CN101597297B - Renewable fluorescent probe for selectively detecting bioactive sulfhydryl compound in cell and synthetic method and application thereof - Google Patents
Renewable fluorescent probe for selectively detecting bioactive sulfhydryl compound in cell and synthetic method and application thereof Download PDFInfo
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- CN101597297B CN101597297B CN2008101144122A CN200810114412A CN101597297B CN 101597297 B CN101597297 B CN 101597297B CN 2008101144122 A CN2008101144122 A CN 2008101144122A CN 200810114412 A CN200810114412 A CN 200810114412A CN 101597297 B CN101597297 B CN 101597297B
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a reproducible fluorescent probe for selectively detecting bioactive sulfhydryl compounds in cells, a synthetic method and application thereof. Will carry R1，R2，R3、R4And R5Dissolving coumarin-3-carbonyl compound with substituent group in dry organic solvent, and slowly dripping the compound with R dissolved in the organic solvent under reflux stirring6A substituted thiosemicarbazide; stirring for reaction, filtering, removing the organic solvent, and drying in vacuum to obtain a ligand; dissolving the obtained ligand in dry organic solvent, and slowly dropwise adding HgX dissolved in organic solvent2(ii) a And (3) carrying out reflux stirring reaction to obtain solid precipitate, filtering, and drying the solid in vacuum to obtain the fluorescent probe shown in the formula (I). The fluorescent probe can be used for detecting bioactive sulfhydryl compounds in a biological system, analyzing and detecting bioactive sulfhydryl compounds in biological living cells and living tissues and detecting the bioactive sulfhydryl compounds in pathological tissues in clinical medicine.
Description
Technical field
The present invention relates to the renewable fluorescent probe and the preparation method and use thereof of selectively detecting bioactive sulfydryl compound in cell.
Background technology
Modern medicine study shows, the ANOMALOUS VARIATIONS of certain amino acid concentration also is the root that causes numerous diseases to produce in the body fluid, the meeting of rising promotes atherosclerosis as homocysteine in the blood (Hcy), and then it is closely related with the ischemia heart, cerebrovascular disease, the detecting homocysteine is an independent reference factor of this type of disease danger of diagnosis in the variation of blood middle concentration at present, and the content that therefore detects homocysteine in the body fluid has exactly become one of important factor of control disease development.At present the detection method of using is loaded down with trivial details and detection speed is slower, develops a kind of convenience, sensitivity, detection means is very important fast.
At present, the assay determination of bioactive sulfhydryl compound generally is after utilizing its reductibility and reacting with some organic reagent, carry out assay determination with electrochemistry, spectrophotometry, chemoluminescence method, catalytic kinetics method and fluorescent method, these method complicated operations, sensitivity is not high and selectivity is relatively poor.
Optochemical sensor is a new problem in science that develops rapidly in recent years, its appearance obviously and supramolecule advancement of science such as molecule assembling, host-guest chemistry, noncovalent interaction, hydrogen bond action, hydrophobic interaction etc. and photoinduction transfer transport (PET) process, intramolecularly conjugated charge transfer compound structure and the characteristics of luminescence thereof etc. study closely related, its development also with many science and technology field such as biological chemistry, clinical medicine, pharmaceutical chemistry and environmental science in a large amount of practical problemss of proposing closely related.For all the foregoing reasons, strong promotion the progress of Optochemical sensor.Optochemical sensor mainly is divided into fluorescent probe (Fluorescent probe) and is called fluorescence chemical sensor (Fluorescent Chemosensor) and colorimetric chemical sensor (Colorimetric Chemosensor) again by the difference of its signal detecting method.Fluorescent probe mainly is that to rely on fluorescent signal be detection means, the moving of enhancing, quencher or emission wavelength of fluorescence arranged usually, and the colorimetric chemical sensor mainly is the variation by means of tone, just can detect by visual inspection, makes things convenient for practical application.Being used for the design and the research of fluorescent probe that alien species are detected, is a problem in science that is subjected to extensive concern in recent years.The testing process of fluorescent probe mainly is by device receptor (Receptor) part alien species (to be comprised positively charged ion, negatively charged ion and neutral molecule etc.) selectivity admit, then through different mechanism of action, shift (ICT) as photoinduction transfer transport (PET) or energy transfer, metal-ligand charge transfer (MLCT), intramolecular charge, the signal report section by device provides the information change of relevant device in admitting the species process again.Fluorescent probe is because its selective good, highly sensitive, advantage such as the time of response is fast, obtained good application in the context of detection of microchemistry species.
