ANSTO :: Browsing by Author "Yepuri, NR"

Browsing by Author "Yepuri, NR"

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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.
The Australian National Deuteration Facility for structure function applications using neutrons
(International Conference on Neutron Scattering, 2017-07-12) Darwish, TA; Yepuri, NR; Heuer, AK; Duff, AP; Wilde, KL; Holden, PJ
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 National Deuteration Facility of the Australian Nuclear Science and Technology Organisation. Recent high-impact case studies in the fields of molecular electronics, structural biology, and biotechnology will be presented which reveal the exciting and diverse characterisation studies which are now available for the neutron community.
Cephalosporin-3'-diazeniumdiolates: targeted no-donor prodrugs for dispersing bacterial biofilms
(Wiley-V C H Verlag GMBH, 2012-01-01) Barraud, N; Kardak, BG; Yepuri, NR; Howlin, RP; Webb, JS; Faust, SN; Kjelleberg, S; Rice, SA; Kelso, MJ
Cereal starch granules with high (>50%) amylose content are a promising source of nutritionally desirable resistant starch, i.e. starch that escapes digestion in the small intestine, but the structural features responsible are not fully understood. We report the effects of partial enzyme digestion of maize starch granules on amylopectin branch length profiles, double and single helix contents, gelatinisation properties, crystallinity and lamellar periodicity. Comparing results for three maize starches (27, 57, and 84% amylose) that differ in both structural features and amylase-sensitivity allows conclusions to be drawn concerning the rate-determining features operating under the digestion conditions used. All starches are found to be digested by a side-by-side mechanism in which there is no major preference during enzyme attack for amylopectin branch lengths, helix form, crystallinity or lamellar organisation. We conclude that the major factor controlling enzyme susceptibility is granule architecture, with shorter length scales not playing a major role as inferred from the largely invariant nature of numerous structural measures during the digestion process (XRD, NMR, SAXS, DSC, FACE). Results are consistent with digestion rates being controlled by restricted diffusion of enzymes within densely packed granular structures, with an effective surface area for enzyme attack determined by external dimensions (57 or 84% amylose - relatively slow) or internal channels and pores (27% amylose - relatively fast). Although the process of granule digestion is to a first approximation non-discriminatory with respect to structure at molecular and mesoscopic length scales, secondary effects noted include (i) partial crystallisation of V-type helices during digestion of 27% amylose starch, (ii) preferential hydrolysis of long amylopectin branches during the early stage hydrolysis of 27% and 57% but not 84% amylose starches, linked with disruption of lamellar repeating structure and (iii) partial B-type recrystallisation after prolonged enzyme incubation for 57% and 84% amylose starches but not 27% amylose starch. © 2012, Wiley-VCH Verlag GmbH & Co. KGaA
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.
Deuterated phospholipids to study the structure, function and dynamics of membrane proteins using neutron scattering
(Australian Nuclear Science and Technology Organisation, 2021-11-26) Yepuri, NR; Moir, M; Krause-Heuer, AM; Darwish, TA
Contrast matching and contrast variation in neutron scattering provide unparalleled power for understanding the structure, function, and dynamics of a selected component in a multicomponent system. A sophisticated contrast study often requires the availability of deuterated molecules in which deuterium atoms are introduced in a predictable and controlled fashion to replace protons. This can be achieved by direct deuteration of precursors followed by custom chemical synthesis, for which expertise and capabilities have been developed at facility (NDF), ANSTO. It this paper we will discuss recent high impact research output using deuterated phospholipids produced by NDF/ANSTO. We will describe the synthesis and applications of selectively or perdeuterated unsaturated phospholipids to contrast match out the whole lipid bilayer or nano disks within a multicomponent system. Further, we also describe their role in investigations related to membrane lipoproteins (ApoE) exchange in relation to lipid unsaturation,[1] effect of membrane composition,[2] and conformational analysis Mg+2 channel by neutron scattering techniques.[2, 3]
