CN111621289A - Light-operated double-channel fluorescent dye and preparation method and application thereof - Google Patents
Light-operated double-channel fluorescent dye and preparation method and application thereof Download PDFInfo
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- 238000000034 method Methods 0.000 claims description 14
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- 238000003756 stirring Methods 0.000 claims description 9
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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
- 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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
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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
- C09B57/08—Naphthalimide dyes; Phthalimide dyes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
The invention discloses a light-operated double-channel fluorescent dye and a preparation method and application thereof, belonging to the technical field of organic light functional materials, wherein the structural formula of the light-operated double-channel fluorescent dye is shown as a formula (I): the molecule of the new compound obtained by the invention has a 1, 8-naphthalimide skeleton capable of emitting green fluorescence and two spiropyran units capable of emitting red fluorescence after illumination, so that the new compound has obvious photochromic and light-regulated bicolor fluorescence properties, and the active ester group of the succinimide can be stably bonded with amino, thereby being used as a dual-channel co-positioned biomoleculeA fluorescent labeling dye;
Description
Technical Field
The invention belongs to the technical field of organic optical functional materials, and particularly relates to a light-operated double-channel fluorescent dye and a preparation method and application thereof.
Background
In recent years, the technology of fluorescence labeling of biological macromolecules has been developed rapidly, and after fluorescence labeling of biological molecules, sensitive and accurate fluorescence methods such as living cell imaging and detection of substances inside and outside cells and nucleic acids can be applied, so that the fluorescence labeling technology plays an important role in the fields of biological and medical research.
Fluorescent dye labeling protein or polypeptide technology is a common biomacromolecule in-vitro labeling technology, and since amino groups are very common in proteins and polypeptides, and N-hydroxysuccinimide ester (NHS ester) can react with the amino groups to form stable amido bonds, the NHS ester is commonly used for carrying out fluorescent labeling on biomacromolecules containing the amino groups.
Several commercial fluorescent dyes that are currently available for labeling antibodies or proteins have been developed in succession. The naphthalimide compound has strong fluorescence performance due to the existence of a rigid conjugated structure and good light and heat stability, the compound has high fluorescence quantum efficiency, most of the compounds can emit strong green or yellow fluorescence, namely, strong fluorescence emission is generated in the range of 500-550nm, but some organisms and tissues thereof can generate self fluorescence emission in the wavelength range under the excitation of visible light, which may affect the sensitivity and accuracy of the fluorescence detection of biological samples; the self-fluorescence intensity of the biological sample in a near-infrared light area is very small, which can reduce the background interference of fluorescence detection of the biological sample, and meanwhile, the near-infrared fluorescent dye also has the advantages of strong tissue penetration capability, low phototoxicity, small light scattering and the like, so the biological sample is favored; however, the existing near-infrared fluorescence labeling dye has low fluorescence quantum yield and poor photochemical stability, and cannot completely meet the use requirement, so that a new compound with fluorescence property still needs to be continuously researched.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides a light-operated dual-channel fluorescent dye and a preparation method and application thereof, the molecule of the novel compound provided by the invention has a 1, 8-naphthalimide skeleton capable of emitting green fluorescence and two spiropyran units capable of emitting red fluorescence after illumination, the fluorescence intensity of an open ring body of the novel compound is improved as a result of the synergistic ring opening of the two spiropyran units, so that the fluorescence resonance energy transfer efficiency of the novel compound is improved, the closed ring isomerization does not cause the fluorescence quenching after illumination is stopped, and the defects of low fluorescence quantum yield and poor photochemical stability of the general near-infrared fluorescence labeling dye are avoided; the N-hydroxysuccinimide ester introduced into the molecule can be used for stably bonding with amino groups in biomolecules, and the function of double-channel co-localization fluorescent labeling of the biomolecules can be realized by emitting green fluorescence before illumination, emitting red fluorescence after illumination and reducing the fluorescence intensity of a green light region.