Coumarin kind compound is because of its bigger molar extinction coefficient and high fluorescence quantum yield, as fluorescent marker certain application is being arranged aspect the biotechnology, but its also rarely found report of example as the chemical sensitisation molecule that directly is used for discerning specific object.In the present invention, we design and have synthesized based on the derivative of the tonka bean camphor thiosemicarbazide fluorescent probe as selectively detecting bioactive sulfydryl compound in cell, compare (1 with forefathers' work, Oleksandr Rusin, Nadia N.St.Luce, Rezik A.Agbaria, Jorge O.Escobedo, Shan Jiang, Isiah M.Warner, Fareed B.Dawan, KunLian, and Robert M.Strongin.Visual Detection of Cysteine and Homocysteine.J.Am.Chem.Soc.2004,126,438～439.2, Weihua Wang, Oleksandr Rusin, Xiangyang Xu, Kyu Kwang Kim, Jorge O.Escobedo, Sayo O.Fakayode, Kristin A.Fletcher, Mark Lowry, Corin M.Schowalter, Candace M.Lawrence, Frank R.Fronczek, Isiah M.Warner, and Robert M.Strongin.Detection of Homocysteineand Cysteine.J.Am.Chem.Soc.2005,127,15949～15958.), it has such advantage: when fluorescent probe molecule has recognition reaction to bioactive sulfhydryl compound, make fluorescent emission that bigger enhancing be arranged, and can be further used for the fluorescence imaging of bioactive sulfydryl compound in cell, this diagnosis for relative disease in the clinical medicine provides reference.
Summary of the invention
One of purpose of the present invention is to overcome the performance and the structural weak point of prior art fluorescent probe, and a kind of fluorescent probe of selectively detecting bioactive sulfydryl compound in cell of excellent property is provided.
Two of purpose of the present invention provides the synthetic method of the fluorescent probe of selectively detecting bioactive sulfydryl compound in cell.
Three of purpose of the present invention provides the purposes of the fluorescent probe of selectively detecting bioactive sulfydryl compound in cell.
The fluorescent probe of selectively detecting bioactive sulfydryl compound in cell of the present invention has the structure shown in the formula (I):
R in the formula (I)
1, R
2, R
5, R
6Respectively or be hydrogen, alkyl, substituted alkyl or aryl simultaneously; R
3, R
4Respectively or be hydrogen, alkyl, substituted alkyl, aryl or halogen simultaneously; X is nitrate radical, sulfate radical, perchlorate, thiocyanate ion or halogen.
Described R
1, R
2, R
3, R
4, R
5, R
6In alkyl be: the cycloalkyl of the alkyl of 1 to 50 carbon atom or 3 to 50 carbon atoms.
Described R
1, R
2, R
3, R
4, R
5, R
6In substituted alkyl be: the alkyl of the alkyl of 1 to 50 carbon atom that the alkyl of 1 to 50 carbon atom that aryl replaces, ω-hydroxyl replace, 1 to 50 carbon atom of ω-carboxyl substituted or the alkyl of 1 to 50 carbon atom that ω-ester group replaces.
Described R
1, R
2, R
3, R
4, R
5, R
6In aryl be: adjacent, to the alkyl phenyl of, 1 to 50 carbon atom in a position, adjacent, to, a position halogenophenyl, adjacent, to, 1 to 50 carbon atom in a position replaces or unsubstituted alkoxyl phenyl or neighbour, to, a position aminocarbonyl phenyl.
Described R
3, R
4, the halogen among the X is: fluorine, chlorine, bromine or iodine.
Described R
1, R
2, R
3, R
4, R
5, R
6In the alkyl of 1 to 50 carbon atom be selected from methyl respectively or simultaneously, ethyl, propyl group, allyl group, sec.-propyl, butyl, isobutyl-, amyl group, isopentyl, hexyl, 2-methyl amyl, heptyl, 2-methyl hexyl, octyl group, 2-methylheptyl, nonyl, the 2-Methyl Octyl, decyl, 2-methyl nonyl, undecyl, dodecyl, tridecyl, a kind of in tetradecyl and the pentadecyl.
Described R
1, R
2, R
3, R
4, R
5, R
6In the cycloalkyl of 3 to 50 carbon atoms be selected from the cyclopropane base respectively or simultaneously, tetramethylene base, pentamethylene base, a kind of in cyclohexyl and the suberane base.
Described R
1, R
2, R
3, R
4, R
5, R
6In the alkyl of 1 to 50 carbon atom replacing of aryl be selected from arylmethyl, aryl ethyl, arylpropyl respectively or simultaneously, the aryl allyl group, aryl sec.-propyl, aryl butyl, the aryl isobutyl-, aryl amyl group, aryl isopentyl, the aryl hexyl, 2-methyl aryl amyl group, aryl heptyl, 2-methyl aryl hexyl, the aryl octyl group, aryl 2-methylheptyl, aryl nonyl, 2-methyl aryl octyl group, the aryl decyl, 2-methyl aryl nonyl, aryl undecyl, aryl dodecyl, the aryl tridecyl, a kind of in aryl tetradecyl and the aryl pentadecyl.
Described R
1, R
2, R
3, R
4, R
5, R
6In the alkyl of 1 to 50 carbon atom replacing of ω-hydroxyl be selected from ω-hydroxymethyl respectively or simultaneously, ω-hydroxyethyl, ω-hydroxypropyl, ω-hydroxyl sec.-propyl, ω-hydroxybutyl, ω-hydroxyl isobutyl-, ω-hydroxyl amyl group, ω-hydroxyl isopentyl, ω-hydroxyl hexyl, ω-hydroxy-2-methyl amyl group, ω-hydroxyl heptyl, ω-hydroxy-2-methyl hexyl, ω-hydroxyl octyl group, ω-hydroxyl heptyl, ω-hydroxyl nonyl, ω-hydroxy-2-methyl octyl group, ω-hydroxyl decyl, ω-hydroxyl-2-ω-hydroxymethyl nonyl, ω-hydroxyl undecyl, ω-hydroxyl dodecyl, ω-hydroxyl tridecyl, a kind of in ω-hydroxyl tetradecyl and ω-hydroxyl pentadecyl.
