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WO2020124688A1 - Colorants fluorescents à haute stabilité, à haute luminosité et à spectre complet, et synthèse et application de ceux-ci - Google Patents

Colorants fluorescents à haute stabilité, à haute luminosité et à spectre complet, et synthèse et application de ceux-ci Download PDF

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WO2020124688A1
WO2020124688A1 PCT/CN2019/000193 CN2019000193W WO2020124688A1 WO 2020124688 A1 WO2020124688 A1 WO 2020124688A1 CN 2019000193 W CN2019000193 W CN 2019000193W WO 2020124688 A1 WO2020124688 A1 WO 2020124688A1
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徐兆超
乔庆龙
周伟
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Dalian Institute of Chemical Physics of CAS
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    • C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
    • C09B11/10—Amino derivatives of triarylmethanes
    • C09B11/24—Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
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Definitions

  • the invention belongs to the field of fluorescent dyes, and in particular relates to the synthesis and application of a full-spectrum high-brightness, high-stability fluorescent dye.
  • Organic fluorescent dyes can be divided into Resonant dyes and Charge (Transfer, CT) dyes from the fluorescence structure-activity relationship, and the fluorescent properties are determined by the electron transfer between the ground state and the excited state, so that the study of the structure-activity relationship can be Achieve structural modification with different light performance requirements, or accurately predict the luminous performance of known structures.
  • Fluorescein, rhodamine, Bodipy, and most cyanine dyes are all classified as resonance dyes, which have narrow absorption and emission peaks, solvent insensitivity, small Stokes shift, high molar extinction coefficient, and high quantum yield.
  • charge transfer dyes such as 1,8-naphthalimide and coumarin etc.
  • charge transfer dyes have a clear electron donor and electron acceptor, which has a wide absorption and emission peak, solvent sensitivity, large Stokes Displacement, relatively low molar absorption coefficient and other luminous properties.
  • the spectral properties of charge transfer dyes are greatly affected by the strong polar environment, and the fluorescence quantum yield in the near infrared region is extremely low.
  • the small organic fluorescent dyes used for fluorescent labeling mainly focus on fluorescein, rhodamine, Bodipy and cyanine dyes with high quantum yield and narrow half-width.
  • fluorescein was first used for immunofluorescence labeling, and then a variety of organic small molecule fluorescent dyes such as rhodamine dyes were gradually developed and should be used for fluorescent labeling.
  • organic fluorescent dyes and different properties each dye has obvious disadvantages.
  • the luminescent form of fluorescein is a negatively charged fluorophore. This form is extremely sensitive to pH and has poor stability. And this negatively charged form has poor cell permeability, which seriously affects its labeling of target molecules in living cells.
  • rhodamine dye is a positive ion fluorescent dye with good membrane permeability and stability. Due to the pain in the mitochondria, this positive ion form can easily enter the mitochondria for non-specific labeling. Cyanine dyes are often used for imaging of living bodies and tissues with their near-infrared fluorescence emission wavelength, which has strong penetration ability in tissues and shields autofluorescence. However, the light stability and low quantum yield of cyanine dyes have always been a barrier limiting their applications.
  • One of the objectives of the present invention is to provide a full-spectrum, high-brightness, high-stability fluorescent dye.
  • the fluorescent dye of this system has extremely high biocompatibility, and can stain living cells within 20 seconds to 5 minutes. Functionalized fluorescent molecules with such dyes can be able to specifically label target molecules or organelles.
  • Another object of the present invention is to provide a method for synthesizing fluorescent dyes with high brightness and high stability, which has the advantages of simple operation, easy derivation, and easy purification.
  • the invention provides a high-brightness and high-stability fluorescent dye, which takes naphthalimide and perylene imide as fluorescent groups, and introduces two amino substituents at one end of the power supply group to greatly improve the stability and fluorescent brightness.
  • these dyes are insensitive to external microenvironments such as viscosity, pH, and temperature.
  • the invention provides a class of functionalized fluorescent molecules based on novel fluorescent dyes, which have high cell permeability, can quickly stain a variety of living cells, and are successfully used in the fields of fluorescent labeling, fluorescent imaging, and the like.
  • the invention provides a full-spectrum, high-brightness, high-stability fluorescent dye whose excitation wavelength covers the full wavelength band.
  • the dye suppresses intramolecular twisting at the end of the electron donor through the adjustment of the rigid cyclic amine structure, achieves an increase in fluorescence quantum efficiency, and stabilizes light
  • the dye is improved by 4-amide substituted naphthalimide dyes, dialkoxy substituted naphthalimide fluorescent dyes, diamino substituted naphthalimide fluorescent dyes, 9,10-bisamino
  • One or more of the substituted perylene imides, six-membered ring-rhodamine dyes, five-membered ring-rhodamine dyes, and silicon-based rhodamine-based dyes are mixed in any ratio.
  • the bisalkoxy substituted naphthalimide fluorescent dye has an absorption wavelength at 390 nm and a fluorescence emission wavelength at 405 nm excitation, and its structural formula is as follows:
  • R 3 and R 4 are respectively independent If R 3 and R 4 are not independent, it is Exist as a whole structure p is an integer from 0-2.