The first purpose of the invention is to provide a light-operated double-channel fluorescent dye, which has a structural formula shown as a formula (I):
the second purpose of the invention is to provide a preparation method of the light-operated double-channel fluorescent dye, which comprises the following steps:
s1, preparing an intermediate 1 by taking absolute ethyl alcohol as a solvent and 4-bromo-1, 8-naphthalic anhydride and glycine tert-butyl ester as raw materials through condensation reaction under stirring and reflux temperature;
s2, dissolving the intermediate 1 prepared in the S1 in ethylene glycol monomethyl ether, adding diethanol amine, stirring, and carrying out nucleophilic substitution reaction at a reflux temperature to prepare an intermediate 2;
s3, mixing the intermediate 2 prepared in the S2, 1-carboxyethyl spiropyran, dicyclohexylcarbodiimide, 4-dimethylaminopyridine and dichloromethane, and preparing an intermediate 3 through an esterification reaction at room temperature under a dark condition;
s4, firstly carrying out deprotection reaction on the intermediate 3 prepared in the step S3, then mixing the intermediate 3 with N-hydroxysuccinimide, dicyclohexylcarbodiimide, 4-dimethylaminopyridine and dichloromethane, and carrying out esterification reaction at room temperature to prepare a compound shown in the formula (I), namely the light-operated dual-channel fluorescent dye (NISP-NHS for short);
the synthetic route is as follows:
preferably, in S1, the molar ratio of 4-bromo-1, 8-naphthalic anhydride to glycine tert-butyrate is 1: the use amount ratio of 1-1.5, 4-bromo-1, 8-naphthalic anhydride to absolute ethyl alcohol is 1 mol: 20-60L, and the reaction time is 3.5-4.5 h.
Preferably, in S2, the ratio of the intermediate 1 to the diethanolamine is 4 mol: 3-4L, wherein the volume ratio of ethylene glycol monomethyl ether to diethanolamine is 40-80: 3, the reaction time is 9.5-10.5 h.
Preferably, in S3, intermediate 2: 1-carboxyethyl spiropyran: the molar ratio of dicyclohexylcarbodiimide is 1: 2-2.2: 2 to 2.4; intermediate 2: the using ratio of dichloromethane is 1 mol: 50-75L; intermediate 2: the molar ratio of the 4-dimethylaminopyridine is 20: 1-2; the reaction time is 11.5-12.5 h.
Preferably, in S4, the reagent for the deprotection reaction is a mixed solution of trifluoroacetic acid and dichloromethane in a volume ratio of 1:1.
Preferably, in S4, the deprotection reaction specifically comprises: the intermediate 3 obtained in S3 was added to a mixed solution of trifluoroacetic acid and dichloromethane, and after stirring at room temperature for 1 hour, the solvent was distilled off.
Preferably, in S4, intermediate 3: the using ratio of the mixed solution of trifluoroacetic acid and dichloromethane is 1 mol: 20L.
Preferably, in S4, intermediate 3: n-hydroxysuccinimide: the mol ratio of dicyclohexylcarbodiimide is 1: 1-1.2, and the content of the intermediate 3: the mass ratio of the 4-dimethylaminopyridine is 19: 1-2, and the reaction time is 11.5-12.5 h.
The third purpose of the invention is to provide the application of the light-operated double-channel fluorescent dye in the multi-generation fluorescent labeling of living biological cells.
Compared with the prior art, the invention has the following beneficial effects:
(1) the novel compound molecule provided by the invention has a 1, 8-naphthalimide skeleton capable of emitting green fluorescence and two spiropyran units capable of emitting red fluorescence after illumination, so that the novel compound molecule has obvious photochromic and light-regulated bicolor fluorescence properties, and the active ester group of succinimide can be stably bonded with amino, so that the novel compound molecule can be used as a fluorescent marking dye of a dual-channel co-located biomolecule, and the mechanism is as follows:
the existence of the double-spiropyran units increases the near-infrared fluorescence emission intensity of the compound, and after illumination, the two spiropyran units are cooperated to open the ring, so that the fluorescence intensity of the open ring body is obviously improved, the fluorescence resonance energy transfer efficiency of the open ring body is improved, and the defect of low fluorescence quantum yield of the existing near-infrared fluorescence labeling dye is overcome; the near-infrared fluorescence emitted by the spiropyran unit can be recovered to a non-fluorescence state after the ultraviolet irradiation is stopped, so that the possibility of fluorescence quenching can be effectively avoided, and the photochemical stability of the near-infrared region is improved; n-hydroxysuccinimide ester introduced into a compound molecule can be used for stably bonding with amino in biological molecules, and through the synergistic effect of the spiropyran and the 1, 8-naphthalimide, the compound emits green fluorescence before ultraviolet irradiation and red fluorescence after ultraviolet irradiation, and simultaneously the fluorescence intensity of a green light area is reduced, so that the function of double-channel co-localization fluorescence labeling of the compound on the biological molecules is realized, and the compound can be used for multi-generation fluorescence labeling of biological living cells;
