CN110804322B - Fluorescent dye based on 1, 2-diaza-benzoperylene as well as preparation method and application thereof - Google Patents
Fluorescent dye based on 1, 2-diaza-benzoperylene as well as preparation method and application thereof Download PDFInfo
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- CN110804322B CN110804322B CN201911133621.6A CN201911133621A CN110804322B CN 110804322 B CN110804322 B CN 110804322B CN 201911133621 A CN201911133621 A CN 201911133621A CN 110804322 B CN110804322 B CN 110804322B
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 30
- UFVJGSSZLJMZOI-UHFFFAOYSA-N 16,17-diazahexacyclo[11.9.1.114,18.02,7.09,23.022,24]tetracosa-1(23),2,4,6,8,10,12,14,16,18,20,22(24)-dodecaene Chemical group C1=CC(C=2C3=CC=CC=C3C=C3C=2C2=CC=C3)=C3C2=CN=NC3=C1 UFVJGSSZLJMZOI-UHFFFAOYSA-N 0.000 title claims abstract description 12
- 238000002360 preparation method Methods 0.000 title claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000003384 imaging method Methods 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims abstract description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 15
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 14
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 10
- 230000015572 biosynthetic process Effects 0.000 claims description 7
- 229940125782 compound 2 Drugs 0.000 claims description 7
- 238000003786 synthesis reaction Methods 0.000 claims description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- HFBMWMNUJJDEQZ-UHFFFAOYSA-N acryloyl chloride Chemical compound ClC(=O)C=C HFBMWMNUJJDEQZ-UHFFFAOYSA-N 0.000 claims description 5
- 229940125904 compound 1 Drugs 0.000 claims description 5
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 5
- 235000018417 cysteine Nutrition 0.000 claims description 5
- 239000003960 organic solvent Substances 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 4
- 238000004440 column chromatography Methods 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 3
- 238000004113 cell culture Methods 0.000 claims description 2
- 239000012046 mixed solvent Substances 0.000 claims description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 2
- 125000003647 acryloyl group Chemical group O=C([*])C([H])=C([H])[H] 0.000 abstract description 3
- 230000008033 biological extinction Effects 0.000 abstract description 3
- 238000002845 discoloration Methods 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 239000012528 membrane Substances 0.000 abstract description 2
- 239000002798 polar solvent Substances 0.000 abstract description 2
- 150000003384 small molecules Chemical class 0.000 abstract description 2
- 238000007614 solvation Methods 0.000 abstract description 2
- 238000013341 scale-up Methods 0.000 abstract 1
- 239000000975 dye Substances 0.000 description 7
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 2
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000006862 quantum yield reaction Methods 0.000 description 2
- 238000010898 silica gel chromatography Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- FNQJDLTXOVEEFB-UHFFFAOYSA-N 1,2,3-benzothiadiazole Chemical compound C1=CC=C2SN=NC2=C1 FNQJDLTXOVEEFB-UHFFFAOYSA-N 0.000 description 1
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 239000005964 Acibenzolar-S-methyl Substances 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 238000004847 absorption spectroscopy Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 125000005605 benzo group Chemical group 0.000 description 1
- 238000012984 biological imaging Methods 0.000 description 1
- 229960000956 coumarin Drugs 0.000 description 1
- 235000001671 coumarin Nutrition 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 125000000664 diazo group Chemical group [N-]=[N+]=[*] 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Substances OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 239000001022 rhodamine dye Substances 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 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
- C09B57/00—Other synthetic dyes of known constitution
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D237/00—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
- C07D237/26—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings condensed with carbocyclic rings or ring systems
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- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B57/00—Other synthetic dyes of known constitution
- C09B57/001—Pyrene dyes
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- 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
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- 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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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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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- 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
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- G01N2021/6417—Spectrofluorimetric devices
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- 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"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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Abstract
The invention discloses a fluorescent dye based on 1, 2-diaza-benzoperylene.an acryloyl group is introduced into a 1, 2-diaza-benzoperylene parent, and the structural formula of the acryloyl group is shown as a formula I. 1, 2-diaza-benzoperylene-based organic small-molecule fluorescent dye is easy to combineThe yield is high, and the method is suitable for scale-up production. The yield of fluorescence quantum in water is high and reaches 40.81%, the molar extinction coefficient is large, the biological membrane has good penetrability and biocompatibility, is not easy to be subjected to solvation and discoloration in a polar solvent, has good stability, can be used for cell imaging, and has wide application prospect in a biological system.
