CN116478553A - Red fluorescent dye based on pyran quinoline structure - Google Patents
Red fluorescent dye based on pyran quinoline structure Download PDFInfo
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 29
- PVDZKJZSOKXQRY-UHFFFAOYSA-N 2h-pyran;quinoline Chemical group C1OC=CC=C1.N1=CC=CC2=CC=CC=C21 PVDZKJZSOKXQRY-UHFFFAOYSA-N 0.000 title claims description 10
- 239000000975 dye Substances 0.000 abstract description 17
- 238000003384 imaging method Methods 0.000 abstract description 6
- HGFMFKSDOFFKRV-UHFFFAOYSA-N 8h-pyrano[2,3-h]quinoline Chemical group C1=CC=NC2=C(C=CCO3)C3=CC=C21 HGFMFKSDOFFKRV-UHFFFAOYSA-N 0.000 abstract description 4
- 238000005415 bioluminescence Methods 0.000 abstract description 3
- 230000029918 bioluminescence Effects 0.000 abstract description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 18
- 239000002904 solvent Substances 0.000 description 14
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 10
- 241000252212 Danio rerio Species 0.000 description 7
- 238000010992 reflux Methods 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000004440 column chromatography Methods 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000002189 fluorescence spectrum Methods 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 2
- SMWDFEZZVXVKRB-UHFFFAOYSA-N Quinoline Chemical compound N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- NUJOXMJBOLGQSY-UHFFFAOYSA-N manganese dioxide Chemical compound O=[Mn]=O NUJOXMJBOLGQSY-UHFFFAOYSA-N 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 125000004309 pyranyl group Chemical group O1C(C=CC=C1)* 0.000 description 2
- 238000006862 quantum yield reaction Methods 0.000 description 2
- 125000002943 quinolinyl group Chemical group N1=C(C=CC2=CC=CC=C12)* 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- SHAHPWSYJFYMRX-GDLCADMTSA-N (2S)-2-(4-{[(1R,2S)-2-hydroxycyclopentyl]methyl}phenyl)propanoic acid Chemical compound C1=CC([C@@H](C(O)=O)C)=CC=C1C[C@@H]1[C@@H](O)CCC1 SHAHPWSYJFYMRX-GDLCADMTSA-N 0.000 description 1
- AXKGIPZJYUNAIW-UHFFFAOYSA-N (4-aminophenyl)methanol Chemical compound NC1=CC=C(CO)C=C1 AXKGIPZJYUNAIW-UHFFFAOYSA-N 0.000 description 1
- XXFUZSHTIOFGNV-UHFFFAOYSA-N 1-bromoprop-1-yne Chemical compound CC#CBr XXFUZSHTIOFGNV-UHFFFAOYSA-N 0.000 description 1
- GBJXKRIRSDZFGG-UHFFFAOYSA-N 2-(2-methylchromen-4-ylidene)propanedinitrile Chemical compound C1=CC=C2OC(C)=CC(=C(C#N)C#N)C2=C1 GBJXKRIRSDZFGG-UHFFFAOYSA-N 0.000 description 1
- MGADZUXDNSDTHW-UHFFFAOYSA-N 2H-pyran Chemical compound C1OC=CC=C1 MGADZUXDNSDTHW-UHFFFAOYSA-N 0.000 description 1
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 description 1
- XDCOYBQVEVSNNB-UHFFFAOYSA-N 4-[(7-naphthalen-2-yl-1-benzothiophen-2-yl)methylamino]butanoic acid Chemical compound OC(=O)CCCNCc1cc2cccc(-c3ccc4ccccc4c3)c2s1 XDCOYBQVEVSNNB-UHFFFAOYSA-N 0.000 description 1
- 229910021595 Copper(I) iodide Inorganic materials 0.000 description 1
- 238000005698 Diels-Alder reaction Methods 0.000 description 1
- TZYWCYJVHRLUCT-VABKMULXSA-N N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucinal Chemical compound CC(C)C[C@@H](C=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)OCC1=CC=CC=C1 TZYWCYJVHRLUCT-VABKMULXSA-N 0.000 description 1
- TZCCKCLHNUSAMQ-DUGSHLAESA-N NC(=O)C[C@H](NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](Cc2ccc(F)cc2)NC(=O)[C@H](Cc3c[nH]c4ccccc34)NC(=O)Cc5cccs5)C(=O)N Chemical compound NC(=O)C[C@H](NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](Cc2ccc(F)cc2)NC(=O)[C@H](Cc3c[nH]c4ccccc34)NC(=O)Cc5cccs5)C(=O)N TZCCKCLHNUSAMQ-DUGSHLAESA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 125000005605 benzo group Chemical group 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000012984 biological imaging Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000010351 charge transfer process Methods 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- LSXDOTMGLUJQCM-UHFFFAOYSA-M copper(i) iodide Chemical compound I[Cu] LSXDOTMGLUJQCM-UHFFFAOYSA-M 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 125000001664 diethylamino group Chemical group [H]C([H])([H])C([H])([H])N(*)C([H])([H])C([H])([H])[H] 0.000 description 1