Fluorine-18 radiolabelling and in vitro / in vivo metabolism of [18F]D4-PBR111
(John Wiley & Sons, Inc, 2019-05-26) Wyatt, NA; Safavi-Naeini, M; Wotherspoon, ATL; Arthur, A; Nguyen, AP; Parmar, A; Hamze, H; Day, CM; Zahra, D; Matesic, L; Davis, E; Rahardjo, GL; Yepuri, NR; Shepherd, R; Murphy, RB; Pham, TQ; Nguyen, VH; Callaghan, PD; Holden, PJ; Grégoire, MC; Darwish, TA; Fraser, BH
Objectives The purinergic receptor P2X ligand-gated ion channel type 7 (P2X7R) is an adenosine triphosphate (ATP)-gated ion-channel, and P2X7R is a key player in inflammation. P2X7R is an emerging therapeutic target in central nervous system (CNS) diseases including Alzheimer's disease (AD) and Parkinson's disease (PD), because P2X7R also plays a pivotal role in neuroinflammation. P2X7R represents a potential molecular imaging target for neuroinflammation via biomedical imaging technique positron emission tomography (PET), and several radioligands targeting P2X7R have been developed and evaluated in animals. In our previous work, we have developed and characterized [11C]GSK1482160 as a P2X7R radioligand for neuroinflammation,2 clinical evaluation of [11C]GSK1482160 in healthy controls and patients is currently underway, and the estimation of radiation dosimetry for [11C]GSK1482160 in normal human subjects has been reported.3 Since the half-life (t1/2) of radionuclide carbon-11 is only 20.4 min, it is attractive for us to develop derivatives of [11C]GSK1482160, which can be labeled with the radionuclide fluorine-18 (t1/2, 109.7 min), and a fluorine-18 ligand would be ideal for widespread use.4 To this end, a series of [18F]fluoroalkyl including [18F]fluoromethyl (FM), [18F]fluoroethyl (FE), and [18F]fluoropropyl (FP) derivatives of GSK1482160 have been prepared and examined as new potential P2X7R radioligands. © 2019 The Authors
The influence of glass transitions on diffusion in OLED stacks
(Australian Institute of Nuclear Science and Engineering, 2016-11-29) McEwan, JA; Clulow, AJ; Shaw, PE; Nelson, A; Yepuri, NR; Darwish, TA; Burn, PL; Gentle, IR
Of all of the organic electronic devices thus far conceived, organic light emitting diodes (OLEDs) have been the most successfully applied in a commercial setting. With OLED displays now available in the television and portable device markets, the appetite for their continued development continues to garner considerable research interest. Optimised OLED device architectures typically comprise a number of organic layers with thicknesses between 10 nm and 100 nm sandwiched between inorganic electrodes. Each of the organic layers used in the device is sequentially deposited in an order that optimises charge transport and capture, and light emission from the devices. The fidelity and stability of these multilayer organic stacks is therefore of paramount importance in determining the efficiencies and operational lifetimes of OLED devices. Neutron reflectometry is a powerful technique for probing the layered structures found within OLEDs by utilising selective deuteration to provide contrast between or within the layers.1–3 Modelling the changes in the neutron reflectivity profiles of the OLED stacks deposited onto smooth substrates allows for the visualisation of changes in the layered structure in a non destructive manner. In this talk we will outline our recent efforts to relate the thermal properties of the organic materials used in OLED devices with their diffusion behaviour under thermal stress. Our collaboration with the National Deuteration Facility has led to the synthesis of a number of previously unobtainable deuterated analogues of semiconducting molecules typically used in OLEDs and that have a range of thermal characteristics.3–5 These molecules were used in time-resolved reflectometry experiments that have allowed us to systematically build up an understanding of the importance of glass transitions for the stability of OLED stacks.