Description
[ technical field ] A method for producing a semiconductor device
The invention belongs to the field of synthesis of novel fluorescent dyes, and particularly relates to a fluorescent dye based on 1, 2-diaza-benzoperylene as well as a preparation method and application thereof.
[ background of the invention ]
In recent years, small-molecule organic fluorescent materials are widely used in the fields of luminescent materials, solar sensitized cells, biological imaging and the like. Common dye mother nucleus structures are generally classified into coumarin dyes, cyanine dyes, BODIPY dyes, rhodamine dyes, benzothiadiazole dyes and the like, but each of the common dye mother nucleus structures has respective disadvantages such as: short absorption emission wavelength, poor light stability, small Stokes shift and the like. The design of near-infrared fluorescent dyes with long absorption wavelength, low toxicity and stable optical properties has attracted the interest of researchers.
The 1, 2-diaza-benzoperylene derivative is a novel fluorescent dye. The combination of the benzo skeleton and the diazo group obviously changes the original electronic structure and photophysical properties of the compound, so that the compound has longer emission wavelength and large Stokes shift. Unfortunately, most fluorescent dyes have the defects of obviously reduced molar extinction coefficient, broadened wavelength, obvious fluorescence quenching, poor stability and the like in water, which seriously limits the performance of the fluorescent dyes in biological applications.
[ summary of the invention ]
Aiming at the technical analysis and problems, the invention provides an organic fluorescent dye with high quantum yield in water and good biocompatibility, and a preparation method and application thereof.
The technical scheme of the invention is as follows:
the invention takes 1, 2-diaza-benzoperylene as a matrix, acryloyl groups are introduced into the matrix, and a fluorescent dye based on 1, 2-diaza-benzoperylene is designed and synthesized, and the structural formula of the fluorescent dye is shown as a formula I:
the method comprises the following specific steps:
(1) dissolving the compound 1 and triethylamine in dichloromethane, dropwise adding acryloyl chloride under an ice bath condition, reacting at room temperature for 3-6 hours after dropwise adding, extracting by using dichloromethane, and purifying by column chromatography to obtain a compound 2;
(2) dissolving the compound 2 and cysteine in an organic solvent, stirring for 3-5 hours at room temperature, and purifying by column chromatography to obtain a compound I.
In the synthesis step (1), the synthesis method is characterized in that: the molar ratio of the compound 1, triethylamine and acryloyl chloride is 1mol: 4-8 mol.
In the synthesis step (2), the synthesis method is characterized in that: the organic solvent is ethanol, dimethyl sulfoxide or N, N-dimethylformamide, and the molar ratio of the compound 2 to cysteine is 1mol: 2-8 mol.
The application of fluorescent dye based on 1, 2-diaza-benzoperylene is characterized in that: for imaging cells, the fluorescent dye can be dissolved in an organic solvent such as tetrahydrofuran, ethanol, acetonitrile, dimethyl sulfoxide, N-dimethylformamide or a mixed solvent of water and the above solvent in any proportion, and the solution in which the fluorescent dye is dissolved can be used for cell culture.
The 1, 2-diaza-benzoperylene-based organic micromolecule fluorescent dye provided by the invention is easy to synthesize and high in yield. The yield of fluorescence quantum in water is high and reaches 40.81%, the molar extinction coefficient is large, the biological membrane has good penetrability and biocompatibility, is not easy to be subjected to solvation and discoloration in a polar solvent, has good stability, can be used for cell imaging, and has wide application prospect in a biological system.
[ description of the drawings ]
FIG. 1 is the NMR spectrum of intermediate 2.
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of the fluorescent dye of the present invention.
FIG. 3 shows fluorescence emission spectra of the fluorescent dye of the present invention in water.
FIG. 4 shows the UV absorption spectrum of the fluorescent dye of the present invention in water.
FIG. 5 shows the use of the fluorescent dye of the present invention for confocal fluorescence imaging in cells
[ detailed description ] embodiments
The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.
The experimental methods described in the following examples are conventional unless otherwise specified; the drug and the solvent can be purchased from commercial sources or synthesized in the literature.
