Nowadays, Biocatalysis is a deeply investigated field and is importance is well established also for chemical industries. With the aim of developing more and more efficient synthetic strategies, enzymes represent now a reliable solution due to multiple factors. Enzymes are biodegradable and non-toxic, moreover are able to catalyze a wide range of reactions with excellent chemo-, regio-, and stereoselectivity. Their ability of working under mild conditions (room temperature, pH and atmospheric pressure) and producing low amount of wastes and by-products (effectively fitting in modern Green Chemistry and sustainability guidelines) is greatly appreciated, especially for the production in the field of fine chemicals and bioactive molecules. In addition, the enzymes, with respect to classical catalysis, are generally suited to be integrated in cascade processes, since they operate typically in very similar reaction conditions. Last but not least, the set-up of multi-enzymatic processes is appealing because avoids possible substrate/product inhibitions. The aim of my PhD research has been the exploitation of biocatalysts (microorganisms and isolated enzymes) both for the production of various fine chemical products (such as pharmaceutical ingredients, flavors and fragrances) and for developing more sustainable synthesis strategies with respect to traditional organic chemistry, avoiding the use of metal-based catalysts, toxic reagents and harmful operative conditions. The bioreduction of “activated” C=C bonds, i.e. those conjugated to one or more electron-withdrawing groups, is a widespread activity in nature and its applicability to preparative organic synthesis has been early recognized in last decades. Most of the enzymes responsible for this kind of activity that have been identified in the last years belong to the well-known and thoroughly characterised family of ene-reductases (ERs), nicotinamide-dependent flavoproteins which mediate the addition of a hydride and a proton to the C=C bond, with an anti mechanism. New applications of ERs for the stereoselective synthesis of active pharmaceutical ingredients and fragrances (or their key precursors) have been reported in this thesis work, in order to add such bioreductions to the toolbox available for asymmetric synthesis and to implement them in industrial manufacturing processes. Different classes of substrates have been investigated, gaining insight into the stereoelectronic factors that control the reactions. In multiple cases, ERs have been exploited in cascade reactions with other classes of enzymes (in particular alcohol dehydrogenases), in order to design even more appealing and promising synthetic applications of these biocatalysts. Considering modern needs and trends in industrial manufacturing processes, primary attempts of using ERs as immobilized biocatalyst, or in continuous-flow reactor, have been reported too, furtherly establishing the great flexibility of these enzymes in productive contexts. The limited availability of non-renewable petroleum-based sources and the increasing attempts to reduce waste and energy consumption (all key aspects of the modern concept of Sustainability) have prompted the international community to move towards the optimization of greener manufacturing processes, recovering unavoidable waste and promoting the market of secondary raw materials and by-products. Fats and oils represent an important class of renewable feedstock from which the so-called oleochemicals are obtained. They are abundant in nature, biodegradable, and possess nontoxic properties. In collaboration with an industrial partner, new chemo-enzymatic strategies for the valorization of soapstock, the by-product obtained from the refining process of vegetable oils, have been successfully developed, achieving the production of carboxylic and dicarboxylic acids of high commercial value.
Oggigiorno, la Biocatalisi rappresenta un ambito molto studiato e la sua importanza è ben consolidata anche per il settore dell’industria chimica. Con l'obiettivo di sviluppare strategie di sintesi sempre più efficienti, gli enzimi rappresentano una soluzione affidabile grazie a molteplici fattori. Gli enzimi sono biodegradabili e non tossici, inoltre sono in grado di catalizzare un'ampia gamma di reazioni con un'eccellente chemo, regio e stereoselettività. La loro capacità di lavorare in condizioni blande (temperatura ambiente, pH e pressione atmosferica) e di produrre una bassa quantità di rifiuti e sottoprodotti ( rispondendo efficacemente anche alle moderne linee guida della Chimica Verde e della sostenibilità) è molto apprezzata, soprattutto nel campo della sintesi di molecole bioattive. Inoltre, gli enzimi, rispetto alla catalisi classica, sono generalmente adatti ad essere integrati nei processi a cascata, poiché operano tipicamente in condizioni di reazione molto simili. Ultimo ma non meno importante, l'allestimento di processi multienzimatici è rilevante perché evita possibili inibizioni del substrato/prodotto. L'obiettivo della mia ricerca di dottorato è stato lo sfruttamento di biocatalizzatori (microrganismi ed enzimi isolati) sia per la produzione di vari prodotti della chimica fine (come intermedi farmaceutici, aromi e fragranze) sia per lo sviluppo di strategie di sintesi più sostenibili rispetto alla chimica organica tradizionale , evitando l'uso di catalizzatori a base metallica, reagenti tossici e condizioni operative di maggior rischio.La bioriduzione di legami C=C "attivati", cioè quelli coniugati a uno o più gruppi elettron-attrattori, è un'attività diffusa in natura e la sua applicabilità alla sintesi organica (anche su scala preparativa) è stata riconosciuta negli ultimi decenni. La maggior parte degli enzimi responsabili di questo tipo di attività che sono stati identificati negli ultimi anni appartengono alla famiglia ben nota e completamente caratterizzata delle ene-reduttasi (ER), flavoproteine nicotinamide-dipendenti che mediano l'aggiunta di un idruro e di un protone al legame C=C, con un meccanismo di addizione anti. In questo