Efficient Production of (S)-Naproxen with (R)-Substrate Recycling Using an Overexpressed Carboxylesterase BsE-NP01

An (S)-enantioselective esterase from Bacillus subtilis ECU0554, named BsE-NP01, has been cloned and over-expressed in a heterologous host Escherichia coli BL21. BsE-NP01 was shown to be a carboxylesterase with a molecular mass of about 32 kDa, and temperature and pH optima at 50 °C and 8.5, respectively. It could catalyze the selective hydrolysis of the (S)-enantiomer of racemic naproxen methyl ester, giving optically pure (S)-naproxen with 98% enantiomeric excess. A mechanic-grinding approach to substrate dispersion was also reported, which was considered to be an alternative to take the place of deleterious surfactants such as Tween-80, with improved performance of the hydrolysis reaction. Batch production of (S)-naproxen was repeatedly carried out in a solid-water biphasic system at 2-L scale, achieving an average total yield of about 85% after ten runs with complete recycling of (R)-substrate.

Equilibrium studies on reactive extraction of naproxen enantiomers using hydrophilic ��-cyclodetrin derivatives extractants

A simple and low-cost method using liquid?Cliquid extraction coupled with complexation reactive technique has been developed for enantioselective separation of naproxen enantiomers. Three kinds of modified ??-cyclodextrins including methyl-??-cyclodextrin (Me-??-CD), hydroxyethyl-??-cyclodextrin (HE-??-CD) and hydroxypropyl-??-cyclodextrin (HP-??-CD), were selected as hydrophilic chiral selectors for extraction naproxen from organic phase to aqueous phase. A systematic study of the factors affecting chiral separation performance were investigated. The experiment results obtained show that, HP-??-CD, HE-??-CD and Me-??-CD has stronger recognition abilities for S-naproxen than those for R-naproxen. Among the ??-CD derivatives studied, HP-??-CD has the strongest ability for chiral recognition and separation. Excellent enantioselectivity (a) of 1.59 is obtained under the optimal conditions of pH of 2.5 and temperature of 5 °C.     

Lipase-catalyzed synthesis of (S)-naproxen ester prodrug by transesterification in organic solvents

A lipase-catalyzed enantioselective transesterification process was developed for the synthesis of (S)-naproxen 2-N-morpholinoethyl ester prodrug from racemic 2,2,2-trifluoroethyl naproxen ester in organic solvents. By selecting isooctane and 37°C as the best solvent and temperature, the apparent fits of the initial conversion rates for transesterification and hydrolysis side reaction suggest a ping-pong Bi-Bi enzymatic mechanism with the alcohol as a competitive enzyme inhibitor. Improvements in the initial conversion rate and the productivity for the desired (S)-ester product were obtained after comparing with the result of an enantioselective esterification process. Studies of water content in isooctane and alcohol containing various N,N-dialkylamino groups on the enzyme activity and enantioselectivity, as well as the recovery of (S)-ester product by using extraction, were also reported.     

Enhancement of the Activity and Enantioselectivity of Lipase by Sol–Gel Encapsulation Immobilization onto β-cyclodextrin-Based Polymer

Candida rugosa lipase was encapsulated within a chemically inert sol–gel support prepared by polycondensation with tetraethoxysilane and octyltriethoxysilane in the presence of β-cyclodextrin-based polymer. The catalytic activity of the encapsulated lipases was evaluated both in the hydrolysis of p-nitrophenylpalmitate and the enantioselective hydrolysis of racemic Naproxen methyl ester. It has been observed that the percent activity yield of the encapsulated lipase was 65 U/g, which is 7.5 times higher than that of the covalently immobilized lipase. The β-cyclodextrin-based encapsulated lipases had higher conversion and enantioselectivity compared with covalently immobilized lipase. The study confirms an excellent enantioselectivity (E >300) for the encapsulated lipase with an enantiomeric excess value of 98% for S-naproxen.     

Measurement and Identification of (R)- and (S)-6-O-Desmethylnaproxen Enantiomers as Diastereomeric O-Ethoxycarbonyl/(R)-(+)-1-Phenylethylamides in Urine by GC and GC-MS

Enantioseparations of (R)- and (S)-6-O-desmethylnaproxens as O-ethoxycarbonyl/(R)-(+)-1-phenylethylamides and (R)- and (S)-naproxens as (R)-(+)-1-phenylethylamides were achieved by achiral gas chromatography in a single run within 11 min. The characteristic mass spectral patterns facilitated easier peak identification. The method for determinations of (R)-naproxen (1.0 to 50 ng) and (S)-naproxen (100 to 2000 ng) was linear with acceptable repeatability and accuracy. The enantiomeric composition ratios between (R)- and (S)-6-O-desmethylnaproxens and between (R)- and (S)-naproxens in urine sample collected six hours after oral administration of (S)-naproxen tablets were measured to be 2.2 (± 0.2):97.8 (± 0.2) and 2.1 (± 0.1):97.9 (± 0.1), respectively.     

