CN102061103A - Type I boron fluoride complex dipyrromethene dye, and preparation method and application thereof - Google Patents
Type I boron fluoride complex dipyrromethene dye, and preparation method and application thereof Download PDFInfo
- Publication number
- CN102061103A CN102061103A CN2009102625656A CN200910262565A CN102061103A CN 102061103 A CN102061103 A CN 102061103A CN 2009102625656 A CN2009102625656 A CN 2009102625656A CN 200910262565 A CN200910262565 A CN 200910262565A CN 102061103 A CN102061103 A CN 102061103A
- Authority
- CN
- China
- Prior art keywords
- pyrroles
- dyestuff
- methine
- fluorescence
- reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
The invention relates to a type I boron fluoride complex dipyrromethene fluorescent dye. The fluorescent dye is shown as a general formula I, wherein R1, R2, R3, R5, R6 and R7 are H or C1-C8 alkyl respectively; R8 is a radical of a structural formula X, Y, Z or P; M is H, Na, K, N (R9R10R11R12) or N-succinimido; and R9, R10, R11 and R12 are H, C1-C8 alkyl or C1-C8 alkyl with substituent group-OH, an ether bond or carboxyl. The dye is synthesized by directly reacting substituted pyrrole with anhydride, has a high photo-physical property, stability, cell penetration capability and intracellular dissolving capability and is particularly suitable for bioluminescence analysis and biological marking.
Description
Technical field
The present invention relates to can be used for the bioluminescence analysis field the novel boron fluoride complexing two pyrroles's methine fluorochromes of a class, and its production and application.
Background technology
Fluorescence analysis is as a kind of traditional analytical procedure, particularly in recent years along with the appearance of novel fluorescence analytical technology and application and obtained significant progress.These new technologies have advantages such as highly sensitive, that selectivity good, sampling amount is few, method is fast and convenient, have become and have carried out a kind of important tool of analyzing on trace and ultra-trace even the molecular level in the multiple research field.Fluorescence analysis method can provide and comprise multiple physical parameters such as excitation spectrum, emmission spectrum and three-dimensional spectrum and fluorescence intensity, fluorescence efficiency, fluorescence lifetime.These parameters have reflected the various characteristics of molecule, and the microtexture information of research object can be provided from different perspectives.
As everyone knows, biomacromolecules such as protein and DNA are carried out qualitative analysis and quantitative assay is the analysis content that often relates in biological chemistry and the life science, the method for fluorescent probe quantitative assay of setting up highly sensitive, low detectability is still significant; In addition, relate to modern fluorometric analysis detection technique at dna sequencing, protein labeling, new drug development, the composition measurement of cell inner tissue, biological medicine invariably for fields such as tracking and analysis and disease pathogenesis researchs.And fluorescence dye is as the precondition in the fluorescence analysis, and the quality of its performance will directly influence even determine sensitivity, reliability and the practical value of whole fluoroscopic examination.Therefore, exploitation excellent performance, the fluorescence dye that is fit to the bioluminescence analytical applications seem and are even more important.
In general, the fluorescence dye of suitable fluorometric analysis application will possess following main character usually:
One, the fluorescence quantum yield of dyestuff is higher, preferably should be greater than 0.5.The fluorescence quantum yield height will help to improve the sensitivity of detection, make limit of detection maintain the level of low concentration.The height of fluorescence quantum yield is one of whether good greatest factor of fluorescence dye of measurement.
Two, dyestuff should have long maximum absorption and emission wavelength, is preferably in visible region.Because there is a lot of organic conjugate small molecules in the organism, for example purine, pyrimidine, each seed amino acid, protein molecule etc., they have absorption and emmission spectrum at ultraviolet region, if the absorbing wavelength of fluorescence dye will reduce the sensitivity of fluorescent probe in ultraviolet or shorter wavelength zone; And the autofluorescence of some disturbing molecule can be covered the emmission spectrum of probe, makes the result of detection deviation occur easily; In addition, uv excitation light is radiated in the biological tissue and the scattered light that causes has also constituted very big obstacle to the detection sensitivity of fluorescence.
Three, fluorescence dye should have advantages of higher stability.No matter be under illumination, under the acid-base condition, still under comparatively high temps, the chemical property of dyestuff should be stablized: serious photoxidation phenomenon can not take place, can not have that bigger fluctuation takes place spectrum under the situation of less change at acid-base condition, should not be under the high slightly temperature certainly yet and just promptly decompose.
Four, dye molecule should have cell-penetrating ability and intracellular dissolving power preferably.Only in this way can design the fluorescent probe that is adapted at using in the cell.It is certain water-soluble that dye molecule should have certain oil soluble to have again, and oil soluble is for the ease of permeates cell membranes, and water-soluble is well to disperse in cell in order to help.Two kinds of character of this of dye molecule can be by introducing suitable wetting ability or the lipophilicity group is regulated.
Five, dye groups should not have or only have minimum murder by poisoning to organism.This only has an effect to specific target with regard to requiring fluorescent probe, and should not produce destruction owing to other active mass in the former thereby pair cell of self structure, otherwise has just lost the biologic applications value of fluorescent probe.And little as the toxic action of the certain pair cell of fluorophore of fluorescent probe important component part, the activity of the interference cell of trying not.
In fluorescence dye commonly used, pyrene class and coumarins fluorescence dye absorbing wavelength are shorter, are unfavorable for improving the sensitivity of detection in bioanalysis; Though cyanine dyes has long emission wavelength, its light stability is relatively poor, and fluorescence quantum yield is also lower.Therefore, exploitation has the novel fluorescence dyestuff of good fluorescence spectrum property, remains the key of fluorescence analysis development.
Boron fluoride complexing two pyrroles's methines (being called for short BODIPY) fluorochrome is nearly twenties years class novel fluorescent compound that just grow up and be subjected to and extensively pay attention to.Because the BODIPY fluorochrome has: the fluorescence quantum yield height, molar extinction coefficient is big, spectral quality is highly stable (not being vulnerable to the influence of solvent polarity and pH value), the fluorescence spectrum peak width of dyestuff is narrower and the light stability very physicochemical property of excellence such as better, and obtained extensive application at numerous areas such as biomolecules fluorescent mark, detection of nucleic acids, pathological analysis and cell imagings recent years.Have the given activity group and synthesize, as amino, carboxyl, new reactive derivatives such as hydroxyl and specific identification group are the bases that this fluorochrome is further used in fields such as biological chemistry and life sciences.
To being applied to the fluorescence dye in bioanalysis field, normally introducing the active function groups of band carboxyl, and, be directly used in and carry out analyzing and testing in biomolecules or the Living organism by after the activation of N-maloyl imines at the parent of fluorescence dye.
For example: have bibliographical information to introduce the aliphatic chain carboxylic acid in No. 2 positions, side of BODIPY dye matrix structure, synthetic route reached more than six steps, and productive rate has only 12%, and production cost is very high.[be published in Bioconjugate Chem, 2007 referring to people such as Ludmila A.Alexandrova and Marina K.Kukhanova; 18:886-93. article " New Triphosphate Conjugates Bearing Reporter Groups:Labelingof DNA Fragments for Microarray Analysis "].
In addition, people such as Niko by containing free carboxyl group the pyrroles and the substituted azole of another molecule asymmetric condensation takes place, finally generate the BODIPY fluorochrome that has pendant carboxylic group, but that this method raw material is difficult to is synthetic, and the entire reaction process is not easy to operate yet, and yield is also lower.Referring to Niko J.Meltola, Rina Wahlroos and Aleksi E.Soini. are published in J.Fluorescence.Vol.14, No.5, the article of 635-647.
Summary of the invention
In sum, demand adopts convenient-to-running novel method to come the BODIPY fluorochrome that has the carboxylic acid active group of synthesizing new in the industry, promote such dyestuff faster and better be applied to the bioluminescence analysis field.
The present invention uses substituted azole and different anhydride reactions, has synthesized to simple possible a series of novel BODIPY fluorochromes that have the carboxylic acid active group.Reactions steps is simple, and a few step reactions are finished for one pot, mild condition, and productive rate is higher.
The inventive method is different from traditional synthetic method of utilizing aldehydes or acyl chloride compound and this series dyes of substituted azole prepared in reaction, but adopt the direct and substituted azole reaction of acid anhydrides, and synthetic method is improved and optimized, thereby make productive rate be up to 25%.
The synthetic dyestuff has following general formula I:
In the formula:
R
1-R
3And R
5-R
7H or C respectively do for oneself
1-8Alkyl;
R
8Group for structural formula X, Y, Z or P;
M is H, Na, K, N (R
9R
10R
11R
12) or the N-succinimido
R wherein
9, R
10, R
11And R
12H, C respectively do for oneself
1-8The C of substituted radicals such as alkyl or band OH, ehter bond, carboxyl
1-8Alkyl.