  • step (1) the mass ratio of polyol and sodium block is 2-1:1; the mass ratio of polyol and N-butyl-4-bromo-5-nitro-1,8-naphthalimide is 2:1-12; the volume ratio of polyol to tetrahydrofuran is 1-10:1mg/mL;
  • the diamino-substituted naphthalimide fluorescent dye has an absorption wavelength of 440-490nm, can be excited by 450nm and 488nm lasers, and can be used as the following dyes by changing the targeting group: mitochondrial fluorescent dye, SNAP- Tag fluorescent dyes, Halo-tag fluorescent dyes, reactive ester fluorescent dyes, drug targeting fluorescent dyes, etc., the structural formula is as follows:
  • R 2 is H, C1-16 alkyl, aryl, substituted aryl, (CH 2 CH 2 O) n H, (CH 2 ) m COOMe and (CH 2 ) m SO 3 H, heteroaryl or substituted Heteroaryl, biological targeting groups such as N-ethylmorpholine, benzylguanine, n-hexane-triphenylphosphine, folic acid, colchicine, paclitaxel, 6-chlorohexane and so on.
  • biological targeting groups such as N-ethylmorpholine, benzylguanine, n-hexane-triphenylphosphine, folic acid, colchicine, paclitaxel, 6-chlorohexane and so on.
  • R 5 and R 6 are respectively independent One of them, if R 5 and R 6 are not independent, it is Exist as a whole structure,
  • R 7 and R 8 are each independently H, C1-4 alkyl, (CH 2 CH 2 O) n H; if R 7 is not H, R 8 must be a non-H substituent;
  • Y is sulfone group, sulfoxide group, dimethylsilyl group, boryl group;
  • n are integers from 0-4.
  • step (1) the mass ratio of 4-bromo-5-nitro-1,8-naphthalic anhydride: primary fatty amine is 1:0.5-2; 4-bromo-5-nitro-1,8-naphthalic anhydride The volume ratio of the mass to absolute ethanol is 1:20-80g/mL.
  • Fatty primary amines include linear alkyl amines such as methylamine, ethylamine, butylamine, n-dodecylamine, n-hexadecylamine, benzylamine analogs, amino-substituted alkyl sulfonic acids, amino alcohols, and the like.
  • step (2) the mass ratio of N-alkyl-4-bromo-5-nitro-1,8-naphthalimide to alicyclic amine is 1:1-3; N-alkyl-4-bromo The volume ratio of -5-nitro-1,8-naphthalimide to ethylene glycol methyl ether is 1:50-200g/mL;
  • Alicyclic amines are aziridine, azetidine, tetrahydropyrrole, piperidine, cycloheximide, ethylenediamine derivatives and cyclohexanediamine derivatives.
  • a full-spectrum, high-brightness, high-stability fluorescent dye that is disubstituted with 9,10 bisamino-substituted peryleneimide dyes. It is characterized by being used for 680nm and 710nm lasers. Its structure is as follows:
  • R 11 is
  • R 9 and R 10 are respectively independent One of them, if R 9 and R 4 are not independent, it is Exist as a whole structure,
  • R 7 and R 8 are each independently H, C1-4 alkyl, (CH 2 CH 2 O) n H; if R 7 is not H, R 8 must be a non-H substituent; n is an integer of 0-4.
  • step (1) the mass ratio of the 9,10-dibromo-1,6,7,12-tetrachloroperyleneimide to the primary alcohol amine or aliphatic primary amine is 1-10:1; the 9 , The mass-volume ratio of 10-dibromo-1,6,7,12-tetrachloroperyleneimide and N-methylpyrrolidone is 1:20-120g/mL; the N-methylpyrrolidone and glacial acetic acid The volume ratio is 1-3:3-4;
  • step (2) the mass ratio of the N-alkyl-9,10-dibromo-1,6,7,12-tetrachloroperyleneimide to fatty amine is 1:6-8; the fat The mass to volume ratio of amine to ethylene glycol methyl ether is 5-120:1mg/mL; the fatty amines include ammonia, aziridine, azetidine, tetrahydropyrrole, piperidine or cyclohexanediamine derivatives Wait.
  • a full-spectrum, high-brightness, high-stability fluorescent dye used in the excitation of 532nm six-membered ring rhodamine dyes, its structure is as follows:
  • R 12 is a 5-position parallel six-membered ring or a 7-position parallel six-membered ring
  • R 13 is H or C1-4 alkane.
  • step (1) the mass ratio of the intermediate N-alkyl-5-hydroxytetrahydroquinolinyl keto acid and phthalic anhydride is 1:1-2, and the intermediate N-alkyl-5-hydroxyl
  • the mass ratio of tetrahydroquinolinyl benzophenone acid to toluene is 1:40-80g/mL;
  • step (2) the intermediate N-alkyl-5-hydroxytetrahydroquinoline (or its analog N-substituted-7-hydroxytetrahydroquinoline) and the intermediate N-alkyl-5-hydroxytetrahydroquinoline
  • the mass ratio of quinolinyl benzophenic acid is 1:2-4; the volume ratio of trifluoroacetic acid and methanesulfonic acid is 1:1-5; the intermediate N-alkyl-5-hydroxytetrahydroquinoline (or its The volume ratio of the analog N-substituted-7-hydroxytetrahydroquinoline) to trifluoroacetic acid is 1:30-80 g/mL.
  • a full-spectrum, high-brightness, high-stability fluorescent dye with five-membered ring rhodamine dyes, its structure is as follows:
  • R 13 is H or C1-4 alkane.
  • step (1) the mass ratio of intermediate N-alkyl-4hydroxyindoline to phthalic anhydride is 1:1-2, and the mass of intermediate N-alkyl-4hydroxyindoline and toluene The volume ratio is 1:20-40g/mL;
  • step (2) the mass ratio of the intermediate N-alkyl-4hydroxyindoline to the intermediate N-alkyl-4-hydroxyindoline benzophenone acid is 1:2-3, trifluoroacetic acid and The volume ratio of methanesulfonic acid is 1:1-5, and the volume ratio of the mass of intermediate N-alkyl-4hydroxyindoline to trifluoroacetic acid is 1:10-30g/mL.
