Browsing by Author "Recsei, C"

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    Deuterated squalene and sterols from modified Saccharomyces cerevisiae
    (Royal Society of Chemistry (RSC), 2023-07-31) Recsei, C; Russell, RA; Cagnes, MP; Darwish, TA
    Uniformly deuterated sterols and biosynthetically related materials are important for neutron, NMR, tracing and bioanalysis studies as well as critical tools for the creation of improved lipid nanoparticle formulations. The production of sufficient quantities of materials relies not only on the engineering of microorganisms to selectively accumulate desired materials but also methods for the isolation, purification and characterisation of these materials to ensure their usefulness. Uniformly deuterated squalene, the universal precursor to sterols in biological systems, has been produced and characterised. Cholesterol has been produced with controlled levels of uniform deuteration, increased biosynthetic yield and a methodology developed for the extraction and purification of this material without HPLC. Two sterols, not previously produced in deuterated forms, have been prepared with uniform deuteration: 22,23-dihydrobrassicasterol and 24-methylenecholesterol. This report triples the number of sterols that have been produced with uniform deuteration, purified and characterised and provides a silylation/silver ion chromatography protocol for the separation of sterols which differ by the degree of unsaturation. The techniques for the 13C NMR analysis of deuterated sterols, site-specific deuteration levels and an analysis of key biosynthetic steps based on these data are reported. © 2023 The Royal Society of Chemistry, Open Access, published under a CC-BY-NC licence.
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    Deuteration for biological SANS: case studies, success and challenges in chemistry and biology
    (Elsevier, 2022-11) Duff, AP; Cagnes, MP; Darwish, TA; Krause-Heuer, AM; Moir, M; Recsei, C; Rekas, A; Russell, RA; Wilde, KL; Yepuri, NR
    Small angle neutron scattering is a powerful complementary technique in structural biology. It generally requires, or benefits from, deuteration to achieve its unique potentials. Molecular deuteration has become a mature expertise, with deuteration facilities located worldwide to support access to the technique for a wide breadth of structural biology and life sciences. The sorts of problems well answered by small angle scattering and deuteration involve large (> 10 Å) scale flexible movements, and this approach is best used where high-resolution methods (crystallography, NMR, cryo-EM) leave questions unanswered. This chapter introduces deuteration, reviewing biological deuteration of proteins, lipids and sterols, and then steps through the ever-expanding range of deuterated molecules being produced by chemical synthesis and enabling sophisticated experiments using physiologically relevant lipids. Case studies of recent successful use of deuteration may provide illustrative examples for strategies for future experiments. We discuss issues of nomenclature for synthesised molecules of novel labeling and make recommendations for their naming. We reflect on our experiences, with cost associated with achieving an arbitrary deuteration level, and on the benefits of experimental co-design by user scientist, deuteration scientist, and neutron scattering scientist working together. Although methods for biological and chemical deuteration are published in the public domain, we recommend that the best method to deuterate is to engage with a deuteration facility. © 2022 Elsevier