COLLINS Sebastian Enrique
congresos y reuniones científicas
Influence of water on enzymatic esterification of racemic ketoprofen in a solventless system
Santa Fe
Conferencia; VI San Luis School and Conference on Surfaces, Interfaces and Catalysis; 2018
Institución organizadora:
Ketoprofen belongs to the non-steroidal anti-inflammatory NSAIDs group. It is a racemic compound and its pharmacological activity resides only in the S-enantiomer, dexketoprofen. For this reason, administration of pure dexketoprofen would reduce the side effects considering that dose and toxicity will be reduced1. One of the most popular methodologies reported to produce dexketoprofen is enzymatic kinetic resolution, and lipases are the most popular enzymes2. These enzymes need a certain amount of water to retain the catalytically active three-dimensional structure. Water may have different effects on the rate of lipase-catalyzed reactions. In general, a positive effect is enzymatic activation by water, due to increased internal flexibility, active site polarity and proton conductivity of the enzyme. However, some negative aspects of water are inhibition (interference with substrate binding), formation of a diffusion barrier for hydrophobic substrates and, when esterification is the main reaction, water interferes as it is a substrate in reversed hydrolysis of the formed ester3. Additionally, water can influence lipase enantioselectivity by binding to substrate-binding pockets thereby selectively interfering in the binding of one of the enantiomers of the substrate4. Optimal amount of water depends on the solvent and substrates, the type of enzyme and operative conditions5.In this context, the optimal amount of water was studied in the esterification of R/S-ketoprofen with ethanol in a solventless system catalyzed by the commercial biocatalyst Novozym® 435 (lipase B from Candida antarctica immobilized on a macroporous resin). Results obtained show that enzyme activity increases together with water content up to 10 % (v/v) when enantioselectivity decays. So, the optimal amount of water for this specific reaction was found to be 9% (v/v). Additionally, infrared studies were performed in order to elucidate the effect of water content on the lipase in terms of enzyme flexibility. Kinetics of the isotope (D2O) exchange followed by infrared spectroscopy in Attenuated Total Reflection (ATR) can provide information of protein structure and dynamics at a molecular level6. Intensity changes in the amide regions are related to the exchange of amide protons, whose exchange rates are determined by solvent accessibility and the conformational flexibility of the protein. Analyses show that the enzyme in contact with the alcohol is more flexible and therefore the active site is more accessible. Water content increment in the reaction mixture is accompanied by a decrease in enzyme flexibility indicating a competition with the substrates for the active site7. These results are in agreement with those obtained for lipase activity with increasing amounts of water exposed above.