Calix[n]arene Carboxylic Acid Derivatives as Regulators of Enzymatic Reactions: Enhanced Enantioselectivity in Lipase-Catalyzed Hydrolysis of (R/S)-Naproxen Methyl Ester

Candida rugosa lipase was immobilized with a sol–gel encapsulation procedure in the presence and absence of a calix[n]arene carboxylic acid derivative grafted onto magnetic nanoparticles or in the presence of the calix[n]arene carboxylic acid derivative with Fe₃O₄ magnetic nanoparticles as an additive. Through the enantioselective hydrolysis of racemic naproxen methyl ester and the hydrolysis of p-nitrophenylpalmitate, the relative enzyme activity was evaluated and tested. These results show that the encapsulated lipase without supports has lower conversion and enantioselectivity compared to the Calix[n]COOH-based encapsulated lipase. It has also been observed that the Calix[4]COOH-based encapsulated lipase has excellent enantioselectivity (enantiomeric ratio (E)?>?400) as compared to encapsulated-free lipase enantioselectivity (E?=?137), and it also has an enantiomeric excess value of ~98 % for S-naproxen.     

‘Gelozymes’ in organic synthesis. Part 2: Candida rugosa lipase mediated synthesis of enantiomerically pure (S)-cyano(3-phenoxyphenyl)methyl butyrate

Significant changes in enantioselectivity (E) have been observed when the butanoate ester of (±)-1-hydroxy-1-(3-phenoxyphenyl)acetonitrile was subjected to hydrolysis in acetate buffer (pH 4.5, E=6.4) and alcoholysis with 1-butanol in hexane catalysed by Candida rugosa lipase (E=45). Enantiomerically pure (S)-butanoate ester so obtained (e.e. 98.4%) was converted to the corresponding (S)-cyanohydrin using Pseudomonas cepacia (Amano Ps) gelozyme. This strategy overcomes the problem of separation of the unwanted (R)-ester from the (S)-cyanohydrin.     

Hydrolase-catalyzed preparation of (R)- and (S)-4-hydroxy-2,6,6-trimethyl-2-cyclohexen-1-ones (phorenols), the key synthetic intermediates for abscisic acid

Preparation of both the enantiomers of 4-hydroxy-2,6,6-trimethyl-2-cyclohexen-1-one (phorenol), which are versatile synthetic intermediates for abscisic acid and carotenoids, was achieved by hydrolase-catalyzed hydrolysis of the corresponding chloroacetate. The hydrolysis with esterase SNSM-87 (Nagase) enriched the (S)-ester, while lipase P (Amano) afforded the (R)-ester.     

Efficient resolution of profen ethyl ester racemates by engineered Yarrowia lipolytica Lip2p lipase

Enzyme-catalyzed enantiomer discrimination is still a great challenge for the development of industrial pharmaceutical processes. For the resolution of ibuprofen, naproxen and ketoprofen racemates, three major anti-inflammatory drugs, only lipases from Candida rugosa present a high selectivity if solvent and surfactant use is discarded. However, their catalytic activities are too low. In the present work, we demonstrate that the lipase Lip2p from the yeast Yarrowia lipolytica has a higher catalytic activity than C. rugosa lipases to hydrolyze the ethyl esters of ibuprofen, naproxen and ketoprofen, but its selectivity is not sufficient [E = 52 (S); 11 (S) and 1.5 (R) respectively]. The enantioselectivity was further improved by site-directed mutagenesis, targeted at the substrate binding site and guided by molecular modelling studies. By investigating the binding modes of the (R)- and (S)-enantiomers in the active site, two amino acid residues located in the hydrophobic substrate binding site of the lipase, namely residues 232 and 235, were identified as crucial for enantiomer discrimination and enzyme activity. The (S) enantioselectivity of Lip2p towards ethyl ibuprofen esters was rendered infinite (E ? 300) by replacing V232 by an A or C residue. Substitution of V235 by C, M, S, or T amino acids led to a great increase in the (S)-enantioselectivity (E ? 300) towards naproxen ethyl ester. Finally, the variant V232F enabled the efficient kinetic resolution of ethyl ketoprofen ester enantiomers [(R)-enantiopreference; E ? 300]. In addition to the increase in selectivity, a remarkable increase in velocity by 2.6, 2.7 and 2.5 times, respectively, was found for ibuprofen, naproxen and ketoprofen ethyl esters.     