Example 1
Synthesis of intermediate 2
Compound 1(0.10g,0.16mmol) and triethylamine (0.13g,0.64mmol) were added to 5mL of dichloromethane, and the mixed solution was stirred in an ice bath for 20 minutes. Acryloyl chloride (0.12g,0.64mmol) was slowly added dropwise to the mixed solution while stirring, and after 1 hour of reaction, the mixture was allowed to warm to room temperature for another 6 hours. The resulting reaction solution was extracted with dichloromethane (3X 5mL), washed with water (3X 15mL), and dried over anhydrous sodium sulfate. The solvent was removed on a rotary evaporator and the crude product was purified by silica gel column Chromatography (CH)2Cl2MeOH 50:1) yielded 0.126g of red solid (83% yield). 1H NMR (500MHz, CDCl)3)δ9.40(d,J＝9.0Hz,1H),8.01(d,J＝3.7Hz,1H),7.81(d,J＝9.1Hz,1H),7.77(d,J＝8.8Hz,1H),7.68-7.65(m,2H),7.33(d,J＝2.3Hz,1H),7.31(d,J＝2.3Hz,1H),7.26-7.23(m,1H),6.59(dd,J＝7.7,1.3Hz,1H),6.56(dd,J＝7.8,1.3Hz,1H),6.43-6.36(m,2H),6.05-6.01(m,2H),5.88(d,J＝3.7Hz,1H).13C NMR(500MHz,DMSO)δ164.27,160.53,147.24,146.78,134.35,131.08,130.30,130.05,129.85,129.16,128.62,128.30,128.05,127.95,123.75,119.25,114.30,112.53,111.28,110.96,95.74.
Synthesis of fluorescent dye I
Compound 2(47mg,0.10mmol) and cysteine were added to ethanol, and the mixed solution was reacted at room temperature for 6 hours. The resulting reaction solution was subjected to solvent removal on a rotary evaporator, and the crude product was purified by silica gel column chromatography to obtain 36mg of a red solid (yield 98%).1H NMR(500MHz,DMSO)δ9.04(d,J＝9.0Hz,1H),8.88(d,J＝3.5Hz,1H),7.76(d,J＝8.8Hz,1H),7.70(d,J＝9.0Hz,1H),7.58(m,3H),7.00(dd,J＝16.0,8.6Hz,2H),5.93(d,J＝3.5Hz,1H)。
Example 2
Spectral properties of fluorescent dyes
All tests were carried out in PBS buffer (10mM, pH 7.4, 40% ethanol) with a fluorescent dye concentration of 10-5And M, performing fluorescence emission spectroscopy and ultraviolet absorption spectroscopy. From FIG. 3, it was found that a significant fluorescence emission peak occurred at 540 nm. The maximum absorption peak is found to occur at 320nm in FIG. 4. The fluorescence quantum yield was determined to be 40.81%.
Example 3
Confocal fluorescence imaging in cells
The cells used in the experiment were HeLa cells, first 5% CO at 37 ℃2Incubate in a wet box for 24 hours. The medium was then replaced with fresh medium, and the cells were incubated with fluorescent dye (5 μ M) containing medium for 30 minutes at 37 ℃ and washed 3 times with PBS to wash off excess dye, and placed in a confocal microscope for fluorescence imaging. For control experiments, cells were washed three times with PBS. The cell morphology is intact as can be seen from the bright field in FIG. 5, which proves that the cell can survive under the experimental condition, and the contrast shows that obvious fluorescent signal appears in the TIA cell added with the probeNumber (n).
Claims (5)
1. A 1, 2-diaza-benzoperylene-based fluorescent dye of the formula:
2. a method for preparing the fluorescent dye of claim 1, wherein the synthetic route is as follows:
the method comprises the following specific steps: (1) dissolving the compound 1 and triethylamine in dichloromethane, dropwise adding acryloyl chloride under an ice bath condition, reacting at room temperature for 3-6 hours after dropwise adding, extracting by using dichloromethane, and purifying by column chromatography to obtain a compound 2;
(2) dissolving the compound 2 and cysteine in an organic solvent, stirring for 3-5 hours at room temperature, and purifying by column chromatography to obtain a compound I.
3. The preparation method according to claim 2, wherein the molar ratio of the compound 1, triethylamine and acryloyl chloride in the synthesis step (1) is 1mol: 4-8 mol.
4. The preparation method according to claim 2, wherein the organic solvent in the synthesis step (2) is ethanol, dimethyl sulfoxide or N, N-dimethylformamide, and the molar ratio of the compound 2 to cysteine is 1mol: 2-8 mol.
5. Use of a fluorescent dye according to claim 1, characterized in that: for imaging cells, the fluorescent dye can be dissolved in tetrahydrofuran, ethanol, acetonitrile, dimethyl sulfoxide, N, N-dimethylformamide or a mixed solvent of water and the above solvent in any proportion, and then the solution in which the fluorescent dye is dissolved is used for cell culture.
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