(2) the novel compound provided by the invention is simple in preparation method and easy in raw material obtaining, and the prepared compound has excellent near-infrared fluorescence performance, is less interfered by the fluorescence background of biomolecules (particularly biomolecules in a living cell system) when being used as a biomolecule marker, is suitable for multi-generation fluorescence labeling of living biological cells, and provides a possibility for the improvement of a biomacromolecule fluorescence labeling technology.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph showing the color change of NISP-NHS prepared in example 1 before and after irradiation with polyethylene glycol;
(a) before illumination; (b) the fluorescence changes after the light starts to be excited and the illumination lasts for 10 min; (c) color after illumination;
FIG. 2 is a graph showing the change in color and fluorescence of NISP-NHS prepared in example 1 before and after irradiation with light in polymethylmethacrylate; (a) before illumination; (b) the light begins to excite the fluorescent color; (c) fluorescent color after illumination for 6 min; (d) color after 6min after illumination;
FIG. 3 is an absorption spectrum of NISP-NHS prepared in example 1 in ethanol; a, before illumination; b-h line is the thermal decoloring process after illumination;
FIG. 4 is an absorption spectrum of NISP-NHS prepared in example 1 in polymethyl methacrylate; a, before illumination; line b-h is the thermal decoloring process after illumination.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the scope of the present invention. Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention. Unless otherwise specifically stated, the various starting materials, reagents, instruments and equipment used in the following examples of the present invention are either commercially available or prepared by conventional methods. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.
The invention provides a light-operated double-channel fluorescent dye, which has a structural formula shown as a formula (I):
the preparation method of the light-operated double-channel fluorescent dye comprises the following steps:
s1, preparing an intermediate 1 by taking absolute ethyl alcohol as a solvent and 4-bromo-1, 8-naphthalic anhydride and glycine tert-butyl ester as raw materials through condensation reaction under stirring and reflux temperature;
s2, dissolving the intermediate 1 prepared in the S1 in ethylene glycol monomethyl ether, adding diethanol amine, stirring, and carrying out nucleophilic substitution reaction at a reflux temperature to prepare an intermediate 2;
s3, mixing the intermediate 2 prepared in the S2, 1-carboxyethyl spiropyran, dicyclohexylcarbodiimide, 4-dimethylaminopyridine and dichloromethane, and preparing an intermediate 3 through an esterification reaction at room temperature under a dark condition;
s4, firstly carrying out deprotection reaction on the intermediate 3 prepared in the step S3, then mixing the intermediate 3 with N-hydroxysuccinimide, dicyclohexylcarbodiimide, 4-dimethylaminopyridine and dichloromethane, and carrying out esterification reaction at room temperature to prepare a compound shown in the formula (I), namely NISP-NHS;
the synthetic route is as follows:
the above-mentioned production method is specifically described below by way of the following examples.
Example 1
A preparation method of a light-operated double-channel fluorescent dye comprises the following steps:
(1) synthesis of intermediate 1
Equal amounts of 4-bromo-1, 8-naphthalic anhydride and glycine tert-butyl ester were stirred at reflux in absolute ethanol for 4 hours, the ratio of the amounts of 4-bromo-1, 8-naphthalic anhydride and absolute ethanol being 1 mmol: 30mL of the intermediate, cooling to room temperature, pouring the cooled intermediate into deionized water, adding ethyl acetate for extraction, drying an organic layer by using anhydrous magnesium sulfate, filtering out a drying agent, adding activated carbon for decolorization, evaporating most of the solvent, cooling and standing, and filtering to obtain a light yellow solid, namely the intermediate 1.
1H NMR[CDC13,300MHz]＝1.49(s,9H),4.83(s,2H)，7.85(t,1H)，7.04(d,1H),8.42(d,1H),8.58(d,1H),8.66(d,1H).
(2) Synthesis of intermediate 2
Dissolving 1.65g (4mmol) of the intermediate 1 in 50mL of ethylene glycol monomethyl ether, adding 3mL of diethanolamine, stirring, heating, refluxing, reacting for 10 hours, cooling, filtering, and performing column chromatography separation to obtain an orange solid, namely the intermediate 2.