The synthetic method of general formula I dyestuff of the present invention comprises the steps:
(1) substituted azole and organic dibasic acid acid anhydride generation condensation, in condensation course, bimolecular substituted azole and a part acid anhydrides carry out condensation, and wherein the substituted azole of a part is for having substituent R
1, R
2And R
3The pyrroles, the substituted azole of another molecule is for having substituent R
5, R
6And R
7The pyrroles, described organic dibasic acid acid anhydride is selected from: Succinic anhydried, Pyroglutaric acid, adipic anhydride or Tetra hydro Phthalic anhydride; The molar ratio of pyrroles and anhydride reaction raw material is 0.5-10: 1, and temperature of reaction is controlled at 15-120 ℃, and reaction obtains product two pyrroles's methine structural compounds;
Two pyrroles's methine structural compounds that (2) will obtain react with the boron trifluoride compound again, and add the organic amine neutralizing agent, form compound of Formula I after taking off hydrogen fluoride, purify then; Described boron trifluoride compound is selected from: the etherate of gaseous state boron trifluoride, boron trifluoride or other can discharge the compound of boron trifluoride under the normal temperature solution state; Temperature of reaction is-10 ℃-100 ℃; The mole add-on of boron trifluoride compound be two pyrroles's methine structural compounds molar weight 0.1-10 doubly.
Its reaction formula is as follows:
Synthetic of the present invention contains boron fluoride complexing two pyrroles's methine fluorochromes of carboxylic acid or carboxylic acid derivative, have good optical physics characteristic, comprising: higher fluorescence quantum yield and molar extinction coefficient, narrow bandwidth of an emission, good light stability, less etc. to solvent and environmental influence.Fluorescence quantum yield is between 0.6-0.8, and maximal ultraviolet attraction and fluorescent emission wavelength are respectively at 495 ± 10nm and 510 ± 10nm, and Stokes shift is between 10-20nm.And have good stability and cell-penetrating ability and intracellular dissolving power, organism is not almost poisoned.Be highly suitable for fluorometric analysis, especially bioluminescence analysis and biomarker.
When the M among fluorescence dye II, III or the IV is the hydrogen proton, because carboxylic acid group's existence makes such dyestuff at lower concentration (1 * 10
-5M) do not have the generation of autohemagglutination phenomenon the time, solution is clarified and is had bright green fluorescence, and dyestuff is scattered in the water medium fully.This phenomenon has been expanded the Application Areas of such dyestuff greatly.
R in fluorescence dye II, III or IV
1=R
3=R
5=R
7During=H, they have high light stability in water.
Description of drawings
Fig. 1 is the single crystal diffraction structure iron (for clarity sake, omission hydrogen atom mark and part of atoms are annotated and shown) of the dyestuff IVa of observed from different perspectives embodiment 3.
Fig. 2 is the maximal ultraviolet absorption spectrum of BODIPY dyestuff in methylene dichloride that contains carboxylic acid, the dyestuff IIIb of dyestuff IVa, embodiment 4 of dyestuff IIIa, embodiment 3 of dyestuff IIa, embodiment 2 of the embodiment of the invention 1 and the dyestuff IVb of embodiment 5 have been shown, the maximum absorption curve in methylene dichloride.X-coordinate is represented absorbing wavelength, and unit is a nanometer; Ordinate zou is represented relative absorption intensity (having done normalized); The dyestuff volumetric molar concentration is 1 * 10
-5M, sample concentration are 2 * 10
-6M.For showing difference, indicate with different curves.
Fig. 3 is the fluorescence emission spectrum of BODIPY dyestuff in methylene dichloride that contains carboxylic acid, has shown the fluorescent emission curve of dyestuff IIa, IIIa, IVa, IIIb and the IVb in the embodiment of the invention in methylene dichloride.X-coordinate is represented wavelength, and unit is a nanometer; Ordinate zou is represented relative intensity of fluorescence (having done normalized); The dyestuff volumetric molar concentration is 2 * 10
-6M, sample concentration are 1 * 10
-5M.For showing difference, indicate with different curves.
Fig. 4 is the light stability test comparison of the BODIPY dyestuff that contains carboxylic acid in acetonitrile that exemplifies out, is dyestuff IIa, IIIa, IVa, IIIb and IVb in the embodiment of the invention light stability test in the acetonitrile medium.The dyestuff volumetric molar concentration is 1 * 10
-5M, sample concentration are 1 * 10
-5M, X-coordinate is represented light application time, unit is hour; Ordinate zou is represented residual absorption per-cent, is 1 to calculate with initial absorption intensity; Arrow is represented the dyestuff trend that absorption intensity weakens along with the increase of light application time.
Fig. 5 is the BODIPY dyestuff that contains carboxylic acid that exemplifies out (contains 0.1% ethanol) in deionized water a light stability test comparison, the light stability test that (contains 0.1% ethanol) for dyestuff IIa of the present invention, IIIa, IVa, IIIb and IVb in deionized water medium.The dyestuff volumetric molar concentration is 1 * 10
-5M, sample concentration are 1 * 10
-5M, X-coordinate is represented light application time, unit is hour; Ordinate zou is represented residual absorption per-cent, is 1 to calculate with initial absorption intensity; Arrow is represented the dyestuff trend that absorption intensity weakens along with the increase of light application time.
Fig. 6 is dye molecule IIIa of the present invention after the activation of N-maloyl imines, is directly used in the vertical panel polyacrylamide gel isolated protein electrophoresis experiment figure of BSA mark.BSA and activation dyestuff IIIa-NHS (1 * 10 from left to right
-4M) molar concentration rate was followed successively by 1: 5, and 1: 10,1: 20,1: 40,1: 100; Electrophoresis time is 1.5 hours; Buffer system is the damping fluid (SDS 2.5%, sucrose 1%) of boric acid-borax of pH=8.7; Adopt the sky, Shanghai to take pictures by gel imaging system (Tanon GIS 2010).A top white point is the fluorescence dye electrophoretic migration band of BSA on the mark among the figure; The bottom is the reactive dyestuffs IIIa-NHS of the unmarked BSA of going up and the electrophoretic migration band of the dyestuff IIIa that inactivation goes bad.
Fig. 7 is illustrated in the interpretation of result graphic representation of activation dyestuff IIIa-NHS mark BSA under the different pH values.X-coordinate is represented different pH values among the figure; Ordinate zou represents that protein labeling finishes relative intensity of fluorescence (Int) size of rear electrophoresis band (can gel imaging system (Tanon GIS 1D) analysis software carried out data logging and calculate by the sky, Shanghai).
Relative intensity of fluorescence was with pH value changing trend diagram after Fig. 8 represented to activate dyestuff IIIa-NHS mark BSA.X-coordinate is represented different pH values among the figure; Ordinate zou represents that protein labeling finishes the product of the relative intensity of fluorescence (Int) of rear electrophoresis band (can gel imaging system (Tanon GIS 1D) analysis software carried out data logging and calculate by the sky, Shanghai) and area (Pix).
The experimental result picture of optimum response mol ratio when Fig. 9 represents dyestuff IIIb activation back generation IIIb-NHS mark BSA.This figure is the design sketch of dyestuff and BSA behind the gel electrophoresis separation marking, among the figure white fluorescent point be the activation dyestuff with the BSA covalent attachment after electrophoresis band, each electrophoresis band is represented different IIIb-NHS/BSA mark mol ratios, is followed successively by from left to right: 10: 1; 15: 1; 20: 1; 25: 1; 30: 1.
The investigation of when Figure 10 is activation dyestuff IVb activation back generation IVb-NHS mark BSA marker detection being limit is figure as a result.The white fluorescent point is the electrophoresis band after activation dyestuff and the BSA covalent attachment among the figure, and each electrophoresis band adopts different BSA consumptions.Its optimized flag condition is: the volumetric molar concentration of activation dyestuff is 0.8 * 10
-9During mol, when boric acid-borax buffer system pH is 9.0, when 40 ℃ of labeled reactant temperature, reaction times 40min, adopt sds polyacrylamide gel electrophoresis discontinuous (10% concentrated glue and 5% separation gel) to separate, when the input amount of BSA is respectively from left to right: 10ng; 30ng; When 50ng and 0.2ug, its fluorescently-labeled detectability can reach 10
-8G.
The single crystal structure synoptic diagram of the activated derivatives IIIa-NHS of Figure 11 dyestuff IIIa (, omitting the mark of the hydrogen atom in the crystalline structure) for conveniently watching.
Embodiment
In general formula I dyestuff of the present invention, R
1-R
3And R
5-R
7H or C respectively do for oneself
1-8Alkyl, be preferably H or C
1-6Alkyl, more preferably H or C
1-4Alkyl.Be preferably H again.
R
8Be the group of structural formula X, Y, Z or P, the group of preferred structure formula X, Y or Z.
M is H, Na, K, N (R
9R
10R
11R
12) or the N-succinimido, preferred H, Na, K or N (R
9R
10R
11R
12), more preferably H, Na or K.
R
9, R
10, R
11, R
12H, C respectively do for oneself
1-8The C of groups such as alkyl or band OH, ehter bond, carboxyl
1-8Alkyl.H or C more preferably respectively do for oneself
1-6Alkyl, H or C more preferably respectively do for oneself
1-4Alkyl.
In the dyestuff of above-mentioned general formula I, the dyestuff of preferred structure formula II, III or IV:
In formula II: R
1=R
3=R
5=R
7=H; In formula III and IV: R
1, R
3, R
5, R
7H or CH respectively do for oneself
3Identical in the implication of M and the general formula I wherein.
In the process of synthetic general formula I fluorescence dye, the present invention adopts the direct and substituted azole reaction of acid anhydrides, and by the ring-opening reaction of acid anhydrides, acid anhydrides self promptly participates in forming the parent formation of BODIPY fluorochrome, also generate the active group that has free carboxylic acid simultaneously, kill two birds with one stone; Generate the 8-position and contain the dyestuff of carboxylic acid functional.
