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A kind of acid-fast bacteria super-resolution imaging dye and its synthesis method and application

A technology of super-resolution imaging and synthesis method, which is applied in the field of fluorescent dyes and imaging, and can solve the problems of inappropriateness, acid-activated fluorescence interference, and biological phototoxicity unfavorable for live cell super-resolution imaging.

Active Publication Date: 2020-12-04
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although rhodamine spiroamide as a light-activated dye can be used for super-resolution fluorescence imaging, there are still some shortcomings of this kind of dye molecules that need to be improved. The first is acid-activated fluorescence interference. Usually, both acid-activated and light-activated can be used Ways to open the rhodamine amide spirocycle
There are many acidic environments in cells (such as lysosomes, acidic proteins, etc.), when rhodamine spiroamide dyes are used in these acidic environments, the fluorescence generated by acid activation will seriously interfere or even lead to complete failure of the photoactivation performance, so Fluorescent probes based on such dyes are currently unsuitable for super-resolution fluorescence imaging in acidic environments
In addition, the optimal pH for the growth of some acidophilic microorganisms is often below 4. Typical examples are acidophilic microorganisms such as chemoautotrophic sulfur oxidizing bacteria in acid mine water and thermoacidophilic bacteria in self-heating coal piles and acid hot springs. Bacteria, etc., and the mystery of these acidophilic bacteria has not yet been solved. If super-resolution imaging is to be applied in this field, the traditional acid-activated rhodamine spiramide dyes are obviously not suitable.
Regrettably, most of the rhodamine spiroamides reported so far can only be photoactivated by irradiation with ultraviolet light (<375nm), and ultraviolet light is phototoxic to organisms, which is not conducive to super-resolution imaging of living cells
Although S.W.Hell et al. used a long-wavelength two-photon laser to activate the fluorescence of rhodamine spiramide and applied it to super-resolution imaging, the power of the two-photon laser is several orders of magnitude larger than that of the single-photon laser, which will also affect the imaged organisms. irreparable photodamage
The visible light-activated dye developed by W.E.Moerner et al. has a maximum absorption wavelength of about 380nm, and only has a little absorption band edge at about 405nm, so it cannot efficiently use 405nm laser to achieve photoactivation

Method used

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  • A kind of acid-fast bacteria super-resolution imaging dye and its synthesis method and application
  • A kind of acid-fast bacteria super-resolution imaging dye and its synthesis method and application
  • A kind of acid-fast bacteria super-resolution imaging dye and its synthesis method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Intermediate molecule (P1) synthetic route and product structure are as follows:

[0025]

[0026] Synthesis steps and characterization: 3-Nitrorhodamine (2.92g, 6mmol) and phosphorus oxychloride (5.6mL, 60mmol) were placed in 1,2-dichloroethane (150mL), heated to 84°C under reflux, stirred After 2 hours the solvent was evaporated to give a dark purple oily liquid. The crude acid chloride product was dissolved in dichloromethane (100mL), then added dropwise to a mixed solution of triethylamine (3mL) and 6-(4-aminophenylethynyl)naphthalene anhydride (1.88g, 6mmol), and stirred at room temperature for 24 hours Afterwards, the solvent was evaporated under reduced pressure, and the residue was separated by column chromatography (silica gel, dichloromethane / ethyl acetate, 30:1 v / v) to obtain a yellow powder product P1 (2.44 g, 52%). The yellow powder product was characterized by NMR and mass spectrometry:

[0027] 1 H NMR (400MHz, CDCl 3 )δ8.75(d, J=8.4Hz, 1H), 8.65(d...