1H NMR＝1.42(s,9H),3.58(t,4H),3.65(t,4H),4.67(s,2H),4.71(t,2H),7.41(t,1H),7.75(q,1H),8.34(d,1H),8.44(dd,1H),8.73(dd,1H).
(3) Synthesis of intermediate 3
Adding 0.42g (1mmol) of the intermediate 2 and 0.76g (2mmol) of 1-carboxyethyl spiropyran into a three-necked bottle, adding 0.44g (2.2mmol) of dicyclohexylcarbodiimide and 0.06g of 4-dimethylaminopyridine, adding 50mL of dichloromethane, reacting at room temperature in a dark place for 12h, filtering, evaporating a solvent, and performing column chromatography separation to obtain a yellow solid, namely the intermediate 3.
1HNMR＝1.10(s,6H),1.25(s,6H),1.51(s,9H),2.40-2.46(m,2H),2.52-2.58(m,2H),3.37-3.42(m,2H),3.51-3.56(m,2H),3.60(t,4H),4.12-4.18(m,4H),4.83(s,2H),5.79(d,2H),6.50(d,2H),6.71(d,2H),6.88-6.90(m,4H),7.08(d,2H),7.16(t,2H),7.27(d,1H),7.61(t,1H),7.98-8.00(m,4H),8.37(d,1H),8.47(d,1H),8.58(d,1H).
Hrms (esi): calculated value C64H62N6O14[M+H]+1139.4397, Experimental value 1139.4396.
(4) Synthesis of target Compound NISP-NHS
0.57g (0.5mmol) of the intermediate 3 is added into 10mL (v/v ═ 1:1) of trifluoroacetic acid in dichloromethane, the mixture is stirred for 1h at normal temperature, the solvent is completely distilled off, 0.5mmol of N-hydroxysuccinimide, 0.5mmol of dicyclohexylcarbodiimide and a catalytic amount of 0.03g of 4-dimethylaminopyridine are added, 20mL of dichloromethane are added, the mixture is reacted for 12h at room temperature, the mixture is filtered, and the target product is separated by column chromatography to obtain a yellow-green color.
1HNMR＝1.09(s,6H),1.24(s,6H),2.38-2.44(m,2H),2.50-2.56(m,2H),2.84(s,4H),3.35-3.41(m,2H),3.49-3.53(m,2H),3.61(t,4H),4.13-4.16(m,4H),5.25(s,2H),5.77(d,2H),6.48(d,2H),6.70(d,2H),6.87-6.90(m,4H),7.08(d,2H),7.14(t,2H),7.27(d,1H),7.62(t,1H),7.97-7.99(m,4H),8.38(d,1H),8.49(d,1H),8.61(d,1H).
13CNMR:19.8,25.6,25.7,33.4,34.0,39.0,52.2,52.9,61.4,106.6,106.7,115.5,116.6,118.4,118.6,120.0,121.8,122.0,122.6,122.8,125.9,126.1,127.4,127.8,128.4,130.3,130.7,132.1,132.4,136.0,141.2,146.1,154.6,159.3,162.8,163.4,164.3,168.5,171.5.
HRMS(ESI)C64H57N7O16[M+H]+Calculated 1180.3935, experimental 1180.3926.
Example 2
A preparation method of a light-operated double-channel fluorescent dye comprises the following steps:
the procedure is as in example 1, except that:
in step (1), the molar ratio of 4-bromo-1, 8-naphthalic anhydride to glycine tert-butyl ester is 1: 1.5, 4-bromine-1, 8-naphthalic anhydride and absolute ethyl alcohol in a dosage ratio of 1 mol: 20L, the reaction time is 3.5 h;
in the step (2), the dosage ratio of the intermediate 1 to the diethanol amine is 1 mol: 1L, the volume ratio of ethylene glycol monomethyl ether to diethanolamine is 40: 3, the reaction time is 9.5 h;
in step (3), intermediate 2: 1-carboxyethyl spiropyran: dicyclohexylcarbodiimide: the using ratio of dichloromethane is 1 mmol: 2.2 mmol: 2.4 mmol: 75 mL; intermediate 2: the mol ratio of the 4-dimethylamino pyridine is 10:1, and the reaction time is 11.5 h;
in step (4), intermediate 3: n-hydroxysuccinimide: dicyclohexylcarbodiimide molar ratio 1:1.2:1.2, intermediate 3: the mass ratio of the 4-dimethylamino pyridine is 19:2, and the reaction time is 11.5 h.
