Amélie Ducret

Lipase-catalyzed enantioselective esterification of ibuprofen in organic solvents under controlled water activity

The enantioselective esterification of ibuprofen (2-(4-iso-butylphenyl)propionic acid), catalyzed by Candida antarctica lipase (type B), was performed in various organic solvents. The reactions were conducted in controlled water activity atmosphere, thereby permitting the influence of the solvents to be separated from their ability to strip water from the solid enzymes. Even in these constant water activity conditions, hydrophobic solvents allow higher enzyme activity than hydrophilic ones, indicating that hydrophilic solvents impede enzyme activity in essence and not because they strip water from the enzyme. The highest enantioselectivity was obtained in solvents leading to low reaction rates. Different properties were used to describe the solvents, namely the hydrophobicity, quantified by log P; ϵ, the dielectric constant; and ETN, a normalized electron acceptance index. None gave a clear and predictive portrait of the influence of the solvent, however, the hydrophobicity was the most satisfactory. This could be linked to the solubility of ibuprofen, which varies linearly with the value of log P. The highest enantioselectivity was observed at low water activities. This is caused by different effects of water activity on the reaction rates with the two isomers. The activity toward the less favored one decreased after reaching a maximum while the rate with the other isomer continued to rise when aW decreased.

Characterization of enzymatically prepared biosurfactants

Various fatty monoesters of sugars and sugar alcohols were prepared enzymatically in organic solvent. Water produced during esterification was removed by refluxing through a dessicant under reduced pressure. Surface properties of these esters such as surface and interfacial tensions and their ability to stabilize emulsions at 30°C were evaluated: oleate esters of glucose, fructose, and sorbitol show similar behavior in reduction of surface and interfacial tensions, and values for the critical micelle concentration are about 8·10−5 M. It was also observed with sorbitol esters that the shorter the alkyl chain, the higher the critical micelle concentration. Generally, emulsions prepared with the emulsifier dissolved in the water or in the oil phase lead to oil-in-water or water-in-oil emulsions, respectively. Sorbitol monolaurate significantly increased the stability of oil-in-water emulsions, with only 5% separation of the phases after 48h at 30°C, compared to 10% for chemically prepared sorbitan monolaurate under the same conditions. Sorbitol monoerucate was very efficient in stabilizing water-in-oil emulsions, with only 1% separation of the phases.

Synthesis of octyl glucopyranoside by almond β-glucosidase adsorbed onto Celite R-640®

Abstract The synthesis of octyl glucoside from p -nitrophenyl glucopyranoside ( p -NPG) and 1-octanol was carried out with almond β-glucosidase adsorbed onto Celite R-640®. The influence of the amount of water added to the system as well as the addition of co-solvents have been studied. The presence of small percentages of DMF has a strong denaturing effect on the enzyme adsorbed onto Celite R-640®. Denaturation is less pronounced with other co-solvents such as acetonitrile, 2-methyl-2-butanol or ethyl acetate.

Enantioselective esterification of racemic ketoprofen in non-aqueous solvent under reduced pressure

The enantioselective esterification of (R,S)-ketoprofen catalyzed by Candida antarctica lipase (type B) has been performed with dodecanol in solvent media under reduced pressure. The nature of the solvent, either aromatic, ether or ketone, affects the activity of the lipase and its enantioselectivity, defined as the ratio of the initial rates of reaction for each enantiomer. Faster reactions are obtained in hydrophobic solvents whereas the enantioselectivity remains constant across all solvents, except for ketones where the enantioselectivity increases with a decrease of the Log P. When the same reaction is performed with 1-propanol in xylenes, an inhibition by the nucleophile is observed, which is not detected when using dodecanol under the same conditions.

