Development of sustainable biocatalytic processes for organic synthesis Daniela Monti Istituto di Chimica del Riconoscimento Molecolare Consiglio Nazionale delle Ricerche Milano, Italy
Sustainable Chemistry at ICRM • Reduction of the use of toxic reagents and waste production • Overall improvement of process productivity Green Chemistry • Biocatalyst (enzyme) production and development • Synthetic applications in the synthesis of chiral synthons, drugs, flavors, fragrances… Biocatalysis • Modification of natural and synthetic polymers • Biomasses exploitation by development of optimized pretreatment processes • Production of valuable chemicals by bioresource utilization Biorefineries
Allylic oxidation using the ‘green’ oxidant TBHP Green Chemistry Oxidation of allylic and benzylic methylene functional group to the corresponding conjugated carbonyl derivative by a one-pot oxidation protocol based on two sequential steps In the first step , carried out at low temperature, MnO 2 catalyses the oxidation of the methylene group. This is followed by a second step where reaction temperature is increased, allowing MnO 2 both to catalyse the decomposition of unreacted TBHP and to oxidize allylic alcohols that could possibly be formed. O OH R''' R'' R''' R'' -30 °C, 2 h reflux + OH R R 5 h -10 °C, 15 h R' R' CH 2 Cl 2 O + + recycling MnO 2 MnO 2 Serra S. (2015) MnO 2 /TBHP: A Versatile and User-Friendly Combination of Reagents for the Oxidation of Allylic and Benzylic Methylene Functional Groups . European Journal of Organic Chemistry , DOI: 10.1002/ejoc.201500829
Lignocellulosic bio-refinery Biorefineries Organosolv pretreatment for biomass destructuration Gianluca Ottolina & Stefano Gandolfi VELICA Project
Are our sugars mixture fermentable? C5 and C6 stream Biorefineries Bacillus coagulans XZL4 42 g of L-(+) LA were obtained from 100 g of raw lignocellulosic biomass ( hemp hurds ) Gianluca Ottolina & Stefano Gandolfi VELICA Project
Biocatalysis: most used enzyme classes Biocatalysis
Biocatalysts sources • Commercially available enzymes (hydrolases, laccases …) • Bacteria, yeasts and fungi from culture collections • Enzymes with known sequences: cloning from wild-type strains (bacteria, yeasts) synthetic genes (< 1 Euro/amino acid) • Novel enzymes: derived from wild-type enzymes (mutagenesis) obtained by screening of microbial collections and metagenomes
In-house Biocatalysts Production by Heterologous Expression • Easily culturable host strains (E. coli…) • Small/medium-scale cultures for biocatalyst characterization • Overexpression of the desired activities with low/none contaminants E. coli transformation + vector pExpress Target gene E. coli growth in medium containing a suitable inducer
Discovery of Novel Biocatalysts by Metagenomic Screening “Traditional” screening of culture collections: access to defined, but limited microbial strains Metagenomic screening : access to “any” sequence present in environmental samples • “ unculturable ” microorganisms can be up to 99% of the total microbial population • suitable for samples from extreme environments The HotZyme Project Systematic screening for novel hydrolases from hot environments
Enzymes applications Enzymatic modification of natural and synthetic polymers Natural polymers Enzymes Selectively modified polymers L. Merlini, A. C. Boccia, R. Mendichi, Y. M. Galante Yves M. Galante “Enzymatic and chemical oxidation of polygalactomannans SUSCHEM Project from the seeds of a few species of leguminous plants and characterization of the oxidized products”, J. Biotechnol ., 198, 31-43 (2015) POLIBIO Project Collaborations with ISMAC and ISPA , Milano
Enzymes applications Synthetic applications in the synthesis of chiral synthons, drugs, flavors, fragrances PRAMIPEXOL lipase (Parkinson) H 2 O S. Riva, P. Fassi, M. Scalpellini, P. Allegrini, G. Razzetti “ Synthesis of intermediates for the preparation of pramipexol ” US 7,662,610 B2 Collaboration with DIPHARMA , production (200 kg/year) active since 2006