Described R
1, R
2, R
3, R
4, R
5, R
6In the alkyl of 1 to 50 carbon atom of ω-carboxyl substituted be selected from ω-carboxyl methyl respectively or simultaneously, ω-carboxy ethyl, ω-carboxyl propyl group, ω-carboxyl sec.-propyl, ω-carboxybutyl, ω-carboxyl isobutyl-, ω-carboxy pentyl, ω-carboxyl isopentyl, ω-carboxyl hexyl, ω-carboxyl-2-methyl amyl, ω-carboxyl heptyl, ω-carboxyl-2-methyl hexyl, ω-carboxyl octyl group, ω-carboxyl heptyl, ω-carboxyl nonyl, ω-carboxyl-2-Methyl Octyl, ω-carboxy decyl, ω-carboxyl-2-ω-carboxyl methyl nonyl, ω-carboxyl undecyl, ω-carboxyl dodecyl, ω-carboxyl tridecyl, a kind of in ω-carboxyl tetradecyl and ω-carboxyl pentadecyl.
Described R
1, R
2, R
3, R
4, R
5, R
6In the alkyl of 1 to 50 carbon atom replacing of ω-ester group be selected from ω-ester group methyl respectively or simultaneously, ω-ester group ethyl, ω-ester group propyl group, ω-ester group sec.-propyl, ω-ester group butyl, ω-ester group isobutyl-, ω-ester group amyl group, ω-ester group isopentyl, ω-ester group hexyl, ω-ester group-2-methyl amyl, ω-ester group heptyl, ω-ester group-2-methyl hexyl, ω-ester group octyl group, ω-ester group heptyl, ω-ester group nonyl, ω-ester group-2-Methyl Octyl, ω-ester group decyl, ω-ester group-2-ω-ester group methyl nonyl, ω-ester group undecyl, ω-ester group dodecyl, ω-ester group tridecyl, a kind of in ω-ester group tetradecyl and ω-ester group pentadecyl.
Described R
1, R
2, R
3, R
4, R
5, R
6In the neighbour, to the alkyl phenyl of, 1 to 50 carbon atom in a position be respectively or simultaneously adjacent, to, a position substituent methyl benzyl, adjacent, a, position is replaced Ethylbenzyl, adjacent, to, a position substituted propyl benzyl or neighbour, a, position is replaced isopropyl benzyl.
Described R
1, R
2, R
3, R
4, R
5, R
6In the neighbour, to, a position halogenophenyl be respectively or simultaneously adjacent, to, a position fluoro benzyl, adjacent, to, a position chloro benzyl, adjacent, to, a position benzyl bromide or neighbour, to, a position benzyl iodide.
Described R
1, R
2, R
3, R
4, R
5, R
6In neighbour, alkoxyl phenyl that 1 to 50 carbon atom in a, position is replaced be respectively or simultaneously adjacent, a, position is replaced methoxybenzyl, adjacent, a, position is replaced ethoxy benzyl or neighbour, a, position is replaced the propoxy-benzyl.
The synthetic method of the renewable fluorescent probe of selectively detecting bioactive sulfydryl compound in cell of the present invention may further comprise the steps:
1) will have R
1, R
2, R
3, R
4And R
5Substituent tonka bean camphor-3-carbonyl compound is dissolved in the exsiccant organic solvent, refluxes to stir slowly to drip the R that has that is dissolved in the organic solvent down
6Substituent thiosemicarbazide wherein has R
1, R
2, R
3, R
4And R
5Substituent tonka bean camphor-3-carbonyl compound with have a R
6The mol ratio of substituent thiosemicarbazide is 1: 1～4; 1～4 hour after-filtration of stirring reaction is removed organic solvent, gets part after the vacuum-drying;
2) part that step 1) is obtained is dissolved in the exsiccant organic solvent, slowly drips the HgX that is dissolved in the organic solvent
2(X is NO
3 -, SO
4 2-, ClO
4 -, SCN
-Or F
-, Cl
-, Br
-, I
-), wherein part and HgX
2Mol ratio be 1: 1～2; The backflow stirring reaction gets the orange solids precipitation after 4～16 hours, filter the final vacuum drying solid and obtain orange crystal, the fluorescent probe of formula promptly of the present invention (I).
Described substituent R
1, R
2, R
3, R
4, R
5, R
6, X definition with described in the above-mentioned summary of the invention.
Described organic solvent is selected from methylene dichloride, trichloromethane, dimethyl sulfoxide (DMSO), N, dinethylformamide, 1, more than one in 2-ethylene dichloride, methyl alcohol, ethanol, ether, acetonitrile, acetone, benzene and the toluene etc.