The synthesis of (R)- and (S)-α-trifluoromethyl-α-hydroxycarboxylic acids via enzymatic resolutions

Kinetic resolution of 1,1,1-trifluoro-2-alkanone cyanohydrin acyl derivatives with Candida rugosa lipase afforded the remaining (R)-enantiomer in high selectivity (E from 30 to >200). Candida rugosa lipases from several suppliers were compared and found to differ remarkably in their selectivity. The (R)-enantiomer was hydrolyzed in one step to yield optically pure (R)-α-trifluoromethyl-α-hydroxycarboxylic acids in excellent yield. The (S)-acids were obtained in good e.e. by subtilisin-catalyzed resolution of the corresponding racemic esters followed by chemical hydrolysis of the remaining (S)-esters.     

A comparative study on the acylative kinetic resolution of racemic fluorinated and non-fluorinated 2-methyl-1,2,3,4-tetrahydroquinolines and 3,4-dihydro-3-methyl-2H-[1,4]benzoxazines

The acylative kinetic resolution of racemic 6-fluoro-2-methyl-1,2,3,4-tetrahydroquinoline, 7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine, and their non-fluorinated analogues with (S)-naproxen and N-phthaloyl-(S)-amino acyl chlorides has been carried out. It has been shown that the presence of fluorine atoms in the aromatic fragment of a heterocyclic amine results in the increasing stereoselectivity of acylation with (S)-naproxen acyl chloride and in a decrease in the efficiency of acylative kinetic resolution using N-phthaloyl-(S)-amino acyl chlorides. A method for the preparation of enantiopure (S)-6-fluoro-2-methyl-1,2,3,4-tetrahydroquinoline (ee >99%) was developed.     

Kinetic resolution of poly(ethylene glycol)-supported carbonates by enzymatic hydrolysis

The enzyme-mediated enantioselective hydrolysis of poly(ethylene glycol) (PEG)-supported carbonates is disclosed. The water-soluble carbonates were prepared by immobilization of a racemic secondary alcohol (4-benzyloxy-2-butanol) onto low-molecular weight (av MW 550 and 750) monomethoxy PEG through a carbonate linker. For the screening of the hydrolytic enzymes, the substrate was enantioselectively hydrolyzed by commercially available lipase from porcine pancreas (PPL; Type II, Sigma) to afford the optically active compounds. In this system, the separation of the remaining (S)-substrate and the resulting (R)-alcohol was achieved by an extraction process without a laborious column chromatography. The (S)-carbonate was easily hydrolyzed with K₂CO₃ to afford the corresponding (S)-alcohol. Other MPEG-supported substrates were also hydrolyzed to afford the corresponding optically active alcohols.     

Preparation of New Calix[4]arene-Immobilized Biopolymers for Enhancing Catalytic Properties of Candida rugosa Lipase by Sol–Gel Encapsulation

The article describes preparation of new calixarene biopolymers consisting of the immobilization of convenience calixarene derivative onto cellulose and chitosan biopolymers, and the encapsulation of these calixarene biopolymers with Candida rugosa lipase within a chemical inert sol–gel supported by polycondensation with tetraethoxysilane and octyltriethoxysilane. The catalytic properties of immobilized lipase were evaluated into model reactions employing the hydrolysis of p-nitrophenylpalmitate and the enantioselective hydrolysis of naproxen methyl esters from racemic prodrugs in aqueous buffer solution/isooctane reaction system. The resolution studies using sol–gel support have observed more improvement in the enantioselectivity of naproxen E?=?300 with Cel-Calix-E than with encapsulated lipase without calixarene-based materials. Furthermore, the encapsulated lipase (Cel-Calix-E) was still retained about 39 % of their conversion ratios after the fifth reuse in the enantioselective reaction.     

Tuning Immobilized Commercial Lipase Preparations Features by Simple Treatment with Metallic Phosphate Salts

Four commercial immobilized lipases biocatalysts have been submitted to modifications with different metal (zinc, cobalt or copper) phosphates to check the effects of this modification on enzyme features. The lipase preparations were Lipozyme^®TL (TLL-IM) (lipase from Thermomyces lanuginose), Lipozyme^®435 (L435) (lipase B from Candida antarctica), Lipozyme^®RM (RML-IM), and LipuraSelect (LS-IM) (both from lipase from Rhizomucor miehei). The modifications greatly altered enzyme specificity, increasing the activity versus some substrates (e.g., TLL-IM modified with zinc phosphate in hydrolysis of triacetin) while decreasing the activity versus other substrates (the same preparation in activity versus R- or S- methyl mandelate). Enantiospecificity was also drastically altered after these modifications, e.g., LS-IM increased the activity versus the R isomer while decreasing the activity versus the S isomer when treated with copper phosphate. Regarding the enzyme stability, it was significantly improved using octyl-agarose-lipases. Using all these commercial biocatalysts, no significant positive effects were found; in fact, a decrease in enzyme stability was usually detected. The results point towards the possibility of a battery of biocatalysts, including many different metal phosphates and immobilization protocols, being a good opportunity to tune enzyme features, increasing the possibilities of having biocatalysts that may be suitable for a specific process.     