  • a full-spectrum, high-brightness, high-stability fluorescent dye of silicon-based rhodamine dyes, its structure is as follows:
  • R 13 is H, C1-4 alkyl.
  • step (1) the mass ratio of intermediate Si-keto to 2-bromobenzoic acid tert-butyl ester is 1:4-8, and the mass ratio of intermediate Si-keto to butyllithium solution is 10-20: 1mg/mL;
  • step (2) the mass ratio of intermediate Si-ketos to tert-butyl 2-bromobenzoate is 1:4-7, the volume ratio of butyllithium solution to tetrahydrofuran is 1:30-50, intermediate Si- The volume ratio of ketos to butyllithium is 1:10-20mg/ ⁇ L.
  • the above-mentioned fluorescent dyes used in a class of high brightness and high stability have high biocompatibility. After functionalization, they can perform real-time fluorescence imaging of different organelles and different protein targets of living cells, and super-resolution in STED and SIM. It can be used in fluorescence microscopy.
  • a full-spectrum high-brightness, high-stability fluorescent dye is used in the field of fluorescent imaging of living cells, tissues, and living bodies.
  • a full-spectrum high-brightness, high-stability fluorescent dye is used in the field of SNAP-tag and Halo-tag identification tags.
  • a full-spectrum high-brightness, high-stability fluorescent dye is used in the field of fluorescent imaging of living cells, tissues, and living bodies.
  • the invention has the following characteristics:
  • the dyes involved in the present invention have the advantages of simple synthetic methods, cheap raw materials and easy functionalization.
  • Some dyes involved in the present invention have a fluorescence emission half-peak width of less than 40nm in different organic solvents, and the narrowest can reach 25nm; the fluorescence quantum yield is significantly improved, and can reach 0.80 in water; the light stability is significantly higher than fluorescein, rhodamine, Fluoroboron pyrrole dyes.
  • the functionalized molecules based on this kind of dye fluorescent precursor have high biocompatibility, and can complete the fluorescent labeling of cells within 20s-5min.
  • the mitochondrial series of fluorescent probes can completely label mitochondria of various living cells within 2 minutes.
  • the lipid droplet dye can accurately locate lipid droplets in various cell lines such as HT29 (colon cancer cells), MCF (breast cancer cells), and fat cells; at the same time, it can mark the zebrafish living lipid metabolism center (liver) And fluorescence imaging.
  • the improvement of the light stability of the dye involved in the present invention enables the dye to achieve super-resolution fluorescence imaging, which is more light stable than the traditional fluorescent dye Alexa 488.
  • FIG. 1 is the hydrogen spectrum of the NMR spectrum of Lyso-DAze prepared in Example 23.
  • FIG. 1 is the hydrogen spectrum of the NMR spectrum of Lyso-DAze prepared in Example 23.
  • Example 2 is a nuclear magnetic spectrum hydrogen spectrum of the Nu-DAC prepared in Example 24.
  • Example 3 is a hydrogen spectrum of the NMR spectrum of the CM-DAze prepared in Example 30.
  • FIG. 4 is a hydrogen spectrum of the NMR spectrum of the NHSB-DAC prepared in Example 40.
  • Example 5 is a hydrogen spectrum of the nuclear magnetic spectrum of BuLD-DAze prepared in Example 49.
  • FIG. 6 is a nuclear magnetic spectrum hydrogen spectrum of the dye SiR-1 prepared in Example 60.
  • FIG. 6 is a nuclear magnetic spectrum hydrogen spectrum of the dye SiR-1 prepared in Example 60.
  • Example 7 is a high-resolution mass spectrum of the DTX-DAC prepared in Example 46.
  • FIG. 8 is the normalized fluorescence excitation spectrum and fluorescence emission spectrum of the dye material BuAN-DAze prepared in Example 5 in ethanol.
  • the abscissa is the wavelength
  • the ordinate is the fluorescence intensity
  • the concentration of the fluorescent dye is 10 ⁇ M.
  • FIG. 9 is the normalized fluorescence excitation spectrum and fluorescence emission spectrum of the dye material OLD-710 prepared in Example 52 in ethanol.
  • the abscissa is the wavelength
  • the ordinate is the fluorescence intensity
  • the concentration of the fluorescent dye is 10 ⁇ M.
  • 10 is a normalized fluorescence excitation spectrum and fluorescence emission spectrum of the dye material SiR-1 prepared in Example 60 in ethanol, the abscissa is the wavelength, the ordinate is the fluorescence intensity, and the concentration of the fluorescent dye is 10 ⁇ M.
  • FIG. 11 is a graph of the fluorescence intensity of the dye material BuAN-DAze prepared in Example 5 at 495 nm as a function of time under the irradiation of a 500 W tungsten lamp.
  • Commercial green mitochondrial dye, rhodamine 123, fluorescein, and Bodipy were selected as reference dyes.
  • FIG. 12 is a fluorescence confocal imaging of live cells of RWPE cells prepared with the dye Mito-DAze prepared in Example 20.
  • FIG. 12 is a fluorescence confocal imaging of live cells of RWPE cells prepared with the dye Mito-DAze prepared in Example 20.
  • FIG. 13 is a light-illuminated micro-imaging image of the living cell structure of the RWPE cell of the dye Mito-DAze prepared in Example 20.
  • FIG. 13 is a light-illuminated micro-imaging image of the living cell structure of the RWPE cell of the dye Mito-DAze prepared in Example 20.
  • FIG. 14 is a fluorescence confocal imaging diagram of HeLa cell live cell fluorescent dye Mito-DAC prepared in Example 19.