The dyestuff that generates can be directly used in fluorometric analysis or biomolecular labeling.All right activated formation activated derivatives, for example NHS fat (as N-maloyl imines or N, N '-two succinimidyl carbonate activated derivatives) is used further to biochemical analysis.
Boron fluoride complexing two pyrroles's methine fluorochromes synthetic that contains hydroxy-acid group
The synthetic method that contains boron fluoride complexing two pyrroles's methine fluorochromes of carboxylic acid of the present invention is based on that such mechanism carries out: ring-opening reaction easily takes place in acid anhydrides under given conditions, and nitrogen ortho-hydrogens atom is active higher and condensation easily takes place on the pyrrole ring.
Synthetic 8-position contains boron fluoride complexing two pyrroles's methine fluorochromes of hydroxy-acid group and the entire reaction of activated derivatives comprises the steps:
The first step: two molecule pyrroles (substituent R
1-R
3And R
5-R
7Be H) or substituted azole (substituent R
1-R
3And R
5-R
7Be not H) and a part acid anhydrides generation condensation, wherein the mol ratio that feeds intake of reaction raw materials pyrroles or substituted azole and acid anhydrides can be 0.1-1000: 1, be preferably 0.5-100: 1; More preferably 0.5-10: 1, preferred again 1-5: 1, the best is 2: 1.Acid anhydrides is open loop after reaction, and free carboxyl group is provided.
Reaction can be carried out in organic solvent, absolute anhydrous organic solvent.Be preferably absolute anhydrous organic solvent.The volumetric molar concentration of pyrroles's raw material in solvent is 0.1-20%.Organic solvent is including, but not limited to methylene dichloride, tetrahydrofuran (THF) or acetonitrile etc.In reaction process, the active intermediate original position of generation is taken off a part water and is formed carbon-carbon double bond-methine key, obtains two pyrroles's methine structures.This structure still is an active intermediate, because unstable products can directly be carried out next step reaction without separating, reaction process can be passed through the terminal point that thin-layer chromatography (TLC) is judged reaction, just, after the new product dot generation, can carry out next step reaction when the disappearance of raw material point.
Because pyrroles's molecule itself is met very easily polymerization of acid, the volumetric molar concentration of raw material can not be too big when therefore reacting, and should be controlled at 0.1-20%, is preferably 1-15%, and the best is 4-8%.
Table 1 is the influence of the volumetric molar concentration of example explanation pyrroles when reacting to productive rate to generate series compound IIa.The structural formula of Compound I Ia is referring to embodiment 1.
Because pyrroles's molecule is easy to take place auto-polymerization when high temperature, generate thick brown material, thereby influence main reaction, reduces yield greatly.And when temperature of reaction is too low, then need long time to react fully.Table 2 has been listed the response situation of Compound I Ia under differing temps, and as can be seen, temperature of reaction should be controlled at 15 ℃-120 ℃, is preferably 30 ℃-100 ℃, more preferably 30 ℃-80 ℃, most preferably be 40 ℃-60 ℃, and yield is higher.
Primary condition and the yield of the synthetic IIa of table 1
The reaction yield of table 2 Compound I Ia under differing temps
Second step: the compound of two pyrroles's methine structures reacts with the boron trifluoride compound again, adds organic amine such as triethylamine and neutralizes, and forms target compound after taking off hydrogen fluoride.The boron trifluoride compound that adds can be etherate or other boron trifluoride compound of gaseous state boron trifluoride, boron trioxide, and other boron trifluoride compound refers to: any compound that can discharge boron trifluoride under the solution of normal temperature.Boron trifluoride compound add-on be two pyrroles's methine structural compounds mole number 0.1-10 doubly; Be preferably 0.5-3 doubly.
Temperature of reaction is-10 ℃-100 ℃, preferred 0-80 ℃, and more preferably 5-60 ℃, more preferably 10-40 ℃; Need add organic amine when adding boron trifluoride compound (for example complex compound of gaseous state boron trifluoride or boron trioxide) and promote the carrying out that react.The preferred triethylamine of organic amine.
This reaction process is the final process that generates of dyestuff, by the ultra violet lamp reaction mother liquor of 365nm, can see tangible green glow fluorescence, and the new product that can judge generation is a fluorescent substance; It is the essential attribute of this type of dyestuff that the intermediate complexing that boron trifluoride compound and the first step reaction obtain generates the material that has fluorescence.And the dyestuff that generates has carboxyl functional group, and it often is the shorter peak shape of broad in nucleus magnetic hydrogen spectrum, and appears at the bigger zone of chemical shift on the nuclear magnetic spectrogram.In addition, carry out structure elucidation by X-ray single crystal diffraction, can identify the fluorescence dye of generation the most intuitively, accompanying drawing 1 has provided the single crystal structure of dyestuff IVa.
The method of purification that contains boron fluoride complexing two pyrroles's methine fluorochromes of carboxylic acid and carboxylic acid derivative of the present invention adopts ordinary method, is not particularly limited.Usually, reaction back mother liquor through the extraction separatory, after the drying, is obtained target product through silica gel column chromatography separation and recrystallization.
Above-mentioned fluorescence dye of the present invention is activation further, forms active very high activated derivatives, is directly used in the covalent labeling of biomolecules.The preparation process of its activated derivatives is exemplified below.
With fluorescence dye (substituent R of the present invention
2=R
6=H), in anhydrous organic medium, with N-hydroxy-succinamide (being abbreviated as NHS) activator and carbonization imide class dehydration activator (as N, N '-dicyclohexylcarbodiimide is abbreviated as DCC) reaction; Or and N, N '-two succinimidyl carbonate (being abbreviated as DSC) activator, reaction under the effect of organic amine such as triethylamine (being abbreviated as TEA), original position forms the activation dyestuff (DYE-NHS as shown below) that contains the NHS ester.Temperature of reaction is 0-80 ℃, preferred 0-60 ℃, and more preferably 10-50 ℃; Containing the dyestuff of carboxylic acid and the mol ratio of activator is 1: 0.1-100 is preferably 0.5-10; The mol ratio of dyestuff and carbonization imide class dehydration activator is 1: 0.1-100 is preferably 0.5-10.The dyestuff activated derivatives that the activity that reaction in obtains is very high can be directly used in the covalent labeling of biomolecules without separation.
The BODIPY fluorescence dye that contains carboxylic acid of the present invention, the used reagent of its activation is NHS or DSC; Dewatering agent can be DCC, organic amine such as monoethylamine (being abbreviated as MEA), diethylamine (being abbreviated as DEA), triethylamine (being abbreviated as TEA) etc.; Adoptable solvent is methylene dichloride, tetrahydrofuran (THF), acetonitrile, DMF etc.
It is as shown below that the activated derivatives that obtains is used for the process of mark:
R in the following formula
2=R
6=H removes R
13Outward, consistent among the definition of other substituted radical and the general structure I.
The activated derivatives product that obtains can qualitatively judge by nuclear-magnetism, because the introducing of NHS ester group, have more a N-succimide structure on the dye structure, two methylene radical are arranged on this structure, they generally about 2.9ppm, generate the new activated derivatives that have NHS ester thereby can qualitatively judge in the chemical shift of the hydrogen on nuclear-magnetism spectrum.
Preparation method of the present invention is different from traditional synthetic method of utilizing aldehydes or acyl chloride compound and substituted azole prepared in reaction dyestuff, but adopt the direct and substituted azole reaction of acid anhydrides, and synthetic method is improved and optimized, thereby make productive rate be up to 25%.
The boron fluoride complexing two pyrroles's methine fluorochromes that contain carboxylic acid and derivative thereof of the present invention, the fluorescence quantum yield of its fluorescence dye is between 0.6-0.8, maximal ultraviolet absorption and fluorescent emission wavelength are respectively at 495 ± 10nm and 510 ± 10nm, and Stokes shift is between 10-20nm.
The boron fluoride complexing two pyrroles's methine fluorochromes that contain carboxylic acid and derivative thereof of the present invention are when the M among its fluorescence dye II, III or the IV is the hydrogen proton, because carboxylic acid group's existence makes such dyestuff at lower concentration (1 * 10
-5M) do not have the generation of autohemagglutination phenomenon the time, solution is clarified and is had bright green fluorescence, and dyestuff is scattered in the water medium fully.This phenomenon has been expanded the Application Areas of such dyestuff greatly.
The boron fluoride complexing two pyrroles's methine fluorochromes that contain carboxylic acid and derivative thereof of the present invention, the R in fluorescence dye II, III or IV
1=R
2During=H, they have quite high light stability in water.
The boron fluoride complexing two pyrroles's methine fluorochromes of carboxylic acid and derivative thereof that contain of the present invention are because its good good optical physics characteristic: higher fluorescence quantum yield and molar extinction coefficient, narrow bandwidth of an emission, good light stability, less etc. to solvent and environmental influence, can be used particularly well in the bioluminescence analysis field.
The spectrum change of dyestuff of the present invention in the solvent of opposed polarity is less, and the fluctuation (shown in accompanying drawing 2 and accompanying drawing 3) about 15nm of absorption and emmission spectrum is especially smaller to the influence of absorption spectrum.These five dyestuff Stokes shifts (Stokes Shift) are little relatively, are no more than 20nm.Its fluorescence quantum yield is all higher, and all greater than 0.6, the BODIPY dye fluorescence quantum yield that has methyl reaches more than 0.8, and its molar extinction coefficient is also more than 80,000; And for not methylic BODIPY dyestuff, because the dye matrix cloud density is little, its fluorescence quantum yield and molar extinction coefficient are all lower.
