemic γ-butyrothiolactones were hydrolyzed by several lipases with low enantioselectivity, whereas a lipase from Candida antarctica (CAL) showed moderate enantioselectivity for (S)-form (>99% ee_S at 76% conversion, E = 11) in the kinetic resolution of racemic -methyl-γ-butyrothiolactone (rac-MBTL). Computer-aided molecular modeling was also performed to investigate the enantioselectivites and activities of PCL toward β-propiothiolactones. The computer modeling results suggest that the alkyl side chains of β-propiothiolactones and γ-butyrothiolactones interact with amino acid residues around hydrophobic crevice, which affects the activity of PCL.     

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).     

Lipase catalysed acylation of hydroxylamine and hydrazine derivatives

The lipase catalysed acylation of hydroxylamine-and hydrazine as well as their derivatives by octanoic acid is very efficient. Cross-linked crystals of Candida rugosa lipase (ChiroCLEC-CR) mediated the conversion of racemic ibuprofen into (S)-ibuproxam. A number of lipases also catalysed the condensation of hydrazine with an excess of octanoic acid giving N,N′-dioctanoylhydrazine. The hydrazide of 2-(4-isobutylphenyl)propanoic acid (ibuprofen), prepared by non-enzymatic reaction of ibuprofen methyl ester with hydrazine, acted as nucleophile towards several lipases that do not accept ibuprofen derivatives as acyl donor.     

Microfluidic separation of (S)-ibuprofen using enzymatic reaction

This study was investigated for the enantioselective separation of (S)-ibuprofen using the ionic liquid in the microfluidic device. A stable and thin ionic liquid flow (ILF) was made by controlling the flow rate of the ILF in the microfluidic channel. In addition, coupling lipase as a biocatalyst with the ILF based on the microfluidic device showed the facilitative and selective transport of (S)-ibuprofen across the ILF, indicating successful optical resolution of a racemic mixture. Subsequently, the enantioselectivity was evaluated in the transport ratio (η) of (R)- and (S)-ibuprofen, the optical resolution ratio () and enantiomeric excess of (S)-ibuprofen (ee_S).     

Selective lipase-catalyzed preparation of diol monobenzoates by transesterification and alcoholysis reactions in organic solvents

Lipases from Mucor miehei (MML) and Candida antarctica (CAL) are able to catalyze the monobenzoylation of the primary hydroxy group of 1,2- 1,4- or 1,5-diols with vinyl benzoate in an organic solvent, the reaction proceeding with high regioselectivity and moderate enantioselectivity. The lipase-catalyzed debenzoylation of 1,2-propanediol dibenzoate by alcoholysis with 1-octanol most satisfactorily occurred with Pseudomonas cepacia lipase absorbed onto celite that allowed also to prepare (R)-1-benzoyloxy-2-methylpropan-3-ol from 2-methyl-1,3-propanediol dibenzoate, a result complementary to MML-catalyzed benzoylation of 2-methyl-1,3-propanediol that affords the (S)-monobenzoate.     

Recognition of a 10 base pair sequence of DNA and stereochemical control of the binding affinity of chiral hairpin polyamide-Hoechst 33258 conjugates

Chiral hairpin polyamides linked to a Hoechst 33258 analogue at the -position of the hairpin turn amino acid (1, 2) were synthesized on solid phase by adopting Fmoc and ivDde techniques. The DNA-binding properties of enantiomeric conjugates 1 and 2, and N-terminal linked conjugate 3 for 8–14 bp sequences were determined by spectrofluorometric and thermal melting studies. Conjugates 1 and 2 recognize a 10 bp sequence, while conjugate 3 recognizes a 9 bp sequence. Interestingly, R-enantiomer 1 exhibited 10- to 30-fold higher binding affinities than S-enantiomer 2 for the DNA sequences studied. These binding differences were accounted for by molecular modeling studies, which revealed that the amide proton nearest to the chiral center in R-conjugate 1 is better positioned to form hydrogen bonds to the DNA bases, while S-conjugate 2 does not.     

Candida antarctica lipase B catalysed amidation of pyroglutamic acid derivatives. A reaction survey

Pyroglutamic acid esters, both (S)- and (R)-enantiomers, have been studied as substrates of the Candida antarctica lipase B catalyzed amidation in anhydrous organic solvents. They behaved as very good substrates when primary amines or ammonia were used as nucleophiles, affording the corresponding secondary and primary amides, respectively, but did not react with secondary amines. The reaction was enantioselective for the (R)-enantiomer of chiral amines although little kinetic difference was observed between (S)- and (R)-pyroglutamates as acyl donors. As an example of an infrequent reaction, free (S)-pyroglutamic acid may also act as a substrate of the reaction, but is much less reactive than its esters.     

Lipase-catalyzed production of optically active (S)-flurbiprofen in aqueous phase reaction system containing chiral succinyl β-cyclodextrin

The lipase-catalyzed production of optically active (S)-flurbiprofen was carried out in a dispersion reaction-system induced by chiral succinyl β-cyclodextrin (suβ-CD). The optimal reaction conditions were 500 mM (R,S)-flurbiprofen ethyl ester ((R,S)-FEE), 600 units of Candida rugosa lipase per 1 mmol of (R,S)-FEE, and 1000 mM suβ-CD at 37 °C for 72 h. An extremely high enantiomeric excess of 0.98 and conversion yield of 0.48 were achieved in the dispersed aqueous phase reaction system containing chiral suβ-CD added as a dispenser and chiral selector. The inclusion complex formability of the immiscible substrate (S)- and (R)-form of FEE with suβ-CD was compared using a phase-solubility diagram, DSC, and ¹H NMR. (S)-Isomer formed a more stable and selective inclusion complex with chiral suβ-CD. It was hydrolyzed much more selectively by lipase from C. rugosa, due to the selective structural modification through inclusion complexation with chiral suβ-CD.     

Absolute configurations of pittosporatobiraside A and B from Pittosporum tobira

The absolute configuration at C-12 of pittosporatobiraside A and B isolated from the leaves of Pittosporum tobira was determined to be S on the basis of the exciton chirality of their dibenzoate derivative. The structures of the two glycosides were thus established to be (1S,9S,10S,11S,12S,14R,16R)-12-[(Z)-2-methyl-1-oxo-2-butenyl]-6,14-dimethyl-2-methylene-9-(1-methylethyl)-15,17-dioxatricyclo[8.7.0.0^11,16]heptadec-5-en-13-one and (1S,9S,10S,11S,12S,14R,16R)-12-(3-methyl-1-oxo-2-butenyl)-6,14-dimethyl-2-methylene-9-(1-methylethyl)-15,17-dioxatricyclo [8.7.0.0^11,16]heptadec-5-en-13-one, respectively.