Michael Trani

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.

Lipase-catalyzed production of wax esters

The lipase (triacylglycerol acylhydrolase, E.C. 3.1.1.3) catalyzed synthesis of wax esters has been investigated via two different approaches. All studies were performed using an immobilized 1,3-specific lipase [Lipozyme from Novo Industries (Montréal, Québec, Canada)]. The first approach involves reacting stoichiometric amounts of a fatty acid and stearyl alcohol in the presence of lipase. The medium is solvent-free, which allows for high substrate concentrations (1.55 M) and use of 5% (w/w) Lipozyme. In this reaction, maximum wax ester synthesis was found to be dependent upon the efficient removal of the water produced by the reaction. Under optimal conditions, yields of 100% were routinely reached after only 2 hr. The medium was then exclusively composed of the wax and the enzyme, no purification was required. The second method involves alcoholysis of a triglyceride, in this case triolein, with stearyl alcohol to produce 1,2-diolein, 2-monoolein and the wax ester of oleic acid. Again, no organic solvent was used. The wax ester yield was found to be directly dependent upon the alcohol concentration that was used to modulate the outcome of the reaction towards either the wax or the partial glycerides. The process was applied to the synthesis of waxes from high erucic acid rapeseed oil.

Solvent free triglyceride synthesis using lipozymeTM IM-20

SummaryThe synthesis of triglycerides using LipozymeTM IM-20 (Mucor miehei lipase immobilized on weak anion exchange resins by Novo) is described. Use of pure substrates in stoichiometric amounts in the absence of any organic solvent enables high conversion to be obtained with the addition of molecular sieves to remove water produced by the reaction.

Use of lipases in multiphasic systems solely composed of substrates

Three lipase-catalyzed reactions have been investigated in relation to specificity and water dependence. The reactions in question include: the synthetic reaction between oleic acid and glycerol; the enzymatic hydrolysis of triolein; and alcoholysis/glycerolysis transesterification reactions. All reactions were carried out under solventfree conditions. In each case, the medium composition and reaction conditions were optimized in order to work at elevated substrate concentrations and to minimize the production of by-products. Different lipase preparations have been tested in each reaction. In the synthetic reaction, the effective removal of produced water was found to be vital for the production of triolein. With water removal and glycerol amounts not higher than required by the stoichiometry of the reaction, 95% of the available oleic acid was converted to triolein in 48 hr. The production of triolein was also found to be dependent on the availability of the 1,2-diglyceride to react with oleic acid. In the hydrolysis reaction, best conversion yields of triolein towards monoolein, diolein and free fatty acid were obtained when water was considered simply as a substrate of the reaction. In glycerolysis reactions, the reaction of triolein to give monoolein and diolein followed much the same pattern as for hydrolysis, when water was replaced by glycerol. It was shown again that near stoichiometric amounts of substrates led to the best conversion to mono- and diglycerides. A small excess of glycerol was found to be very inhibitory to the reaction. All possible isomers were formed during the reaction. Conversely, in alcoholysis reactions between triolein and stearyl alcohol the specificity of the lipase was upheld. Excess alcohol in this instance was found to be beneficial.

Effect of lipase specificity on triglyceride synthesis

SummaryEnzymatic triglyceride synthesis from free fatty acid and glycerol is shown to be catalysed by both 1,3-specific lipase and non-specific lipase. This paper elucidates the mechanism of the reaction, showing that, with a 1,3-specific lipase, the 1,3-diolein enzymatically formed has to isomerise spontaneously to 1,2-diolein in presence of oleic acid, before being transformed to triolein by the enzyme.

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).