The renewable fluorescent probe of selectively detecting bioactive sulfydryl compound in cell of the present invention can be used for the detection of bioactive sulfhydryl compound in the living things system, the analyzing and testing and the fluorescence imaging of the bioactive sulfhydryl compound in biological viable cell and the living tissue detect, and the detection of bioactive sulfhydryl compound in the pathological tissues on the clinical medicine.
The invention provides a kind of be parent with the tonka bean camphor, be used for the renewable fluorescent probe of selectively detecting bioactive sulfydryl compound in cell.This fluorescent probe is to reduced glutathion, halfcystine, acetylcysteine, homocystine, bioactive sulfhydryl compounds such as gsh have good selectivity, other amino acid, protein and enzyme are to detecting not influence, and the concentration of fluorescence intensity of solution and bioactive sulfhydryl compound is 1 * 10
-6M to 1 * 10
-5In the scope of M certain linear is arranged, shown favorable actual application; This series fluorescent probe molecule is simple in structure, and synthetic method is simple and efficient is high, easily is extended to actual detected.
Description of drawings
Fig. 1. species Ser (Serine) in the fluorescent probe of the embodiment of the invention 1 (I-1) pair cell, Met (methionine(Met)), Lys (Methionin), Arg (arginine), Gly (glycine), Glu (L-glutamic acid), Asp (aspartic acid), Phe (phenylalanine), His (Histidine), Pro (proline(Pro)), Cys (halfcystine), AMP (single adenosine phosphate), ATP (Triphosaden), Mantose (seminose), BSA (bovine serum albumin), the selectivity identification of Glucose (glucose); Wherein:
Fig. 1 a is the fluorescence spectrum of species effect in fluorescent probe and the cell; Can find out that from fluorescence curve (top the figure) fluorescent probe (I-1) has very single-minded recognition effect to Cys (halfcystine); Can find out from fluorescence curve (below the figure),, (be followed successively by from top to bottom: Ser (Serine) species in other cells, Met (methionine(Met)), Lys (Methionin), Arg (arginine), Gly (glycine), Glu (L-glutamic acid), Asp (aspartic acid), Phe (phenylalanine), His (Histidine), Pro (proline(Pro)), AMP (single adenosine phosphate), ATP (Triphosaden), Mantose (seminose), BSA (bovine serum albumin), Glucose (glucose)) no recognition effect;
Fig. 1 b is the fluorescence intensity column diagram after the species effect in 520nm place fluorescent probe (I-1) pair cell.
Fluorescent probe (I-1) is to fluorescent probe (I-1) cycle index after the identification of biological sulfhydryl compound and the relation of fluorescence intensity in Fig. 2 a. embodiment of the invention 1.
The recyclability photo of fluorescent probe (I-1) after biological sulfhydryl compound is discerned in Fig. 2 b. embodiment of the invention 1.
Fig. 3. the fluorescence intensity of fluorescent probe in the embodiment of the invention 1 (I-1) is to the linear relationship (little figure is the linear dependence that match obtains) of bioactive sulfhydryl compound semicystinol concentration.
Bioactive sulfhydryl compound imaging photo in Fig. 4 a, Fig. 4 b. embodiment of the invention 1 in fluorescent probe (I-1) pair cell; Wherein Fig. 4 a is the HepG2 cell under the light field; Fig. 4 b is adding the HepG2 cell fluorescence image of fluorescent probe after 10 minutes, and cell presents very strong fluorescent emission.
Embodiment
With 5.4g 7-N, N-dimethyl tonka bean camphor-3-aldehyde is dissolved in the 10ml exsiccant dimethyl formamide (DMF), slowly drips the 4g thiosemicarbazide and be dissolved in 100ml alcoholic acid solution under refluxing.Stir 3 hours after-filtration, remove dimethyl formamide and ethanol, get part after the vacuum-drying.Then the 5g part is dissolved in the 10ml exsiccant dimethyl sulfoxide (DMSO) (DMSO), slowly drips 3g HgCl
2Be dissolved in the solution of 100ml acetonitrile.Stirring and refluxing 3 hours, obtaining orange bulk crystals is fluorescent probe (I-1).
EI-MS，m/e，561.1[M+1]
+.λ
ab. max/nm＝470nm，Φ＝0.033。
The structure of fluorescent probe (I-1)
Fluorescent probe (I-1) is joined respectively in the interior species of various cells, and by Fig. 1 a, 1b can find out that fluorescent probe (I-1) has very single-minded recognition effect to Cys (halfcystine).
Fluorescent probe is seen Fig. 2 a and Fig. 2 b to the selectivity identification of bioactive sulfhydryl compound Cys (halfcystine), and (from Fig. 2 a, X-coordinate 1 alternately adds biological sulfhydryl compound (X-coordinate 2,4,6,8,10) and HgCl in fluorescent probe (I-1)
2(X-coordinate 3,5,7,9) can cause fluorescence generation alternative " on-off " to change, and also can embody alternately to add biological sulfhydryl compound and HgCl in photo Fig. 2 b
2" on-off " of back solution fluorescence changes.