The light stability that contains carboxyl BODIPY dyestuff that the present invention provides is good.In the acetonitrile medium, the light stability of BODIPY dyestuff that contains fat carboxyl is better, and the light stability of BODIPY dyestuff that contains phenyl ring is relatively poor, this mainly is because phenyl ring is subjected to the steric restriction effect (as shown in Figure 1) of the parent of BODIPY dyestuff, cause dyestuff when being stimulated, the efficient of the internal conversion of speeding to produce by intramolecules vibration Henan reduces, so fluorescence quantum yield improves the therefore photic decomposition of also easier generation (as shown in Figure 4); And have the BODIPY dyestuff of methyl, because electron density increases on the dye matrix, this effect is strengthened, so the relatively also easier photoxidation of dyestuff is rotten.Need to prove that this phenomenon is more obvious when testing in deionized water, its reason contains more singlet oxygen in the water probably, in being subjected to the optical excitation process, and the easier generation photodegradation of dyestuff (as shown in Figure 5).
Boron fluoride complexing two pyrroles's methine fluorochromes of the present invention can also be used for the probe biomolecule of complex sign protein or DNA.Biomolecules behind the mark can be inquired into physiological phenomenon and interaction mechanism with the research life entity by analytical procedures such as electrophoretic analysis, capillary electrophoresis analysis, liquid-phase chromatographic analysis, flow cytometry, fluorescent microscope analysis, fluorometric analysis, fluoroimmunoassays on the cell yardstick.
After the BODIPY fluorescence dye that contains carboxylic acid of the present invention is activated, be directly used in proteinic mark, can be applicable to fluoroimmunoassay, protein labeling, dna marker, polypeptide marker, antigen or antibody labeling, anti-antibody mark etc.The mol ratio of the NHS ester of protein and dyestuff is 1: 0.1-100, best 1: 1-50; The NHS ester of protein and dyestuff can carry out in the environment of pH 4-11, is preferably pH 7-10, available pH buffered soln control pH, and temperature of reaction is preferably in 25-60 ℃ at 10-80 ℃; The time of reaction is 1-100 minute, preferred 10-60 minute, is preferably 20-50 minute.
Protein behind the mark can be used for fluorescently-labeled applied analysis fields such as electrophoretic analysis, capillary electrophoresis analysis, liquid-phase chromatographic analysis, flow cytometry, fluorescent microscope analysis, fluorometric analysis, fluoroimmunoassay.
With labelled protein BSA is example, utilizes the concrete steps of optimum mark mol ratio of electrophoresis detection reactive dyestuffs DYE-NHS and BSA mark as follows:
The first step: the polyacrylamide separation gel of preparation 10% and 5% concentrated glue, make the sample hole, after solidifying, place stand-by.
Second step: BSA sex change experiment.Add the damping fluid (SDS 2.5%, sucrose 1%) of boric acid-borax of the pH=8.7 of 2ul in 5ug (1ul) BSA, the 1ul deionized water boils 5min.
The 3rd step: add the DYE-NHS tetrahydrofuran solution of 1ul among the BSA after sex change, the mol ratio of preparing BSA and dyestuff respectively is: BSA/DYE-NHS=1: 5,1: 10,1: 20,1: 40,1: 100.30 ℃ of following lucifuge reaction 30-40min.
The 4th step: negate should be gone up sample for the back mixed solution successively, fix electrophoresis apparatus after, connect power supply, under 100 volts of voltages, when dyestuff to be activated moves to bottom the gel 1-1.5cm, cut off the electricity supply.
The 5th step: unload gel film, take pictures after the handled (as shown in Figure 6), and do data logging and analysis.
By above-mentioned experiment, we find out more excellent protein labeling condition and are: BSA is 1: 20 with the mol ratio of activation dyestuff DYE-NHS.
In orthogonal experiment subsequently, when changing different pH of buffer (8.0; 8.4; 8.7; 9.0; 9.3; 9.6) time, final experimental result shows when pH is 9.0, the electrophoresis of resulting protein labeling swimming band strength the highest (as shown in Figure 7), and the product value maximum (as shown in Figure 8) of intensity that is calculated and area.
Proof after the BODIPY fluorescence dye that contains carboxylic acid of this invention is activated, has broad application prospects at biochemical analysis and life science by experiment.For example under optimal conditions: the volumetric molar concentration of activation dyestuff is 4 * 10
-9During mol, when boric acid-borax buffer system pH is 9.0, when 27 ℃ of labeled reactant temperature, reaction times 40min, adopt sds polyacrylamide gel electrophoresis discontinuous (10% concentrated glue and 5% separation gel) to separate, when the input amount of BSA is respectively: 5ng; 10ng; 30ng; When 50ng and 0.1ug, its fluorescently-labeled detectability can reach 10
-8G.
The salt (metal cation salt and ammonium salt) of boron fluoride complexing two pyrroles's methine fluorochromes of the present invention is after synthesizing the corresponding carboxylic acid dyestuff, reacts resulting salt with mineral alkali (as NaOH, KOH) or organic amine again.The stable performance of this salt structure can be used for biochemical marker, or after adding the activating reagent activation, is used for biochemical marker.
Embodiment
Compound I Ia's is synthetic:
In exsiccant 250ml single necked round bottom flask, add 204mg (1.8mmol) Pyroglutaric acid successively, 201mg (3mmol) pyrroles, under nitrogen protection, inject the tetrahydrofuran (THF) 75ml of absolute anhydrous new steaming then with syringe, control reaction temperature is 55 ℃, magnetic force stirs fast and spends the night reaction solution brown of light color; After being cooled to normal temperature and steaming the solvent of half, under ice bath, slowly add 1.2g (12mmol) anhydrous triethylamine, violent stirring dripped 1.28g (9mmol) boron trifluoride ether solution after 10 minutes.Keep stirring fast, after reaction solution recovers normal temperature, continue to stir 4-8 hour.Entire reaction course is followed the tracks of with TLC.After reacting completely, mother liquor is poured in the separating funnel, use dichloromethane extraction, deionized water wash is repeatedly after three times, merge organic phase, behind the anhydrous magnesium sulfate drying, 200 order silica gel column chromatographies separate, and elutriant is: petrol ether/ethyl acetate=1: 1-1: 2 (v/v), the sorrel solid that obtains after the solvent evaporated gets brick-red powder 96mg with methylene dichloride and normal hexane recrystallization.
Yield: 23%.Mp：136-137℃。HRMS (TOF MS EI
+) calculated value C
13H
13BF
2N
2O
2: 278.1038.Measured value: 278.1031.
1H?NMR(CDCl
3，400MHz)δ：10.73(s，1H，-COOH)，7.88(s，2H，pyrrole-H)，7.63(d，2H，pyrrole-H，J＝3.6Hz)，6.61(d，2H，pyrrole-H，J＝4.0Hz)，3.16(t，2H，-CH
2，J＝8.4Hz)，2.51(t，2H，-CH
2，J＝7.6Hz)，2.08-2.00(m?2H，-CH
2)。
13C?NMR(CDCl
3，400MHz)：173.5，151.2，143.8，135.4，128.8，118.3，32.8，29.8，29.1。
Gained IIa dyestuff (10mg) is dissolved in the methyl alcohol (20ml), with the methanol solution NH (C of 10% triethylamine
2H
5)
3); In like manner, add the trolamine of equivalent, can get the triethylamine salt (M=NH (C of IIa
2H
4OH)
3); The 1%NaOH methanol solution that adds equivalent neutralizes, and can get the sodium salt (M=Na) of IIa; Add the 1%KOH methanol solution neutralization of equivalent, can get the sylvite (M=K) of IIa etc.
The Compound I Ia that generates can be directly used in biochemical marker and analysis, and its activated derivatives IIa-NHS synthetic sees embodiment 6.Activated derivatives IIa-NHS is used to investigate the effect of BSA mark optimum mole ratio and sees embodiment 10.
Compound III a's is synthetic:
In the 250mL single necked round bottom flask, add 160mg (1.6mmol) Succinic anhydried successively, 201mg (3mmol) 1-hydrogen pyrroles, under nitrogen protection, inject the DCM of 10mL exsiccant acetonitrile and 50mL then respectively with syringe, magnetic force stirs fast and makes solid dissolving (also can utilize ultrasonic wave or heating to promote dissolving), cost condensing works then and use nitrogen replacement reaction system 3-5 time under vacuumizing, oil bath heating and back flow reaction are spent the night; After TLC followed the tracks of and detects no raw material residue, question response mother liquor cool to room temperature was placed in the ice-water bath, slowly drips 2.4g (24mmol) anhydrous triethylamine, and violent stirring then dripped 2.56g (18mmol) boron trifluoride ether solution after 10 minutes.Keep stirring fast, reaction solution slowly is heated to 50 ℃ after recovering normal temperature, continues to stir till no more fluorescent substance generates.Entire reaction course is followed the tracks of with TLC.After reaction is finished, mother liquor is poured in the separating funnel, with dichloromethane extraction (3 * 50mL), the dilute hydrochloric acid of 2N and saturated common salt water washing separatory, final merging organic phase, and use anhydrous magnesium sulfate drying, after the solvent evaporated, use a small amount of silica gel mixed sample, silica gel column chromatography separates, and elutriant is: petrol ether/ethyl acetate=1: 1-2: 1 (v/v), the product that obtains after the solvent evaporated methylene dichloride and normal hexane mixed solvent cold process recrystallization, get scarlet powder 91mg, yield: 23%.Product structure is identified:
Mp:142-144 ℃ of .HRMS (TOF MS EI
+) calculated value C
12H
11BF
2N
2O
2: 264.0882; Measured value: 264.0871.
