A New Approach in Lipase-Octyl-Agarose Biocatalysis of 2-Arylpropionic Acid Derivatives.

The use of lipase immobilized on an octyl-agarose support to obtain the optically pure enantiomers of chiral drugs in reactions carried out in organic solvents is a great challenge for chemical and pharmaceutical sciences. Therefore, it is extremely important to develop optimal procedures to achieve a high enantioselectivity of the biocatalysts in the organic medium. Our paper describes a new approach to biocatalysis performed in an organic solvent with the use of CALB-octyl-agarose support including the application of a polypropylene reactor, an appropriate buffer for immobilization (Tris base-pH 9, 100 mM), a drying step, and then the storage of immobilized lipases in a climatic chamber or a refrigerator. An immobilized lipase B from Candida antarctica (CALB) was used in the kinetic resolution of (R,S)-flurbiprofen by enantioselective esterification with methanol, reaching a high enantiomeric excess (eep = 89.6 ± 2.0%). As part of the immobilization optimization, the influence of different buffers was investigated. The effect of the reactor material and the reaction medium on the lipase activity was also studied. Moreover, the stability of the immobilized lipases: lipase from Candida rugosa (CRL) and CALB during storage in various temperature and humidity conditions (climatic chamber and refrigerator) was tested. The application of the immobilized CALB in a polypropylene reactor allowed for receiving over 9-fold higher conversion values compared to the results achieved when conducting the reaction in a glass reactor, as well as approximately 30-fold higher conversion values in comparison with free lipase. The good stability of the CALB-octyl-agarose support was demonstrated. After 7 days of storage in a climatic chamber or refrigerator (with protection from humidity) approximately 60% higher conversion values were obtained compared to the results observed for the immobilized form that had not been stored. The new approach involving the application of the CALB-octyl-agarose support for reactions performed in organic solvents indicates a significant role of the polymer reactor material being used in achieving high catalytic activity.

Ligand fishing using new chitosan based functionalized Androgen Receptor magnetic particles.

Superparamagnetic nanoparticles with chemically modified chitosan has been proposed as a potential support for the immobilization of the androgen receptor (AR). The study involved comparison of different AR carriers like commercially available magnetic beads coated with silica (BcMag) and chitosan coated nanoparticles with different amount of amino groups. The immobilization was carried out through covalent immobilization of the AR through the terminal amino group or through available carboxylic acids. The initial characterization of the AR coated magnetic beads was carried out with dihydrotestosterone, a known AR ligand. Subsequently, chitosan modified nanporticles with long-distanced primary amino groups (Fe3O4CS-(NH2)3) (upto 8.34mM/g) were used for further study to isolate known AR ligands (bicalutamide, flutamide, hydroxyflutamide and levonogestrel) from a mixture of tested compounds in ammonium acetate buffer [10mM, pH 7.4]. The results showed that the selected nanoparticles are a promising semi-quantitative tool for the identification of high affinity compounds to AR and might be of special importance in the identification of novel agonists or antiandrogens.

High enantioselective Novozym 435-catalyzed esterification of (R,S)-flurbiprofen monitored with a chiral stationary phase.

Lipases form Candida rugosa and Candida antarctica were tested for their application in the enzymatic kinetic resolution of (R,S)-flurbiprofen by enantioselective esterification. Successful chromatographic separation with well-resolved peaks of (R)- and (S)-flurbiprofen and their esters was achieved in one run on chiral stationary phases by high-performance liquid chromatography (HPLC). In this study screening of enzymes was performed, and Novozym 435 was selected as an optimal catalyst for obtaining products with high enantiopurity. Additionally, the influence of organic solvents (dichloromethane, dichloroethane, dichloropropane, and methyl tert-butyl ether), primary alcohols (methanol, ethanol, n-propanol, and n-butanol), reaction time, and temperature on the enantiomeric ratio and conversion was tested. The high values of enantiomeric ratio (E in the range of 51.3-90.5) of the esterification of (R,S)-flurbiprofen were obtained for all tested alcohols using Novozym 435, which have a great significance in the field of biotechnological synthesis of drugs. The optimal temperature range for the performed reactions was from 37 to 45 °C. As a result of the optimization, (R)-flurbiprofen methyl ester was obtained with a high optical purity, eep = 96.3 %, after 96 h of incubation. The enantiomeric ratio of the reaction was E = 90.5 and conversion was C = 35.7 %.

Kinetic resolution of profens by enantioselective esterification catalyzed by Candida antarctica and Candida rugosa lipases.

Profens (2-arylpropionic acids) are known as one of the major nonsteroidal antiinflammatory drugs (NSAIDs) used in the treatment of inflammation associated with tissue injury. The inflammatory activity of profens is mainly due to their (S)-enantiomer, whereas they are commercially available not only as pure enantiomers, but as racemates as well. There are several methods widely used in order to obtain enantiomerically pure compounds, however, the kinetic resolution with the application of lipases as biocatalysts may have an added advantage in the production of optically pure active pharmaceutical ingredients, such as milder reaction conditions, reduced energy requirements, and production costs. The aim of this study was to compare the results described in the literature in the case of the influence of reaction medium, alcohol moiety, and reaction temperature on the catalytic activity of lipases from Candida antarctica and Candida rugosa.