The fluorescence intensity of fluorescent probe (I-1) is seen Fig. 3 (little figure is the linear dependence that match obtains) to the linear relationship of bioactive sulfhydryl compound semicystinol concentration.
Fluorescent probe (I-1) can carry out cell imaging by the interior bioactive sulfhydryl compound of pair cell, sees Fig. 4 a and Fig. 4 b; Wherein Fig. 4 a is the HepG2 cell under the light field; Fig. 4 b is adding the HepG2 cell fluorescence image of fluorescent probe after 10 minutes, and cell presents very strong fluorescent emission.
Embodiment 2
With 5.6g 7-N, N-diallyl-4-methylcoumarin-3-aldehyde is dissolved in the 10ml exsiccant dimethyl sulfoxide (DMSO) (DMSO), and slowly Dropwise 5 g thiosemicarbazide is dissolved in 100ml alcoholic acid solution under refluxing.Stir 3 hours after-filtration, remove dimethyl sulfoxide (DMSO) and ethanol, get part after the vacuum-drying.Then the 5g part is dissolved in the 10ml exsiccant dimethyl sulfoxide (DMSO), slowly drips 4g HgCl
2Be dissolved in the solution of 100ml acetonitrile.Stirring and refluxing 3 hours, obtaining orange bulk crystals is fluorescent probe (I-2).
EI-MS，m/e，621.1[M+1]
+.λ
ab. max/nm＝467nm，Φ＝0.032。
The structure of fluorescent probe (I-2)
Embodiment 3
With 6g 7-N, N-diethyl tonka bean camphor-3-aldehyde is dissolved in the 10ml exsiccant acetonitrile, slowly drips 4g 1-phenyl thiosemicarbazide and be dissolved in 100ml alcoholic acid solution under refluxing, and stirs 3 hours after-filtration, removes acetonitrile and ethanol, gets part after the vacuum-drying.Then the 5g part is dissolved in the 10ml exsiccant dimethyl sulfoxide (DMSO), slowly drips 4g HgCl
2Be dissolved in the solution of 100ml acetone.Stirring and refluxing 3 hours, obtaining orange bulk crystals is fluorescent probe (I-3).
EI-MS，m/e，666.1[M+1]
+.λ
ab. max/nm＝468nm，Φ＝0.031。
The structure of fluorescent probe (I-3)
Embodiment 4
With 5.6g 7-N, N-two (2-methylheptyl)-4-methoxy coumarin-3-aldehyde is dissolved in the 10ml exsiccant dimethyl formamide, slowly Dropwise 5 g 1-phenyl thiosemicarbazide is dissolved in 100ml alcoholic acid solution under refluxing, stir 3 hours after-filtration, remove dimethyl formamide and ethanol, get part after the vacuum-drying.Then the 5g part is dissolved in the 10ml exsiccant dimethyl formamide, slowly drips 4g HgCl
2Be dissolved in the solution of 100ml acetonitrile.Stirring and refluxing 3 hours, obtaining orange amorphous solid is fluorescent probe (I-4).
EI-MS，m/e，864.3[M+1]
+.λ
ab. max/nm＝466nm，Φ＝0.031。
Embodiment 5
With 6g 7-N, N-two (ω-methoxycarbonyl heptyl) amino-4-methoxy coumarin-3-aldehyde is dissolved in the 10mL exsiccant trichloromethane, under refluxing, slowly drip 4g 1-propyl dithiocarbamate Urea,amino-and be dissolved in 100mL alcoholic acid solution, stir 3 hours after-filtration, remove trichloromethane and ethanol, get part after the vacuum-drying.Then the 6.4g part is dissolved in the 10mL exsiccant trichloromethane, slowly drips 4g HgCl
2Be dissolved in the solution of 100mL acetone.Stirring and refluxing 3 hours, obtaining orange oily liquids is fluorescent probe (I-5).EI-MS，m/e，958.2[M+1]
+.λ
ab. max/nm＝467nm，Φ＝0.031。
Embodiment 6
With 5g 7-N, N-diheptyl-4-bromine 6-ethyl coumarin-3-aldehyde is dissolved in the 10ml exsiccant acetone, and slowly Dropwise 5 g 1-propyl dithiocarbamate Urea,amino-is dissolved in 100ml alcoholic acid solution under refluxing, and stirs 4 hours after-filtration, remove acetone and ethanol, get part after the vacuum-drying.Then the 6.5g part is dissolved in the 10ml exsiccant methylene dichloride, slowly drips 4g HgCl
2Be dissolved in the solution of 100ml acetonitrile.Stirring and refluxing 4 hours, obtaining orange solids is fluorescent probe (I-6).
EI-MS，m/e，880.2[M+1]
+.λ
ab. max/nm＝472nm，Φ＝0.028。
The structure of fluorescent probe (I-6)
Embodiment 7
With 5.8g 7-N, N-two (ω-hydroxybutyl) tonka bean camphor-3-aldehyde is dissolved in the 10ml exsiccant trichloromethane, slowly drips 4g 1-propyl dithiocarbamate Urea,amino-and be dissolved in 100ml alcoholic acid solution under refluxing, and stirs 3 hours after-filtration, remove trichloromethane and ethanol, get part after the vacuum-drying.Then the 5.8g part is dissolved in the 10ml exsiccant acetone, slowly drips 6g HgCl
2Be dissolved in the solution of 100ml acetonitrile.Stirring and refluxing 3 hours, obtaining orange oily liquids is fluorescent probe (I-7).
