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Composition and methods for measuring ion channel activity in a cell

A technology of potassium ion channels and cells, applied in the field of compositions and methods for measuring ion channel activity in cells, capable of solving problems such as insufficient sensitivity

Active Publication Date: 2019-04-26
LIFE TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the problem faced by existing fluorescent dyes is that they are not sensitive enough to detect very low levels of thallium (eg, below about 100-500 μM) in HTS assays

Method used

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  • Composition and methods for measuring ion channel activity in a cell
  • Composition and methods for measuring ion channel activity in a cell
  • Composition and methods for measuring ion channel activity in a cell

Examples

Experimental program
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example 1

[0117] Bis(acetoxymethyl)2,2'-((4-(3',6'-diacetoxy-2',7'-difluoro-3-oxo-3H-spiro[isobenzo Synthesis of furan-1,9'-xanthene]-6-formylamino)-2-methoxyphenyl)azanediyl)diacetic acid

[0118] For the synthesis of diacetate bis(acetoxymethyl) 2,2'-((4-(3',6'-diacetoxy-2',7'-difluoro-3-oxo-3H- The reaction scheme for spiro[isobenzofuran-1,9'-xanthene]-6-carboxamido)-2-methoxyphenyl)azanediyl)ester is shown in Figure 12A and Figure 12B middle. 3',6'-diacetoxy-2',7'-difluoro-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (236 mg, 0.475 mmol) was suspended in 15 mL of anhydrous DCM and the suspension was cooled in an ice-water bath. Triethylamine (66 μL, 0.47 mmol) was added to the suspension, followed by isobutyl chloroformate (73 μL, 0.56 mmol), and the resulting solution was stirred in a cooling bath for 20 minutes. Afterwards, the solution was concentrated and placed under vacuum to provide 3',6'-diacetoxy-2',7'-difluoro-3-oxo-3H-spiro[isobenzofuran-1,9'-xanth...

example 2

[0121] Bis(acetoxymethyl)2,2'-((4-(3',6'-diacetoxy-2',7'-difluoro-3-oxo-3H-spiro[isobenzo Synthesis of furan-1,9'-xanthene]-5-formylamino)-2-methoxyphenyl)azanediyl)diacetic acid

[0122] For the synthesis of diacetate bis(acetoxymethyl) 2,2'-((4-(3',6'-diacetoxy-2',7'-difluoro-3-oxo-3H- The reaction scheme for spiro[isobenzofuran-1,9'-xanthene]-5-carboxamido)-2-methoxyphenyl)azanediyl)ester is shown in Figure 13A and Figure 13B middle. 3',6'-diacetoxy-2',7'-difluoro-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-carboxylic acid (283 mg, 0.570 mmol) was suspended in 20 mL of anhydrous DCM and the suspension was cooled in an ice-water bath. Triethylamine (79 μL, 0.56 mmol) was added to the suspension, followed by isobutyl chloroformate (88 μL, 0.67 mmol), and the resulting solution was stirred in a cooling bath for 20 minutes. The solution was then concentrated and subjected to vacuum to provide 3',6'-diacetoxy-2',7'-difluoro-3-oxo-3H-spiro[isobenzofuran-1,9'- Xanthene]-...

example 3

[0125] Synthesis of bis(acetoxymethyl)-2,2'-((5-amino-2-methoxyphenyl)azanediyl) diacetate

[0126] The reaction scheme for the synthesis of bis(acetoxymethyl)-2,2'-((5-amino-2-methoxyphenyl)azanediyl) diacetate is shown in Figure 14 middle. Dimethyl 2,2'-((2-methoxyphenyl)azanediyl)diacetate (315 mg; 1.178 mmol) was dissolved in 96% sulfuric acid (0.86 mL; 15 mmol) and cooled in an ice-water bath The solution was cooled for 15 minutes. Powdered potassium nitrate (119 mg; 1.17702 mmol) was added to the mixture in portions over 15 minutes. The reaction mixture was stirred in the cooling bath for 2 h, then quenched with ice, and the product was extracted with EtOAc (3 x 50 mL). The combined extracts were washed with saturated NaHCO3, water, brine, washed with Na 2 SO 4 Dry and evaporate. The crude material was purified on a silica gel column with an ethyl acetate-hexane gradient (0-40%) to afford 2,2'-((2-methoxy-5-nitrophenyl)azanediyl) Dimethyl diacetate (220 mg, 60%)....

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Abstract

Compositions and methods for detecting the activity of an ion channel in a cell are described. The methods include providing a loading buffer solution to the cell, where the loading buffer includes athallium ion indicator and optionally chloride ions, and providing a stimulus buffer that includes thallium ions to the cell. Providing the stimulus buffer can cause thallium ion influx into or effluxout of the cell through the ion channel. After providing the stimulus buffer, a change in at least one optical property of the thallium ion indicator is detected in response to thallium influx or efflux, thereby detecting the activity of the ion channel.

Description

technical field [0001] Compositions, methods and kits for measuring the activity of ion channels in cells are described. Background technique [0002] Thallium influx can be used as a surrogate indicator of potassium channel activity in clonal cell lines loaded with the calcium indicator benzothiazole calcium acetoxymethyl (BTCAM) ester or thallium-sensitive fluorescent dyes. Current assays for monitoring potassium channels use thallium(I) ions that selectively enter open potassium channels and bind BTC, giving an optical readout of potassium channel activity. This method can be used to study the activation and / or inhibition of ion channels by drugs tested in a high-throughput screening (HTS) format from compound libraries. However, methods based on incorporation into BTC have significant drawbacks. That is, when the assay is performed in the presence of chloride ions, thallium (I) ions form thallium chloride (TlCl), which is poorly soluble and will be released from soluti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68C07D493/10
CPCC07D493/10C07D311/90C07C229/18C07C251/30C07C2603/24C09B11/24C09B1/00G01N33/6872C09B11/245G01N2021/7786G01N21/77G01N33/48G01N33/84
Inventor D·比查姆K·吉A·卢卡维什尼科夫
Owner LIFE TECH CORP
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