  • FIG. 14 is a fluorescence confocal imaging diagram of HeLa cell live cell fluorescent dye Mito-DAC prepared in Example 19.
  • FIG. 15 is a fluorescent confocal imaging image of adipose cell live cell fluorescence of the lipid droplet dye OLD-DAze prepared in Example 50.
  • FIG. 15 is a fluorescent confocal imaging image of adipose cell live cell fluorescence of the lipid droplet dye OLD-DAze prepared in Example 50.
  • Example 16 is a fluorescent confocal imaging of HeLa cells transfected with HALO-H2B dye prepared in Example 14 by the dye Halo-DAze, and the concentration of the fluorescent probe is 1 ⁇ M.
  • Example 17 is a fluorescent confocal imaging of HeSN cells of pSNAP f -H2B transfected with the dye SNAP-DAC prepared in Example 17, and the concentration of the fluorescent probe is 1 ⁇ M.
  • Example 18 is a microscopic image of stimulated radiation loss of HeLa cells of pSNAP f -H2B transfected with the dye SNAP-DAC prepared in Example 17, and the concentration of the fluorescent probe is 1 ⁇ M.
  • FIG. 19 is a structured light illumination microcolor imaging of RWPE cells with Rho-4 prepared in Example 58 and Nu-DAC prepared in Example 33
  • FIG. 20 is a structured light illumination microscope multicolor imaging diagram of OLD-DAze prepared in Example 50 and Nu-DAC prepared in Example 33 on HT29 cells.
  • the dye has strong absorption at 405 nm and can be used for excitation at 405 nm.
  • the fluorescence emission wavelength of OEOAN in acetonitrile, chloroform, ethanol, dimethyl sulfoxide, and water is 420-450nm.
  • the fluorescence wavelength does not basically change with the polarity of the solvent, and the half-width is less than 50nm. It avoids the interference of different polar environments on the fluorescence signal in fluorescence imaging and detection.
  • OEOAN's ultraviolet absorption wavelength in acetonitrile, chloroform, ethanol, dimethyl sulfoxide, and water has strong absorption at 405nm, which is convenient for excitation to obtain high-brightness fluorescence.
  • the dye is less affected by changes in polarity.
  • the dye has strong absorption at 405 nm and can be used for excitation at 405 nm.
  • the dye has strong absorption at 405nm and can be used for excitation at 405nm.
  • N-butyl-4-bromo-5-nitro-1,8-naphthalimide (100 mg, 0.26 mmol) was dissolved in 20 mL of ethylene glycol methyl ether, and azetidine (300 mg) was added thereto , 5.26mmol).
  • the reaction solution was slowly heated to 120°C and reacted for 24h.
  • the nuclear magnetic spectrum hydrogen spectrum and carbon spectrum of BuAN-DAze prepared in Example 8 are shown in Figures 3 and 4, respectively. The specific data are:
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value calcd for C 22 H 26 N 3 O 2 [M+H] + 364.2025, measured value 364.2035.
  • the fluorescence excitation and emission spectrum of BuAN-DAze is shown in Fig. 34:
  • the excitation wavelength of BuAN-DAze in ethanol is 480nm
  • the fluorescence emission wavelength is 488nm
  • the half-width of the fluorescence emission is only 32nm. This shows that BuAN-Daze can be applied to multi-color fluorescence imaging.
  • the nuclear magnetic spectrum hydrogen spectrum of BuAN-DAzo prepared in Example 9 is shown in FIG. 5, and the specific data of hydrogen spectrum and carbon spectrum are:
  • the high-resolution mass spectrometry data is as follows: the theoretical value of the resolution mass spectrum C 24 H 30 N 3 O 2 [M+H1 + 392.2338, the actual value is 392.2343.
  • the fluorescence excitation and emission spectrum of BuAN-DAzo is shown in Figure 35:
  • the excitation wavelength of BuAN-DAzo in ethanol is 485nm
  • the fluorescence emission wavelength is 495nm
  • the fluorescence emission half-width is only 40nm, which is suitable for 488nm excitation.
  • N-butyl-4-bromo-5-nitro-1,8-naphthalimide (80 mg, 0.21 mmol) was dissolved in 15 mL of ethylene glycol methyl ether, and 400 mg of hexamethyleneimine was added thereto .
  • the reaction solution was slowly heated to 120°C and reacted for 20h.
  • the nuclear magnetic spectrum hydrogen spectrum of the BuAN-DHMI prepared in Example 11 is shown in FIG. 7, and the specific data are:
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 28 H 38 N 3 O 2 [MH + ] 448.2964, found value 448.2973.
  • the hydrogen spectrum of the nuclear magnetic spectrum of BuAN-450 prepared in Example 12 is shown in FIG. 8, and the specific data of hydrogen spectrum and carbon spectrum are:
  • the high-resolution mass spectrometry data is as follows: high-resolution mass spectrometry theoretical value C 24 H 30 N 3 O 2 [M+H] + 392.2338, the actual value is 392.2352.
  • the fluorescence emission wavelength in methylene chloride is 475nm
  • the data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 18 H 18 BrN 2 O 2 [M+H] + 373.0554, the actual value is 373.0561.
  • the hydrogen spectrum of the NMR spectrum of BuAN-AzeAzi prepared in Example 13 is shown in FIG. 9, and the specific data are:
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 21 H 24 N 3 O 2 [M+H] + 350.1869, actual value 350.1872.
  • the fluorescence emission wavelength in water is 530nm
  • the absorption wavelength reaches 468nm, which is suitable for excitation by a 450nm laser.