Example 3
A preparation method of a light-operated double-channel fluorescent dye comprises the following steps:
the procedure is as in example 1, except that:
in step (1), the molar ratio of 4-bromo-1, 8-naphthalic anhydride to glycine tert-butyl ester is 1: 1.5, 4-bromine-1, 8-naphthalic anhydride and absolute ethyl alcohol in a dosage ratio of 1 mol: 60L, and the reaction time is 4.5 h; (ii) a
In the step (2), the dosage ratio of the intermediate 1 to the diethanol amine is 1 mol: 1L, the volume ratio of ethylene glycol monomethyl ether to diethanolamine is 80: 3, the reaction time is 10.5 h;
in step (3), intermediate 2: 1-carboxyethyl spiropyran: dicyclohexylcarbodiimide: the using ratio of dichloromethane is 1 mmol: 2.2 mmol: 2 mmol: 75 mL; intermediate 2: the mol ratio of the 4-dimethylamino pyridine is 10:1, and the reaction time is 12.5 h;
in step (4), intermediate 3: n-hydroxysuccinimide: dicyclohexylcarbodiimide molar ratio 1:1.2:1.2, intermediate 3: the mass ratio of the 4-dimethylamino pyridine is 19:2, and the reaction time is 12.5 h.
The fluorescent properties of the NISP-NHS prepared in examples 1-3 were determined using the following example 1 alone.
First, the compound NISP-NHS photochromism in organic solvents as well as in solid media.
Taking polyethylene glycol as an example, observing the color change of NISP-NHS in polyethylene glycol before and after illumination, and the result is shown in figure 1, wherein (a) is the color of the compound before illumination and is light yellow; (b) the fluorescence changes after light excitation and illumination for 10min, the compound emits green fluorescence when the light excitation is started, and emits red fluorescence after the illumination for 10min, and the color of the compound changes into light red after the illumination, so that the compound has obvious photochromic phenomenon in an organic solvent, the fluorescence color changes remarkably, and the research of double-channel co-localization fluorescence labeling on living cells in a solution can be realized.
In the case of polymethyl methacrylate, the color and fluorescence of NISP-NHS were observed before and after exposure to polymethyl methacrylate, and the results are shown in fig. 2, which shows that: the compound changes the color from yellow green to purple before and after the illumination in the polymer film, and simultaneously, the fluorescence also changes from green before the illumination to pink after the illumination, namely, the colors from (b) to (c) change.
Secondly, the absorption spectrum and fluorescence spectrum of the compound NISP-NHS in organic solvents and in solid media.
The absorption spectrum of NISP-NHS in ethanol, as shown in figure 3, is obtained from figure 3: before illumination (line a), the compound has no obvious absorption at more than 480nm, after illumination for 3min, the compound has obvious absorption at 556nm (line b) in a visible light area, after an ultraviolet light source is removed, the absorption peak value of the visible light area is gradually reduced along with the time lapse (line b-h), and the spiropyran open ring body can be restored to the original structure of the closed ring body, namely, the process is reversible.
The absorption spectrum of NISP-NHS in pmma is shown in fig. 4, from which fig. 4 it is derived: before the ultraviolet irradiation, the high polymer film doped with the compound has strong fluorescence emission at 485nm (line a), after the ultraviolet irradiation is carried out for 10min, the fluorescence value of the compound at 485nm is obviously reduced, a new fluorescence emission peak is arranged at 600-650nm (line b), after the ultraviolet irradiation is stopped, the peak value corresponding to green fluorescence is gradually recovered along with the time lapse, and the red fluorescence value is gradually reduced (line b-h), which indicates that the compound has the performance of the fluorescence resonance energy transfer type fluorescent molecular switch which can be adjusted by light.
In conclusion, the novel compound molecule provided by the invention has a 1, 8-naphthalimide skeleton capable of emitting green fluorescence and two spiropyran units capable of emitting red fluorescence after illumination, so that the novel compound molecule has obvious photochromic and light-regulation bicolor fluorescence properties, emits green fluorescence before illumination of ultraviolet light, emits red fluorescence after illumination of ultraviolet light, and simultaneously reduces the fluorescence intensity of a green light region, thereby realizing the function of carrying out double-channel co-positioning fluorescence labeling on biomolecules by the compound, and being used for multi-generation fluorescence labeling of biological living cells.