Intrinsically disordered stress protein COR15A resides at the membrane surface during dehydration
(Biophysical Society, 2017-08-08) Bremer, A; Kent, B; Hauß, T; Thalhammer, A; Yepuri, NR; Darwish, TA; Garvey, CJ; Bryant, G; Hincha, DK
Plants from temperate climate zones are able to increase their freezing tolerance during exposure to low, above-zero temperatures in a process termed cold acclimation. During this process, several cold-regulated (COR) proteins are accumulated in the cells. One of them is COR15A, a small, intrinsically disordered protein that contributes to leaf freezing tolerance by stabilizing cellular membranes. The isolated protein folds into amphipathic α-helices in response to increased crowding conditions, such as high concentrations of glycerol. Although there is evidence for direct COR15A-membrane interactions, the orientation and depth of protein insertion were unknown. In addition, although folding due to high osmolyte concentrations had been established, the folding response of the protein under conditions of gradual dehydration had not been investigated. Here we show, using Fourier transform infrared spectroscopy, that COR15A starts to fold into α-helices already under mild dehydration conditions (97% relative humidity (RH), corresponding to freezing at −3°C) and that folding gradually increases with decreasing RH. Neutron diffraction experiments at 97 and 75% RH established that the presence of COR15A had no significant influence on the structure of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. However, using deuterated POPC we could clearly establish that COR15A interacts with the membranes and penetrates below the headgroup region into the upper part of the fatty acyl chain region. This localization is in agreement with our hypothesis that COR15A-membrane interaction is at least, in part, driven by a hydrophobic interaction between the lipids and the hydrophobic face of the amphipathic protein α-helix. © 2017 Biophysical Society - Open access
Mild conditions for deuteration of primary and secondary arylamines for the synthesis of deuterated optoelectronic organic molecules
(Multidisciplinary Digital Publishing Institute, 2014-11-13) Krause-Heuer, AM; Yepuri, NR; Darwish, TA; Holden, PJ
Deuterated arylamines demonstrate great potential for use in optoelectronic devices, but their widespread utility requires a method for large-scale synthesis. The incorporation of these deuterated materials into optoelectronic devices also provides the opportunity for studies of the functioning device using neutron reflectometry based on the difference in the scattering length density between protonated and deuterated compounds. Here we report mild deuteration conditions utilising standard laboratory glassware for the deuteration of: diphenylamine, N-phenylnaphthylamine, N-phenyl-o-phenylenediamine and 1-naphthylamine (via H/D exchange in D2O at 80 °C, catalysed by Pt/C and Pd/C). These conditions were not successful in the deuteration of triphenylamine or N,N-dimethylaniline, suggesting that these mild conditions are not suitable for the deuteration of tertiary arylamines, but are likely to be applicable for the deuteration of other primary and secondary arylamines. The deuterated arylamines can then be used for synthesis of larger organic molecules or polymers with optoelectronic applications. © 2014, Creative Commons.
Monitoring the phase evolution in LiCoO2 electrodes during battery cycles using in-situ neutron diffraction technique
(John Wiley & Sons, Inc, 2019-12-03) Jena, A; Lee, PH; Pang, WK; Hsiao, KC; Peterson, VK; Darwish, TA; Yepuri, NR; Wu, SH; Chang, H; Liu, RS
LiCoO2 (LCO) with average particle distribution of 8 μm (LCO-A) and 11 μm (LCO-B) exhibit substantial differences in cycle performance. The half-cells have similar first-cycle discharge capacities of 173 and 175 mAh/g at 0.25 C, but after 100 cycles, the discharge capacities are substantially different, that is, 114 and 141 mAh/g for LCO-A and LCO-B, respectively. Operando neutron powder diffraction of full LCO||Li4Ti5O12 batteries show differences in the LCO reaction mechanism underpinning the electrochemical behavior. LCO-A follows a purely solid solution reaction during cycling compared to the solid solution and two-phase reaction mechanism in LCO-B. The absence of the two-phase reaction in LCO-A is consistent with a homogeneous distribution of Li throughout the particle. The two-phase reaction in LCO-B reflects two distinguishable distributions of Li within the particles. The faster capacity decay in LCO-A is correlated to an increase in electrode cracking during battery cycles. © 2019 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co.