  • the hydrogen spectrum of the NMR spectrum of BuAN-AzeAzo prepared in Example 15 is shown in FIG. 10, and the specific data are:
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 23 H 28 N 3 O 2 [M+H] + 378.2182, actual value 378.2093.
  • the fluorescence emission wavelength in water is 493nm
  • the absorption wavelength reaches 481nm, which is suitable for 488nm laser excitation.
  • the hydrogen spectrum of the nuclear magnetic spectrum of BuAN-EDA prepared in Example 17 is shown in FIG. 12, and the specific data of the hydrogen spectrum and the carbon spectrum are:
  • the nuclear magnetic spectrum hydrogen spectrum of the BuAN-DAC prepared in Example 18 is shown in FIG. 13, and the specific data are:
  • the fluorescence emission wavelength in water is 488nm
  • the absorption wavelength reaches 481nm, which is suitable for excitation by a 488nm laser.
  • N-butyl-4-bromo-5-nitro-1,8-naphthalimide (100 mg, 0.27 mmol) was dissolved in 10 mL of ethylene glycol methyl ether, and N,N'-dimethyl was added thereto Cyclohexanediamine 350mg.
  • the reaction solution was slowly heated to 120°C and reacted for 12h.
  • the hydrogen spectrum data of the nuclear magnetic spectrum is as follows:
  • the data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 22 H 26 N 3 O 4 [M+H] + 396.1923, found value 396.1919.
  • the hydrogen spectrum of the Halo-DAze NMR spectrum prepared in Example 20 is shown in FIG. 14, and the specific data of the hydrogen spectrum and the carbon spectrum are as follows:
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 28 H 37 ClN 3 O 4 [M+H] + 514.2473, the actual value is 514.2477.
  • the structure is as shown in the above formula Halo-DAze, the ultraviolet absorption wavelength in water is 484nm, and the fluorescence emission wavelength is 493nm, which can be used for Halo-tag fluorescent labeling.
  • the data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 22 H 26 N 3 O 4 [M+H] + 396.1923, found value 396.1919.
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 28 H 37 ClN 3 O 4 [M+H] + 514.2473, the actual value is 514.2477.
  • Halo-DAC has a water light emission wavelength of about 490nm, an excitation wavelength of 480nm, and a fluorescence half-width of only 40nm.
  • the NMR spectrum hydrogen spectrum is shown in Figure 1, the specific data are as follows:
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 26 H 26 N 3 O 3 [M+H] + 428.1974, actual value 428.1997.
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 31 H 29 N 8 O 3 [M+H] + 561.2363, actual value 561.2380.
  • SNAP-DAze has a light emission wavelength of about 490nm in acetonitrile, chloroform, dimethyl sulfoxide, ethanol, and water, and fluoresces with the change of polarity There is no obvious change in emission wavelength and fluorescence peak shape.
  • the data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 31 H 29 N 8 O 3 [M+H] + 561.2363, actual value 561.2380.
  • BA-NBr 300 mg, 0.68 mmol was dissolved in 30 mL of ethylene glycol methyl ether, and 39 mg of azetidine was added thereto.
  • the reaction solution was slowly heated to 50°C and reacted for 1 h.
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 27 H 28 N 3 O 3 [M+H] + 442.2131, the actual value is 442.2142.
  • High-resolution mass spectrometry data are as follows: C 42 H 43 N 10 O 21 P + [M] + calculated value: 652.3087, experimental value: 652.3128.
  • Mito-DAC suitable for imaging of live cell mitochondria in a variety of physiological states and the light performance is not affected by the microenvironment, high brightness and strong stability can meet the long-term dynamics of mitochondria by super-resolution imaging Tracking, the fluorescence emission wavelength is around 481nm.
  • High-resolution mass spectrometry data are as follows: C 42 H 43 N 3 O 2 P + [M] + calculated value: 652.3088, experimental value: 652.3109.
  • the structure of the above product is Mito-DAze, the compound can be quickly and accurately located in the mitochondria in live cell imaging experiments, with high brightness and strong stability.
  • BA-DMEDA 50 mg, 0.12 mmol was dissolved in 15 mL of dimethyl sulfoxide, and 350 mg of triethylamine was added thereto.
  • the reaction solution was slowly heated to 140°C and reacted for 12h.
  • the data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 19 H 20 N 3 O 2 [M+H] + 336.1712, actual value 336.1733.
  • the nuclear magnetic spectrum hydrogen spectrum of Lyso-DAze prepared in Example 32 is shown in FIG. 18, and the specific data are:
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 24 H 29 N 4 O 3 [M+H] + 421.2240, the actual value is 421.2251.
  • the nuclear magnetic spectrum hydrogen spectrum of the Nu-DAC prepared in Example 33 is shown in FIG. 19, and the specific data of the hydrogen spectrum and the carbon spectrum are as follows:
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 24 H 29 N 4 O 3 [M+H] + 421.2240, the actual value is 421.2248.
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 19 H 20 N 3 O 5 S[M+H] + 402.1124, actual value 402.1140.
  • the nuclear magnetic spectrum hydrogen spectrum of the Tro-DAC prepared in Example 38 is shown in FIG. 21, and the specific data are:
  • the data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 32 H 32 N 4 O 6 Na[M+Na] + 591.2220, actual value 591.2284.
  • PhAN-DAze (20 mg, 0.03 mmol) was dissolved in 20 mL of ethanol, and 200 ⁇ L of an aqueous hydrazine hydrate solution (85%) was added thereto. After the reaction solution was refluxed for 3 h, ethanol was removed under reduced pressure, and the residue was dissolved in 20 mL of dichloromethane. The organic phase was washed with 3 ⁇ 50 mL of saturated brine, and the organic phase was dried over anhydrous sodium sulfate overnight.
