52 TESTING N O CI D D D N CI O N N P450 tandem MS 0
Assay Time (minutes)
15 D O O N N P450
Intensity (cps)
1 >1
422
Ratio of MS transitions 349/418 345
metabolic assay (in vitro) MS transition
Figure 6: Schematic of the method to determine the difference in metabolic stability between deuterated and non-deuterated isotopologues, as applied to the imaging agent PBR111; (a) metabolic assay of a 1:1 mixture of the isotopologues in one-pot, (b) tandem MS (blue and red bars), (c) calculate the ratio of the MS transitions of the isotopologues with time (green bars), (d) deviation of the ratio from the initial value with time indicates an observable DKIE.
Germany, in collaboration with RMIT University, has allowed investigation of the mechanism of these sugars in protecting cells against damage under dry or freezing conditions, and the localisation of the sugar molecules in cell membranes. This not only adds to our understanding of such fundamental processes but is relevant to ultra-low temperature storage of biological material.3,4
In thin-film nanotechnology, functional devices containing deuterated components from the NDF have been studied by the University of Queensland using neutron scattering at OPAL. The use of selected combinations of protonated and deuterated components provides contrast between the layers of organic light emitting diode devices enabling detection of diffusion between the layers when the devices become hot, which adversely impacts their performance.5 Semi-solid systems based on lipids such as glycerides in water are attracting interest for their potential as (a) controlled-release drug-delivery agents (Monash University) and (b) systems for crystallisation of protein as structures for drug targets (National Institute for Standards and Technology, U.S.A.). Deuterated lipids have a role to play as subjects for neutron scattering experiments, to provide information about the structures, interactions, and kinetics of these systems.
Deuteration to enhance the properties of materials Heavier than hydrogen by a single neutron, deuterium might not seem to have much chemical effect. But this increase in mass atom makes a massive difference in the reactivity of hydrogen versus its isotope deuterium. Deuterium-carbon bonds are generally about six to ten times more stable than the corresponding hydrogen carbon bonds (Fig 4). These stronger bonds are more difficult to break, which can slow the rate of bond cleavage. This effect upon rate
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is called the deuterium kinetic isotope effect (KIE), which the NDF and the Human Health teams at ANSTO have been investigating in a number of applications that ranges from molecular electronics to healthcare products. For example, once inside the body, a radiotracer (molecule which enable the medical imaging of diseases) or a drug compound must escape the ambush of our metabolic system as it races towards its biological target (Fig 5). The body’s devotion to rapidly breaking foreign materials into tiny inactive species and then ejecting them means that some radiotracers will not reach their targets or drugs need to be taken more frequently than they otherwise would. The NDF has already started to explore these valuable opportunities, in which deuterium modification can provide an important industrial, medical and technological advantage, owing to the superior properties of some deuterated materials.
Development of a rapid screening method for new deuterated molecules
Evaluating the difference in metabolism between deuterated and non-deuterated analogues is typically performed separately for both isotopologues because they cannot be distinguished by methods involving liquid chromatography or radiolabelling. We recently developed a new screening
which combines an in vitro metabolism assay with tandem mass spectrometry to rapidly determine if the site of deuteration has resulted in an improvement to metabolic stability, in a one-pot approach. As the mass (unique to each molecule) is being monitored, there is no need to analyse the compounds independently.
method,6
A 1:1 ratio of both the deuterated and non-deuterated molecule is subjected to
metabolism, and the relative rate of consumption of both isotopologues was determined by using MS/MS transitions unique to both molecules. A deviation of the ratio of the MS transitions from the initial starting point pre-metabolism with time indicates a KIE. The process is shown in Figure 6.
While this method was applied to the imaging agent PBR111, it could be applied to any pair of deuterated and non- deuterated isotopologues, including pharmacologically active molecules, to aid in determining the suitability of the chosen site of deuteration.
PC
References: 1.
https://www.ansto.gov.au/research/
facilities/national-deuteration-facility
2.
https://www.ansto.gov.au/research/ publications/deuteration-publications
3. Kent B, Hauß T, Demé B, Cristiglio V, Darwish TA, Hunt T, Bryant G, Garvey C. Direct comparison of disaccharide interaction with lipid membranes at reduced hydrations. Langmuir 2015; 31(33): 9134-41.
4. Kent B, Hunt T, Darwish TA, Hauß T, Garvey CJ, Bryant G. Localization of trehalose in partially hydrated DOPC bilayers: insights into cryoprotective mechanisms. Journal of the Royal Society Interface 2014; 11: 95.
5. Smith AR, Ruggles JL, Cavaye H, Shaw PE, Darwish TA, James M, Gentle IR, and Burn PL. (2011) Investigating morphology and stability of fac-tris(2-phenylpyridyl) iridium(III) films for OLEDs. Advanced Functional Materials. 21, 2225-31.
6. Murphy RB, Wyatt NA, Fraser BH, Yepuri NR, Holden PJ, Wotherspoon ATL, Darwish TA. 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. Analytica Chimica Acta 2019;
https://doi.org/10.1016/j.aca.2019.02.025
September 2019
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