A rapid MS/MS method to assess the deuterium kinetic isotope effect and associated improvement in the metabolic stability of deuterated biological and pharmacological molecules as applied to an imaging agent
(Elsevier B. V., 2019-08-08) Murphy, RB; Wyatt, AN; Fraser, BH; Yepuri, NR; Holden, PJ; Wotherspoon, ATL; Darwish, TA
The deuterium kinetic isotope effect has been known for a period of 40 years, but it is only relatively recently that new drug entities (NDEs) incorporating deuterium demonstrating beneficial pharmacokinetics, pharmacodynamics, and toxicology have arrived to market. Determination of the precise location to deuterate and subsequently any evaluation for a kinetic isotope effect (KIE) is challenging. Typically, such an evaluation would be performed in an in vitro metabolic assay (e.g. liver microsomes) in separate reaction media for both the deuterated and non-deuterated analogues. Here, we have devised an approach whereby we incubate a 1:1 ratio of both the deuterated and protio-form of an imaging agent together in the same liver microsomal assay and determine the relative rate of consumption of both moieties, based upon specific MS-MS transitions unique to both molecules without the need for liquid chromatography-mass spectrometry (LC-MS) separation and quantification. Any deviation of the ratio of the MS transitions from the initial starting point indicated an observable KIE. A site specific deuteration of PBR111, a neuroinflammation imaging agent, was chosen for a proof-of-concept study. Based upon prior mechanistic knowledge of PBR111, two locations were selected for deuteration; an active and inactive site, to corroborate that there was no significant KIE for the inactive site and confirm the efficacy of the developed methodology. Crown Copyright © 2019 Published by Elsevier B.V.
Realtime structural characterisation of thin film OLED stacks during thermally induced diffusion - the importance of glass transitions
(International Conference on Neutron Scattering, 2017-07-12) Nelson, A; McEwan, JA; Clulow, AJ; Shaw, PE; Darwish, TA; Yepuri, NR; Burn, PL; Gentle, IR
Organic Light Emitting Diode (OLED) devices are ubiquitous in the displays of many modern electronic devices, including televisions and mobile phones. High efficiency OLEDs are typically made as a sequentially deposited multilayer, with different organic semiconductor layers being required for hole/electron injection/transport, as well as light emission. The operational lifetime, efficiency and fidelity of these OLEDs depends on the structural stability of this multilayer stack, and understanding the factors that affect this stability are crucial in developing new devices. We have systematically characterised the kinetics of structural evolution in these systems as they experience thermally induced diffusion. These leading edge experiments are performed on the Platypus neutron reflectometer, which has been pioneering event mode acquisition techniques to capture quickly changing reflection signals as the multilayers inter-diffuse. Selective deuteration of these semiconductor materials is also critical in providing neutron contrast between each of the layers, without which the experiment could not be carried out. Our investigations reveal the importance of glass transition temperatures on the stability of these OLED systems and provide clear guidelines for material choices when designing new devices. Indeed, with knowledge of each of the T \'s one can predict the way in which diffusion occurs. For example, use of a high Tg emissive layer does not necessarily prevent diffusion from taking place.
Stereoselective synthesis of perdeuterated phytanic acid, its phospholipid derivatives and their formation into lipid model membranes for neutron reflectivity studies
(Elsevier, 2014-10-01) Yepuri, NR; Holt, SA; Moraes, G; Holden, PJ; Hossain, KR; Valenzuela, SM; Darwish, TA; James, M
We describe a straightforward method, for synthesis of large scale (gram quantities) of highly deuterated phytanic acid from commercially available phytol while preserving the stereochemistry around the chiral centres. The subsequent synthesis of tail-deuterated analogues of the archeabacterial membrane lipids 1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-phosphocholine (DPEPC) and 1,2-di(3RS,7R,11R-phytanoyl)-sn-glycero-3-phosphocholine (DPhyPC) from perdeuterated phytanic acid is also described. Both lipids were employed in construction of two different model membranes, namely Langmuir monolayers and a tethered bilayer membrane (TBM) on a solid substrate, characterised by pressure area isotherm and neutron reflectometry techniques. At 10 mN/m pressure the head-group thickness of both monolayers was similar while the thickness of the tail region was significantly larger for tail-deuterated DPhyPC, which was evident from a smaller area per molecule. At 20 mN/m the thickness of the head and tail regions in both lipids was comparable, yet the area per molecule of tail-deuterated DPhyPC was 10% smaller than tail-deuterated DPEPC. In the TBM bilayer model membrane, the thickness of the lipid tails in both inner and outer leaflets was 8.2 Å, giving a total of 16.4 Å. Deuteration enabled unambiguous determination of the relative proportion of the hydrogenous tether, phospholipid and subphase. © 2014, Elsevier Ireland Ltd.
