- Email: [email protected]
Asymmetric acylation of (±)-2-amino-1-phenylethanols with vinyl acetate by immobilized Mucor miehei lipase
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Poster presentations / Current Opinion in Biotechnology 24S (2013) S48–S143
for the immobilization of the Gluconobacter sp. cells, offering a protective environment for the cells even for toxic organic precursors (2-phenylethanol) was developed. As a result, increased yield of phenylacetic acid during the repeated bioconversions was observed. This is the first time when encapsulated whole cell biocatalyst was used for natural flavour production. Acknowledgements: This work was supported by the Slovak Research and Development Agency under the contract No. APVV-0302-10. This contribution is the result of the project implementation: Applied research in the field of industrial biocatalysis, ITMS code: 26240220079 supported by the Research & Development Operational Programme funded by the ERDF. http://dx.doi.org/10.1016/j.copbio.2013.05.152 Screening of fungal strains to be used as biocatalysts in bioconversion processes based on cycloaddition reactions Georgiana Parfene 1 , Ioana Otilia Ghinea 1 , Bianca Furdui 2 , Gabriela Bahrim 1 , Martine Demeunynck 3 , Rodica Mihaela Dinica 2 1
Department of Food Science, Food Engineering and Applied Biotechnology, “Dunarea de Jos” University of Galati, 111 Domneasca Street, Galati 800201, Romania 2 Department of Chemistry, Physics and Environment, “Dunarea de Jos” University of Galati, 111 Domneasca Street, Galati 800201, Romania 3 Département Pharmacochimie Moléculaire, UMR 5063 & FR 2607, CNRS/Université de Grenoble, 38041 Grenoble cedex 9, France E-mail address: [email protected] (I.O. Ghinea). Microorganisms and their enzymes are widely exploited as biocatalysts in bioconversion with applications in production of a broad spectrum of fine chemicals, pharmaceuticals and their building blocks, as well as commodity and agrochemicals. Recently, plenty of studies have clearly demonstrated that whole cells and enzymes have catalytic promiscuity, which allows adaptation of their selectivity for non-natural functional groups. Therefore, remarkable applications in synthetic chemistry have been largely expanded with the development of green chemistry. Herein, we report the use of whole cells of microorganisms and their enzymes as biocatalysts in organic reactions. Previously, a total of twenty microbial strains were tested to determine their biocatalytic activity in reactions of cycloaddition by employing screening techniques. Yarrowia lipolitica MIUG RB105 lipase-producing selected yeast strain was selected to be used as biocatalyst in cycloaddition reactions for indolizine synthesis in aqueous medium, in which the best result was achieved. The reactions were monitored by TLC, HPLC/MS and FTIR. This procedure represents an eco-friendly single step “green” synthesis of fluorescent indolizines. Acknowledgement: This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCSUEFISCDI, project number PN-II-ID-PCE-2011-3-0226. http://dx.doi.org/10.1016/j.copbio.2013.05.153
Cross linked enzyme aggregates of Prunus armeniaca hydroxynitrile lyase for synthesis of enantiopure (R)-cyanohydrins Dilek Alagöz, Deniz Yıldırım, Seyde Seyhan Tükel, Özlem Alptekin Chemistry Department, C¸ukurova University, Adana, Turkey E-mail address: [email protected] (D. Alagöz). Cross linked enzyme aggregates (CLEAs) have emerged as interesting biocatalysts design for immobilization since this technique may integrate in a single/or reduce operation: enzyme purification and immobilization. In this study, (R)-hydroxynitrile lyase
was purified from Prunus armeniaca seeds (PaHNL) and then immobilized as cross-linked enzyme aggregates (PaHNL-CLEA). PaHNL-CLEA was used for the preparation of industrially important enantiopure cyanohydrins such as (R)-mandelonitrile, (R)3,4-dihydroxymandelonitrile, (R)-2-chloromandelonitrile. CLEAs of PaHNL were prepared based on the reported procedure. The carboligation activity of PaHNL-CLEA was determined using HPLC equipped with a Nucleocel Delta chiral column (4.6 × 250 mm). The formed products were detected by a diode array detector at 220 nm. The maximum yield and enantiomeric excess (ee) values were obtained as 100% and >98% for (R)mandelonitrile and (R)-3,4-dihydroxymandelonitrile, respectively. (R)-2-Chloromandelonitrile was synthesized with 100% yield whereas its ee value was 21%. The CLEA methodology combined purification and immobilization of PaHNL into one step. The results of enantiopure cyanohydrin syntheses showed that PaHNL-CLEA showed good enantioselectivity towards the addition of HCN to benzaldehyde and 3,4-dihydroxybenzaldehyde, however, ortho