It should be noted that when the following claims refer to numerical ranges, it should be understood that both ends of each numerical range and any value between the two ends can be selected, and since the steps and methods used are the same as those of the embodiments, the preferred embodiments and effects thereof are described in the present invention for the sake of avoiding redundancy, but once the basic inventive concept is known, those skilled in the art may make other changes and modifications to the embodiments. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. A light-operated double-channel fluorescent dye is characterized in that the structural formula is shown as the formula (I):
2. the method for preparing the light-controlled dual-channel fluorescent dye according to claim 1, characterized by comprising the following steps:
s1, preparing an intermediate 1 by taking absolute ethyl alcohol as a solvent and 4-bromo-1, 8-naphthalic anhydride and glycine tert-butyl ester as raw materials through condensation reaction under stirring and reflux temperature;
s2, dissolving the intermediate 1 prepared in the S1 in ethylene glycol monomethyl ether, adding diethanol amine, stirring, and carrying out nucleophilic substitution reaction at a reflux temperature to prepare an intermediate 2;
s3, mixing the intermediate 2 prepared in the S2, 1-carboxyethyl spiropyran, dicyclohexylcarbodiimide, 4-dimethylaminopyridine and dichloromethane, and preparing an intermediate 3 through an esterification reaction at room temperature under a dark condition;
s4, firstly carrying out deprotection reaction on the intermediate 3 prepared in the step S3, then mixing the intermediate 3 with N-hydroxysuccinimide, dicyclohexylcarbodiimide, 4-dimethylaminopyridine and dichloromethane, and carrying out esterification reaction at room temperature to prepare a compound shown in the formula (I), namely the light-operated dual-channel fluorescent dye;
the synthetic route is as follows:
3. the method for preparing the light-operated dual-channel fluorescent dye according to claim 2, wherein in the S1, the molar ratio of the 4-bromo-1, 8-naphthalic anhydride to the glycine tert-butyl ester is 1: the use amount ratio of 1-1.5, 4-bromo-1, 8-naphthalic anhydride to absolute ethyl alcohol is 1 mol: 20-60L, and the reaction time is 3.5-4.5 h.
4. The method for preparing the light-controlled two-channel fluorescent dye according to claim 2, wherein in the step S2, the dosage ratio of the intermediate 1 to the diethanol amine is 4 mol: 3-4L, wherein the volume ratio of ethylene glycol monomethyl ether to diethanolamine is 40-80: 3, the reaction time is 9.5-10.5 h.
5. The method for preparing the light-controlled two-channel fluorescent dye according to claim 2, wherein in S3, the ratio of the intermediate 2: 1-carboxyethyl spiropyran: the molar ratio of dicyclohexylcarbodiimide is 1: 2-2.2: 2 to 2.4; intermediate 2: the using ratio of dichloromethane is 1 mol: 50-75L; intermediate 2: the molar ratio of the 4-dimethylaminopyridine is 20: 1-2; the reaction time is 11.5-12.5 h.
6. The method for preparing the optically controlled dual-channel fluorescent dye according to claim 2, wherein in S4, the reagent used in the deprotection reaction is a mixed solution of trifluoroacetic acid and dichloromethane in a volume ratio of 1:1.
7. The method for preparing the light-controlled dual-channel fluorescent dye according to claim 6, wherein in S4, the deprotection reaction comprises the following specific steps: the intermediate 3 obtained in S3 was added to a mixed solution of trifluoroacetic acid and dichloromethane, and after stirring at room temperature for 1 hour, the solvent was distilled off.
8. The method for preparing the light-controlled two-channel fluorescent dye according to claim 7, wherein in S4, the ratio of intermediate 3: the using ratio of the mixed solution of trifluoroacetic acid and dichloromethane is 1 mol: 20L.
9. The method for preparing the light-controlled two-channel fluorescent dye according to claim 2, wherein in S4, the ratio of the intermediate 3: n-hydroxysuccinimide: the mol ratio of dicyclohexylcarbodiimide is 1: 1-1.2, and the content of the intermediate 3: the mass ratio of the 4-dimethylaminopyridine is 19: 1-2, and the reaction time is 11.5-12.5 h.
10. The light-controlled dual-channel fluorescent dye as claimed in claim 1, which is used for multi-generation fluorescent labeling of living biological cells.
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