- Efficient resolution of profen ethyl ester racemates by engineered Yarrowia lipolytica Lip2p lipase
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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.
- Gérard, Doriane,Guéroult, Marc,Casas-Godoy, Leticia,Condoret, Jean-Stéphane,André, Isabelle,Marty, Alain,Duquesne, Sophie
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p. 433 - 441
(2017/03/24)
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- Biocatalyzed irreversible esterification in the preparation of S-naproxen
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Dimethyl carbonate was used as alcohol donor in the esterification of naproxen in the presence of immobilised lipase B from Candida antarctica (Novozym 435). The conjugation of hydrolysis of dimethyl carbonate and esterification of acid, created irreversible operative conditions so permitting the recovery of S-naproxen in high ee (>98%). The adopted procedure has a general use.
- Morrone,D'Antona,Lambusta,Nicolosi
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experimental part
p. 49 - 51
(2010/12/19)
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- (R,S)-azolides as novel substrates for lipase-catalyzed hydrolytic resolution in organic solvents
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Azolides, that is, N-acylazoles, as versatile acylation reagents are well characterized in the literature, in which the azole structure can not only act as a better leaving group but also make the carbonyl carbon more electrophilic and susceptible to nucleophilic attack. It is therefore desirable to combine this unique property and lipase resolution ability in the development of a new resolution process for preparing optically pure carboxylic acids. With the Candida antarctica lipase B (CALB)-catalyzed hydrolysis of (R,S)-N- profenylazoles in organic solvents as the model system, (R,S)-N-profenyl-l,2,4- triazoles instead of their corresponding ester analogues were exploited as the best substrates for preparing optically pure profens, i.e., 2-arylpropionic acids. The structure-reactivity correlations for the (R,S)-azolides in water-saturated methyl tert-butyl ether (MTBE) at 45°C coupled with a thorough kinetic analysis were further employed for elucidating the rate-limiting formation of a tetrahedral adduct without C-N bond breaking or with moderate C-N bond breaking concerted with C-O bond formation in the acylation step. The advantages of easy substrate preparation, high enzyme reactivity and enantioselectivity, and easy recovery of the product and remaining substrate by aqueous extraction demonstrate the potential of using (R,S)-azolides as novel substrates for the enzymatic resolution process.
- Wang, Pei-Yun,Chen, Ying-Ju,Wu, An-Chi,Lin, Yi-Sheng,Kao, Min-Fang,Chen, Jin-Ru,Ciou, Jyun-Fen,Tsai, Shau-Wei
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supporting information; scheme or table
p. 2333 - 2341
(2009/12/27)
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- Influence of {single bond}OR ester group length on the catalytic activity and enantioselectivity of free lipase and immobilized in membrane used for the kinetic resolution of naproxen esters
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Lipases are suitable catalysts for the kinetic resolution of racemic mixtures due to their ability to discriminate between enantiomers. For this reason, they have been studied largely to develop reactors for the production of optically pure enantiomers. The main problem in these productive systems is enzyme stability and enantiocatalytic selectivity as a function of time. In this work, the enantiocatalytic properties of lipase as a function of the {single bond}OR group length were studied. The methyl, butyl, and octyl esters of naproxen were synthesized and used as reagents. The lipase was used as a free agent and immobilized in a polymeric membrane reactor. The results show that selectivity and stability of the enzyme improved while catalytic activity decreased with the {single bond}OR length group. The immobilized enzyme had higher activity compared with the free enzyme.
- Giorno,D'Amore,Drioli,Cassano,Picci
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p. 194 - 200
(2008/03/12)
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- Enzymatic resolution of naproxen
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Trichosporon sp. (TSL), a newly found strain isolated from a locally fermented cottage cheese has been found to be highly stereoselective in the resolution of (S)-(+)-naproxen (ee >99%, E~500) from the corresponding racemic methyl ester. The process of resolution using whole cells has been scaled up to multi-kg level. Optimization of experimental conditions including downstream processing at 80-100 g/L substrate concentration with >90% recovery has been achieved. Changes in the physical parameters such as the particle size of the substrate play an important role in the resolution kinetics. A new strain of Trichosporon sp. having high cell density in cultivation (>60 g dry cell mass L-1 in 14-16 h) is found to be sufficiently stable for two years in dry powder form at 5-8°C. The viability of the resolution process has been further improved by the development of a simple racemization process for the enriched (R)-(-)-ester.
- Koul, Surrinder,Parshad, Rajinder,Taneja, Subhash C.,Qazi, Ghulam N.
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p. 2459 - 2465
(2007/10/03)
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- Process for preparing naproxen
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Process for preparing Naproxen via a sequence of stereospecific reactions which comprises: condensation of 1-chloro-2-methoxy-naphthalene with a (S)-2-halo-propionyl halide according to the Friedel-Crafts reaction, ketalization of the thus obtained (S)-2-halo-1-(5'-chloro-6'-methoxy-2'-naphthyl)-propan-1-one, rearrangement of said ketal to afford an ester, hydrolysis of the ester and removal of the chlorine atom in 5-position by hydrogenolysis. New (S) 2-halo-1-(5'-chloro-6'-methoxy-2'-naphthyl)-propan-1-ones.
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- ESTERS OF ARYLPROPIONIC ACIDS WITH 1,2:5,6-DI-O-ISOPROPYLIDENE- AND 1,2-O-ISOPROLYLIDENE-α-D-GLUCOFURANOSE
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On fractional crystallization of 3-O-(2-(2-fluoro-4-biphenylyl)propionyl-, 3-O-(2-(4-isobutylphenyl)propionyl)- and 3-O-(2-(6-methoxy-2-naphthyl)propionyl)-1,2:5,6-di-O-isopropylidene-α-D-glucofuranoses V - VII optically pure R-diastereoisomers were isolated.The derivatives of 1,2-O-isopropylidene-α-D-glucofuranose obtained on partial deacetylation of esters V - VII were separated chromatographically to R and S-diastereoisomers.Their hydrolysis or transesterification afforded optically pure arylpropionic acids or their methyl esters, respectively.Kinetic resolutionof the acids gives rise to esters V - VII enriched in R-diastereoisomer.
- Svoboda, Jiri,Capek, Karel,Palecek, Jaroslav
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p. 766 - 774
(2007/10/02)
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