Synthesis of cephalosporin-3'-diazeniumdiolates: biofilm dispersing no-donor prodrugs activated by b-lactamase
(Royal Society of Chemistry, 2013-01-01) Yepuri, NR; Barraud, N; Mohammadi, NS; Kardak, BG; Kjelleberg, S; Rice, SA; Kelso, MJ
Use of biofilm dispersing NO-donor compounds in combination with antibiotics has emerged as a promising new strategy for treating drug-resistant bacterial biofilm infections. This paper details the synthesis and preliminary evaluation of six cephalosporin-3[prime or minute]-diazeniumdiolates as biofilm-targeted NO-donor prodrugs. Each of the compounds is shown to selectively release NO following reaction with the bacteria-specific enzyme [small beta]-lactamase and to trigger dispersion of Pseudomonas aeruginosa biofilms in vitro. © 2013, Royal Society of Chemistry
Synthesis of deuterated [D-32]oleic acid and its phospholipid derivative [D-64]dioleoyl-sn-glycero-3-phosphocholine
(Wiley-Blackwell, 2013-07-16) Darwish, TA; Luks, E; Moraes, G; Yepuri, NR; Holden, PJ; James, M
Oleic acid and its phospholipid derivatives are fundamental to the structure and function of cellular membranes. As a result, there has been increasing interest in the availability of their deuterated forms for many nuclear magnetic resonance, infrared, mass spectroscopy and neutron scattering studies. Here, we present for the first time a straightforward, large-scale (gram quantities) synthesis of highly deuterated [D32]oleic acid by using multiple, yet simple and high yielding reactions. The precursors for the synthesis of [D32]oleic acid are [D14]azelaic acid and [D17]nonanoic acid, which were obtained by complete deuteration (>98% D) of their 1H forms by using metal catalysed hydrothermal H/D exchange reactions. The oleic acid was produced with ca. 94% D isotopic purity and with no contamination by the trans-isomer (elaidic acid). The subsequent synthesis of [D64]dioleoyl-sn-glycero-3-phosphocholine from [D32]oleic acid is also described. Copyright © 2013 John Wiley & Sons, Ltd.© 2013, Wiley-Blackwell.
Synthesis of per-deuterated alkyl amines for the preparation of deuterated organic pyromellitamide gelators
(Pergamon-Elsevier Science Ltd, 2013-05-15) Yepuri, NR; Jamieson, SA; Darwish, TA; Rawal, A; Hook, JM; Thordarson, P; Holden, PJ; James, M
A general, direct and scalable synthesis of per-deuterated alkyl amines is reported, together with their incorporation into pyromellitamides, which form self-assembled gels in cyclohexane. The deuterium labelling of these gelators allows the study of the dynamic intermolecular interactions in these materials using solid-state 2H NMR spectroscopy.© 2013, Elsevier Ltd.
Synthesis of perdeuterated 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine ([D82]POPC) for neutron studies on bilayer lipid membranes
(International Conference on Neutron Scattering, 2017-07-12) Yepuri, NR; Darwish, TA; Leung, A; Krause-Heuer, AM; Wacklin, HP; Delhom, R; Holden, PJ
The complexity of the chemical synthesis of completely deuterium-labelled unsaturated lipids has meant that most neutron experiments, to date, have been restricted to saturated phospholipid species, the behaviour of which under physiological conditions may not be representative of the unsaturated varieties found in biological membranes. Unsaturated lipids occur widely in nature and are crucial for the fluidity of cell membranes. Biologically relevant phospholipids such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) are typically asymmetric and cis-9 unsaturated in the sn-2-acyl chain, where as the sn-1 chain is often saturated. Commercially available sn-1 chain deuterated [D31]1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine([D31]POPC) does not provide enough contrast for detailed structural investigation through neutron studies, and is nearly perfectly contrast-matched to most proteins and membrane binding peptides in heavy water, which makes them neutron invisible. With this background in mind, we have developed a method for the synthesis of perdeuterated POPC and its partially labelled form.[1] The structure of a supported bilayer membrane formed from these lipids was determined by measuring neutron reflectivity in a series of solvent contrasts, which will be discussed in this paper.