  • DDAN-NBr (0.25g, 0.51mmol) was dissolved in 20mL ethylene glycol methyl ether, and 1,2-cyclohexanediamine (0.35g, 3.1mmol) was added thereto, and the reaction solution was slowly heated to 130°C, And react for 18h.
  • the specific data of the nuclear magnetic spectrum hydrogen spectrum and carbon spectrum are as follows:
  • the data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 34 H 50 N 3 O 2 [M+H] + 532.3903, actual value 532.3930.
  • HexAN-DAC After testing, its structure is as shown in the above formula HexAN-DAC, its ultraviolet absorption wavelength in ethanol is 475nm, the fluorescence emission wavelength is 485nm, has a high brightness and light stability, is not sensitive to the environment and can accurately locate the cell membrane of living cells .
  • the hydrogen spectrum of the nuclear magnetic spectrum is shown in FIG. 23, and the specific data of the hydrogen spectrum and the carbon spectrum are as follows:
  • the high-resolution mass spectrum of MBSO3-DAC prepared in Example 42 is shown in FIG. 31, and the mass spectrum data is: theoretical value of high-resolution mass spectrum C 35 H 53 N 4 O 5 S [M+H] + 641.3737, measured value 641.3762.
  • CFAN-NBr 150 mg, 0.33 mmol was dissolved in 20 mL of ethylene glycol methyl ether, and 1,2-cyclohexanediamine (400 mg, 3.54 mmol) was added thereto, and the reaction solution was slowly heated to 130° C. for 18 h. .
  • the data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:
  • the data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:
  • the hydrogen spectrum data of the nuclear magnetic spectrum is as follows:
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 49 H 69 N 4 O 4 [M+H] + 777.5319, actual value 777.5365.
  • CMN-DAC ultraviolet absorption wavelength in ethanol is 475nm, and its fluorescence emission wavelength is 485nm. It has high brightness and light stability, is not sensitive to the environment, and can accurately locate the cell membrane of living cells. .
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 22 H 26 N 3 O 3 [M+H] + 364.2025, actual value 364.2082.
  • the hydrogen spectrum data of the nuclear magnetic spectrum is as follows:
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 20 H 20 N 3 O 4 [M+H] + 366.1454, found value 366.1440.
  • the emission wavelength of COOH-DAze in different solvents is 480-495nm, the half-width of fluorescence emission is less than 35nm, and the fluorescence wavelength does not change with the change of polarity.
  • the ultraviolet absorption wavelength of COOH-DAze in different solvents is 470-485nm, and the absorption wavelength does not change with the change of polarity, which can keep the fluorescence signal stability as much as possible.
  • the high-resolution mass spectrometry data is as follows: high-resolution mass spectrometry theoretical value C 22 H 24 N 3 O 4 [M+H] + 394.1767, the actual value is 394.1788.
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 20 H 20 N 3 O 4 [M+H] + 366.1454, found value 652.3109.
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 24 H 28 N 3 O 4 [M+H] + 422.2080, the actual value is 422.2108.
  • BCOMe-DAC (80 mg, 0.19 mmol) was dissolved in 5 mL of methanol, and 8 mL of 2M sodium hydroxide solution was slowly added dropwise to the reaction solution. After the dropwise addition was completed, the reaction liquid was reacted at room temperature for 1 h, and methanol was distilled off under reduced pressure. The turbid liquid was filtered and the filter cake was washed with 5 mL of water and dried to obtain 65 mg of BCOOH-DAC, with a yield of 87%.
  • the data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:
  • the high-resolution mass spectrometry data is as follows: high-resolution mass spectrometry theoretical value C 22 H 24 N 3 O 4 [M+H] + 394.1767, the actual value is 394.1824.
  • the structure is as shown in the above formula BOOH-DAC, its ultraviolet absorption wavelength in water is 481nm, the fluorescence emission wavelength is 489nm, and the fluorescence quantum yield is as high as 0.80.
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 26 H 27 N 4 O 6 [M+H] + 491.1931, the actual value is 491.1981.
  • the hydrogen spectrum data of the nuclear magnetic spectrum is as follows:
  • the hydrogen spectrum data of the nuclear magnetic spectrum is as follows:
  • the hydrogen spectrum data of the nuclear magnetic spectrum is as follows:
  • the high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C 25 H 28 N 3 O 4 [M+H] + 434.2080, the measured value is 434.2108.
  • NHSB-DAC (20 mg, 0.04 mmol) and aminocolchicine (15 mg, 0.04 mmol) were placed in a 5 mL Shrek bottle and replaced with nitrogen three times. 5 ⁇ L of diisopropylethylamine (DIPEA) was dissolved in 2 mL of dimethyl sulfoxide (DMSO), and then the mixture was added to the reaction bottle.
  • DIPEA diisopropylethylamine
  • DMSO dimethyl sulfoxide
  • the high-resolution mass spectrum of Col-DAC prepared in Example 55 is shown in FIG. 32, and the mass spectrum data is: high-resolution mass spectrometry theoretical value C 42 H 45 N 4 O 8 [M+H] + 733.3237, the measured value 733.3220.
  • the high-resolution mass spectrum of DTX-DAC prepared in Example 56 is shown in FIG. 33, and the mass spectrum data are: high-resolution mass spectrometry theoretical value C 60 H 67 N 4 O 15 [M+H] + 1083.4603, measured value 1083.4603.