Embodiment 2

[0030] Intermediate molecule (P2) synthetic route and product structure are as follows:

[0031]

[0032]Synthesis steps and characterization: P1 (1.56g, 2mmol), stannous chloride dihydrate (1.80g, 8mmol) and concentrated hydrochloric acid (9mL) were placed in absolute ethanol (50mL), heated to 78°C and refluxed, stirred for 8 After 1 hour, the solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography (silica gel, ethyl acetate / petroleum ether, 1:3 v / v) to obtain yellow solid P2 (1.27 g, 85%). The yellow solid product was characterized by NMR and mass spectrometry:

[0033] 1 H NMR (400MHz, CDCl 3 )δ8.75(d, J=8.3Hz, 1H), 8.64(d, J=7.2Hz, 1H), 8.54(d, J=7.7Hz, 1H), 7.90(d, J=7.7Hz, 1H) ,7.85(t,J=7.8Hz,1H),7.44(d,J=8.5Hz,2H),7.22(t,J=7.7Hz,1H),7.13(d,J=8.6Hz,2H),6.76 (d, J=8.5Hz, 2H), 6.60(d, J=8.0Hz, 1H), 6.37(d, J=7.4Hz, 1H), 6.35–6.24(m, 4H), 5.44(s, 2H) , 3.32 (q, J=7.0Hz, 8H), 1.16 (t, J=7.0Hz, 12H). 13 C NMR (101MH...

Embodiment 3

[0036] The synthetic route and product structure of intermediate P3 are as follows:

[0037]

[0038] Synthesis steps and characterization: P2 (0.75g, 1mmol) and acetyl chloride (0.12g, 1.5mmol) were mixed in dichloromethane (10mL), stirred for 2 hours, and the solvent was evaporated under reduced pressure, and the crude product was passed through column chromatography (silica gel, Ethyl acetate / petroleum ether, 1:3 v / v) isolated the product P3 as a yellow powder (0.76 g, 96%). The yellow powder product was characterized by NMR and mass spectrometry:

[0039] 1 H NMR (400MHz, CDCl 3 )δ10.58(s,1H),8.75(d,J=8.2Hz,1H),8.65(d,J=7.2Hz,1H),8.55(d,J=7.7Hz,1H),8.51(d, J=8.2Hz,1H),7.92(d,J=7.7Hz,1H),7.90–7.82(m,1H),7.56–7.43(m,3H),7.00(d,J=8.5Hz,2H), 6.81(d, J=7.6Hz, 1H), 6.67(d, J=8.8Hz, 2H), 6.37–6.26(m, 4H), 3.33(q, J=7.0Hz, 8H), 2.31(s, 3H ), 1.17 (t, J=7.0Hz, 12H). 13 CNMR (100MHz, CDCl 3 )δ169.31,168.94,160.38,160.11,153.44,152.94,148.99,137.79,137.43,134.99,133.82,133....

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Abstract

A super-resolution imaging dye for acid-fast bacteria and its synthesis method and application. The specific molecular structure of the dye is rhodamine spiramide substituted with succinimide active ester as the basic structural unit, and its structural formula is shown in (1): The super-resolution imaging dye for acid-fast bacteria not only has acid-resistant performance, but also retains the visible light activation performance. Therefore, this kind of acid-fast bacteria super-resolution imaging dye can be applied in super-resolution imaging technology without interference from acidic environment.

Description

technical field [0001] The invention belongs to the field of fluorescent dyes and imaging, and in particular relates to an acid-resistant bacteria super-resolution imaging dye and its synthesis method and application. Background technique [0002] In recent years, a series of ultra-high-resolution imaging techniques have been developed, among which photoactivated localization microscopy (PLAM) and stochastic optical reconstruction microscopy (STORM or dSTORM) based on single-molecule localization have made the spatial resolution of optical microscopy unprecedented. the height of. At present, super-resolution microscopy imaging technology has been widely used in life science research. However, although super-resolution microscopy imaging technology has made great progress, the spatial resolution of fluorescence microscopy has been advanced to 20 nanometers. [0003] The best way to develop single-molecule localized super-resolution fluorescent dyes for biological imaging is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C09B57/08C09K11/06C07D491/107G01N21/64
CPCC07D491/107C09B57/08C09K11/06C09K2211/1007C09K2211/1029C09K2211/1088G01N21/6486
Inventor 徐兆超祁清凯
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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