substituent caused probably a steric hindrance for the addition of HCN to 2-chlorobenzaldehyde. The PaHNL-CLEA offers many economic and environmental benefits in the context of industrial biocatalysis due to its relatively simple and large-scale preparation and effectual usage for the synthesis of (R)-mandelonitrile and (R)-3,4-dihydroxymandelonitrile. http://dx.doi.org/10.1016/j.copbio.2013.05.154 Asymmetric acylation of (±)-2-amino-1-phenylethanols with vinyl acetate by immobilized Mucor miehei lipase Deniz Yildirim, Dilek Alagöz, Seyde Seyhan Tükel, Özlem Alptekin Chemistry Department, C¸ukurova University, Adana, Turkey E-mail address: [email protected] (D. Alagöz). Beta-adrenergic receptor blocking agents (-blockers) are important drugs consumed for the treatment of high blood pressure, heart failure, hypertension and myocardial ischemic diseases. In this study, a facile approach for the preparation of enantiopure 2-amino-1-phenylethanols which may be used as potential new -blockers was provided by the asymmetric acylation of (±)-2-amino-1-phenylethanols with vinyl acetate catalyzed by immobilized Mucor miehei lipase. The immobilized lipase preparations were prepared according to literature. The hydrolytic activity was determined using p-nitrophenyl palmitate. The synthetic activity of immobilized lipase preparation was determined using chiral HPLC. The formed products were detected by a UV–visible detector at 220 nm. The (±)-2-amino-1phenylethanols used were (±)-2-(methylamino)-1-phenylethanol, (±)-2-(ethylamino)-1-phenylethanol, (±)-2-(propylamino)1-phenylethanol, (±)-2-(butylamino)-1-phenylethanol and (±)-2-(hexylamino)-1-phenylethanol. The optimal conditions were determined as bulk water content of 2%, reaction temperature of 50◦ C, initial molar ratio of vinyl acetate to amino alcohol of 1.5, and immobilized lipase loading of 40 mg mL−1 . (R)-Enantiomers of tested amino alcohols were preferentially acylated and the reaction purely took place on the hydroxyl group of 2-amino-1-phenylethanols. The increase of alkyl chain length substituted onto nitrogen atom caused an increase in the acylation yield and ee values of (S)-enantiomers. Our results demonstrated that the immobilized lipase is a promising biocatalyst for the preparation of (S)-2-amino-1-phenylethanols and their corresponding (R)-esters via O-selective acylation of (±)-2-amino-1-phenylethanols with vinyl acetate. http://dx.doi.org/10.1016/j.copbio.2013.05.155
Poster presentations / Current Opinion in Biotechnology 24S (2013) S48–S143
for the immobilization of the Gluconobacter sp. cells, offering a protective environment for the cells even for toxic organic precursors (2-phenylethanol) was developed. As a result, increased yield of phenylacetic acid during the repeated bioconversions was observed. This is the first time when encapsulated whole cell biocatalyst was used for natural flavour production. Acknowledgements: This work was supported by the Slovak Research and Development Agency under the contract No. APVV-0302-10. This contribution is the result of the project implementation: Applied research in the field of industrial biocatalysis, ITMS code: 26240220079 supported by the Research & Development Operational Programme funded by the ERDF. http://dx.doi.org/10.1016/j.copbio.2013.05.152 Screening of fungal strains to be used as biocatalysts in bioconversion processes based on cycloaddition reactions Georgiana Parfene 1 , Ioana Otilia Ghinea 1 , Bianca Furdui 2 , Gabriela Bahrim 1 , Martine Demeunynck 3 , Rodica Mihaela Dinica 2 1
Department of Food Science, Food Engineering and Applied Biotechnology, “Dunarea de Jos” University of Galati, 111 Domneasca Street, Galati 800201, Romania 2 Department of Chemistry, Physics and Environment, “Dunarea de Jos” University of Galati, 111 Domneasca Street, Galati 800201, Romania 3 Département Pharmacochimie Moléculaire, UMR 5063 & FR 2607, CNRS/Université de Grenoble, 38041 Grenoble cedex 9, France E-mail address: [email protected] (I.O. Ghinea). Microorganisms and their enzymes are widely exploited as biocatalysts in bioconversion with applications in production of a broad spectrum of fine chemicals, pharmaceuticals and their building blocks, as well as commodity and agrochemicals. Recently, plenty of studies have clearly demonstrated that whole cells and enzymes have catalytic promiscuity, which allows adaptation of their selectivity for non-natural functional groups. Therefore, remarkable applications in synthetic chemistry have been largely expanded with the development of green chemistry. Herein, we report the use of whole cells of microorganisms and their enzymes as biocatalysts in organic reactions. Previously, a total of twenty microbial strains were tested to determine their biocatalytic activity in reactions of cycloaddition by employing screening techniques. Yarrowia lipolitica MIUG RB105 lipase-producing selected yeast strain was selected to be used as biocatalyst in cycloaddition reactions for indolizine synthesis in aqueous medium, in which the best result was achieved. The reactions were monitored by TLC, HPLC/MS and FTIR. This procedure represents an eco-friendly single step “green” synthesis of fluorescent indolizines. Acknowledgement: This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCSUEFISCDI, project number PN-II-ID-PCE-2011-3-0226. http://dx.doi.org/10.1016/j.copbio.2013.05.153