  • NHSB-DAC (30 mg, 0.06 mmol) and 4-(4-methyl-2,3,5,6-azaphenyl)benzylamine hydrochloride (19 mg, 0.06 mmol) were placed in a 5 mL Shrek bottle, Replace with nitrogen 4 times. 20 L of diisopropylethylamine (DIPEA) was dissolved in 2 mL of dimethyl sulfoxide (DMSO), and then the mixture was added to the reaction bottle.
  • DIPEA diisopropylethylamine
  • the structure is shown in the above formula BuLD-DAze, and the fluorescence emission wavelength in ethanol is around 720nm, which has reached the near infrared region.
  • LD-DBr 200 mg, 0.28 mmol
  • azetidine 104 mg, 1.42 mmol
  • the structure is shown in the above formula OLD-DAze, and the fluorescence emission wavelength in ethanol is about 750nm, which has reached the near infrared region.
  • the structure is shown in the above formula BuLD-710, and the fluorescence emission wavelength in ethanol is about 750nm, which has reached the near infrared region.
  • Dissolve the dye in DMSO solution prepare 2mM mother liquor, prepare test solutions with different concentrations according to the needs, detect the change of fluorescence spectrum and fluorescence imaging of cells and in vivo lipid droplets.
  • Fluorescence excitation and emission spectrum test in ethanol Each time, 20 ⁇ L of the mother liquor of dye was added to 4 mL of ethanol to prepare a 10 ⁇ M fluorescent probe test solution, and fluorescence excitation and emission spectrum tests were performed.
  • the excitation and emission spectra of OLD-710 in ethanol are shown in Figure 36: Normalized fluorescence spectra of OLD-710 in ethanol.
  • the fluorescence emission wavelength in OLD-710 ethanol reached 750nm, the excitation wavelength was 712nm, and the excitation and emission wavelength reached the near infrared emission wavelength.
  • ELD-DBr 200 mg, 0.27 mmol
  • 1,2-cyclohexanediamine 228 mg, 2.00 mmol
  • ethylene glycol methyl ether 20 mL
  • 1,2-cyclohexanediamine 228 mg, 2.00 mmol
  • the hydrogen spectrum data of the nuclear magnetic spectrum is as follows:
  • N-butyl-1,6,7,12-tetrachloro-9,10-dibromo-3,4-peryleneimide BuL]D-DBr 200mg, 0.30mmol
  • aziridine 100mg, 2.32 mmol
  • 20 mL of ethylene glycol methyl ether 20 mL
  • the hydrogen spectrum data of the nuclear magnetic spectrum is as follows:
  • the structure is as shown in the above formula BuLD-DAzi, the fluorescence emission wavelength in ethanol is about 730nm, and the emission wavelength has reached the near infrared region.
  • Rho-1 which can be used to image mitochondria in cells, and its optical properties are as follows:
  • Rho-1 dye molecule Rho-1 The absorption and emission spectrum of Rho-1 dye molecule Rho-1 in ethanol.
  • the rhodamine dye molecule Rho-1 obtained in Example 64 was dissolved in DMSO to prepare a 2 mM stock solution. 20 ⁇ L of the mother liquor was dissolved in 4 mL of ethanol and configured as a test solution with a final concentration of 10 ⁇ M, and its absorption and emission spectra were measured.
  • Rho-1 in ethanol The absorption and emission spectra of Rho-1 in ethanol are shown in Figure 37: The absorption of Rho-1 in ethanol is 533nm and the emission wavelength is 558nm. The fluorescence quantum yield in ethanol is calculated to be 0.91.
  • Rho-2 After examination, the structure is as shown in the above formula Rho-2, and its optical properties are as follows: the dye Rho-2 absorbs in ethanol at 534 nm and the emission wavelength at 559 nm, and the fluorescence quantum yield in ethanol is calculated to be 0.85.
  • Rho-3 After testing, its structure is as shown in the above formula Rho-3, and its optical properties are as follows: The absorption and fluorescence emission wavelengths of Rho-3 in ethanol are: absorption wavelength is 538nm, emission wavelength is 561nm, and the fluorescence quantum in ethanol is calculated The yield was 0.81.
  • Rho-5 which can be used to image mitochondria in cells.
  • the optical properties are as follows: Rho-5 absorption in ethanol is 551nm, emission wavelength is 574nm, and Stokes shift is 23nm. The half-width of the emission spectrum is 26nm, and the fluorescence quantum yield in ethanol is calculated to be 0.93.
  • the absorption and emission wavelengths of Rho-5 in ethanol are: absorption wavelength is 553nm, emission wavelength is 577nm, and the fluorescence quantum yield in ethanol is calculated to be 0.90.
  • SiR-1 which can be used to image mitochondria in cells, and its optical properties are as follows:
  • the crude product was separated through a silica gel column.
  • the hydrogen spectrum data of the nuclear magnetic spectrum is as follows:
  • the absorption and fluorescence wavelengths of SiR-4 in methanol are: absorption wavelength is 660nm, emission wavelength is 681nm, and the calculated quantum yield is 0.31 .
  • the present invention involves dissolving the dyes in DMSO solution and preparing 2mM mother liquors of different dyes, and preparing different concentrations of test solutions as needed to detect changes in their fluorescence spectra and intracellular fluorescence imaging.
  • BuAN-DAze tested the fluorescence intensity with time under the irradiation of 500W tungsten lamp. Take 20 ⁇ L of BuAN-DAze and commercial dye stock solution into 4mL PBS (Phosphate buffer, pH 7.4), and then add it to the threaded cuvette. Place it on the front at 50cm of the tungsten lamp. Take 0, 0.5, 1, 1.5, 2, respectively. 3,4,6,8,10h are the time nodes to carry out the fluorescence spectrum test, and the fluorescence emission peak of each dye is selected and the time is plotted.