1H NMR (CDCl
3, 400MHz): δ=7.88 (s, 2H, pyrrole-H), 7.32 (d, 2H, pyrrole-H, J=3.6Hz), 6.57 (d, 2H, pyrrole-H, J=3.2Hz), 3.28 (t, 2H ,-CH
2, J=8.0 Hz), 2.85 (t, 2H ,-CH
2, J=8.0Hz).
13C NMR (CDCl
3, 100.518MHz): δ=177.0,147.4,144.5,135.0,128.2,118.7,36.7,25.7.
Gained IIIa dyestuff (10mg) is dissolved in the methyl alcohol (20ml), with the methanol solution NH (C of 10% triethylamine
2H
5)
3); In like manner, add the trolamine of equivalent, can get the triethylamine salt (M=NH (C of IIIa
2H
4OH)
3); The 1%NaOH methanol solution that adds equivalent neutralizes, and can get the sodium salt (M=Na) of IIIa; Add the 1%KOH methanol solution neutralization of equivalent, can get the sylvite (M=K) of IIIa etc.Can be directly used in the synthetic embodiment of opinion 7 of the compound III a-NHS ester of biochemical marker and analysis, activated derivatives IIIa-NHS is used to investigate the effect of BSA mark optimum mole ratio and sees embodiment 11.
Compound IV a's is synthetic:
In exsiccant 100mL single necked round bottom flask, add 237mg (1.6mmol) Tetra hydro Phthalic anhydride successively, 201mg (3mmol) pyrroles, inject 50mL exsiccant acetonitrile with syringe then under nitrogen protection, magnetic force stirs fast, and 65 ℃ of reactions are spent the night, after TLC detects no pyrroles's residue, mother liquor is cooled to normal temperature, slowly adds 1.5g (15mmol) anhydrous triethylamine, violent stirring; After 15 minutes, drip 2.19g (16mmol) boron trifluoride ether solution.Keep stirring fast, till no more fluorescent substance generates.Entire reaction course is followed the tracks of with TLC.The reaction finish after, mother liquor is poured in the separating funnel, use dichloromethane extraction, the dilute hydrochloric acid of 2N and saturated common salt water washing separatory, extract three times repeatedly after, the merging organic phase, anhydrous sodium sulfate drying; Column chromatography for separation then, elutriant is: petrol ether/ethyl acetate=2: 1-1: 1 (v/v), the thick product methylene dichloride/normal hexane that obtains after the solvent evaporated (5/95, v/v) recrystallization, obtain having the brick-red powder 121mg of metalluster, productive rate 26%.Product structure is identified:
Mp:170-172 ℃ of .HRMS (TOF MS EI
+) calculated value C
16H
11BF
2N
2O
2: 312.0882; Measured value: 312.0886.
1H NMR (CD
3COCD
3, 400MHz): δ=11.48 (s, 1H ,-COOH), 8.19 (d, 1H, Ar-H, J=7.6Hz), 7.99 (s, 2H, pyrrole-H), 7.80 (t, 1H, Ar-H, J=6.4Hz), 7.78 (t, 1H, Ar-H, J=6.8Hz), 7.62 (dd, 1H, Ar-H, J=7.6Hz), 6.79 (d, 2H, pyrrole-H, J=4.0Hz), 6.59 (d, 2H, pyrrole-H, J=4.0Hz).
13C NMR (CD
3COCD
3, 100.518MHz): δ=167.1,149.0,144.8,136.4,134.9,132.9,132.6,132.0,131.5,131.0,130.9,119.3.
Gained IVa dyestuff (10mg) is dissolved in the methyl alcohol (20ml), with the methanol solution NH (C of 10% triethylamine
2H
5)
3); In like manner, add the trolamine of equivalent, can get the triethylamine salt (M=NH (C of IVa
2H
4OH)
3); The 1%NaOH methanol solution that adds equivalent neutralizes, and can get the sodium salt (M=Na) of IVa; Add the 1%KOH methanol solution neutralization of equivalent, can get the sylvite (M=K) of IVa etc.
Fluorescence dye IVa is (reactivation process of deriving, mode and condition are similar to embodiment 1) behind derivatize, are used for biochemical analysis.Under the flag condition after the optimization: the volumetric molar concentration of activation dyestuff is 1.8 * 10
-9During mol, when boric acid-borax buffer system pH is 9.0, when 40 ℃ of labeled reactant temperature, reaction times 40min, adopt sds polyacrylamide gel electrophoresis discontinuous (10% concentrated glue and 5% separation gel) to separate, when the input amount of BSA is respectively: 10ng; 30ng; 50ng, when 200g and 0.5ug, its fluorescently-labeled detectability can reach 3 * 10
-8G.
Compound III b's is synthetic:
In exsiccant 250ml single necked round bottom flask; add 180mg (1.8mmol) Succinic anhydried successively; 285mg (3mmol) 2; the 4-dimethyl pyrrole; under nitrogen protection, inject the methylene dichloride of 7.5ml exsiccant acetonitrile and 67.5ml then respectively with syringe; magnetic force stirs fast; back flow reaction is spent the night; TLC detect not to have 2, behind the 4-dimethyl pyrrole, after mother liquor is cooled to normal temperature and steams the solvent of 40ml; under ice bath; slowly add 1.2g (12mmol) anhydrous triethylamine, violent stirring dripped 1.28g (9mmol) boron trifluoride ether solution after 10 minutes.Keep stirring fast, after reaction solution recovered normal temperature, 40 ℃ were continued to stir till no more fluorescent substance generates down.Entire reaction course is followed the tracks of with TLC.After reaction is finished, mother liquor is poured in the separating funnel, use dichloromethane extraction, deionized water wash, extract three times repeatedly after, merge organic phase, behind the anhydrous magnesium sulfate drying, 200 order silica gel column chromatographies separate, and elutriant is: petrol ether/ethyl acetate=1: 1-1: 2 (v/v), the sorrel solid that obtains after the solvent evaporated gets yellowish red color powder 101mg with methylene dichloride and normal hexane recrystallization.
Yield: 21%.Mp：173-174℃。HRMS (TOF MS EI
-) calculated value C
16H
19BF
2N
2O
2: 319.1429.Actual measurement: 319.1435, (TOF MS EI
+Na): 343.0405.
1H?NMR(CDCl
3，400MHz)δ：6.08(s，2H，pyrrole-H)，3.34(t，2H，-CH
2，J＝8.8Hz)，2.67(t，2H，-CH
2，J＝8.8Hz)，2.53(s，6H，-CH
3)2.85(s，6H，-CH
3)。
13C?NMR(CDCl
3，400MHz)：176.4，155.5，142.8，140.7，131.4，122.2，35.1，23.5，16.6，14.7。
Gained IIIb dyestuff (10mg) is dissolved in the methyl alcohol (20ml),, can gets the triethylamine salt (M=NH (C of IIIb with the methanol solution neutralization of 10% triethylamine
2H
5)
3); In like manner, add the trolamine of equivalent, can get the triethylamine salt (M=NH (C of IIIb
2H
4OH)
3); The 1%NaOH methanol solution that adds equivalent neutralizes, and can get the sodium salt (M=Na) of IIIb; Add the 1%KOH methanol solution neutralization of equivalent, can get the sylvite (M=K) of IIIb etc.
This dyestuff is used for the orthogonal experiment of labelled protein BSA in the activation back (reactivation process of deriving, mode and condition are similar to embodiment 1) of deriving, and under optimal condition: the volumetric molar concentration of activation dyestuff is 1 * 10
-9During mol, when boric acid-borax buffer system pH is 8.8, when 30 ℃ of labeled reactant temperature, reaction times 40min, adopt sds polyacrylamide gel electrophoresis discontinuous (10% concentrated glue and 5% separation gel) to separate, when the consumption mol ratio of BSA and dyestuff is 1: 25, its fluorescently-labeled sensitivity best (seeing accompanying drawing 9).
Compound IV b's is synthetic:
In exsiccant 250ml single necked round bottom flask; add 267mg (1.8mmol) Tetra hydro Phthalic anhydride successively; 285mg (3mmol) 2; the 4-dimethyl pyrrole; under nitrogen protection, inject 75ml exsiccant acetonitrile then with syringe; magnetic force stirs fast; 60 ℃ of reactions are spent the night; TLC detect not to have 2, behind the 4-dimethyl pyrrole, after mother liquor is cooled to normal temperature and steams the solvent of 30ml; under ice bath; slowly add 1.2g (12mmol) anhydrous triethylamine, violent stirring dripped 1.28g (9mmol) boron trifluoride ether solution after 10 minutes.Keep stirring fast, after question response liquid recovers normal temperature, continue to stir till no more fluorescent substance generates.Entire reaction course is followed the tracks of with TLC.The reaction finish after, mother liquor is poured in the separating funnel, use dichloromethane extraction, deionized water wash, extract three times repeatedly after, the merging organic phase, anhydrous magnesium sulfate drying; Separate with 200 order silica gel column chromatographies then, elutriant is: petrol ether/ethyl acetate=2: 1-1: 1 (v/v), the sorrel solid that obtains after the solvent evaporated, thick product obtain having the brick-red powder 138mg of metalluster with methylene dichloride and normal hexane recrystallization.
