The synthesis of tritium-labelled glycine transporter 1 inhibitor Org24598 is reported. Because of the instability of the Org24598 skeleton under hydrogenation conditions, a synthetic approach using an in-house prepared tritium-labelled alkylating agent ([3 H]MeI, SA = 26.2 Ci/mmol) was employed. Alternative methods of labelling are discussed.
A straightforward methodology of fluorine substitution by tritium/deuterium is reported. The described method is selective towards the F─C (sp3 ) group and leaves both the aromatic F─C (sp2 ) and F2 ─C (sp3 ) moieties unaffected. Alkylfluorides, readily synthesized from appropriate alcohols by treatment with diethylaminosulfur trifluoride (DAST) reagent in an overall yield up to 76%, undergoes activation with the boron-based Lewis acid B(C6 F5 )3 , and stoichiometric in situ reduction with a tritide/deuteride reagent-the [TMP2(3) H][2(3) HB(C6 F5 )3 ] system of frustrated Lewis pair. This methodology provides an isolated yield of up to 93% of regio-specifically labeled small organic compounds with superior 2 H-enrichment of over 95%. The specific activity of prepared 1-(2-[3 H]-ethyl)naphthalene was determined at 29.0 Ci/mmol. The site selectivity of the Lewis acid/ [TMP2(3) H][2(3) HB(C6 F5 )3 ] approach is orthogonal to currently used methods and allows for isotopic labeling of complementary positions in molecules. Reported labeling methodology proceeds well at ultra-mild reaction conditions (220 mbar of T2 ), allowing very low consumption of the radioactive source (4.2 Ci/156 GBq), and producing limited amount of radioactive waste.
Deuterium- and tritium-labeled compounds play a principal role in tracing of biologically active molecules in complicated biochemical systems. The state-of-the-art techniques using noble metal catalysts or strong reducing agents often suffers from low functional group tolerances, poor selectivity, tricky or multistep synthesis of reagents, and low specific activity of the labeled product. Herein, we demonstrate a mild and nonmetallic technique of deuteration and tritiation of polarized double bonds, such as carbonyl compounds, yielding labeled alcohols of high specific activities. This one-pot synthesis uses carrier-free hydrogen gas in situ activated by a freshly prepared frustrated Lewis pair, generating reducing reagents. This labeling strategy shows better selectivity and functional group tolerances compared with current reductive methods. Reported is an example of the selective reduction of the aldehyde moiety of 3-acetylbenzaldehyde. What makes this technology groundbreaking is its mildness, selectivity, and generation of limited amount of radioactive waste as almost no byproducts were generated after use of (B(C6 F5 )33 H)(3 HTMP) reducing reagent. Radiochemical purity of desired 3 H-labeled product in a crude reaction mixture was determined of over 94%. This work provides, to the community of radiochemists, a practical protocol for frustrated Lewis pairs (FLP)-assisted deuterium/tritium labeling technology.
Xanomeline (3-(Hexyloxy)-4-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-1,2,5-thiadiazole) is a muscarinic agonist that is considered to be functionally selective for the M1/M4 receptor subtypes. Part of xanomeline binding is resistant to washing. Wash-resistant xanomeline activates muscarinic receptors persistently, except for the M5 subtype. Mutation of leucine 6.46 to isoleucine at M1 or M4 receptors abolished persistent activation by wash-resistant xanomeline. Reciprocal mutation of isoleucine 6.46 to leucine at the M5 receptor made it sensitive to activation by wash-resistant xanomeline. Lowering of membrane cholesterol made M1 and M4 mutants and M5 wild type receptors sensitive to activation by wash-resistant xanomeline. Molecular docking revealed a cholesterol binding site in the groove between transmembrane helices 6 and 7. Molecular dynamics showed that interaction of cholesterol with this binding site attenuates receptor activation. We hypothesize that differences in cholesterol binding to this site between muscarinic receptor subtypes may constitute the basis for xanomeline apparent functional selectivity and may have notable therapeutic implications. Differences in receptor-membrane interactions, rather than in agonist-receptor interactions, represent a novel possibility to achieve pharmacological selectivity. Our findings may be applicable to other G protein coupled receptors.
