Radiolabelled Reagents

Patent Radioligands and Their Use for Identifying Potassium Channel 

Altenbach etal, US Patent 6,632,418 (Octobet 14, 2003) Assignee Abbott Laboratories 

Utility Radiolabeled Reagent for Characterizating Channels in Tissues 

Sodium hydride (1.25 mol) was mixed in 800 ml THF at 0°C then added to propargyl alcohol (1.5 mol) dissolved in 175 ml THF. The mixture was stirred one hour, treated with methyl bromoacetate dissolved in 250 ml THF, stirred at 0°C 30 minutes, 2 hours at ambient temperature, and treated with 800 ml 2M HCl. The organic layer was isolated and the aqueous layer extracted twice with 500 ml EtOAc. The combined organics were washed with 200 ml brine, dried, filtered, concentrated, distilled, and 83 g product isolated, bp = 75-85°C at 20 torr. H-NMR data supplied. 

The product from Step 1 (0.44 mol) dissolved in 1.5 L methyl alcohol was treated with Hg(OAc)2 (44 mmol) and 3 ml concentrated H2SO4 then refluxed one hour. The mixture was cooled to ambient temperature, concentrated to a volume of 200 ml, treated with 500 ml 1M HCl, and extracted twice with 300 ml CH2CI2. The combined CH2CI2 extracts were dried, filtered, concentrated, distilled, and the product isolated, bp = 65-95°C at 1 torr. H-NMR data supplied. 

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Another technique which has much potential in analysis, but which to date has had only limited use, is the formation of radioactive derivatives of non-radioactive compounds for quantitation by radiocounting. A radiolabeled reagent is used to form the derivative. This approach has been of use in combination with chromatography. The advantage of this technique is that it avoids problems of sample background which are often associated with spectrophotometric methods. The 14C-methylation of carboxylic acids and the 14 C-acetylation of hydroxyl groups have been studied [39,40]. These methods are quantitative and the sensitivity is dependent on the activity of the radioactive group added to the molecules. The radioactive derivatization of lipids has been reviewed [41]. 

Chloramphenicol acetyl transferase (CAT). This bacterial enzyme was the first reporter protein used for studying the transcriptional activity of eukaryotic regulatory sequences (Gorman et al., 1982). CAT inactivates chloramphenicol, an inhibitor of prokaryotic protein synthesis, by converting it to the mono- or di-acetylated species. Measurement of CAT activity requires a 14C-radiolabeled chloramphenicol or acetyl-CoA and, therefore, an additional step is neccessary to separate the radio-labeled reactant from the product. Novel detection methods based on fluorescent substrates or ELISA assays, which do not use radiolabeled reagents, have been described more recently (Bullock and Gorman, 2000). 

The preparation of tritium radiolabelled reagents using the reducing agent, LiEt3BT, (Supertritide), is described (4). 

Many derivatives of histidine are not stable to acid hydrolysis and are not discussed here (however, see 2.12.2 for those that occur naturally in proteins). Brief mention should be made of the iodination of histidyl residues by HOI ( 3.7.2). The mono- and diiodohistidines can be identified and distinguished from the iodinated tyrosines by high voltage paper electrophoresis in 1 M formic acid (Roholt and Pressman 1972) after complete enzymic hydrolysis of the protein or peptide ( 2.11 Roholt and Pressman 1972). Quantitation and identification are facilitated by the use of radiolabeled reagent. 

BPN, a potent opioid partial agonist , 277, has been labelled with carbon-11 at the 6-methoxy position, [ C PPN], using a precursor 278 synthesized in high yield (89%) from BPN 277 in two steps (equation Ilia) and employing [ C]iodomethane as the radiolabelling reagent (equation lib). 

Aliquots (10 pL) of each radiolabeled reagent are retained and counted with the tissue samples for subsequent calculation of percent injected dose per gram of the tissue samples. 

V. USE OF RADIOLABELED REAGENTS TO FOLLOW THE CHANGES IN RESIN FUNCTIONALITIES... 

An interesting approach for the analysis of the functionalization of soluble polymer supports could be the introduction of radiolabeled reagents. For a detailed description of the application of these methods see the respective chapters in [50]. 

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