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Visible-light-controlled SNAP protein tag type acid-resistant fluorescent molecular switch and synthesis method thereof

A fluorescent molecule, visible light technology, applied in fluorescence/phosphorescence, material analysis by optical means, material analysis, etc., can solve the problem of biological phototoxicity, adverse living cell super-resolution imaging, acid-activated fluorescence interference, photoactivation performance failure, etc. question

Active Publication Date: 2019-09-24
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-controlled SNAP protein tag type acid-resistant fluorescent molecular switch and synthesis method thereof
  • Visible-light-controlled SNAP protein tag type acid-resistant fluorescent molecular switch and synthesis method thereof
  • Visible-light-controlled SNAP protein tag type acid-resistant fluorescent molecular switch and synthesis method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Intermediate molecule (P1) synthetic route and product structure are as follows:

[0028]

[0029] 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:

[0030] 1 H NMR (400MHz, CDCl 3 )δ8.75(d, J=8.4Hz, 1H), 8.65(d...

Embodiment 2

[0033] Intermediate molecule (P2) synthetic route and product structure are as follows:

[0034]

[0035] 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:

[0036] 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 (101M...

Embodiment 3

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

[0040]

[0041] 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:

[0042] 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

The invention provides a visible-light-controlled SNAP protein tag type acid-resistant fluorescent molecular switch and a synthesis method thereof. The specific structure of the molecular switch adopts a 3-amino or acetylamino substituted rhodamine spiroamide as a basic structure, an SNAP protein tag recognition group benzyl guanine (BG) is covalently linked to a rhodamine spiroamide fluorescent switch molecule, the structure of the visible-light-controlled SNAP protein tag type acid-resistant fluorescent molecular switch is represented by formula (1), the SNAP tag-linked fluorescent switch probe is specifically labeled on an intracellular tubulin, and super-resolution imaging of the tubulin is reconstructed by overlapping using an STORM technology. The visible-light-controlled SNAP protein tag type acid-resistant fluorescent molecular switch has acid resistance, and also retains the visible light activation property, so the visible-light-activated acid-resistant fluorescent switch dye can be applied to a super-resolution imaging technology without being interfered by the acidic environment.

Description

technical field [0001] The invention belongs to the field of molecular switches, and in particular relates to a SNAP protein-labeled acid-resistant fluorescent molecular switch controlled by visible light and its synthesis. 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, advancing the spatial resolution of fluorescence microscopy to 20 nanometers, super-resolution microscopy imaging technology There are still many technical problems, one of which is that the performa...

Claims

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Application Information

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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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