Cyclopropane Synthase Enzymes as Biocatalysts for Green Cyclopropanation Reactions

Projektinformationen

CycloproBio

ID Finanzhilfevereinbarung: 101027045

DOI

10.3030/101027045

Projekt abgeschlossen

EK-Unterschriftsdatum 17 März 2021

Startdatum 1 Januar 2022

Enddatum 16 Januar 2024

Finanziert unter

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Gesamtkosten

€ 224 933,76

EU-Beitrag

€ 224 933,76

Koordiniert durch

UNIVERSITY COLLEGE LONDON
United Kingdom

Periodic Reporting for period 1 - CycloproBio (Cyclopropane Synthase Enzymes as Biocatalysts for Green Cyclopropanation Reactions)

Berichtszeitraum: 2022-01-01 bis 2023-12-31

Zusammenfassung vom Kontext und den Gesamtzielen des Projekts

Cyclopropane rings are a structural element naturally occurring in biologically active natural products compounds and pharmaceutical ingredients.The rigidity of its constrained structure and its reactivity make it a unique structural motif of interest in pharmaceutical science, synthetic chemistry, chemical biology. Due to their wide interest, several different synthetic methodologies have been developed over time, to prepare cyclopropanes, as racemates or in enantiomerically pure form, including, among the most important, the Simmons-Smith and the Corey–Chaykovsky reactions. More recently, enzymatic cyclopropanation methodologies ,using engineered variants of the cytochrome P450-BM3, myoglobin-based catalyst, for highly selective cyclopropanation reactions of different compounds. However, all this methodologies still suffers major synthetic and environmental limitations due to the use of harsh reaction conditions, stoichiometric metal mediators or chiral auxiliaries and hazardous diazo-compound reagents. Ultimately, cyclopropanation is still a major challenge in organic chemistry. To tackle this issue in terms of "green chemistry" and industrial application, Cyclopropane Synthase (CS) enzymes constitute an interesting class of enzymes, naturally catalysing the cyclopropanation of unsaturated lipids in bacteria and plants , operating under milder conditions and without using hazardous diazo-carbene co-substrates.
Cycloprobio has characterised new CS enzymes and paved the way to their exploitation as biocatalysts for selective synthesis of cyclopropanated fatty acids and phospholipids. This ultimately is expected to contribute in reducing the environmental impact of a variety of chemical and pharmaceutical manufacturing processes. The project also firmly aligns with the key European research priorities of “green chemistry” and “sustainable manufacturing processes”.

Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse

Over the course of the project the initial focus was understanding the biochemistry of the cyclopropanating enzyme E.coli CFAS in order two develop a new biocatalyst. First GC-MS based assay was used to unambiguously prove ecCFAS catalyzed stereoselective biocatalytic synthesis of cyclopropyl lipids. DLS and LC-MS studies disclosed the selectivity of the enzymatic transformation of phosphatidylglycerol and the impact of phospholipid vesicles on the catalytic activity. The substrate scope of the enzyme demonstrated PG being the preferred substrate of ecCFAS. The toolbox of cyclopropanating enzymes was expanded to members of the CFAS and MACS families of enzymes and these new enzymes were compared in term of substrate scope and selectivity. This unvealed new insights in the sequence/selectivity relationship in this class of under explored enzymes.
Mutagenesis and in-silico experiments confirmed the role of key amino acids involved in the catalytic cyclopropanation and provided an insight into the lipid substrates preference of ecCFAS. Follow up work of semi-rational engineering of ecCFAS is currently ongoing to expand the substrate scope of the enzyme to new non-natural substrates. The results of the project is being disseminated to the scientific community via peer review open access publications and via specialized congresses contributions and will be shared with the broader public after publication, via social media.

Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts)

CycloproBio firmly aligns with the key European research priorities of “green chemistry” and “sustainable manufacturing processes” and with the European and global chemical and pharmaceutical industries’ strategic interests in the development of new environmental friendly alternative manufacturing chemical processes. The cyclopropane ring is a highly strained structural element widely present in natural products, pharmaceuticals, and other biologically active molecules. Its presence often confers unique physiochemical properties to those compounds. This unique chemical functionality can also be easily manipulated further in organic synthesis with a variety of chemical methods. Despite the fact that several methods are available for their preparation, as racemate and enantioenriched form, their preparation is still challenging from the environmental impact and safety perspectives, to be applied in industry . The research conducted during this project has addressed this pressing issue in the field of synthetic and industrial chemistry with the characterization of enzymes that can potentially be exploited for the environmental friendly and sustainable manufacturing of cyclopropanated chemicals and pharmaceutical ingredients.

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