Cross linked enzyme aggregates of Prunus armeniaca hydroxynitrile lyase for synthesis of enantiopure (R)-cyanohydrins Dilek Alagöz, Deniz Yıldırım, Seyde Seyhan Tükel, Özlem Alptekin Chemistry Department, C¸ukurova University, Adana, Turkey E-mail address: [email protected] (D. Alagöz). Cross linked enzyme aggregates (CLEAs) have emerged as interesting biocatalysts design for immobilization since this technique may integrate in a single/or reduce operation: enzyme purification and immobilization. In this study, (R)-hydroxynitrile lyase
was purified from Prunus armeniaca seeds (PaHNL) and then immobilized as cross-linked enzyme aggregates (PaHNL-CLEA). PaHNL-CLEA was used for the preparation of industrially important enantiopure cyanohydrins such as (R)-mandelonitrile, (R)3,4-dihydroxymandelonitrile, (R)-2-chloromandelonitrile. CLEAs of PaHNL were prepared based on the reported procedure. The carboligation activity of PaHNL-CLEA was determined using HPLC equipped with a Nucleocel Delta chiral column (4.6 × 250 mm). The formed products were detected by a diode array detector at 220 nm. The maximum yield and enantiomeric excess (ee) values were obtained as 100% and >98% for (R)mandelonitrile and (R)-3,4-dihydroxymandelonitrile, respectively. (R)-2-Chloromandelonitrile was synthesized with 100% yield whereas its ee value was 21%. The CLEA methodology combined purification and immobilization of PaHNL into one step. The results of enantiopure cyanohydrin syntheses showed that PaHNL-CLEA showed good enantioselectivity towards the addition of HCN to benzaldehyde and 3,4-dihydroxybenzaldehyde, however, ortho substituent caused probably a steric hindrance for the addition of HCN to 2-chlorobenzaldehyde. The PaHNL-CLEA offers many economic and environmental benefits in the context of industrial biocatalysis due to its relatively simple and large-scale preparation and effectual usage for the synthesis of (R)-mandelonitrile and (R)-3,4-dihydroxymandelonitrile. http://dx.doi.org/10.1016/j.copbio.2013.05.154 Asymmetric acylation of (±)-2-amino-1-phenylethanols with vinyl acetate by immobilized Mucor miehei lipase Deniz Yildirim, Dilek Alagöz, Seyde Seyhan Tükel, Özlem Alptekin Chemistry Department, C¸ukurova University, Adana, Turkey E-mail address: [email protected] (D. Alagöz). Beta-adrenergic receptor blocking agents (-blockers) are important drugs consumed for the treatment of high blood pressure, heart failure, hypertension and myocardial ischemic diseases. In this study, a facile approach for the preparation of enantiopure 2-amino-1-phenylethanols which may be used as potential new -blockers was provided by the asymmetric acylation of (±)-2-amino-1-phenylethanols with vinyl acetate catalyzed by immobilized Mucor miehei lipase. The immobilized lipase preparations were prepared according to literature. The hydrolytic activity was determined using p-nitrophenyl palmitate. The synthetic activity of immobilized lipase preparation was determined using chiral HPLC. The formed products were detected by a UV–visible detector at 220 nm. The (±)-2-amino-1phenylethanols used were (±)-2-(methylamino)-1-phenylethanol, (±)-2-(ethylamino)-1-phenylethanol, (±)-2-(propylamino)1-phenylethanol, (±)-2-(butylamino)-1-phenylethanol and (±)-2-(hexylamino)-1-phenylethanol. The optimal conditions were determined as bulk water content of 2%, reaction temperature of 50◦ C, initial molar ratio of vinyl acetate to amino alcohol of 1.5, and immobilized lipase loading of 40 mg mL−1 . (R)-Enantiomers of tested amino alcohols were preferentially acylated and the reaction purely took place on the hydroxyl group of 2-amino-1-phenylethanols. The increase of alkyl chain length substituted onto nitrogen atom caused an increase in the acylation yield and ee values of (S)-enantiomers. Our results demonstrated that the immobilized lipase is a promising biocatalyst for the preparation of (S)-2-amino-1-phenylethanols and their corresponding (R)-esters via O-selective acylation of (±)-2-amino-1-phenylethanols with vinyl acetate. http://dx.doi.org/10.1016/j.copbio.2013.05.155