Biocatalytic resolution of cis/trans mixtures of limonene oxide Preparative-scale hydrolysis reactions catalyzed by either cis- or trans-specific epoxide hydrolases provide enantiomerically pure epoxides and diols: 1 1 1 1 2 cis -specific LEH 2 2 2 4 4 4 4 (1 S ,2 R ,4 R ) (1 R ,2 S ,4 R ) (1 S ,2 R ,4 R ) (1 S ,2 S ,4 R ) (+)- trans (+)- trans (+)- cis 1 1 1 1 2 trans -specific LEH 2 2 2 4 4 4 4 (1 R ,2 S ,4 R ) (1 R ,2 S ,4 R ) (1 S ,2 R ,4 R ) (1 S ,2 S ,4 R ) (+)- cis (+)- trans (+)- cis Collaboration with SIGMA-ALDRICH , SUSCHEM Project
Gram-scale solvent-free preparative resolutions of (+)-limonene oxide and (-)-limonene oxide catalyzed by selected epoxide hydrolases • Reactions performed in phosphate buffer, pH 8.0, on neat substrates • Excellent recovery yields for both the unreacted epoxides and the formed diols Process parameter (+)-Limonene oxide (-)-Limonene oxide Re -LEH Tomsk-LEH CH55-LEH Re -LEH Substrate total amount (g) 6.09 1.52 6.09 3.04 Substrate loading (mol L -1 ) 2 0.5 2 1 Enzyme (mg mL -1 ) 0.2 0.7 1.5 0.4 T (°C) 20 30 50 20 Reaction time (h) 4.5 24 6 4.5 Epoxide yield (%) 44 ( 2 ) 33 ( 1 ) 36 ( 5 ) 34 ( 4 ) Diol yield (%) 40 ( 3 ) 59 ( 3 ) 64 ( 6 ) 66 ( 6 ) STY (mmol L -1 h -1 ) 167.3 6.6 120.5 76.0 Specific productivity (µmol mg -1 h -1 ) 837 9.4 80 190 ECN (mg mmol -1 ) 0.26 4.4 2.1 1.1 E. E. Ferrandi, C. Marchesi, C. Annovazzi, S. Riva, D. Monti, R. Wohlgemuth “Efficient epoxide hydrolase-catalyzed resolutions of (+)- and (-)- cis / trans -limonene oxides “ ChemCatChem , 7 , 3171-3178 (2015)
Development of innovative multi-enzymatic processes The sustainability of a chemical process can depend largely on the number of synthetic steps and purification of reaction intermediates. To improve the competitiveness of the catalytic processes of interest, the different steps of the reaction can be coupled in " one-pot " systems, in which the isolation of intermediate products is not necessary, or even in " cascade " processes, in which all the reagents and catalysts are present in the reaction mixture since the beginning of the process.
One-pot enzymatic synthesis of 12-ketoursocholic acid Concomitant oxidation and reduction of bile acids intermediates Exploitation of different cofactor regeneration systems providing the proper driving force to the oxidation and reduction reactions catalyzed by selected HSDHs: Double Selective oxidation reduction 7 -HSDH 7 -HSDH 12 -HSDH NAD + NADP + NADH NADPH Cholic acid 12-Ketoursodeoxycholic acid NADP + -dep DH 2 NAD + -dep DH 1 D. Monti, E. E. Ferrandi, I. Zanellato, L. Hua, F. Polentini, G. Carrea , S. Riva “One -pot multienzymatic synthesis of 12-ketoursodeoxycholic acid: subtle cofactor specificities rule the reaction equilibria of five biocatalysts working in a row” Adv. Synth. Catal. , 351, 1303-1311 (2009) Collaboration with Prodotti Chimici Alimentari S.p.A.
Exploitation of ER-ADH cascade reactions in the stereoselective synthesis of pharmaceutical intermediates Concomitant stereoselective reduction of the C=C double bond and chemoselective reduction of the carbonyl group of the aldehyde/ketone E. Brenna, F. G. Gatti, L. Malpezzi, D. Monti, F. Parmeggiani, A. Sacchetti "Synthesis of robalzotan, ebalzotan and rotigotine precursors via stereoselective multienzymatic cascade reduction of α , β - unsaturated aldehydes" J. Org. Chem. , 78, 4811-4822 (2013)
Cascade coupling of ene-reductases and ω -transaminases Stereoselective synthesis of diastereoisomerically enriched amines The biocatalytic synthesis of diastereomerically enriched ( R )- and ( S )-amines was achieved by one-pot coupling of ene-reductases and ω -transaminases , in sequential and cascade processes . Using α - or β -substituted unsaturated ketones as substrates, up to >99% conversion and >99% de were obtained. D. Monti, M. C. Forchin, M. Crotti, F. Parmeggiani, F. G. Gatti, E. Brenna, S. Riva, ChemCatChem , 7 , 3106-3109 (2015)
Recommend
More recommend
