ANSTO :: Browsing by Author "Cagnes, MP"

Browsing by Author "Cagnes, MP"

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ANSTO’s National Deuteration Facility: recent advancements and an overview on molecular deuteration capabilities for neutron applications
(Australian Institute of Nuclear Science and Engineering (AINSE), 2020-11-11) Wilde, KL; Cagnes, MP; Duff, AP; Klenner, MA; Krause-Heuer, AM; Moir, M; Rekas, A; Russell, RA; Yepuri, NR; Darwish, TA
The National Deuteration Facility (NDF) at the Australian Nuclear Science and Technology Organisation (ANSTO) provides deuteration through both biological and chemical techniques for a diversity of molecules and applications and is the only facility of its type in the Southern Hemisphere with the specialised expertise and infrastructure for both biological and chemical molecular deuteration. Molecular deuteration of organic compounds and biomolecules significantly increases the options in complex structure function investigations using neutron scattering and reflectometry, nuclear magnetic resonance (NMR), mass spectrometry (MS) and other techniques. Deuteration (substitution of the naturally occurring hydrogen stable isotope deuterium (2H or D) for 1H (or H)) can provide contrast and improved resolution to assist investigations into the relationship between molecular structure and function of molecules of both biological and synthetic origin. By developing a suite of capabilities in both in vivo deuteration of biomolecules and chemical deuteration of small organic molecules, the NDF provides access to a broad range of deuterated molecules for research and industry. Variably deuterated proteins can be produced via recombinant expression in Escherichia coli and other microbial systems utilised to produce deuterated cellulose and cholesterol. By tailoring deuteration approaches with the ongoing development of chemical deuteration protocols for a broader range of molecular classes than available commercially, the NDF has increased the range of systems that can be investigated using deuterated molecules. Lipids, phospholipids (including head or tail or head/tail deuterated mono-unsaturated lipids such as POPC and DOPC), heterocyclics, aromatics, surfactants, ionic liquids, saturated and unsaturated fatty acids, sugars and match-out detergents have been deuterated. Common neutron applications include partially deuterated proteins for SANS experiments investigating multiprotein systems, neutron crystallography of perdeuterated proteins, neutron reflectometry of lipid bilayers systems and SANS using saturated lipid vesicles, or detergents amongst others. An overview and update on the NDF will be provided which will include details on the NDF User Program (e.g. information on the available modes of access), recent advancements in custom deuterated molecules available and brief highlights of deuterated molecule utilisation for neutron experiments at ANSTO’s Australian Centre for Neutron Scattering (ACNS). © 2020 The Authors.
Chemical deuteration and neutrons for structure function applications
(Australian Institute of Nuclear Science and Engineering, 2016-11-29) Darwish, TA; Yepuri, NR; Heuer, AK; Cagnes, MP; Holden, PJ
In small angle neutron scattering and neutron reflectometry studies, the use of mixtures of deuterated and hydrogenated solvents to manipulate scattering length density and achieve contrast variation is widespread. This approach, while useful is less effective for multicomponent organic systems containing molecules of similar scattering length densities. In such systems molecular deuteration is necessary to achieve contrast between the different components and it significantly increases the options in structure function investigations. There have been limited global initiatives in the field of molecular deuteration where the majority of these programs focus on biological deuteration of proteins and lipids, while more complex deuterated small molecules haven’t been widely available to the neutron community. This has limited the experiments that can be performed, and formed a bottle neck for advancing the applications of neutron scattering. In this paper we will discuss the recent advancements and the impact of deuteration on the research outcomes achieved by using deuterated molecules produced by the chemical deuteration laboratories at ANSTO’s National Deuteration Facility. Recent high-impact case studies will be presented which reveal the exciting and diverse characterisation studies which are now available for the neutron community. The chemical deuteration of surfactants, sugars, heterocyclic and aromatic compounds has made possible a wide range of investigations systems in the fields of molecular electronics, structural biology, and biotechnology.
Chemical deuteration of ionic liquids and their application in neutron reflectivity
(International Conference on Neutron Scattering, 2017-07-12) Akutsu, K; Darwish, TA; Cagnes, MP; Tamura, K; Kanaya, T
Low melting imidazolium salts are one of the most popular and widely used ionic liquids (ILs). Due to their low vapor pressures and thermal stability, ILs have been widely studied and employed in many applications such as synthesis and catalysis solvents, extraction solvents, electrolyte, and biopolymer solvents. However, in many case, structural analysis of IL samples is difficult because the X-ray and neutron scattering contrast between the ILs and the organic solute is not enough to allow different elements to be clearly discerned. Partial or complete deuteration of organic compounds for contrast variation in the scattering length densities of materials is one of the most effective techniques in the application of neutron scattering analysis. Therefore, this technique is ideally suited for the structural analysis of IL samples using neutron scattering techniques. In this study, we synthesized several kinds of deuterated 1-alkyl-3-methylimidazolium ionic liquids (alkyl = ethyl, butyl, and octyl) for studying the electric double layer structure in ILs using neutron reflectivity technique. For this purpose, we have developed a simple and effective method for the deuteration of imidazolium salts ILs on gram scale, starting from their protonated versions. In this presentation, we will show the summary of deuteration experiments and the results of neutron reflectivity analysis of the electric double layer structure in ILs.