- ZIUSEGSNTOUIPT-UHFFFAOYSA-N ethyl 2-cyanoacetate Chemical compound CCOC(=O)CC#N ZIUSEGSNTOUIPT-UHFFFAOYSA-N 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 150000002391 heterocyclic compounds Chemical class 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- DZFWNZJKBJOGFQ-UHFFFAOYSA-N julolidine Chemical group C1CCC2=CC=CC3=C2N1CCC3 DZFWNZJKBJOGFQ-UHFFFAOYSA-N 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 238000002428 photodynamic therapy Methods 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 239000002379 progesterone receptor modulator Substances 0.000 description 1
- 230000005588 protonation Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 125000000437 thiazol-2-yl group Chemical group [H]C1=C([H])N=C(*)S1 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 229910052724 xenon Inorganic materials 0.000 description 1
- FHNFHKCVQCLJFQ-UHFFFAOYSA-N xenon atom Chemical compound [Xe] FHNFHKCVQCLJFQ-UHFFFAOYSA-N 0.000 description 1
- 108700024526 zebrafish sox32 Proteins 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/0008—Methine or polymethine dyes, e.g. cyanine dyes substituted on the polymethine chain
- C09B23/005—Methine or polymethine dyes, e.g. cyanine dyes substituted on the polymethine chain the substituent being a COOH and/or a functional derivative thereof
- C09B23/0058—Methine or polymethine dyes, e.g. cyanine dyes substituted on the polymethine chain the substituent being a COOH and/or a functional derivative thereof the substituent being CN
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
- C09B23/04—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
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- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/10—The polymethine chain containing an even number of >CH- groups
- C09B23/105—The polymethine chain containing an even number of >CH- groups two >CH- groups
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
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- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
Abstract
The invention discloses a red fluorescent dye based on a pyranoquinoline structure, which belongs to the technical field of bioluminescence imaging, and has the following structure:
Description
Technical Field
The invention belongs to the technical field of bioluminescence imaging, and particularly relates to a red fluorescent dye based on a pyranoquinoline structure and application of the red fluorescent dye in bioluminescence imaging seeds.
Background
The fluorescent dye has wide application in the life science fields of medical diagnosis, photodynamic therapy, fluorescence sensing and the like, so that the development of the novel fluorescent dye with good performance has important significance. The red/near infrared fluorescent dye has the advantages of good penetrability, small background interference, small damage to tissues and the like, and is widely applied to the field of biological imaging; fluorescent dyes with large stokes shifts can reduce self-absorption and increase their sensitivity. . The fluorescent probe developed based on fluorescent dye has the advantages of good selectivity, high sensitivity, convenient operation, real-time nondestructive detection and the like, and becomes an important tool in the technical field of analysis and detection. Therefore, it is interesting to develop new fluorescent dyes with red/near infrared emission and large stokes shift.
