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cytoplasmic fluorescent ion indicator

An indicator and fluorescent technology, applied in the field of fluorophores, can solve problems such as poor water solubility and limited organic solvent ion measurement

Inactive Publication Date: 2016-11-09
ASANTE RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Many chelating agents, such as crown ethers and cryptands, have the disadvantage of poor water solubility, which limits the measurement of ions in organic solvents

Method used

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  • cytoplasmic fluorescent ion indicator
  • cytoplasmic fluorescent ion indicator
  • cytoplasmic fluorescent ion indicator

Examples

Experimental program
Comparison scheme
Effect test

example to ， approach 9

[0267]

[0268] O-anisidine (362 mL) was added along with diisopropylethylamine (233 mL) and 1,2-bis(2-chloroethoxy)ethane (84 mL). The reaction was stirred at 120 °C for 3 days. The crude product was placed on a rotary evaporator with an oil bath and heated to >100°C with high vacuum to remove most of the excess anisidine. After this time, the residue was diluted with ethyl acetate and washed twice with water, then dried over sodium sulfate and evaporated. The crude product was evaporated under vacuum and purified by column chromatography using 6:1 hexane / ethyl acetate with increasing ethyl acetate content.

[0269]

[0270] Diglycolic acid (25 g) was added to a dry flask. To this was added thionyl chloride (85 mL) and the reaction was immediately heated to reflux and stirred at this temperature for 5 hours. Simultaneously, the excess thionyl chloride was distilled at 85° C. under reduced pressure, then at room temperature by high vacuum until the mixture precipitate...

example 1

[0285] Example 1 - Sodium indicator ANG-1

[0286] figure 1 is the sodium-titrated fluorescence emission spectrum of Asante NatriumGreen-1 (ANG-1) salt solution (1M TMACl, 10 mM MOPS, pH=7.10) titrated with 1M sodium chloride solution excited at 517 nm.

[0287] Figure 7 It is shown that HEK293 cells loaded with ANG-1 (AM) express TRPV1 channels. Capsaicin, an agonist of the TRPV1 channel, conducts Na + and Ca 2+ , was used to excite Na + inflow.

[0288] Figure 8 Shown are REF52 cells loaded with ANG-1(AM) and applied Na + The ionophore gramicidin to promote Na + inflow. The obtained Na + An increase in the intracellular concentration resulted in a corresponding increase in ANG-1 fluorescence.

[0289] Figure 9 Shown are astrocytes loaded with ANG-1 (AM), showing Na due to ouabain inhibition of this sodium pump + An increase in intracellular concentration results in an increase in fluorescence from the first box to the second box.

example 2

[0290] Example 2-sodium indicator ANG-2

[0291] figure 2 It is Asante Natrium Green (sodium green)-2 (ANG-2) salt solution (140mM TMACl, 10mM MOPS, pH=7.10) titrated with sodium chloride solution (1M NaCl, 10mM MOPS, pH=7.10) excited at 517nm 7.11), sodium titrated fluorescence emission spectrum.

[0292] Figure 10 Shown are REF52 fibroblasts loaded with ANG-2(AM) and the Na + ionophore SQI-Pr to promote Na + inflow. The obtained Na + An increase in the intracellular concentration resulted in a corresponding increase in ANG-2 fluorescence. Further addition of 20 μΜ amphotericin B gave only a weak increase in fluorescence.

[0293] Figure 11 Shown is REF52 fibroblasts loaded with ANG-2(AM) maintained in 145 mM NMG-gluconate. 50 μM Amphotericin-B depletes Na + and K + of these cells. Following equilibration of intracellular and extracellular sodium concentrations, incremental NaCl was added extracellularly, resulting in a corresponding increase in ANG-2 fluores...

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Abstract

Novel fluorophores for fluorescent ion indicators incorporate water-solubilizing functional groups. These fluorophores enable visible-wavelength analysis of ion concentrations with chelators that are insoluble or poorly soluble in water. A green version of the fluorophore comprises a putative fluorescein with carboxylate appendages. An orange version of the fluorophore comprises a putative rhodamine, also with carboxylate appendages. Attaching the fluorophore to a 12-crown-4, a 15-crown-5, a 18-crown-6, a 21-crown-7, or a 24-crown-8 produces lithium, sodium, potassium, rubidium, and cesium indicator families, respectively. Attaching the fluorophore to diaza [1,1,1], [1,1,2], [1,2,2], [2,2,2], [2,2,3], [2,3,3], or [3,3,3] cryptands produces more selective lithium, sodium, potassium, rubidium, and cesium indicator families, respectively. Attaching the fluorophore to BAPTA, APTRA, or half- BAPTA or their analogs produces new calcium, magnesium, and other metal ions indicator families that strongly resist leakage from the cell.

Description

[0001] related application [0002] This application is related to and claims priority from US Provisional Patent Application No. 61 / 384,695, filed September 20, 2010. This application is related to US Provisional Patent Application No. 61 / 453,664, filed March 17, 2011, which is hereby incorporated by reference. field of invention [0003] The present invention relates to a fluorophore and its use in combination with a chelating moiety to provide novel fluorescent ion indicators. Background of the invention [0004] Fluorescent ion indicators typically include a chelating moiety attached to a fluorophore such that a change in excitation and / or emission wavelength, or a change in excitation and / or emission intensity, or both occurs upon binding of the ion. In order to sense ion concentration in aqueous media, the fluorescent ion indicator should be water soluble. For intracellular measurements, the indicator should not leak out of the cell at a rate that would interfere wit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D413/14
CPCC07D413/14C07D413/10
Inventor 阿夸西·敏塔佩德罗·罗杰利奥·埃斯卡米利亚
Owner ASANTE RES
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