Scale-up of the enantioselective reaction for the enzymatic resolution of (R,S)-ibuprofen

The reaction for the resolution of (R,S)-ibuprofen was scaled-up to yield gram quantities of (S)-ibuprofen. This was accomplished through two enantioselective reactions each catalysed by Novozym 435. In the first reaction, starting from 300 g of racemic ibuprofen, 88.9 g of enantioenriched (S)-ibuprofen with an enantiomeric excess of the order of 85% were produced. In the subsequent reaction, 75 g of the 85 % e.e. material were utilized to produce 38.4 g of (S)-ibuprofen with an enantiomeric excess of 97.5 %.

Chiral high performance liquid chromotography resolution of ibuprofen esters

Two cellulose-based chiral stationary phases (Chiralcel OD and Chiralcel OJ) were compared on their ability to resolve various aliphatic ibuprofen esters. Chiralcel OJ with hexane as the mobile phase allows for the separation of most of the esters. Observed changes in resolution depending on the solute nature (basicity of the solute, esterified alcohol chain length, presence of a double bond) are discussed. An example of the application of this method for following the kinetic resolution of racemic ibuprofen is presented.

Enantioselective Esterification of Racemic Ibuprofen in Solvent Media under Reduced Pressure

The enantioselective esterification of (R,S)-ibuprofen has been performed with Novozym 435TM in solvent media under reduced pressure. The nature of the solvent affects the activity and enantioselectivity of the lipase. High esterification rates are obtained in solvents having high hydrophobicity (log P>4). On the other hand, the best enantioselectivity is obtained with solvents having low hydrophobicity (log P<3). This particular enantioselectivity trend is observed within the individual solvent families investigated (alkanes, ethers and aromatics).

Comparison of two HPLC techniques for monitoring enantioselective reactions for the resolution of (R,S)-ibuprofen: Chiral HPLC vs achiral HPLC linked to an optical rotation detector

Two approaches have been used to monitor the enantioselective esterification of (R,S)-ibuprofen with 1-dodecanol catalyzed by Novozym 435. The first approach involves the use of a chiral column (Chiralcel OD) connected to a UV detector. The second approach involves using a C-18 reversed-phase column connected in series to a RI detector and to an Optical Rotation detector. The two methods yield the same results.

Enzymatic asymmetrization of prochiral 2-benzyl-1,3-propanediol through esterification in solvent media

The enzymatic esterification of the prochiral substrate, 2-benzyl-1,3-propanediol, has been studied in solvent media. Among the five tested lipases, Lipozyme and Novozym 435 led to higher reaction rates. Novozym 435 catalyzed faster reactions at low water activity and in solvents having log P above 2. However, the two positions of the diol, pro-(R) and pro-(S), led to the same reaction rate trends and no prochiral selectivity was obtained. When using Lipozyme in toluene, the reaction rates for the formation of both (R) and (S) products presented an optimum at a water activity of 0.22. In this case, the prochiral selectivity increased with the water activity, from a value of 5 at aw < 0.01, to a value of 8 at aw = 0.22, at which point it remained constant.

Synthesis of nucleosidic bonds using a nucleoside hydrolase in aqueous-organic media

The synthesis of inosine from nonactivated and nonprotected ribose and hypoxanthine was performed by enzyme-catalyzed condensation, using a nucleoside hydrolase from Crithidia fasciculata expressed in Escherichia coli with a synthetic gene. One round of directed evolution was performed in the presence of dimethylformamide, used to lower the water activity in the reaction media, leading to a double mutant (Asp54Asn, Arg137Gly). It afforded a doubling of the specificity constant for the hydrolysis of p-nitrophenol-β-d-riboside in the presence of 40% acetonitrile. In aqueous conditions, concentrations of 0.1 and 0.2 mM inosine were obtained, starting from 25 mM hypoxanthine and 2 or 3 M ribose, respectively. With 20% acetonitrile increases of 95% and 60% were observed. These conversions are very low, and the work exemplifies the difficulties encountered when trying to define conditions for hydrolase-catalyzed condensation and at the same time evolve an enzyme to perform well in these a priori unknown conditions.