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Visible light light-controlled snap protein tag-like acid-resistant fluorescent molecular switch and its synthesis

A fluorescent molecule and visible light technology, applied in fluorescence/phosphorescence, material analysis through optical means, luminescent materials, etc., can solve biological photodamage, biological phototoxicity unfavorable live cell super-resolution imaging, photoactivation performance failure, etc. question

Active Publication Date: 2021-06-11
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 the cell, 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 in Fluorescent probes based on such dyes in acidic environments are currently unavailable for super-resolution fluorescence imaging
In addition, most of the reported rhodamine spiroamides can only be photoactivated by ultraviolet light (<375nm) irradiation, 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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  • Visible light light-controlled snap protein tag-like acid-resistant fluorescent molecular switch and its synthesis
  • Visible light light-controlled snap protein tag-like acid-resistant fluorescent molecular switch and its synthesis
  • Visible light light-controlled snap protein tag-like acid-resistant fluorescent molecular switch and its synthesis

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The intermediate molecule (P1) synthesis route and product structure are as follows:

[0028]

[0029] Synthesis steps and characterization: 3-nitrogamin (2.92 g, 6 mmol) and trichlorochloride (5.6 ml, 60 mmol) were placed in 1,2-dichloroethane (150 mL), warmed to 84 ° C reflux, stir After 2 hours, the solvent was evaporated to give a dark purple oil liquid. The crude chloride product was dissolved in dichloromethane (100 mL), followed by adding triethylamine (3 mL) and 6- (4-aminoethylene-group) naphthamine (1.88 g, 6 mmol) mixed solution, and stirred at room temperature for 24 hours. After evaporation of the solvent, the residue was separated from the column chromatography (ethyl chloride / ethyl acetate, 30: 1V / V) to obtain a yellow powder product P1 (2.44 g, 52%). Characterization of nuclear magnetic and mass spectrometry for yellow powder products:

[0030] 1 H NMR (400MHz, CDCL 3 Δ8.75 (D, J = 8.4 Hz, 1H), 8.65 (D, J = 7.1Hz, 1H), 8.55 (D, J = 7.7 Hz, 1H), 7.92 (D...

Embodiment 2

[0033] The intermediate molecule (P2) synthesis route and product structure are as follows:

[0034]

[0035] Synthesis steps and characterization: P1 (1.56 g, 2 mmol), dihydrochloride (1.80 g, 8 mmol) and concentrated hydrochloric acid (9 ml) were placed in anhydrous ethanol (50 mL), warmed to 78 ° C for reflux, stir 8 After the hour, the solvent was evaporated under reduced pressure, and the crude product was isolated by column chromatography (ethyl acetate / petroleum ether, 1: 3V / V) to obtain yellow solid P2 (1.27 g, 85%). Characterization of nuclear magnetic and mass spectrometry for yellow solid products:

[0036] 1 H NMR (400MHz, CDCL 3 Δ8.75 (D, J = 8.3Hz, 1H), 8.64 (D, J = 7.2 Hz, 1H), 8.54 (D, J = 7.7 Hz, 1H), 7.90 (D, J = 7.7 Hz, 1H) 7.85 (T, J = 7.8 Hz, 1H), 7.44 (D, J = 8.5 Hz, 2H), 7.22 (T, J = 7.7 Hz, 1H), 7.13 (D, J = 8.6Hz, 2H), 6.76 (D, J = 8.5 Hz, 2H), 6.60 (D, J = 8.0 Hz, 1H), 6.37 (D, J = 7.4 Hz, 1H), 6.35-6.24 (M, 4H), 5.44 (S, 2H) 3.32 (q, j = 7.0 Hz, 8h...

Embodiment 3

[0039] The intermediate P3 synthesis route and product structure are as follows:

[0040]

[0041] Synthesis steps and characterization: P2 (0.75 g, 1 mmol) and acetyl chloride (0.12 g, 1.5 mmol) were mixed with dichloromethane (10 mL), and the solvent was evaporated under reduced pressure after stirring for 2 hours. The crude product passed column chromatography (silica gel, Ethyl acetate / petroleum ether, 1: 3V / V) were isolated from yellow powder product P3 (0.76 g, 96%). Characterization of nuclear magnetic and mass spectrometry for yellow powder products:

[0042] 1 H NMR (400MHz, CDCL 3 δ 10.58 (S, 1H), 8.75 (D, J = 8.2 Hz, 1H), 8.65 (D, J = 7.2 Hz, 1H), 8.51 (D, J = 7.7 Hz, 1H), 8.51 (D, J = 8.2Hz, 1H), 7.92 (D, J = 7.7 Hz, 1H), 7.90-7.82 (M, 1H), 7.56-7.43 (M, 3H), 7.00 (D, J = 8.5 Hz, 2H), 6.81 (D, J = 7.6Hz, 1H), 6.67 (D, J = 8.8 Hz, 2H), 6.37-6.26 (m, 4H), 3.33 (Q, J = 7.0 Hz, 8h), 2.31 (s, 3h ), 1.17 (t, j = 7.0 Hz, 12h). 13 CNMR (100MHz, CDCL 3 ) Δ169.31,168.94,16...

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Abstract

The invention provides a visible light-controlled SNAP protein-labeled acid-resistant fluorescent molecular switch and its synthesis. The specific structure of the molecular switch is based on rhodamine spiramide substituted with 3-amino or acetylamino as the basic structure, and the SNAP protein tag recognition group benzylguanine (BG) is covalently connected to the rhodamine spiramide fluorescent switch molecule. The structural formula is shown in (1). The fluorescent switch probe linked with the SNAP tag is specifically labeled on the tubulin in the cell, and the super-resolution imaging of the tubulin is superimposed and reconstructed by STORM technology. In the present invention, the SNAP protein-labeled acid-resistant fluorescent molecular switch controlled by visible light not only has the performance of acid resistance, but also retains the performance of activation by visible light. Therefore, this kind of acid-resistant fluorescent switch dye activated by visible light can be applied in super-resolution imaging technology without interference from acidic environment.

Description

Technical field [0001] The present invention belongs to the field of molecular switch, and in particular to a visible light-controlled SNAP protein label class acid-resistant molecular switch and synthesis thereof. Background technique [0002] A series of ultra-high resolution imaging techniques developed in recent years, where single molecular positioned photosphere positioning micro technology (Plam) and random optical reconstruction micro technology (Storm or DSTORM) have reached an unprecedented space of optical microscope the height of. At present, the super-distinguishing micro-imaging technology has been widely used in life science research, but although the super-distinguishing microscopic imaging technology has made tremendous progress, the spatial resolution of the fluorescent microscope is advanced to 20 nanometers, but super distracted micro-imaging technology Faced with many technical issues, one of the technical issues is that the performance of fluorescent dyes is...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D519/00C09K11/06G01N21/33G01N21/64
CPCC07D519/00C09K11/06C09K2211/1007C09K2211/1029C09K2211/1044C09K2211/1088G01N21/33G01N21/6458G01N21/6486
Inventor 徐兆超祁清凯
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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