EI-MS，m/e，720.1[M+1]
+.λ
ab. max/nm＝465nm，Φ＝0.034。
The structure of fluorescent probe (I-7)
Embodiment 8
With 5.8g 7-N, N-two (ω-hydroxybutyl) tonka bean camphor-3-methyl ketone is dissolved in the 10ml exsiccant acetonitrile, slowly drips 4g 1-propyl dithiocarbamate Urea,amino-and be dissolved in 100ml alcoholic acid solution under refluxing, and stirs 3 hours after-filtration, remove acetonitrile and ethanol, get part after the vacuum-drying.Then the 5.8g part is dissolved in the 10ml exsiccant dimethyl formamide, slowly drips 6g HgBr
2Be dissolved in the solution of 100ml acetone.Stirring and refluxing 3 hours, obtaining orange solids is fluorescent probe (I-8).
EI-MS，m/e，822.1[M+1]
+.λ
ab. max/nm＝465nm，Φ＝0.021。
The structure of fluorescent probe (I-8)
Embodiment 9
With 7g 7-N, N-two (p-chlorobenzyl) tonka bean camphor-3-aldehyde is dissolved in the 10ml exsiccant dimethyl formamide, slowly drips 8g 1-propyl dithiocarbamate Urea,amino-and be dissolved in 100ml alcoholic acid solution under refluxing, and stirs 3 hours after-filtration, remove dimethyl formamide and ethanol, get part after the vacuum-drying.Then the 6.5g part is dissolved in the 10ml exsiccant acetonitrile, slowly drips 6g HgCl
2Be dissolved in the solution of 100ml acetone.Stirring and refluxing 3 hours, obtaining orange crystal is fluorescent probe (I-9).
EI-MS，m/e，824.1[M+1]
+.λ
ab. max/nm＝459nm，Φ＝0.029。
The structure of fluorescent probe (I-9)
Embodiment 10
With 5.8g 7-N, N-two (to methoxybenzyl)-4-ethoxy coumarin-3-aldehyde is dissolved in the 10ml exsiccant acetonitrile, slowly drips 4g 1-propyl dithiocarbamate Urea,amino-and be dissolved in 100ml alcoholic acid solution under refluxing, and stirs 3 hours after-filtration, remove acetonitrile and ethanol, get part after the vacuum-drying.Then the 5.8g part is dissolved in the 10ml exsiccant dimethyl formamide, slowly drips 6g HgBr
2Be dissolved in the solution of 100ml acetonitrile.Stirring and refluxing 3 hours, obtaining orange solids is fluorescent probe (I-10).
EI-MS，m/e，929.3[M+1]
+.λ
ab. max/nm＝457nm，Φ＝0.021。
The structure of fluorescent probe (I-10)
Embodiment 11
With 5.8g 7-N, N-two (p-chlorobenzyl)-4-oxyethyl group-5-bromine tonka bean camphor-3-aldehyde is dissolved in the 10ml exsiccant dimethyl formamide, under refluxing, slowly drip the 4g thiosemicarbazide and be dissolved in 100ml alcoholic acid solution, stir 3 hours after-filtration, remove dimethyl formamide and ethanol, get part after the vacuum-drying.Then the 5.8g part is dissolved in the 10ml exsiccant dimethyl formamide, slowly drips 6g HgBr
2Be dissolved in the solution of 100ml acetonitrile.Stirring and refluxing 3 hours, obtaining orange solids is fluorescent probe (I-11).
EI-MS，m/e，1019.3[M+1]
+.λ
ab. max/nm＝455nm，Φ＝0.021。
The structure of fluorescent probe (I-11)
Embodiment 12
With 5.8g 7-N, N-dibenzyl-4-methoxy coumarin-3-aldehyde is dissolved in the 10ml exsiccant dimethyl formamide, slowly drips the 4g thiosemicarbazide and be dissolved in 100ml alcoholic acid solution under refluxing, and stirs 3 hours after-filtration, remove dimethyl formamide and ethanol, get part after the vacuum-drying.Then the 5.8g part is dissolved in the 10ml exsiccant toluene, slowly drips 6g HgBr
2Be dissolved in the solution of 100ml acetonitrile.Stirring and refluxing 3 hours, obtaining orange solids is fluorescent probe (I-12).
EI-MS，m/e，831.6[M+1]
+.λ
ab. max/nm＝457nm，Φ＝0.024。
The structure of fluorescent probe (I-12)
Embodiment 13
With 6.1g 7-N, N-dibenzyl-4-methoxy coumarin-3-methyl ketone is dissolved in the 10ml exsiccant acetonitrile, slowly drips the 4g thiosemicarbazide and be dissolved in 100ml alcoholic acid solution under refluxing, and stirs 3 hours after-filtration, remove acetonitrile and ethanol, get part after the vacuum-drying.Then the 6g part is dissolved in the 10ml exsiccant toluene, slowly drips 6g HgCl
2Be dissolved in the solution of 100ml acetonitrile.Stirring and refluxing 3 hours, obtaining orange solids is fluorescent probe (I-13).