- MeSH
- Muscarinic Agonists pharmacokinetics MeSH
- Cell Membrane drug effects metabolism MeSH
- CHO Cells cytology MeSH
- Cholesterol metabolism MeSH
- Cricetulus MeSH
- Inositol Phosphates metabolism MeSH
- Magnetic Resonance Spectroscopy MeSH
- Models, Molecular MeSH
- Flow Cytometry MeSH
- Pyridines pharmacokinetics MeSH
- Radioligand Assay MeSH
- Receptors, Muscarinic genetics metabolism MeSH
- Molecular Docking Simulation MeSH
- Thiadiazoles pharmacokinetics MeSH
- Tritium pharmacokinetics MeSH
- Calcium metabolism MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
During the routine operation, nuclear power plants discharge waste water containing a certain amount of radioactivity, whose main component is the artificial radionuclide tritium. The amounts of tritium released into the environment are kept within the legal requirements, which minimize the noxious effects of radioactivity, but the activity concentration is well measurable in surface water of the recipient. This study compares amount of tritium activity in waste water from nuclear power plants and the tritium activity detected at selected relevant sites of surface water quality monitoring. The situation is assessed in the catchment of the Vltava and Elbe Rivers, affected by the Temelín Nuclear Power Plant as well as in the Jihlava River catchment (the Danube River catchment respectively), where the waste water of the Dukovany Nuclear Power Plant is discharged. The results show a good agreement of the amount of released tritium stated by the power plant operator and the tritium amount detected in the surface water and highlighted the importance of a robust independent monitoring of tritium discharged from a nuclear power plant which could be carried out by water management authorities. The outputs of independent monitoring allow validating the values reported by a polluter and expand opportunities of using tritium as e.g. tracer.
- MeSH
- Power Plants MeSH
- Nuclear Power Plants * MeSH
- Water Pollutants, Radioactive analysis chemistry MeSH
- Radioisotopes analysis MeSH
- Rivers MeSH
- Tritium chemistry MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic MeSH
The Dukovany nuclear power plant (NPP Dukovany) releases liquid effluents, including HTO, to the Mohelno reservoir, located in a deep valley. Significantly enhanced tritium activities were observed in the form of non-exchangeable organically bound tritium in the surrounding biota which lacks direct contact with the water body. This indicates a tritium uptake by plants from air moisture and haze, which is, besides the uptake by roots from soil, one of the most important mechanisms of tritium transfer from environment to plants. Results of a pilot study based on four sampling campaigns in 2011-2015 are presented and discussed, with the aim to provide new information on tritium transport in the Mohelno reservoir - Jihlava River - plants ecosystems.
- MeSH
- Water Pollutants, Chemical analysis MeSH
- Nuclear Power Plants MeSH
- Radiation Monitoring * MeSH
- Rivers chemistry MeSH
- Tritium analysis MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic MeSH
A regiospecific and enantiospecific synthesis of tritium-labeled 28-homocastasterone is reported. Appropriate chlorocarbonate, efficiently synthesized from the starting 28-homocastasterone in an overall yield of 46%, undergoes catalytic tritium dechlorination by the T2 /Pd[0]/Et3 N system, providing 28-[3β-3 H]homocastasterone, in a good yield, radiochemical purity (>97%), and with a high specific activity (5.8 Ci/mmol).
- MeSH
- Cholestanones chemistry MeSH
- Isotope Labeling MeSH
- Catalysis MeSH
- Stereoisomerism MeSH
- Tritium chemistry MeSH
- Publication type
- Journal Article MeSH
Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.