Yield: 25%.Mp：235-237℃。HRMS (TOF MS EI
+) calculated value C
20H
19BF
2O
2N
2: 368.1508.Actual measurement: 368.1504.
1H?NMR(CD
3COCD
3，400MHz)δ：11.47(s，1H，COOH)，8.14(dd，J＝8.0Hz，1H)，7.79(dd，
3J＝7.6Hz，
4J＝1.2Hz，1H)，7.69(dd，?
3J＝7.6Hz，
4J＝1.2Hz，1H)，7.42(dd，J＝7.6Hz，1H)，6.03(s，2H)，2.45(s，6H，CH
3)，1.33(s，6H，CH
3)。
13C?NMR(CD
3COCD
3，400MHz)：166.4，154.6，142.9，142.2，136.0，133.4，131.4，131.2，131.0，129.9，129.8，121.0，13.9，13.5。
Gained IVb dyestuff (10mg) is dissolved in the methyl alcohol (20ml),, can gets the triethylamine salt (M=NH (C of IVb with the methanol solution neutralization of 10% triethylamine
2H
5)
3); In like manner, add the trolamine of equivalent, can get the triethylamine salt (M=NH (C of IVb
2H
4OH)
3); The 1%NaOH methanol solution that adds equivalent neutralizes, and can get the sodium salt (M=Na) of IVb; Add the 1%KOH methanol solution neutralization of equivalent, can get the sylvite (M=K) of IVb etc.
Fluorescence dye IVb is (reactivation process of deriving, mode and condition are similar to embodiment 1) behind derivatize, are used for biochemical analysis.Under the flag condition after the optimization: the volumetric molar concentration of activation dyestuff is 0.8 * 10
-9During mol, when boric acid-borax buffer system pH is 9.0, when 40 ℃ of labeled reactant temperature, reaction times 40min, adopt sds polyacrylamide gel electrophoresis discontinuous (10% concentrated glue and 5% separation gel) to separate, when the input amount of BSA is respectively: 10ng; 30ng; When 50ng and 0.2ug, its fluorescently-labeled detectability can reach 5 * 10
-8G (seeing accompanying drawing 10).
Can be directly used in Compound I Ia-NHS (N-hydroxy-succinamide ester) synthetic of biochemical marker and analysis:
In the 25ml single necked round bottom flask; the IIa that successively adds 55.6mg (0.2mmol); 77mg (0.3mmol) N; N '-two succinimidyl carbonate; inject the trolamine of 200ul (1.5mmol) then with microsyringe; add the 5ml dry DMF, under nitrogen protection, the normal temperature lucifuge stirred 24 hours.Available TLC detection reaction process.After treating that dyestuff activates fully, steam solvent, remaining yolk yellow solid is dissolved with methylene dichloride, twice of washing separatory.After the organic layer drying concentrates, quick silica gel column chromatography twice, elutriant is: dichloromethane/ethyl acetate=20: 1-10: 1 (v/v) obtains the garnet solid after evaporating elutriant, thick product obtains the Powdered activatory IIa-NHS of 67mg scarlet with methylene dichloride and normal hexane recrystallization.This method is simple and easy to operate, product after the activation need carry out the silicagel column sharp separation, finally can be directly used in protein labeling, and because only more methylene radical on this dyestuff and IIIa-NHS (embodiment 7) structure, physicochemical property is close, so we think effect basically identical on tag application.
Yield: 90%.HRMS (TOF MS EI
+Na) calculated value C
17H
16BF
2N
3O
4: 398.1097.Actual measurement: 398.1100.
1H?NMR(CDCl
3，400MHz)：7.87(s，2H，pyrrole-H)，7.365(d，2H，pyrrole-H，J＝4.4Hz)，6.552(d，2H，pyrrole-H，J＝3.2Hz)，3.075(t，2H，-CH
2，J＝8.0Hz)，2.887(d，2H，-CH
2，J＝2.8Hz)，2.783(d，2H，-CH
2，J＝6.4Hz)，2.258-2.185(m，2H，-CH
2)。
13C?NMR(CDCl
3，400MHz)：169.2，168.1，148.6，144.2，135.3，128.3，118.6，30.8，29.7，28.3，25.6。
The applicating expedition of the optimum mark mol ratio when Compound I Ia-NHS is directly used in the BSA mark is seen embodiment 10.
Can be directly used in compound III a-NHS ester synthetic of biochemical marker and analysis:
In the 25ml single necked round bottom flask, successively add the IIIa of 80mg (0.3mmol), the N-maloyl imines of 42mg (0.36mmol), the carbodicyclo hexylimide of 155mg (0.75mmol).Under the nitrogen, inject the 8ml anhydrous acetonitrile, shading was stirred 36 hours under the normal temperature.TLC steams solvent after detecting no IIIa residue, remaining yolk yellow solid is dissolved twice of washing separatory with methylene dichloride.After the organic layer drying concentrates, quick silica gel column chromatography twice, elutriant is: dichloromethane/ethyl acetate=20: 1-10: 1 (v/v) obtains the garnet solid after evaporating elutriant, thick product obtains the Powdered IIIa-NHS of 100mg orange with methylene dichloride and normal hexane recrystallization.This method is simple and easy to operate, and the product after the activation need carry out the silicagel column sharp separation, finally can be directly used in protein labeling, and its application and effect thereof are referring to embodiment 11.
Yield: 92%.HRMS (TOF MS EI
+) calculated value C
16H
14BF
2N
3O
4: 361.1045.Actual measurement: 361.1013.
1H?NMR(CDCl
3，400MHz)：7.893(s，2H，pyrrole-H)，7.327(d，2H，pyrrole-H，J＝4.0Hz)，6.583(d，2H，pyrrole-H，J＝3.6Hz)，3.345(t，2H，-CH
2，J＝8.0Hz)，3.069(t，2H，-CH
2，J＝8.0Hz)，2.875(s，2H，-CH
2)。
13C?NMR(CDCl
3，400MHz)：168.94，167.06，145.93，144.91，134.94，128.20，118.97，33.74，25.79，25.55。
This activated derivatives structure is confirmed by X-ray single crystal diffraction, is seen accompanying drawing 11.
For further verifying the good prospects for application of the dyestuff that has carboxylic acid functional, in the present embodiment, get is in their dissolving different solvents (table 3), and the volumetric molar concentration of solution is 1 * 10
-5Mol/L, and measure the absorption and the emmission spectrum of dyestuff by ultraviolet-visible spectrophotometer and fluorophotometric instrument, carry out the spectrum property test.
By measuring the selected uv-absorbing (accompanying drawing 2) of dyestuff under DCM and fluorescent emission (accompanying drawing 3) spectrum that has carboxyl, we can clearly be seen that: their maximal ultraviolet absorption and fluorescent emission wavelength are respectively at 495 ± 10nm and 510 ± 10nm, and Stokes shift is between 10-20nm.This fluorochrome excites in visible-range and launches, and narrower bandwidth of an emission, makes it can avoid the interference of biological background fluorescence effectively, improves the sensitivity that detects, and is the bioanalysis fluorescence dye of a class excellent performance.And the fluorescence quantum yield of dyestuff is higher, have in addition reach 0.83, this character helps improving the detection sensitivity when using.
The selected dyestuff that has carboxyl is dissolved in all kinds of different solvents: hexane (Hexane), methylene dichloride (DCM), ethanol (Ethanol) and water (H
2O) in, the maximum absorption wavelength λ max (abs) that records, maximum emission wavelength λ max (em), Stokes displacement (Δ λ), molar extinction coefficient (E), fluorescein (Φ
f=0.85in 0.1N NaOH) is the measured fluorescence quantum yield (Φ of reference
f) wait and list in table 3.Remove cited part dyestuff (IIa:R in the above-mentioned example in the table 3
1=R
2=M=H; IIIb:R
1=R
2=CH
3, M=H; IVb:R
1=R
2=CH
3, M=H) outside, the structure of other dyestuff is IIIa:R
1=R
2=M=H; IVa:R
1=R
2=M=H.
The photophysical property of selecting in table 3 different solvents that contains carboxyl BODIPY dyestuff
Have the light stability test of the dyestuff of carboxyl:
The light stability test of dyestuff in the acetonitrile medium: sample concentration is 1 * 10
-5M in different light time post analysis dye solution density, calculates residual absorption per-cent, is 1 to calculate with initial absorption intensity; The results are shown in accompanying drawing 4.By the light stability of dyestuff in acetonitrile relatively, we find: not with methyl and to contain its light stability of dyestuff of aliphatic carboxylic acid good, this mainly is because less the causing of cloud density on the dyestuff core texture on the precursor structure.Therefore dyestuff IIIa and IIa have better actual application value.
The light stability test of dyestuff in deionized water medium: sample concentration is 1 * 10
-5M in different light time post analysis dye solution density, calculates residual absorption per-cent, is 1 to calculate with initial absorption intensity.The results are shown in accompanying drawing 5.By the light stability of dyestuff in acetonitrile relatively, we further find: the light stability of dyestuff in water with methyl is ungood, and promptly dyestuff IIIa and IIa's is stable very good, can satisfy the bioanalysis applied research fully.
With labelled protein BSA is example, utilizes the concrete steps of optimum mark mol ratio of electrophoresis detection reactive dyestuffs IIa-NHS and BSA as follows:
The first step: the polyacrylamide separation gel of preparation 10% and 5% concentrated glue, make the sample hole, after solidifying, place stand-by.