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.
Synthesis of perdeuterated linoleic acid-d31 and chain deuterated 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine-d62
(John Wiley & Sons, 2022-08-08) Moir, M; Yepuri, NR; Marshall, DL; Blanksby, SJ; Darwish, TA
Herein, we report a gram-scale synthesis of perdeuterated linoleic acid-d31. The starting materials for the synthesis are two saturated fatty acids, azelaic acid-d14 and pentanoic acid-d9, which can be obtained by metal catalysed hydrothermal hydrogen-deuterium exchange. The synthesis utilises the fatty acids directly via decarboxylative coupling. Copper catalysed coupling of a terminal alkyne intermediate with a propargyl bromide derivative affords a skipped diyne, which can be reduced using P-2 nickel to obtain the desired cis,cis-diene geometry. The subsequent synthesis of the tail-deuterated phospholipid, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine-d62 (PLPC-d62) is also described. Optimised reaction conditions were developed to access this phospholipid and its regioisomeric purity was characterised by two complementary mass spectrometry techniques. © 2022 The Authors - CC- BY Licence
To D or not to D – an in vitro/mass spectrometry screening method to confirm improved metabolic stability in deuterium-labelled radiotracers
(Australian Nuclear Science and Technology Organisation, 2019-09-03) Murphy, RB; Wyatt, NA; Fraser, BH; Yepuri, NR; Holden, PJ; Wotherspoon, ATL; Darwish, TA
Deuteration, where hydrogen within molecules is switched with the non-radioactive and naturally occurring isotope deuterium, can lead to enhanced material properties. For example, the deuterium kinetic isotope effect is well known to improve the metabolic stability of molecules such as drugs, as the C–D bond is stronger and more difficult for enzymes to break than the C–H bond. However, the specific molecular location to deuterate to gain a metabolically favourable outcome may not be clear, without undertaking separate assays of both the deuterated and non-deuterated molecules, followed by separate HPLC-UV/radiometric measurement. In the case of compounds which also contain a radiolabel (e.g. radiotracers for diagnostic medical imaging), specialist infrastructure and teams are required, and chemical synthesis and bioanalysis are time-critical. Our ongoing work with the radiotracer [18F]PBR111, a TSPO ligand showing potential for imaging neuroinflammation1,2,3, demonstrated that [18F]PBR111-d4 (where d4 is incorporated at a specific site) has slower metabolic breakdown4,5 and a decreased rate of formation of polar metabolites in vitro (rat and human liver microsomes) relative to non-deuterated 5,6. Our recently published MS/MS method4 demonstrates the relative difference in metabolic stability without radiolabelling, by analysing different time points from a liver microsome assay which has been administered a 50:50 mixture of deuterated/non-deuterated compound. Pharmacokinetic parameters can also be determined from the data. Deuteration adjacent to the fluoro-alkyl group in PBR111 showed ~50% improvement in the stability of the intact radiotracer relative to non-deuterated using a ratio determination of the analogous MS transitions unique to the deuterated/non-deuterated compounds4. As a control, a second deuterated analogue was also synthesised4 with deuterium purposely incorporated at a site significantly less metabolised than the first site5, which our newly developed method was also able to confirm. We expect this simple and rapid method could be applied to deuterated and non-deuterated analogues of other biologically important molecules to determine the suitability of the chosen site of deuteration. For radiotracers, there is no requirement to radiolabel until studies progress to in vivo PET imaging.

Browsing by Author "Yepuri, NR"

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