- MeSH
- Models, Biological MeSH
- CHO Cells MeSH
- Cricetulus MeSH
- Kinetics MeSH
- Cricetinae MeSH
- Humans MeSH
- Ligands MeSH
- Mutant Proteins chemistry metabolism MeSH
- N-Methylscopolamine chemistry metabolism MeSH
- Protein Domains MeSH
- Receptors, Muscarinic chemistry metabolism MeSH
- Protein Structure, Secondary MeSH
- Molecular Dynamics Simulation MeSH
- Tritium metabolism MeSH
- Protein Binding MeSH
- Binding Sites MeSH
- Acceleration MeSH
- Animals MeSH
- Check Tag
- Cricetinae MeSH
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Apolipoprotein E4 (apoE4) is the most prevalent genetic risk factor for Alzheimer's disease. We utilized apoE4-targeted replacement mice (approved by the Tel Aviv University Animal Care Committee) to investigate whether cholinergic dysfunction, which increases during aging and is a hallmark of Alzheimer's disease, is accentuated by apoE4. This revealed that levels of the pre-synaptic cholinergic marker, vesicular acetylcholine transporter in the hippocampus and the corresponding electrically evoked release of acetylcholine, are similar in 4-month-old apoE4 and apolipoprotein E3 (apoE3) mice. Both parameters decrease with age. This decrease is, however, significantly more pronounced in the apoE4 mice. The levels of cholinacetyltransferase (ChAT), acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) were similar in the hippocampus of young apoE4 and apoE3 mice and decreased during aging. For ChAT, this decrease was similar in the apoE4 and apoE3 mice, whereas it was more pronounced in the apoE4 mice, regarding their corresponding AChE and BuChE levels. The level of muscarinic receptors was higher in the apoE4 than in the apoE3 mice at 4 months and increased to similar levels with age. However, the relative representation of the M1 receptor subtype decreased during aging in apoE4 mice. These results demonstrate impairment of the evoked release of acetylcholine in hippocampus by apoE4 in 12-month-old mice but not in 4-month-old mice. The levels of ChAT and the extent of the M2 receptor-mediated autoregulation of ACh release were similar in the adult mice, suggesting that the apoE4-related inhibition of hippocampal ACh release in these mice is not driven by these parameters. Evoked ACh release from hippocampal and cortical slices is similar in 4-month-old apoE4 and apoE3 mice but is specifically and significantly reduced in hippocampus, but not cortex, of 12-month-old apoE4 mice. This effect is accompanied by decreased VAChT levels. These findings show that the hipocampal cholinergic nerve terminals are specifically affected by apoE4 and that this effect is age dependent.
- MeSH
- Acetylcholine metabolism MeSH
- Apolipoprotein E3 genetics MeSH
- Apolipoprotein E4 genetics metabolism MeSH
- Choline O-Acetyltransferase metabolism MeSH
- Guanosine 5'-O-(3-Thiotriphosphate) pharmacology MeSH
- Hippocampus drug effects metabolism MeSH
- Mice, Inbred C57BL MeSH
- Mice, Transgenic MeSH
- Mice MeSH
- N-Methylscopolamine pharmacology MeSH
- Receptors, Muscarinic metabolism MeSH
- Tritium metabolism MeSH
- Age Factors MeSH
- Vesicular Acetylcholine Transport Proteins metabolism MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
3-Hydroxycyclopent-1-ene-1-carboxylic acid (HOCPCA (1)) is a potent ligand for high-affinity γ-hydroxybutyric acid binding sites in the central nervous system. Various approaches to the introduction of a hydrogen label onto the HOCPCA skeleton are reported. The outcomes of the feasible C─H activation of olefin carbon (C-2) by iridium catalyst are compared with the reduction of the carbonyl group (C-3) by freshly prepared borodeuterides. The most efficient iridium catalysts proved to be Kerr bulky phosphine N-heterocyclic species providing outstanding deuterium enrichment (up to 91%) in a short period of time. The highest deuterium enrichment (>99%) was achieved through the reduction of ketone precursor 2 by lithium trimethoxyborodeuteride. Hence, analogical conditions were used for the tritiation experiment. [3 H]-HOCPCA selectively labeled on the position C-3 was synthetized with radiochemical purity >99%, an isolated yield of 637 mCi and specific activity = 28.9 Ci/mmol.
- MeSH
- Alkenes chemistry MeSH
- Boron chemistry MeSH
- Deuterium chemistry MeSH
- Hydroxybutyrates chemistry MeSH
- Iridium chemistry MeSH
- Isotope Labeling MeSH
- Catalysis MeSH
- Ligands MeSH
- Oxidation-Reduction MeSH
- Tritium chemistry MeSH
- Deuterium Exchange Measurement * MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