Second step: protein denaturation experiment.Add the damping fluid (SDS 2.5%, sucrose 1%) of boric acid-borax of the pH=8.7 of 2ul in 5ug (1ul) BSA, the 1ul deionized water boils 5min.
The 3rd step: add the IIa-NHS tetrahydrofuran solution of 1ul among the BSA after sex change, the mol ratio of preparing BSA and dyestuff respectively is: BSA/IIa-NHS=1: 5,1: 10,1: 20,1: 40,1: 100.30 ℃ of following lucifuge reaction 30-40min.
The 4th step: negate should be gone up sample for the back mixed solution successively, fix electrophoresis apparatus after, connect power supply, under 100 volts of voltages, when dyestuff to be activated moves to bottom the gel 1-1.5cm, cut off the electricity supply.
The 5th step: unload gel film, take pictures after the handled (as shown in Figure 6), and do data logging and analysis.
By above-mentioned experiment, we find out more excellent protein labeling condition and are: BSA is 1: 20 with the mol ratio of activation dyestuff IIa-NHS.
Activation dyestuff IIIa-NHS mark BSA and gel electrophoresis experiment thereof:
With labelled protein BSA is example, utilizes the concrete steps of activation dye marker effect under the different pH of SDS-PAGE (sodium laurylsulfonate-polyacrylamide) detected through gel electrophoresis as follows:
The first step: the polyacrylamide separation gel of preparation 10% and 5% concentrated glue, make the sample hole, after solidifying, place stand-by.
Second step: protein denaturation experiment.Add the damping fluid (SDS 2.5%, sucrose 1%) of boric acid-borax of the pH=8.7 of 2ul in 5ug (1ul) BSA, the 1ul deionized water boils 5min.
The 3rd step: add the IIIa-NHS tetrahydrofuran solution of 1ul among the BSA after sex change, the mol ratio of preparation BSA and dyestuff: BSA/IIIa-NHS=1: 20; Attempt different pH of buffer values (8.0; 8.4; 8.7; 9.0; 9.3; 9.6) carrying out the mark control experiment, marked body ties up to 30 ℃ of following lucifuge reaction 30-40min.
The 4th step: negate should be gone up sample for the back mixed solution successively, fix electrophoresis apparatus after, connect power supply, under 100 volts of voltages, when dyestuff to be activated moves to bottom the gel 1-1.5cm, cut off the electricity supply.
The 5th step: unload gel film, take pictures after the appropriate processing, and do data logging and analysis.
Experimental result shows: when the labeled reactant pH of buffer is 9.0, and the electrophoresis of resulting protein labeling swimming band strength the highest (as shown in Figure 7), and also maximum (as shown in Figure 8) of the product value of the intensity that is calculated and area.
Proof after the BODIPY fluorescence dye that contains carboxylic acid of this invention is activated, has broad application prospects at biochemical analysis and life science by experiment.For example under optimal conditions: the consumption of activation dyestuff is 3.6 * 10
-9Mol (1ug is dissolved in the tetrahydrofuran (THF) of 1ul), when boric acid-borax buffer system pH is 9.0, when 30 ℃ of labeled reactant temperature, reaction times 40min, adopt sds polyacrylamide gel electrophoresis discontinuous (10% concentrated glue and 5% separation gel) to separate, when the input amount of BSA is respectively: 2ng; 5ng; 10ng; 30ng; When 50ng and 0.2ug, its fluorescently-labeled detectability can reach 2ng, and with the naked eye just can see tangible BSA indicia band by gel imaging system under the ultraviolet lamp of 302nm.
The salt formula structure of the IIa that in embodiment 1, obtains, the salt formula structure of the IIIa that in embodiment 2, obtains, and the salt formula structure of the IVa that in embodiment 3, obtains, their effects when being used for the labelled protein experiment, described basic identical with embodiment 11.The salt formula structure of the IIIb that obtains in embodiment 4 has identical effect with compound III b-NHS when being used for the labelled protein experiment; The salt formula structure of the IVb that obtains in embodiment 5 has identical effect with compound IV b-NHS when being used for the labelled protein experiment.
Concrete grammar is: the dye salt that takes by weighing certain mass, add 10 times of normal N, N '-two succinimidyl carbonate, excessive trolamine reaches the following activation of 30 degree water-baths limit marks under the buffer system of pH 7.5-8.5, the mark time is 12 hours, according to the step of embodiment 8, carry out separation detection then, have identical mark effect by SDS-PAGE.
The salt (metal cation salt and ammonium salt) of boron fluoride complexing two pyrroles's methine fluorochromes of the present invention, the effect when being used for biochemical marker is similar to corresponding D YE-NHS, and present embodiment is explanation no longer separately.
Claims (9)
1. a class boron fluoride complexing two pyrroles's methine fluorescence dyes is characterized in that this fluorescence dye has following general structure I:
In the formula:
R
1, R
2, R
3, R
5, R
6, R
7H or C respectively do for oneself
1-8Alkyl;
R
8Group for structural formula X, Y, Z or P;
M is H, Na, K, N (R
9R
10R
11R
12) or the N-succinimido
R wherein
9, R
10, R
11, R
12H, C respectively do for oneself
1-8Alkyl or be with substituent C
1-8Alkyl, described substituting group is selected from OH, ehter bond or carboxyl.
2. boron fluoride complexing two pyrroles's methine fluorochromes as claimed in claim 1, wherein said R
1, R
2, R
3, R
5, R
6, R
7H or C respectively do for oneself
1-4Alkyl.
3. boron fluoride complexing two pyrroles's methine fluorescence dyes as claimed in claim 1, wherein said R
9, R
10, R
11, R
12H or C respectively do for oneself
1-4Alkyl.
4. as each described boron fluoride complexing two pyrroles's methine fluorescence dyes among the claim 1-3, it is characterized in that this dyestuff has following structural formula II, III or IV:
In said structure formula II, R
1=R
3=R
5=R
7=H; In said structure formula III and IV: R
1, R
3, R
5, R
7H or CH respectively do for oneself
3
5. one kind prepares the method for fluorescence dye according to claim 1, comprising:
Condensation takes place in (1) substituted pyrroles and organic dibasic acid acid anhydride in organic solvent or absolute anhydrous organic solvent, in condensation course, bimolecular substituted azole and a part acid anhydrides carry out condensation, and wherein the substituted azole of a part is for having substituent R
1, R
2And R
3The pyrroles, the substituted azole of another molecule is for having substituent R
5, R
6And R
7The pyrroles, the organic dibasic acid acid anhydride is selected from: Succinic anhydried, Pyroglutaric acid, adipic anhydride or Tetra hydro Phthalic anhydride; Organic solvent is selected from methylene dichloride, tetrahydrofuran (THF) or acetonitrile, the volumetric molar concentration of described substituted azole in described organic solvent is 0.1-20%, and the raw material molar ratio of substituted azole and acid anhydrides is 0.5-10: 1, temperature of reaction is controlled at 15-120 ℃, and reaction obtains product two pyrroles's methine structural compounds;
Two pyrroles's methine structural compounds that (2) will obtain react with the boron trifluoride compound again, and add the organic amine neutralizing agent, form compound of Formula I after taking off hydrogen fluoride, purify then; Described boron trifluoride compound is selected from: the etherate of gaseous state boron trifluoride, boron trifluoride or other can discharge the compound of boron trifluoride under the normal temperature solution state; Temperature of reaction is-10 ℃-100 ℃; The add-on of boron trifluoride compound is: the mol ratio that makes two pyrroles's methine structural compounds and boron trifluoride compound is 1: 0.1-10.
6. the purposes of the described boron fluoride complexing two pyrroles's methine fluorescence dyes of claim 1, it is used for the probe biomolecule of complex sign protein or DNA.
7. the purposes of the described boron fluoride complexing two pyrroles's methine fluorescence dyes of claim 1, it is used for bioluminescence analysis or biomolecular labeling.
8. purposes as claimed in claim 7, wherein said biomolecular labeling is: protein labeling, dna marker, polypeptide marker, antigen or antibody labeling or anti-antibody mark.