  • PBS Phosphate buffer, pH 7.4
  • BuAN-DAze fluorescence intensity can still maintain a higher intensity (97%) after 10 hours of illumination, while commercial green mitochondrial dyes, rhodamine 123, fluorescein, Bodipy and other fluorescence intensity are all Significantly reduced, which shows that BuAN-DAze light stability is extremely high.
  • Some dyes in this project are fluorescence confocal imaging and structured light illumination microscopic imaging in living cells (RWPE, HeLa, fat cells, etc.). Take more than 0.5 ⁇ L of the probe mother solution and dissolve it in 1 mL of cell culture solution, and then incubate the cells at 37° C. for 10-30 minutes for fluorescence imaging.
  • RWPE fluorescence confocal imaging and structured light illumination microscopic imaging in living cells
  • Fig. 12 The fluorescence confocal imaging of live cell RWPE cells of the dye Mito-DAze is shown in Fig. 12: accurate dye positioning, clear mitochondrial contours, and a good co-localization effect with the commercial dye MitoTracker Deep.
  • the mitochondrial dye Mito-DAze's RWPE cell live cell structure light illumination microscopic image is shown in Figure 13: RWPE cell mitochondria are clearly line-shaped, and can clearly see the mitochondrial ridge.
  • Mito-DAC imaging of live cell mitochondria is shown in Figure 14: the dye Mito-DAC can specifically label HeLa cell mitochondria and has a high signal-to-noise ratio.
  • the imaging diagram of OLD-DAze on live cell lipid droplets is shown in Figure 15:
  • the lipid droplet dye OLD-DAze can specifically mark lipid droplets in adipocytes, and it can monitor lipid droplet particles of different sizes.
  • the confocal imaging of Halo-DAze on the nucleus of living cells is shown in Figure 16.
  • the dye Halo-DAze can accurately locate the nucleus and specifically react with the histone protein fused with Halo-tag, and the nuclear contour is clear.
  • SNAP-DAze's confocal imaging of the nucleus in living cells is shown in Figure 17:
  • the dye SNAP-DAC can accurately locate the nucleus and react specifically with histones fused with SNAP-tag.
  • the nuclear contour is clear and the signal is noisy Relatively high.
  • SNAP-DAC was STED super-resolution fluorescence imaging in transfected pSNAP f -H2B HeLa cells. Take 0.5 ⁇ L of SNAP-DAC mother liquor dissolved in 1mL cell culture solution, incubate at 37°C, 5% CO 2 for 30 minutes, fix the cells with 4% formaldehyde solution and place in 1mL PBS buffer for STED super resolution Fluorescence imaging.
  • SNAP-DAC can specifically label the nucleus in HeLa cells. Due to the improvement of light stability, SNAP-DAC can perform multiple imaging and reconstruction under GW/cm 2 high-intensity laser to obtain higher resolution images.
  • Rho-4 and Nu-DAC experiments on structured light illumination microcolor imaging of RWPE cells Take more than 0.5 ⁇ L of the probe mother solution and dissolve it in 1 mL of cell culture solution at the same time, and then incubate the cells at 37°C for 10-30 minutes for fluorescence imaging.
  • Rho-4 and Nu-DAC's structured light illumination microcolor imaging of RWPE cells is shown in Figure 19: (a) Rho-4 channel imaging, which can specifically label the cell mitochondria; (b) It is a Nu-DAC channel imaging map, which can specifically stain the cell nucleus; (c) is an overlay of the above two. This shows that Rho-4 and Nu-DAC can be used simultaneously for multi-color fluorescence imaging of living cells.
  • OLD-DAze and Nu-DAC experiments on structured light illumination microcolor imaging of HT29 cells Take more than 0.5 ⁇ L of the probe mother solution and dissolve it in 1 mL of cell culture solution at the same time, and then incubate the cells at 37° C. for 10-30 minutes for fluorescence imaging.
  • OLD-DAze and Nu-DAC's structured light illumination microcolor imaging of HT29 cells is shown in Figure 20: (a) is the OLD-DAze channel imaging map, which can specifically mark cell lipid droplets; (b ) Is the Nu-DAC channel imaging map, which can specifically stain the cell nucleus. This shows that OLD-DAze and Nu-DAC can be used simultaneously for multi-color fluorescence imaging of living cells.

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Abstract

La présente invention concerne des colorants fluorescents à haute stabilité, à haute luminosité et à spectre complet, les colorants étant un ou plusieurs colorants suivants mélangés selon un quelconque rapport : un colorant de naphtalimide substitué par 4-amido, un colorant fluorescent de naphtalimide substitué par bisalcoxy, un colorant fluorescent de naphtalimide substitué par bisamino, un pérylène imide substitué par 9,10-bisamino, un colorant de rhodamine en anneau à six chaînons, un colorant de rhodamine en anneau à cinq chaînons, et un colorant de rhodamine à base de silicium. Par rapport aux colorants commerciaux actuels, les colorants fluorescents de la présente invention présentent une photostabilité supérieure et une largeur totale plus étroite à mi-hauteur (25 nm), et ne sont pas sensibles à divers environnements externes comprenant le pH, la polarité, et la température. Au moyen de l'introduction de groupes actifs tels que des groupes de clics, des étiquettes de protéines, et des molécules de médicament, les molécules fluorescentes fonctionnalisées obtenues présentent une biocompatibilité élevée, et peuvent rapidement et spécifiquement colorer des cellules et des corps vivants. En raison de la photostabilité et de la luminosité fluorescentes accrues, la présente série de colorants peut mettre en œuvre une imagerie par fluorescence à super-résolution dans des modes Tempête, STED et SIM.
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