EI-MS，m/e，800.2[M+1]
+.λ
ab. max/nm＝458nm，Φ＝0.028。
The structure of fluorescent probe (I-13)
Embodiment 14
With 5.8g 7-N, N-dibenzyl-4-Clocoumarol-3-methyl ketone is dissolved in the 10ml exsiccant dimethyl sulfoxide (DMSO), and slowly Dropwise 5 g thiosemicarbazide is dissolved in 100ml alcoholic acid solution under refluxing, and stirs 3 hours after-filtration, remove dimethyl sulfoxide (DMSO) and ethanol, get part after the vacuum-drying.Then the 7g part is dissolved in the 10ml exsiccant dimethyl sulfoxide (DMSO), slowly drips 6g HgBr
2Be dissolved in the solution of 100ml acetonitrile.Stirring and refluxing 3 hours, obtaining orange solids is fluorescent probe (I-14).
EI-MS，m/e，849.9[M+1]
+.λ
ab. max/nm＝455nm，Φ＝0.025。
The structure of fluorescent probe (I-14)
Embodiment 15
With 7g 7-N, N-two (luorobenzyl)-4-methoxy coumarin-3-aldehyde is dissolved in the 10ml exsiccant dimethyl sulfoxide (DMSO), under refluxing, slowly drip 4g 1-propyl dithiocarbamate Urea,amino-and be dissolved in 100ml alcoholic acid solution, stir 3 hours after-filtration, remove dimethyl sulfoxide (DMSO) and ethanol, get part after the vacuum-drying.Then the 5.8g part is dissolved in the 10ml exsiccant dimethyl sulfoxide (DMSO), slowly drips 6g Hg (SCN)
2Be dissolved in the solution of 100ml acetonitrile.Stirring and refluxing 3 hours, obtaining orange solids is fluorescent probe (I-15).
EI-MS，m/e，884.6[M+1]
+.λ
ab. max/nm＝459nm，Φ＝0.030。
The structure of fluorescent probe (I-15)
Be to be understood that, claims have been summarized scope of the present invention, under the guiding of the present invention's design, it should be appreciated by one skilled in the art that, certain change to the various embodiments of the present invention scheme is carried out all will be covered by the spirit and scope of claims of the present invention.
Claims (9)
1. the fluorescent probe of a selectively detecting bioactive sulfydryl compound in cell is characterized in that, the fluorescent probe of this selectively detecting bioactive sulfydryl compound in cell has the structure shown in the formula (I):
R in the formula (I)
1, R
2, R
5, R
6Respectively or be hydrogen simultaneously, alkyl, the cycloalkyl of 3-50 carbon, neighbour, to, a position halogenophenyl, the neighbour, to, bit amino phenyl or substituted alkyl; R
3, R
4Respectively or be hydrogen simultaneously, alkyl, the cycloalkyl of 3-50 carbon, neighbour,, neighbour, to, a bit amino phenyl, substituted alkyl or halogen to, a position halogenophenyl; X is nitrate radical, perchlorate, thiocyanate ion or halogen;
Described R
1, R
2, R
3, R
4, R
5, R
6In alkyl be selected from methyl respectively or simultaneously, ethyl, propyl group, sec.-propyl, butyl, isobutyl-, amyl group, isopentyl, hexyl, 2-methyl amyl, heptyl, a kind of in 2-methyl hexyl and the octyl group;
Described R
1, R
2, R
3, R
4, R
5, R
6In the cycloalkyl of 3-50 carbon be selected from the cyclopropane base respectively or simultaneously, tetramethylene base, pentamethylene base, a kind of in cyclohexyl and the suberane base;
Described R
1, R
2, R
3, R
4, R
5, R
6In substituted alkyl be selected from allyl group respectively or simultaneously, ω-hydroxymethyl, ω-hydroxyethyl, ω-hydroxypropyl, ω-hydroxyl sec.-propyl, ω-hydroxybutyl, ω-hydroxyl isobutyl-, ω-hydroxyl amyl group, ω-hydroxyl isopentyl, ω-hydroxyl hexyl, ω-hydroxy-2-methyl amyl group, ω-hydroxyl heptyl, ω-hydroxy-2-methyl hexyl and ω-hydroxyl octyl group, ω-carboxyl methyl, ω-carboxy ethyl, ω-carboxyl propyl group, ω-carboxyl sec.-propyl, ω-carboxybutyl, ω-carboxyl isobutyl-, ω-carboxy pentyl, ω-carboxyl isopentyl, ω-carboxyl hexyl, ω-carboxyl-2-methyl amyl, ω-carboxyl heptyl, ω-carboxyl-2-methyl hexyl and ω-carboxyl octyl group, adjacent, right, between position substituent methyl benzyl, adjacent, right, between the position replace Ethylbenzyl, neighbour, right, between position substituted propyl benzyl, the neighbour, right, between the position replace isopropyl benzyl, adjacent, right, between position fluoro benzyl, adjacent, right, between position chloro benzyl, the neighbour, right, between the position benzyl bromide, the neighbour, right, between the position benzyl iodide, adjacent, right, between position methoxyl group displacement benzyl, the neighbour, right, between the position replace ethoxy benzyl or neighbour, right, between the position replace a kind of in the propoxy-benzyl;
Described R
3, R
4, the halogen among the X is: fluorine, chlorine, bromine or iodine.
