It has been demonstrated that nitro groups are selectively replaced by deuterium in a variety of organic molecules (e.g., _-nitro ketones, secondary and tertiary nitro hydrocarbons) upon treatment with tributyltin deuteride. It follows that analogous use of high specific activity tributyltin tritide could lead to an extremely useful tritiation method. To demonstrate this approach, we have prepared a variety of _-deuteriated cyclic ketones via radical denitration reactions. The preliminary labelling steps involved the synthesis of _-nitroketones in one or two steps and removal of the _-nitro group with Bu3SnD (TBTD) in a radical reaction using azobisisobutyronitrile (AIBN) as the initiator. As a test of potential labelling substrates, a tertiary cyclic ketone, an _-nitroethyl ester and several primary, secondary, and tertiary _-nitroketones were prepared as follows: a. 1-Nitroadamantane was prepared from 1-aminoadamantane in a one-step oxidation using 3-chloroperoxybenzoic acid (m-CPBA) in dichloroethane at 83oC for 5 hours. The product 1-nitroadamantane was characterized by NMR spectroscopy and gas chromatography. b. Ethyl _-nitromyristate: Ethyl _-bromomyristate was nitrated at the _-position with sodium nitrite in dimethylformamide (DMF) in the presence of phloroglucinol dihydrate. Recrystallization from hexane furnished the product in 67% yield. c. Nitromethyladamantyl ketone: This compound was prepared using the method described in (b) from bromomethyladamantyl ketone. After several steps of purification using the chromatotron, a 34% yield of the desired product was obtained. d. 2-Nitrocholesta-3-one: 5 _-Cholesta-3-one was dissolved in dry tetrahydrofuran (THF) and was added to a suspension of potassium-t-butoxide in THF and t-butanol, at -30oC. A solution of isobutyl nitrate in THF was then added dropwise. The resulting mixture was acidified with HCl (2N), and was extracted with methylene chloride. The yellow residue was recrystallized from hexane:ether to give 2-nitrocholesta-3-one (60%). e. 2-Nitrocyclododecan-1-one: This product was prepared using a two step synthesis (1) 1-Acetoxy-1-cyclododecene: Cyclododecanone was added to a solution of acetic anhydride and p-toluene-sulfonic acid. The solution was heated at 175oC for 3 hours, cooled, and washed with sodium bicarbonate. The residue was purified by flash chromatography using hexane as the eluting solvent. The enol acetate product was isolated in 73% yield. (2) 2-Nitro- cyclododecan-1-one: 1-Acetoxy-1-cyclododecene was nitrated using preground ammonium nitrate, followed by addition of trifluoroacetic anhydride. Recrystallization from hexane gave pure product (48%). f. [2-Nitro-2-(1-propion-1-al)]cyclododecan-1-one: 2-Nitrocyclododecan-1-one was dissolved in THF, triethylamine was added and the mixture was stirred. A mixture of acrolein in THF was added and stirred for 3 hours. Completion of the reaction gave a white product (72%). In our exploratory labelling experiments, the denitration reaction was carried out in the presence of TBTH with AIBN as the radical initiator, in refluxing toluene. Subsequent reactions with TBTD under the same reaction conditions gave deuteriated products. To further demonstrate the utility of this method in tritium labelling chemistry, one of the secondary __nitroketones was converted to a tertiary __nitroketone using a Michael reaction. This reaction demonstrates the role of the nitro group for activating conjugate addition of an activated double bond. The nitro group is then removed with TBTD to regioselectively introduce a deuterium atom into the new product. Deuterium labelling of the only __nitro ethyl ester in the series was also completed to give __deuterio ethyl myristate. All the deuteriated products were analyzed with 2H NMR, mass spectrometry and gas chromatography for the labelling specificity, deuterium content and chemical yields respectively. We have applied this approach to the tritiation of the labelling precursors synthesized in (c) and (e) above. As a first example, radical tritiodenitration of _-nitromethyladamantanyl ketone with TBTT (25% tritium) gave 67% of a product with specific activity 6.7 Ci/mmole. The product was analyzed by LSC, radio-GC and both 1H and 3H NMR spectroscopy to confirm the amount, purity and specificity of tritium labelling. For the second example, the tritiodenitration of 2-nitrocyclododecan-1-one gave a mixture of the desired product and some unidentifiable side products. GC analysis of the mixture, including collection and quantitation of the product peak, showed a specific activity of 5.6 Ci/mmole and a chemical yield of 12%. These two tritiation examples will be included in a detailed manuscript demonstrating the use of denitration reactions as a selective labelling approach.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
3P41RR001237-16S1
Application #
6220425
Study Section
Project Start
1998-08-01
Project End
2000-07-31
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
16
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Lawrence Berkeley National Laboratory
Department
Type
DUNS #
078576738
City
Berkeley
State
CA
Country
United States
Zip Code
94720
Singh, Navneet; Moody, Alan R; Zhang, Bowen et al. (2017) Age-Specific Sex Differences in Magnetic Resonance Imaging-Depicted Carotid Intraplaque Hemorrhage. Stroke 48:2129-2135
Paul-Pletzer, Kalanethee; Yamamoto, Takeshi; Ikemoto, Noriaki et al. (2005) Probing a putative dantrolene-binding site on the cardiac ryanodine receptor. Biochem J 387:905-9
Wang, Huimin; Shimizu, Eiji; Tang, Ya-Ping et al. (2003) Inducible protein knockout reveals temporal requirement of CaMKII reactivation for memory consolidation in the brain. Proc Natl Acad Sci U S A 100:4287-92
Paul-Pletzer, Kalanethee; Yamamoto, Takeshi; Bhat, Manjunatha B et al. (2002) Identification of a dantrolene-binding sequence on the skeletal muscle ryanodine receptor. J Biol Chem 277:34918-23
Westler, William M; Frey, Perry A; Lin, Jing et al. (2002) Evidence for a strong hydrogen bond in the catalytic dyad of transition-state analogue inhibitor complexes of chymotrypsin from proton-triton NMR isotope shifts. J Am Chem Soc 124:4196-7
Tomizawa, M; Wen, Z; Chin, H L et al. (2001) Photoaffinity labeling of insect nicotinic acetylcholine receptors with a novel [(3)H]azidoneonicotinoid. J Neurochem 78:1359-66
Than, C; Morimoto, H; Williams, P G et al. (2001) Preparation, NMR characterization, and labeling reactions of tritiated triacetoxy sodium borohydride. J Org Chem 66:3602-5
Saljoughian, M; Williams, P G (2000) Recent developments in tritium incorporation for radiotracer studies. Curr Pharm Des 6:1029-56
Cianci, C; Yu, K L; Dischino, D D et al. (1999) pH-dependent changes in photoaffinity labeling patterns of the H1 influenza virus hemagglutinin by using an inhibitor of viral fusion. J Virol 73:1785-94
Palnitkar, S S; Bin, B; Jimenez, L S et al. (1999) [3H]Azidodantrolene: synthesis and use in identification of a putative skeletal muscle dantrolene binding site in sarcoplasmic reticulum. J Med Chem 42:1872-80

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