9. purposes as claimed in claim 7, wherein said fluorometric analysis are fluoroimmunoassay, fluorescent microscope analysis or fluorometric analysis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102625656A CN102061103A (en) | 2009-11-11 | 2009-12-25 | Type I boron fluoride complex dipyrromethene dye, and preparation method and application thereof |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910198609 | 2009-11-11 | ||
| CN200910198609.3 | 2009-11-11 | ||
| CN2009102625656A CN102061103A (en) | 2009-11-11 | 2009-12-25 | Type I boron fluoride complex dipyrromethene dye, and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102061103A true CN102061103A (en) | 2011-05-18 |
Family
ID=43996614
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009102625656A Pending CN102061103A (en) | 2009-11-11 | 2009-12-25 | Type I boron fluoride complex dipyrromethene dye, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102061103A (en) |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102520153A (en) * | 2011-12-15 | 2012-06-27 | 安徽师范大学 | Method for quickly detecting dibutyl phthalate in food |
| WO2012083509A1 (en) * | 2010-12-20 | 2012-06-28 | 大连理工大学 | Boron fluoride-dipyrromethene dye, preparation method and application thereof |
| CN102564987A (en) * | 2012-01-12 | 2012-07-11 | 浙江大学 | pH colorimetric detection analysis method based on pyrromethene derivative |
| CN102702768A (en) * | 2012-06-04 | 2012-10-03 | 天津理工大学 | Novel red BODIPY fluorescent dye and preparation method and application thereof |
| CN102993763A (en) * | 2012-12-09 | 2013-03-27 | 大连理工大学 | Single charge boron fluroride complexing dipyrrole methenyl fluorochrome and application thereof |
| WO2015081803A1 (en) * | 2013-12-02 | 2015-06-11 | 大连理工大学 | Boron-dipyrromethene fluorescence probes and manufacturing method and use thereof |
| CN105102464A (en) * | 2012-12-26 | 2015-11-25 | 新加坡国立大学 | Megastokes amino-triazolyl-bodipy compounds and applications to live neuron staining and human serum albumin FA1 drug site probing |
| CN105237570A (en) * | 2014-05-28 | 2016-01-13 | 中国科学院化学研究所 | Biological reagent used for DNA fluorescence in-situ hybridization (FISH), and preparation and applications thereof |
| CN105884806A (en) * | 2016-06-21 | 2016-08-24 | 福州大学 | Preparation method of fluorescent probe and oxytetracycline detection method based on same |
| CN106928261A (en) * | 2017-02-09 | 2017-07-07 | 南京航空航天大学 | A kind of pyrroles's fluorescent chemicals of three fluorine boron two of base ester of HOSu NHS containing N, preparation method and its amido coupled product |
| CN111100153A (en) * | 2019-12-31 | 2020-05-05 | 华侨大学 | Boron dipyrromethene derivative dye ligand and preparation method thereof |
| CN112574239A (en) * | 2019-09-27 | 2021-03-30 | 中国科学院上海药物研究所 | 3-thiazolenyl boron fluoride complex dipyrromethene compound and preparation method and application thereof |
| CN113321671A (en) * | 2021-01-29 | 2021-08-31 | 南京工业大学 | Boron dipyrromethene solid-state luminescent material, preparation method and application thereof, and blue light driven LED |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1944540A (en) * | 2006-09-15 | 2007-04-11 | 大连理工大学 | Near infrared boron fluoride complexed dipyrrolyl methine fluorescent dye for biological analysis |
| CN101550156A (en) * | 2009-05-14 | 2009-10-07 | 浙江大学 | Fluorescence probe, synthetic method and uses thereof |
-
2009
- 2009-12-25 CN CN2009102625656A patent/CN102061103A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1944540A (en) * | 2006-09-15 | 2007-04-11 | 大连理工大学 | Near infrared boron fluoride complexed dipyrrolyl methine fluorescent dye for biological analysis |
| CN101550156A (en) * | 2009-05-14 | 2009-10-07 | 浙江大学 | Fluorescence probe, synthetic method and uses thereof |
Non-Patent Citations (2)
| Title |
|---|
| DONGCHUAN WANG ET AL.: "Carboxyl BODIPY Dyes from Bicarboxylic Anhydrides: One-Pot Preparation, Spectral Properties, Photostability, and Biolabeling", 《J. ORG. CHEM.》 * |
| YANG LIU ET AL.: "TEMPO-based Redox-sensitive Fluorescent Probes and Their Applications to Evaluating Intracellular Redox Status in Living Cells", 《CHEMISTRY LETTERS》 * |
Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012083509A1 (en) * | 2010-12-20 | 2012-06-28 | 大连理工大学 | Boron fluoride-dipyrromethene dye, preparation method and application thereof |
| CN102520153A (en) * | 2011-12-15 | 2012-06-27 | 安徽师范大学 | Method for quickly detecting dibutyl phthalate in food |
| CN102564987A (en) * | 2012-01-12 | 2012-07-11 | 浙江大学 | pH colorimetric detection analysis method based on pyrromethene derivative |
| CN102702768A (en) * | 2012-06-04 | 2012-10-03 | 天津理工大学 | Novel red BODIPY fluorescent dye and preparation method and application thereof |
| CN102702768B (en) * | 2012-06-04 | 2013-12-04 | 天津理工大学 | Novel red BODIPY fluorescent dye and preparation method and application thereof |
| CN102993763A (en) * | 2012-12-09 | 2013-03-27 | 大连理工大学 | Single charge boron fluroride complexing dipyrrole methenyl fluorochrome and application thereof |
| CN102993763B (en) * | 2012-12-09 | 2014-08-27 | 大连理工大学 | Single charge boron fluroride complexing dipyrrole methenyl fluorochrome and application thereof |
| CN105102464A (en) * | 2012-12-26 | 2015-11-25 | 新加坡国立大学 | Megastokes amino-triazolyl-bodipy compounds and applications to live neuron staining and human serum albumin FA1 drug site probing |
| CN105102464B (en) * | 2012-12-26 | 2017-03-29 | 新加坡国立大学 | Prunus mume (sieb.) sieb.et zucc. coffee Stokes aminotriazole(ATA) base BODIPY compounds and the application for live neuron staining and the detection of human serum albumin's FA1 medicines site |
| WO2015081803A1 (en) * | 2013-12-02 | 2015-06-11 | 大连理工大学 | Boron-dipyrromethene fluorescence probes and manufacturing method and use thereof |
| US9862731B2 (en) | 2013-12-02 | 2018-01-09 | Dalian University Of Technology | Difluoroboron dipyrromethene fluorescent probe, production method and application thereof |
| CN105237570A (en) * | 2014-05-28 | 2016-01-13 | 中国科学院化学研究所 | Biological reagent used for DNA fluorescence in-situ hybridization (FISH), and preparation and applications thereof |
| CN105884806A (en) * | 2016-06-21 | 2016-08-24 | 福州大学 | Preparation method of fluorescent probe and oxytetracycline detection method based on same |
| CN106928261A (en) * | 2017-02-09 | 2017-07-07 | 南京航空航天大学 | A kind of pyrroles's fluorescent chemicals of three fluorine boron two of base ester of HOSu NHS containing N, preparation method and its amido coupled product |
| CN112574239A (en) * | 2019-09-27 | 2021-03-30 | 中国科学院上海药物研究所 | 3-thiazolenyl boron fluoride complex dipyrromethene compound and preparation method and application thereof |
| CN112574239B (en) * | 2019-09-27 | 2022-03-25 | 中国科学院上海药物研究所 | 3-thiazolenyl boron fluoride complex dipyrromethene compound and preparation method and application thereof |
| CN111100153A (en) * | 2019-12-31 | 2020-05-05 | 华侨大学 | Boron dipyrromethene derivative dye ligand and preparation method thereof |
| CN113321671A (en) * | 2021-01-29 | 2021-08-31 | 南京工业大学 | Boron dipyrromethene solid-state luminescent material, preparation method and application thereof, and blue light driven LED |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102061103A (en) | Type I boron fluoride complex dipyrromethene dye, and preparation method and application thereof | |
| Knorr et al. | New Red‐Emitting Tetrazine‐Phenoxazine Fluorogenic Labels for Live‐Cell Intracellular Bioorthogonal Labeling Schemes | |
| CN107089937B (en) | The fluorescence probe and its preparation method and application of Mitochondrially targeted measurement viscosity | |
| US20070134737A1 (en) | Fluorophore compounds and their use in biological systems | |
| Starck et al. | Towards libraries of luminescent lanthanide complexes and labels from generic synthons | |
| Xu et al. | A novel “Turn-On” fluorescent probe for F− detection in aqueous solution and its application in live-cell imaging | |
| Aranda et al. | Vinyl-diazine triphenylamines and their N-methylated derivatives: synthesis, photophysical properties and application for staining DNA | |
| CN102735667B (en) | Fluorescence sensing film based on boron dipyrromethene-phenyl boronic acid (BODIPY-PBA), preparation method and application thereof | |
| CN104745177A (en) | Light activated fluorescent probe having protein label positioning function as well as preparation method and application thereof | |
| CN101787218A (en) | Conjugated chain beta-N-substituted cyanine fluorescent dye | |
| CN113683631B (en) | Organic boric acid glucose probe and preparation method and application thereof | |
| CN111303139A (en) | Compound with aggregation-induced emission performance and preparation method and application thereof | |
| CN110256218A (en) | A kind of aggregation-induced emission dye molecule and its synthetic method | |
| CN102344449B (en) | Heterocyclic-fused naphthalimide and preparation method and application thereof | |
| CN110498799A (en) | A kind of fluorescence probe and its preparation method and application | |
| CN109134441A (en) | A kind of novel fluorescence probe and its preparation method and application detecting cysteine | |
| CN105985769A (en) | Preparation and application of fluorescent probe for phiophenol | |
| Szmacinski et al. | Long-lifetime Ru (II) complexes for the measurement of high molecular weight protein hydrodynamics | |
| JP2001002951A (en) | New fluorescent pigment and its use as fluorescent marker | |
| CN114853656B (en) | Carbazole derivative with AEE characteristic, preparation method and application | |
| Yoshizawa et al. | A Bis (phenylethynyl) pyrene‐Based [3] Rotaxane as an Extremely Photostable Fluorescence Probe Suitable for Hard‐Edged Irradiation Experiments | |
| CN107903220B (en) | Fluorescent probe for visually detecting ozone and preparation method thereof | |
| CN110669350B (en) | Piperidyl BODIPY red-light fluorescent dye and preparation method and application thereof | |
| CN104531134B (en) | The fluorescent probe of a kind of polar sensitive, preparation method and application | |
| Kozhevnikov et al. | Strong emission increase of a dicarboxyterpyridene europium (III) complex in the presence of citrate and hydrogen peroxide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20110518 |