2. the synthetic method of the fluorescent probe of a selectively detecting bioactive sulfydryl compound in cell according to claim 1 may further comprise the steps:
1) will have R
1, R
2, R
3, R
4And R
5Substituent tonka bean camphor-3-carbonyl compound is dissolved in the exsiccant organic solvent, refluxes to stir slowly to drip the R that has that is dissolved in the organic solvent down
6Substituent thiosemicarbazide wherein has R
1, R
2, R
3, R
4And R
5Substituent tonka bean camphor-3-carbonyl compound with have a R
6The mol ratio of substituent thiosemicarbazide is 1: 1～4; The stirring reaction after-filtration is removed organic solvent, gets part after the vacuum-drying;
2) part that step 1) is obtained is dissolved in the exsiccant organic solvent, slowly drips the HgX that is dissolved in the organic solvent
2, wherein part and HgX
2Mol ratio be 1: 1～2; Get solid precipitation behind the backflow stirring reaction, filter the fluorescent probe that the final vacuum drying solid obtains formula (I);
Described substituent R
1, R
2, R
3, R
4, R
5, R
6, X definition described with claim 1.
3. method according to claim 2 is characterized in that: the described stirring reaction time of step 1) is 1～4 hour.
4. method according to claim 2 is characterized in that: step 2) the described backflow stirring reaction time is 4～16 hours.
5. method according to claim 2, it is characterized in that: described organic solvent is selected from methylene dichloride, trichloromethane, dimethyl sulfoxide (DMSO), N, dinethylformamide, 1, more than one in 2-ethylene dichloride, methyl alcohol, ethanol, ether, acetonitrile, acetone, benzene and the toluene.
6. the fluorescent probe of a selectively detecting bioactive sulfydryl compound in cell is characterized in that, the structure of the fluorescent probe of this selectively detecting bioactive sulfydryl compound in cell is:
7. the synthetic method of the fluorescent probe of a selectively detecting bioactive sulfydryl compound in cell according to claim 6 is characterized in that:
With 6g 7-N, N-two (ω-methoxycarbonyl heptyl) amino-4-methoxy coumarin-3-aldehyde is dissolved in the 10mL exsiccant trichloromethane, under refluxing, slowly drip 4g 1-propyl dithiocarbamate Urea,amino-and be dissolved in 100mL alcoholic acid solution, stir 3 hours after-filtration, remove trichloromethane and ethanol, get part after the vacuum-drying; Then the 6.4g part is dissolved in the 10mL exsiccant trichloromethane, slowly drips 4g HgCl
2Be dissolved in the solution of 100mL acetone; Stirring and refluxing 3 hours obtains the fluorescent probe shown in the formula (I-5).
8. purposes according to the fluorescent probe of claim 1 or 6 described selectively detecting bioactive sulfydryl compound in cell, it is characterized in that: the fluorescent probe of described selectively detecting bioactive sulfydryl compound in cell is used for the detection of living things system bioactive sulfhydryl compound.
9. purposes according to claim 8, it is characterized in that: the described detection that is used for the living things system bioactive sulfhydryl compound, be analyzing and testing and fluorescence imaging detection to the bioactive sulfhydryl compound in biological viable cell and the living tissue, and the detection of bioactive sulfhydryl compound in the pathological tissues on the clinical medicine.
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| CN102702154B (en) * | 2012-05-31 | 2014-08-27 | 西北师范大学 | Receptor compound for colorimetric detection of copper ions and preparation method and application thereof |
| CN105315986A (en) * | 2014-07-04 | 2016-02-10 | 中国科学院大连化学物理研究所 | Fluorescence system with fluorescence intensity insensitive to temperature |
| KR101797865B1 (en) | 2015-12-30 | 2017-11-15 | 한국과학기술원 | A novel coumarin-based compound for detecting biothiols and a detecting composition using the same and a preparing method for the same |
| KR20240029044A (en) * | 2021-07-05 | 2024-03-05 | 도소 가부시키가이샤 | rare earth complexes |
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Non-Patent Citations (3)
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| Ruilong Sheng, et al.A new colorimetric chemosensor for Hg2+ based on coumarin azine derivative.《Sensors and Actuators B》.2007,第128卷507-511. * |
| Weihua Wang, et al.Detection of Homocysteine and Cysteine.《Journal of the American Chemical Society》.2005,第127卷15949-15958. * |
| Weimin Liu, et al.A Water-Soluble "Switching On"Fluorescent Chemosensor of Selectivity to Cd2.《ORGANIC LETTERS》.2007,第9卷(第19期),2329-2382. |
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