‘Easy-on,easy-off’ resolution of chiral 1-phenylethylamine catalyzed by Candida antarctica lipase B

An ‘easy-on, easy-off’ process for the effective resolution of (±)-1-phenylethylamine was designed using the lipase B of Candida antarctica. This two step lipase-catalyzed process for the resolution of a chiral arylalkylamine involves a high-conversion enantioselective condensation of (R)-(+)-1-phenylethylamine with capric acid (conversion 99%, <24 h), followed by the hydrolysis of the corresponding synthesized (R)-(+)-amide (conversion 98%, 48 h). As a result, this efficient enzymatic process yields both (R)- and (S)-enantiomers of 1-phenylethylamine in high enantiomeric purity.     

A new enzymatic strategy for the preparation of (2R,3S)-3-phenylisoserine: a key intermediate for the Taxol side chain

Burkholderia cepacia lipase PS-IM catalysed the hydrolysis of racemic ethyl 3-amino-3-phenyl-2-hydroxypropionate with excellent enantioselectivity (E >200), when the reaction was performed with added H₂O as a nucleophile, in iPr₂O, at 50 °C. The hydrolysis of the less reactive enantiomeric ethyl 3-amino-3-phenyl-2-hydroxypropionate with 18% HCl afforded the corresponding enantiomerically pure (2R,3S)-3-amino-3-phenyl-2-hydroxypropionic acid hydrochloride, a key intermediate for the Taxol side chain.     

Improvement of catalytic activity of lipase in the presence of wide rim substituted calix[4]arene carboxylic acid-grafted magnetic nanoparticles

Candida rugosa lipase immobilized on calix[4]arene carboxylic acid-grafted magnetic nanoparticles using a sol–gel encapsulation technique was tested for activity, which was assessed both in the enantioselective hydrolysis of racemic Naproxen methyl ester and that of p-nitrophenylpalmitate. It has also been noticed that, compared to the free enzyme (E = 137) with an ee value of >98 %, S-Naproxen calix[4]arene carboxylic acid-grafted magnetic nanoparticles based on encapsulated lipase (Calix-1-MN and Calix-2-MN) offer excellent enantioselectivity (E = 373 and E = 381). Moreover, the results indicated that after the fifth reuse in the enantioselective reaction, the encapsulated lipase (Calix-2-MN) still retained about 43 % of its conversion power.     

Enzymatic resolution of hindered cyanohydrins,key precursors of muscarinic receptor antagonists

Enantiomerically pure 1-cycloalkyl-1-hydroxy-1-phenylcyanohydrins, key precursors in the synthesis of (S)-oxybutynin and other antimuscarinic agents, have been successfully prepared via lipase-catalyzed kinetic resolutions. Pseudomonas cepacia and Candida antarctica lipase B have shown excellent enantioselectivities in hydrolysis and acylation processes, depending on the substrate structure and the reaction conditions. Furthermore, molecular modeling of phosphonate transition state analogue for the C. antarctica lipase B enzymatic hydrolysis resolution step supports these facts, which were experimentally observed.     

Porcine pancreatic lipase mediated regio- and stereoselective hydrolysis: chemoenzymatic synthesis of (2S,3S)-2-amino-3,4-dihydroxybutyric acid

Porcine pancreatic lipase was used in the chemoenzymatic hydrolysis of 2-azido-3-hydroxy-4-methylcarbonyloxybutyl acetate. The reaction occurred with high regio- and stereoselectivity to give enantiomerically pure (2S,3R)-3-azido-2,4-dihydroxy butyl acetate 5 (e.e. >99%) which was easily converted to (2S,3S)-2-amino-3,4-dihydroxybutyric acid 1, an important synthetic intermediate in the synthesis of β-lactam antibiotics and phytosiderophores.     

Enantioselective protonation of prochiral enolates in the asymmetric synthesis of (S)-naproxen

Racemic methyl, iso-propyl, and tert-butyl ester derivatives of naproxen were treated with achiral LDA base to give the corresponding prochiral enolates 2-Li, 3-Li, and 4-Li. Protonation of these enolates with novel chiral proton sources (S)-10 and (S,S)-11, containing the α-phenylethylamino group, proceeded in a highly enantioselective manner. Saponification of enantioenriched ester derivatives 2-4 afforded naproxen, (S)-1, with no loss of enantiopurity.