DOE OSTI2016
The primary focus of this project continues to be the development of radiotracers and radiotracer methodology for studying physiology and biochemistry. The compounds that have been labeled areacylethanolamines and acylglycerols that are, as classes, represented in both in plants and in animals. In the latter, some of these act as ligands for cannabinoid receptors and they are therefore known as endocannabinoids. Cannabinoid receptors are not found in plant genomes so that plants must contain other receptors and signaling systems that use acylethanolamines. Relatively little work has been done on that issue, though acylethanolamines do modulate plant growth and stress resistance, thus possessing obvious relevance to agriculture and energy production. Progress has been described in five peer-reviewed papers and seven meeting abstracts. Preparation of 2-acylglycerol lipid messengers in high purity. A novel enzymatic synthesis was developedthat gave pure 2-acylglycerols free of any rearrrangement to the thermodynamically more stable 1(3)-acylglycerol byproducts. The method utilized 1,3-dibutyryl-2-acylglycerol substrate ethanolysis by a resinimobilized lipase. Thus, pure radiolabeled 2-acylglycerols can now be conveniently prepared just prior to their utilization. These synthetic studies were published in the Journal of Medicinal Chemistry, 2011. Diacylglycerol lipase assay methodology. Diacylglycerol lipases (DAGLs) generate 2- acylglycerols, and are thus potential targets for disease- or growth-modifying agents, by means of reducing formation of 2-acylglycerols. A radioTLC assay of the hydrolysis of radiolabeled diglyceride substrate [1''-carbon-14]2-arachidonoyl-1-stearoyl-sn-glycerol has been implemented, and used to validate a novel, potentially highthroughput fluorescence resonance energy transfer (FRET) based assay. A number of new DAGL inhibitors that have selectivity for DAGLs were synthesized and screened. This work was very recently published in Bioorganic and Medicinal Chemistry Letters.Acylethanolamines. The work initially focused on myristoylethanolamine (MEA) labeled in the acyl moiety with tritium and with carbon-14. Standard coupling conditions were used and gave the desired radiotracers in decent yields. Tritiated product was needed for the search for binding sites of this signaling messenger, and C-14 labeled product for imaging using phosphorimaging screens. Additionally, C-14 MEA was prepared labeled in the ethanolamine moiety. Fluorine-18 labeled acylethanolamines. A multi-step synthesis was performed to obtain unlabeled N-(16- fluorohexadecanoyl)ethanolamide for an HPLC standard via the precursor 16-bromo-N- [2[(tetrahydro-2Hpyran-2-yl)oxy]ethyl]hexadecanoylamide. Near-anhydrous F-18 and the bromo precursor (approximately 2-3 mg) in acetonitrile (0.5 ml) were heated with a PETWave microwave reactor for 10 min at 80 °C. F-18 incorporation was checked using silica-gel TLC (8:92 methanol/chloroform). Deprotection was carried out using TFA. The crude product was purified by semi-preparative ODS HPLC (80:20 AcN/ H2O). The product peak was diluted in water, concentrated on an SPE column, and eluted in 1 mL EtOH. F-18 incorporation to form the THP protected product was >90% with negligible side products observed. Deprotection and HPLC purification proceeded successfully with >99% radiochemical l purity. Brain distribution studies were done in mice. This work was presented at the Society of Nuclear Medicine meeting in June 2012. Subsequent more detailed studies including whole body tissue distribution studies, microPET experiments and radiochromatographic studies were published in ACS Chemical Neurosciences in 2014. Iodine-125 labeled acylethanolamines. The precursor 12-bromo-N-[2[(tetrahydro-2H-pyran-2-yl)oxy]ethyl]dodecanoylamide was prepared and a 1 mg sample was subject to exchange labeling withradioiodide in refluxing acetonitrile for 1 hour. HPLC analysis as above indicated incorporation of about10% of the radioactivity into a compound with the expected retention time of the corresponding iodocompound. Treatment with TFA converted the radioactivity into a compound with the expected retentiontime of 12-iodododenanoyl ethanolamine. Radiotracer studies in plants. One of the aims of this funding was to follow up the studies of Tripathy et al. (Plant Physiol., 2003) who first reported high affinity binding sites for tritiated myristoylethanolamine in plants, and also reported blockage of these sites by the cannabinoid receptor antagonist AM281. Because plant genomes do not contain genes for cannabinoid receptors, this was an intriguing report. I-125 labeled AM281 was therefore prepared, to facilitate identification of binding sites for this compound in Arabodopsis thaliana plants. However, such sites could not be found, the binding studies were repeated in tobacco plants. Again, binding sites for AM281 were not found. Additionally, it has become evident that clear demonstration of binding sites for tritiated MEA is obscured by metabolic incorporation of radioactivity into plant tissues. Studies on this issue are being aggressively pursued. Binding methodologies used in experiments with animal tissues require modification for their optimal use with plant tissues. Detailed studies of [14C]myristoylethanolamine isotopomers and of [14C]arachidonoylethanolamine isotopomers Since the labeled compounds being prepared for experiments with plants may have utility in Nuclear Medicine, studies were conducted with some of these compounds in mice. The brain uptake of C-14 MEA labeled in either acyl or ethanolamine moiety was 3-4 fold higher than uptake of C-14 myristic acid or C-14 ethanolamine. Carrier MEA increased brain uptake. Autoradiographs of MEA showed regionally specific uptake, with somewhat different patterns for acyl and ethanolamine isotopomers. It is hypothesized that these labeled compounds might form the basis of autoradiographic imaging of regional activity of enzymes such as fatty acid amide hydrolase, which may regulate endocannabinoid tone in animals. Early results were presented at the 2011 ICRS meeting, and at the 2012 Society for Neurosciences. Narachidonoylethanolamine is an endocannabinoid signaling messenger in animals and is known as “anandamide”. It is one of several families of signaling molecules derived from arachidonic acid, the principal omega-6 polyunsaturated fatty acids (PUFA’s) in animal species. Other derivatives of arachidonic acid include thromboxanes and prostaglandins. Full details of the studies with the ethanolamide isotopomers were a part of the PhD dissertation of Kun Hu (nee Qian), and were submitted for publication to Nuclear Medicine and Biology in August 2016. Syntheses of [14C]docosahexanoylethanolamine isotopomers and preliminary biological investigations Docosahexaenoic acid (DHA) is the omega-3 PUFA that can be regarded in some respects as the counterpart of arachidonic acid in the omega-6 series. While arachidonic acid is proinflammatory, DHA is anti-inflammatory, and foods high in DHA (or artificially enriched in DHA) are commonly regarded as promoting health. In contrast to the large literature on the Nethanolamide of arachidonic acid (i.e. the endocannabinoid anandamide) as of now (9/25/2016) there are only six papers on the corresponding ethanolamide of DHA, and when our studies under this grant began there were none. Beneficial actions of endogenously produced DHAethanolamine (“synaptamide”) have been indicated, and to help elucidate the possible roles of synaptamide, we have synthesized this molecule for the first time labeled with C-14 in either the ethanolamine moiety or the fatty acid moiety. Studies of the disposition of endogenously administered isotopomers of DHA-ethanolamine are in progress, to complement tissue culture experiments evaluation hypothesized protective effects of this DHA derivative.
38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEA↗