Pyran quinoline derivatives are heterocyclic compounds that are hybridized to the pyran and quinoline rings, which can be synthesized by Diels-Alder reaction of the heterocyclic ring. The current research is mainly focused on the synthesis method and biological activity. It is often used as a progesterone receptor modulator, glucocorticoid modulator and transcriptional activity modulator. From structural analysis, the pyran quinoline parent has a larger conjugated structure. The quinoline ring is an electron-deficient part, the pyran ring is an electron-donating part, and the pyran ring has the characteristic of a donor-acceptor D-A fluorescent dye, and the pyran quinoline is a potential chromophore. In 2012, talukdar et al reported for the first time the photophysical properties (lambda max ＝377nm，λ em =422 nm) (D.Kand, A.M.Kalle, S.J.Varma and P.Talukdar, chem.Commun.,2012,48,2722-2724); in 2014, lin et al have red shifted the wavelength to λ by introducing diethylamino into the pyran quinoline matrix em 483nm, and found to develop under acidic conditions due to protonation of the quinoline ringPeak of lambda em 545nm (w.huang, w.lin and x.guan, tetrahedron lett.,2014,55,116-119); in 2017, we have set the problem of introducing julolidine groups into pyranoquinoline dyes such that the emission wavelength red shifts to the yellow region (λ em =560 nm) (X.Liu, Y.Su, H.Tian, L.Yang, H.Zhang, X.SongandJ.W.Foley, anal.Chem.,2017,89,7038-7045); in 2018, our subject group developed methylated pyranoquinoline fluorescent dyes that can be red shifted to red fluorescence (lambda) em ＝622nm)(X.Liu,Y.Li,X.Ren,Q.Yang,Y.Su,L.He andX.Song,Chem.Commun.,2018,54,1509-1512)。
Based on this, we believe that the emission wavelength of the pyran quinoline fluorescent dye can be further increased and its stokes shift increased by increasing the molecular conjugate plane and increasing the charge transfer process within the molecule. In the invention, the pyran quinoline dye of a large conjugated system is constructed through conjugated double bonds for the first time, so that the emission wavelength is red shifted to far-red light.
Disclosure of Invention
The invention aims to provide a red fluorescent dye based on a pyran quinoline structure.
The fluorescent dye has the following molecular structure:
the fluorescent dye is prepared by the following synthetic route:
(a) Bromopropyne, potassium carbonate, N-dimethylformamide, 25 ℃ for 10h; (b) P-aminobenzyl alcohol, cuprous iodide, N-dimethylformamide, 110 ℃ for 6h; (c) manganese dioxide, methylene chloride, 25 ℃ for 5h; (d) piperidine, acetonitrile, reflux, 6h.
The fluorescent dyes of the present invention have long emission wavelengths and stokes shifts. The fluorescent dye shows long emission wavelength in different solvents, and the emission wavelength reachesRed light region. Maximum absorption peak (lambda) in different solvents abs ) Maximum emission peak (lambda) abs ) Stokes shift (Δss), molar extinction coefficient (ε), quantum yield (Φ) f ) And fluorescence lifetime (τ) are shown in the following table.
The fluorescent dye has lower cytotoxicity. After the HeLa cells and the probes are incubated for 24 hours at 37 ℃, the survival rate within 15.0 mu M is more than 90 percent.
The fluorescent dye can be used for well staining HeLa cells. Cells were stained red for fluorescence by co-culture with HeLa cells with dye (5.0. Mu.M).
The fluorescent dye can be used for imaging zebra fish. After incubation with zebra fish by probe (5.0. Mu.M), zebra fish were stained with red fluorescence.
The probe has good light stability. At 20mW/cm 2 The light degradation rate is less than 5% when the xenon lamp is irradiated for 1 hour.
Drawings
Fig. 1 is a graph of normalized uv-vis absorption spectra of dye 5a in different solvents. The abscissa is wavelength and the ordinate is absorbance.
FIG. 2 is a normalized fluorescence spectrum of dye 5a in different solvents. The abscissa is wavelength and the ordinate is fluorescence intensity.
Fig. 3 is a normalized uv-vis absorption spectrum of fluorescent dye 5b in different solvents. The abscissa is wavelength and the ordinate is absorbance.
FIG. 4 is a normalized fluorescence spectrum of dye 5b in different solvents. The abscissa is wavelength and the ordinate is fluorescence intensity.
Fig. 5 is a normalized uv-vis absorption spectrum of dye 5c in different solvents. The abscissa is wavelength and the ordinate is absorbance.
FIG. 6 is a normalized fluorescence spectrum of dye 5c in different solvents. The abscissa is wavelength and the ordinate is fluorescence intensity.
Fig. 7 is a normalized uv-vis absorption spectrum of dye 5d in different solvents. The abscissa is wavelength and the ordinate is absorbance.
