Green chemistry is a chemical concept, condensed in 12 principles, which aims at directing chemical industries and academic researchers to paths of sustainability, supporting the efforts in the context of the ecological transition. A valuable strategy widely recognized to fulfill most of the 12 principles is the exploitation of biocatalytic methods. Biocatalysis, at the intersection of chemistry and biology, delineates the family of catalytic processes operating in a living biological system to perform chemical modifications on organic compounds. It involves the exploitation of enzymes as highly specialized proteins to be used in vitro to transform selected substrates into valuable products. As molecular catalysts, enzymes are proteins which provide precision and efficiency, enabling these transformations with outstanding stereo- and regioselectivity, working under significantly mild and ecofriendly conditions, thus allowing greener and more efficient synthesis of complex molecules. Biocatalyzed reactions can be performed by exploiting either purified isolated enzymes or lyophilized crude cell lysates. The wide availability of commercial synthetic genes suggests that, at least theoretically, any enzyme with a known amino acid sequence can be obtained recombinantly, thus expanding the range of applications for these catalysts. The first part of this PhD, carried out in the laboratories of SCITEC-CNR, focused on the development of sustainable entries to bioactive compounds, small molecules, and chiral synthons of pharmaceutical and industrial interest, by combining the ability of various biocatalysts alongside traditional organic synthetic protocols. Both commercially available and in-house expressed, recombinant enzymes were considered, exploiting their activities as isolated proteins to produce scaffolds of pharmaceutical interest and compounds endowed with potential antiprotozoal properties and/or cytotoxicity. Chapters I to III provide an overview of the enzymes used and the results obtained. A detailed study on the substrate promiscuity and selectivity features of a panel of alcohol dehydrogenases, including the functional characterization of a newly identified ketoreductase named Is2-SDR, is presented. Additionally, novel chemo-enzymatic strategies were developed to prepare compounds potentially endowed with antiprotozoal activity. An innovative approach, comprising two biocatalyzed step coupled with a chemical oxidation one, was applied to synthesize the promising antiplasmodial scaffolds linear diarylpirazines. Further investigations involved the application of laccases for various purposes: specifically, to study the oxidation of a series of aromatic amines; to prepare enantiopure terpene derivatives and their corresponding atropisomeric C-C dimers; and, finally, to propose an enzymatic approach for the synthesis of biologically active riminophenazines. The third year of my PhD program was spent at Dipharma Francis Srl, co-funder of the doctoral fellowship. The experimental work carried out during this period, described in Chapter IV, involved the design and development of a novel synthetic protocol to prepare a pharmaceutical precursor of industrial interest, starting from a metabolite byproduct of intestinal bacteria.
NOVEL SUSTAINABLE STRATEGIES FOR THE SYNTHESIS OF ACTIVE PHARMACEUTICAL INGREDIENTS (APIS) AND BIOACTIVE MOLECULES THROUGH THE EXPLOITATION OF ISOLATED ENZYMES AS BIOCATALYSTS
TOGNOLI, CHIARA
2025
Abstract
Green chemistry is a chemical concept, condensed in 12 principles, which aims at directing chemical industries and academic researchers to paths of sustainability, supporting the efforts in the context of the ecological transition. A valuable strategy widely recognized to fulfill most of the 12 principles is the exploitation of biocatalytic methods. Biocatalysis, at the intersection of chemistry and biology, delineates the family of catalytic processes operating in a living biological system to perform chemical modifications on organic compounds. It involves the exploitation of enzymes as highly specialized proteins to be used in vitro to transform selected substrates into valuable products. As molecular catalysts, enzymes are proteins which provide precision and efficiency, enabling these transformations with outstanding stereo- and regioselectivity, working under significantly mild and ecofriendly conditions, thus allowing greener and more efficient synthesis of complex molecules. Biocatalyzed reactions can be performed by exploiting either purified isolated enzymes or lyophilized crude cell lysates. The wide availability of commercial synthetic genes suggests that, at least theoretically, any enzyme with a known amino acid sequence can be obtained recombinantly, thus expanding the range of applications for these catalysts. The first part of this PhD, carried out in the laboratories of SCITEC-CNR, focused on the development of sustainable entries to bioactive compounds, small molecules, and chiral synthons of pharmaceutical and industrial interest, by combining the ability of various biocatalysts alongside traditional organic synthetic protocols. Both commercially available and in-house expressed, recombinant enzymes were considered, exploiting their activities as isolated proteins to produce scaffolds of pharmaceutical interest and compounds endowed with potential antiprotozoal properties and/or cytotoxicity. Chapters I to III provide an overview of the enzymes used and the results obtained. A detailed study on the substrate promiscuity and selectivity features of a panel of alcohol dehydrogenases, including the functional characterization of a newly identified ketoreductase named Is2-SDR, is presented. Additionally, novel chemo-enzymatic strategies were developed to prepare compounds potentially endowed with antiprotozoal activity. An innovative approach, comprising two biocatalyzed step coupled with a chemical oxidation one, was applied to synthesize the promising antiplasmodial scaffolds linear diarylpirazines. Further investigations involved the application of laccases for various purposes: specifically, to study the oxidation of a series of aromatic amines; to prepare enantiopure terpene derivatives and their corresponding atropisomeric C-C dimers; and, finally, to propose an enzymatic approach for the synthesis of biologically active riminophenazines. The third year of my PhD program was spent at Dipharma Francis Srl, co-funder of the doctoral fellowship. The experimental work carried out during this period, described in Chapter IV, involved the design and development of a novel synthetic protocol to prepare a pharmaceutical precursor of industrial interest, starting from a metabolite byproduct of intestinal bacteria.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14242/209570
URN:NBN:IT:UNIMI-209570