lavoro di tesi sono state riportate nuove applicazioni delle ER per la sintesi stereoselettiva di molecole farmacologicamente attive e fragranze (o dei loro precursori chiave), al fine di aggiungere tali bioriduzioni all’elenco degli strumenti disponibile per la sintesi asimmetrica, oltre ad implementarle nei processi di produzione industriale. Sono state studiate diverse classi di substrati, ottenendo informazioni sui fattori stereoelettronici che controllano l’andamento delle reazioni. In molti casi, le ER sono state sfruttate in reazioni a cascata con altre classi di enzimi (in particolare le alcol-deidrogenasi), al fine di progettare applicazioni sintetiche ancora più accattivanti e promettenti. Considerando le moderne esigenze e tendenze nei processi di produzione industriale, sono stati effettuati anche dei primi tentativi per l’utilizzo di ER come biocatalizzatori immobilizzati o in reattori a flusso continuo, sottolinenando ulteriormente la grande flessibilità di questi enzimi nei contesti produttivi. La limitata disponibilità di fonti petrolifere non rinnovabili e i crescenti tentativi di riduzione degli sprechi e dei consumi energetici (tutti aspetti chiave del moderno concetto di Sostenibilità) hanno spinto la comunità internazionale a muoversi verso l'ottimizzazione di processi produttivi più ecologici, recuperando i rifiuti inevitabili e promuovendo il mercato delle materie prime secondarie e dei sottoprodotti. Grassi e oli rappresentano un'importante classe di materie prime rinnovabili da cui si ottengono i cosiddetti prodotti oleochimici. Sono abbondanti in natura, biodegradabili e possiedono proprietà non tossiche. In collaborazione con un partner industriale sono state sviluppate con successo nuove strategie chemoenzimatiche per la valorizzazione del soapstock, il sottoprodotto ottenuto dal processo di raffinazione degli oli vegetali, ottenendo la produzione di acidi carbossilici e dicarbossilici ad alto valore commerciale.
Development of new biocatalysts for application in organic synthesis
Colombo, Danilo
2021/2022
Abstract
Nowadays, Biocatalysis is a deeply investigated field and is importance is well established also for chemical industries. With the aim of developing more and more efficient synthetic strategies, enzymes represent now a reliable solution due to multiple factors. Enzymes are biodegradable and non-toxic, moreover are able to catalyze a wide range of reactions with excellent chemo-, regio-, and stereoselectivity. Their ability of working under mild conditions (room temperature, pH and atmospheric pressure) and producing low amount of wastes and by-products (effectively fitting in modern Green Chemistry and sustainability guidelines) is greatly appreciated, especially for the production in the field of fine chemicals and bioactive molecules. In addition, the enzymes, with respect to classical catalysis, are generally suited to be integrated in cascade processes, since they operate typically in very similar reaction conditions. Last but not least, the set-up of multi-enzymatic processes is appealing because avoids possible substrate/product inhibitions. The aim of my PhD research has been the exploitation of biocatalysts (microorganisms and isolated enzymes) both for the production of various fine chemical products (such as pharmaceutical ingredients, flavors and fragrances) and for developing more sustainable synthesis strategies with respect to traditional organic chemistry, avoiding the use of metal-based catalysts, toxic reagents and harmful operative conditions. The bioreduction of “activated” C=C bonds, i.e. those conjugated to one or more electron-withdrawing groups, is a widespread activity in nature and its applicability to preparative organic synthesis has been early recognized in last decades. Most of the enzymes responsible for this kind of activity that have been identified in the last years belong to the well-known and thoroughly characterised family of ene-reductases (ERs), nicotinamide-dependent flavoproteins which mediate the addition of a hydride and a proton to the C=C bond, with an anti mechanism. New applications of ERs for the stereoselective synthesis of active pharmaceutical ingredients and fragrances (or their key precursors) have been reported in this thesis work, in order to add such bioreductions to the toolbox available for asymmetric synthesis and to implement them in industrial manufacturing processes. Different classes of substrates have been investigated, gaining insight into the stereoelectronic factors that control the reactions. In multiple cases, ERs have been exploited in cascade reactions with other classes of enzymes (in particular alcohol dehydrogenases), in order to design even more appealing and promising synthetic applications of these biocatalysts. Considering modern needs and trends in industrial manufacturing processes, primary attempts of using ERs as immobilized biocatalyst, or in continuous-flow reactor, have been reported too, furtherly establishing the great flexibility of these enzymes in productive contexts. The limited availability of non-renewable petroleum-based sources and the increasing attempts to reduce waste and energy consumption (all key aspects of the modern concept of Sustainability) have prompted the international community to move towards the optimization of greener manufacturing processes, recovering unavoidable waste and promoting the market of secondary raw materials and by-products. Fats and oils represent an important class of renewable feedstock from which the so-called oleochemicals are obtained. They are abundant in nature, biodegradable, and possess nontoxic properties. In collaboration with an industrial partner, new chemo-enzymatic strategies for the valorization of soapstock, the by-product obtained from the refining process of vegetable oils, have been successfully developed, achieving the production of carboxylic and dicarboxylic acids of high commercial value.| File | Dimensione | Formato | |
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https://hdl.handle.net/10589/191926