FIG. 8 is a normalized fluorescence spectrum of dye 5d in different solvents. The abscissa is wavelength and the ordinate is fluorescence intensity.
FIG. 9 is a series of photophysical properties of fluorescent dyes of the present invention. Lambda (lambda) abs/nm Is the maximum absorption wavelength lambda em Is the maximum emission wavelength, delta ss Is Stokes shift, epsilon/M -1 cm -1 Is the molar extinction coefficient, Φ is the fluorescence quantum yield, τ/ns is the fluorescence lifetime.
FIG. 10 is a toxicity test of dye 5c of the present invention on HeLa cells. The abscissa indicates probe concentration, and the ordinate indicates cell viability.
FIG. 11 is a fluorescence image of co-staining of dye 5c (2.0. Mu.M) with Hela cells according to the invention. Imaging was performed after incubation for 15min in the dark at 37 ℃.
FIG. 12 is a fluorescent image of zebra fish staining with fluorescent dye 5c (2.0. Mu.M) of the present invention. Imaging after incubation for 15min at 28 ℃.
Examples of the embodiments
Example 1: synthesis of Compound 5a
Compound 4 (10.0 mmol) and 2- (2-methyl-4H-chromen-4-ylidene) malononitrile (10.0 mmol) were dissolved in 10mL of acetonitrile, 10. Mu.L of piperidine was added, stirred under reflux for 6 hours, after the reaction was completed, the solvent was removed under vacuum, and purified by column chromatography to give 5a in 73% yield. HRMS (ESI) m/z calcd.for C 34 H 27 N 4 O 2 [M+H] + 523.2134；found 523.2136. 1 H NMR(400MHz,CDCl 3 )δ8.28(s,1H),8.02(s,1H),7.80(d,J＝8.9Hz,1H),7.73(d,J＝7.4Hz,2H),7.52(dd,J＝9.1,2.1Hz,2H),7.35(d,J＝9.1Hz,2H),7.16(d,J＝16.0Hz,1H),7.06(d,J＝16.1Hz,1H),6.87(s,1H),6.52(d,J＝10.1Hz,1H),6.23(d,J＝2.4Hz,1H),5.29(d,J＝9.3Hz,2H),3.43(q,J＝7.1Hz,4H),1.22(d,J＝7.1Hz,6H). 13 C NMR(100MHz,CDCl 3 )δ175.50,163.35,154.43,152.09,150.23,148.04,145.04,141.73,141.51,139.85,139.64,137.78,135.66,133.56,128.16,125.31,123.42,122.31,119.24,115.49,112.93,111.29,109.64,106.12,104.47,103.78,101.92,95.84,95.54,92.32,66.50,63.21,43.03,15.87。
Example 5: synthesis of Compound 5b
Compounds 4 (10 mmol) and 2- (benzo [ d)]Thiazol-2-yl) acetonitrile (10 mmol) was dissolved in 10mL acetonitrile, 10 μl piperidine was added, stirred under reflux for 6 hours, after the reaction was completed, the solvent was removed under vacuum, and purified by column chromatography to give 5c in 70% yield. HRMS (ESI) m/z calcd.for C 30 H 25 N 4 OS[M+H] + 489.1749；found 489.1742. 1 H NMR(400MHz,CDCl 3 )δ8.29(dt,J＝8.9,7.1Hz,4H),8.09(d,J＝8.0Hz,2H),7.92(d,J＝7.9Hz,1H),7.81(s,1H),7.58–7.50(m,1H),7.47–7.40(m,1H),6.51(dd,J＝9.0,2.4Hz,1H),6.21(d,J＝2.3Hz,1H),5.28(s,2H),3.41(q,J＝7.1Hz,4H),1.22(t,J＝7.1Hz,6H). 13 C NMR(100MHz,CDCl 3 )δ163.03,159.71,153.69,151.91,146.10,143.67,135.00,131.35,131.09,129.70,128.73,127.41,126.94,126.81,126.49,125.94,125.71,124.09,123.56,121.67,121.58,117.29,116.83,107.24,104.69,98.00,68.38,58.47,44.68,18.44,12.71。
Example 6: synthesis of Compound 5c