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.
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
Lattice response of the porous coordination framework Zn(hba) to guest adsorption
(Cambridge University Press, 2017-09-05) Auckett, JE; Dharma, AD; Cagnes, MP; Darwish, TA; Abrahams, BF; Barbarao, R; Hudson, TA; Robson, R; White, KF; Peterson, VK
Analysis of in situ neutron powder diffraction data collected for the porous framework material Zn(hba) during gas adsorption reveals a two-stage response of the host lattice to increasing CO2 guest concentration, suggesting progressive occupation of multiple CO2 adsorption sites with different binding strengths. The response of the lattice to moderate CH4 guest concentrations is virtually indistinguishable from the response to CO2, demonstrating that the influence of host–guest interactions on the Zn(hba) framework is defined more strongly by the concentration than by the identity of the guests. © International Centre for Diffraction Data 2017
New contrast options - diversity and specifity of deuteration
(Australian Institute of Nuclear Science and Engineering (AINSE), 2018-11-19) Holden, PJ; Cagnes, MP; Davydova, N; Duff, AP; Howard, JK; Krause-Heuer, AM; Yepuri, NR; Darwish, TA
The Australian National Deuteration Facility (NDF) continues to develop new capabilities in molecular deuteration. The tailoring of deuteration approach to solve specific contrast problems has greatly increased the range of systems that can be investigated and recent capability development and new applications will be described. The common problem of how to obtain good contrast during SANS of membrane proteins solubilised in detergent micelles has been solved by specific deuteration of detergent head groups and hydrophobic tails to different levels to contrast match at 100% D20 buffer. Likewise, in investigating the lo cation of a purely hydrophobic transmembrane peptide in bicontinuous cubic phase liquid crystals Page 25 ANBUG-AINSE Neutron Scattering Symposium, AANSS 2018 / Book of Abstracts suitable for drug delivery, a mixture of deuterated and hydrogenated phytanoyl monoethanolamide was used to perfectly contrast match out either gyroid or diamond phase lipids to pinpoint the peptides location. In Soft Matter, investigation of structure of the ligands in the shell layer of self-assembled monolayerprotected nanoparticles has been made possible by use of various combinations of immiscible deuterated or hydrogenated Phenylethanethiol (PET) and Dodecanethiol (DDT) and MONSA modelling of SANS data showed the janus and belt like distribution of the ligands on the surface and the effects of nanoparticle core size and ligand ratio on the patterning. Other case studies will include use of deuterated ionic liquids and new previously unavailable deuterated molecules will be highlighted. This work was supported by the National Collaborative Research Infrastructure Strategy © The Authors.
Synthesis of perdeuterated and selectively deuterated phospholipids and lipids for neutron applications
(Australian Institute of Nuclear Science and Engineering, 2016-11-29) Yepuri, NR; Darwish, TA; Krause-Heuer, AM; Leung, A; Cagnes, MP; Holden, PJ
The National Deuteration Facility (NDF) is focused on the provision of deuterated molecules which extends the options for contrast in neutron scattering to encompass not only solvent but molecular deuteration. Over the past few years the NDF has expanded its synthesis capability from simple deuterated fatty acids to complex deuterated molecules including lipids and phospholipids. We are now able to produce head or tail deuterated lipids including phospholipids based on oleic acid with a range of head groups (Fig. 1). These include perdeuterated 1,2-oleoyl-sn-glycero-3-phosphocholine (POPC), selectively deuterated POPC, branched chain (phytanic) phospholipids 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhyPC), and perdeuterated mono oleoyl glycerol and phytanoyl monoethanolamide.[1] These lipids have been extensively used in constructing biologically more relevant model membranes and lipidic matrices for investigations using neutron studies. These include structural and dynamical studies of biomimetic membranes and the encapsulation of biomolecules in lipid-based bicontinuous cubic phases for drug-delivery, membrane protein crystallization, and biosensor applications. Details about design, synthesis and characterisation of these deuterated precursors and final compound will be presented.
Time-resolved small-angle neutron scattering for characterization of molecular exchange in lipid nanoparticle therapeutics
(Elsevier, 2025-01) Hilburg, SL; Sokolova, AV; Cagnes, MP; Pozzo, LD
Hypothesis: Nano-scale dynamics of self-assembled therapeutics play a large role in their biological function. However, assessment of such dynamics remains absent from conventional pharmaceutical characterization. We hypothesize that time-resolved small-angle neutron scattering (TR-SANS) can reveal their kinetic properties. For lipid nanoparticles (LNP), limited molecular motion is important for avoiding degradation prior to entering cells while, intracellularly, enhanced molecular motion is then vital for effective endosomal escape. We propose TRSANS for quantifying molecular exchange in LNPs and, therefore, enabling optimization of opposing molecular behaviors of a pharmaceutical in two distinct environments. Experiments: We use TR-SANS in combination with traditional SANS and small-angle x-ray scattering (SAXS) to experimentally quantify nano-scale dynamics and provided unprecedented insight to molecular behavior of LNPs. Findings: LNPs have molecular exchange dynamics relevant to storage and delivery which can be captured using TR-SANS. Cholesterol exchanges on the time-scale of hours even at neutral pH. As pH drops below the effective pKa of the ionizable lipid, molecular exchange occurs faster. The results give insight into behavior enabling delivery and provide a quantifiable metric by which to compare formulations. Successful analysis of this multicomponent system also expands the opportunities for using TR-SANS to characterize complex therapeutics. © 2024 Elsevier Inc.

Browsing by Author "Cagnes, MP"

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