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

Green Chemistry

• Reduction of the use of toxic reagents and waste production

• Overall improvement of process productivity

Biocatalysis

• Biocatalyst (enzyme) production and development

• Synthetic applications in the synthesis of chiral synthons, drugs, flavors, fragrances…

• Modification of natural and synthetic polymers

Biorefineries

• Biomasses exploitation by development of optimized pretreatment processes

• Production of valuable chemicals by bioresource utilization

Allylic oxidation using the ‘green’ oxidant TBHP

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

-30 °C, 2 h

-10 °C, 15 h

reflux

5 h

R''' R''

R'R

R''' R''

R'R

O

OH

MnO2 MnO2

CH2Cl2

O OH

+ +

+

recycling

Serra S. (2015) MnO2/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

Green Chemistry

In the first step, carried out at low temperature, MnO2 catalyses the oxidation of the methylene group. This is followed by a second step where reaction temperature is increased, allowing MnO2 both to catalyse the decomposition of unreacted TBHP and to oxidize allylic alcohols that could possibly be formed.

Lignocellulosic bio-refinery

Organosolv pretreatment for biomass destructuration

Gianluca Ottolina & Stefano Gandolfi VELICA Project

Biorefineries

Are our sugars mixture fermentable?

C5 and C6 stream

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

Biorefineries

Biocatalysis:

most used enzyme classes Biocatalysis

Biocatalysts sources

• Commercially available enzymes (hydrolases, laccases…)

• 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

• Bacteria, yeasts and fungi from culture collections

Target gene

vector pExpress

E. coli transformation

E. coli growth in medium containing a suitable inducer

+

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

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

Selectively

modified

polymers

Enzymes Natural polymers

Enzymes applications

Enzymatic modification of natural and synthetic polymers

Yves M. Galante SUSCHEM Project POLIBIO Project

L. Merlini, A. C. Boccia, R. Mendichi, Y. M. Galante “Enzymatic and chemical oxidation of polygalactomannans from the seeds of a few species of leguminous plants and characterization of the oxidized products”, J. Biotechnol., 198, 31-43 (2015) Collaborations with ISMAC and ISPA, Milano

Enzymes applications

Synthetic applications in the synthesis of chiral synthons, drugs, flavors, fragrances

lipase

H2O

PRAMIPEXOL (Parkinson)

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

trans-specific LEH1

(1R,2S,4R)

4

(+)-cis

12

(1S,2S,4R)

1

4

2

(1R,2S,4R)

21

4

(+)-cis

2

4

(1S,2R,4R)

(+)-trans

cis-specific LEH 211

(1S,2S,4R)

1

4 4

(1S,2R,4R)

2

(1R,2S,4R)

(+)-trans

21

4

(+)-cis

2

4

(1S,2R,4R)

(+)-trans

Preparative-scale hydrolysis reactions catalyzed by either cis- or trans-specific epoxide hydrolases provide enantiomerically pure epoxides and diols:

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

Re-LEH Tomsk-LEH

(-)-Limonene oxide

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-1h-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)

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.

Development of innovative multi-enzymatic processes

7 -HSDH

12 -HSDH

NAD+ NADH

7 -HSDH

NADP+NADPH

NAD+-dep DH1 NADP+-dep DH2

Cholic acid 12-Ketoursodeoxycholic acid

Doubleoxidation

Selectivereduction

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:

Collaboration with Prodotti Chimici Alimentari S.p.A.

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)

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)