Compound 4 (10 mmol) and 2- (3-cyano-4, 5-trimethylfuran-2 (5H) -ylidene) malononitrile (10 mmol) were dissolved in 10mL of acetonitrile, 10. Mu.L of piperidine was added, stirred under reflux for 6 hours, after completion of the reaction, the solvent was removed under vacuum and purified by column chromatography to give 5c in 85% yield. HRMS (ESI) m/z calcd.for C 32 H 28 N 5 O 2 [M+H] + 514.2243；found 514.2252. 1 H NMR(400MHz,CDCl 3 )δ7.95(s,1H),7.91(s,1H),7.55(d,J＝9.1Hz,2H),6.88(s,1H),6.84(s,1H),6.42(dd,J＝9.1,2.1Hz,2H),6.26(s,1H),4.81(d,J＝2.4Hz,4H),3.49(d,J＝7.1Hz,6H),2.62(t,J＝2.3Hz,2H),1.27(s,6H). 13 C NMR(100MHz,CDCl 3 )δ193.61,180.90,176.54,175.04,167.16,160.21,157.32,153.32,152.72,143.16,131.58,131.50,129.44,127.63,113.05,112.45,112.43,112.25,111.66,108.74,108.68,106.91,99.14,96.60,95.02,93.11,58.48,56.41,45.43,27.03,18.44,12.72。
Example 7: synthesis of Compound 5d
Compound 4 (10 mmol) and ethyl 2-cyanoacetate (10 mmol) were dissolved in 10mL of acetonitrile, 10. Mu.L of piperidine was added, stirred under reflux for 6 hours, after the reaction was completed, the solvent was removed under vacuum and purified by column chromatography to give 5d in 81% yield. HRMS (ESI) m/z calcd.for C 26 H 26 N 3 O 3 [M+H] + 428.1974；found 428.1977. 1 H NMR(400MHz,CDCl 3 )δ8.32(d,J＝7.8Hz,2H),8.29–8.19(m,2H),8.05(d,J＝8.8Hz,1H),7.79(s,1H),6.51(dd,J＝9.0,2.3Hz,1H),6.22(d,J＝2.3Hz,1H),5.27(s,2H),4.40(q,J＝7.1Hz,2H),3.42(q,J＝7.1Hz,4H),1.42(t,J＝7.1Hz,3H),1.22(t,J＝7.1Hz,6H). 13 C NMR(100MHz,CDCl 3 )δ162.83,159.76,154.28,152.57,151.92,132.77,131.06,129.86,129.70,127.73,127.39,126.38,125.76,115.98,110.40,107.21,101.73,97.97,68.38,62.69,44.68,31.51,30.13,29.71,14.22,12.71。
Example 8: dye 5c staining of cells
The HeLa cells were incubated with PBS buffer containing dye 5c (5.0. Mu.M) for 15min, and after rinsing 3 times with PBS buffer, the cells were imaged by fluorescence with a confocal laser fluorescence microscope, and a strong red fluorescence signal was observed.
Example 9: dye 5c fluorescent staining of zebra fish
Zebra fish is cultured in E3 embryo culture medium at 28deg.C, wherein the culture medium comprises 15mM NaCl and 0.5mM MgSO 4 ,1mM CaCl 2 ,0.15mM KH 2 PO 4 ,0.05mM Na 2 HPO 4 ,0.7mM NaHCO 3 And 1% methylene blue. The pH of the medium was 7.5. Fluorescence imaging observations were performed after 3-day-old zebra fish were selected and incubated with dye 5c (5.0 μm) for 30 minutes. Zebra fish can be observed to be stained with red fluorescence.
Claims (1)
1. The red fluorescent dye based on the pyran quinoline structure is characterized by having the structural formula:
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| CN105504860A (en) * | 2014-10-20 | 2016-04-20 | 中南大学 | Synthesis and applications of pyranoquinoline fluorescent dye |
| CN109400616A (en) * | 2018-11-02 | 2019-03-01 | 广西师范大学 | A kind of Sulfur Dioxide-derivatives fluorescence probe and its preparation method and application |
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| CN105504860A (en) * | 2014-10-20 | 2016-04-20 | 中南大学 | Synthesis and applications of pyranoquinoline fluorescent dye |
| CN109400616A (en) * | 2018-11-02 | 2019-03-01 | 广西师范大学 | A kind of Sulfur Dioxide-derivatives fluorescence probe and its preparation method and application |
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