Li, Chuhan; (2025) An innovative and sustainable biocatalytic approach for the cyclopropanation of phospholipids and fatty acids by Cyclopropane Fatty Acid Synthase (CFAS) enzymes. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Cyclopropane moieties serve as valuable pharmacophores in drug design by modulating lipophilicity, enhancing solubility, improving metabolic stability, and restricting conformation. To date, more than sixty drugs containing a cyclopropane subunit have been launched on the market approved by FDA. Traditional chemical synthesis faces limitations including stoichiometric metal mediators, noble-metal catalysts, chiral auxiliaries, hazardous diazo compounds, and harsh conditions. Although carbene transferases developed in last decade overcome some limitations, they still require hazardous diazo precursors. CFAS enzymes provide a greener approach by catalysing phospholipid cyclopropanation under mild conditions using S-adenosyl methionine (SAM) cofactor. This study comprehensively investigated the substrate adaptability, selectivity, and catalytic mechanism of CFAS enzymes. Novel chromatographic methods integrating LC-MS and chiral HPLC were developed, enabling the first quantification of conversion, regioselectivity, and stereoselectivity in CFAS biocatalytic cyclopropanation. Systematic optimization confirmed significant catalytic potential in three wild-type CFAS variants (ec/la/st). Crucially, we synthesized nonphospholipid pseudo substrates and achieved the first CFAS-catalysed cyclopropanation beyond natural phospholipids. Dynamic light scattering (DLS) verified vesicle organization in reaction buffer. Substrate scope investigation concerning polar headgroup confirmed essential anionic headgroups, while an aiding phospholipid strategy with saturated phosphatidylglycerols (PG) effectively enhanced compatibility of substrate with zwitterionic headgroup. Furthermore, optimization of fatty acid chain length and position yielded >99% enantiomeric excess across multiple substrates. Mutational analysis of ecCFAS revealed functional correlations between catalytic centre residues and enzymatic activity. These findings provide an important foundation for the engineering and application of CFAS enzymes.

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