CN102807543B - Reagent and method for detecting biological endogenous oxidants - Google Patents
Reagent and method for detecting biological endogenous oxidants Download PDFInfo
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- CN102807543B CN102807543B CN201210181179.6A CN201210181179A CN102807543B CN 102807543 B CN102807543 B CN 102807543B CN 201210181179 A CN201210181179 A CN 201210181179A CN 102807543 B CN102807543 B CN 102807543B
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Abstract
The invention relates to a chemical reagent and a method for detecting biological endogenous oxidants, in particular to a compound shown as a general formula I or II and a method for detecting the biological endogenous oxidants such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) by using the compound. The compound has high selectivity particularly for peroxynitrous acid (ONOO-) and hypochlorous acid (ClO-) and can be applied to a cell microscopic imaging experiment, a fluorescent dye can be generated, and a sensitive, proportional and multi-channel fluorescent signal can be sent.
Description
Technical field
The present invention relates to chemical reagent and detection method for detection of biological endogenous property oxygenant.
Background technology
Conventionally using active oxygen (ROS) and active nitrogen (RNS) as the very little inorganic or organic molecule with strong oxidizing property.There are various forms of ROS and RNS, comprise free radical, as superoxide radical, hydroxyl radical free radical, nitrogen protoxide, nitrogen peroxide and organic hydroperoxide free radical, and non-free radical kind, as hydrogen peroxide, singlet oxygen, ozone, nitrous acid, peroxynitrite and hypochlorous acid.ROS and RNS are the by products of cellular respiration.Under normal operation, ROS and RNS exist with low-down level, and play a significant role in cell signaling.And during oxidative stress, ROS and RNS level sharply raise, this may cause serious harm as protein, lipid and DNA to various biomolecules.Known can excessive generation ROS and RNS in as cardiovascular disorder, inflammatory diseases, metabolic trouble, cancer and central nervous system disease at many human diseasess.Therefore, in the urgent need to can sensitive and optionally measuring, detect or screen the chemical substance of some ROS and RNS, to explain its physiological action in vitro and in vivo.
In various forms of ROS and RNS, peroxynitrite and hypochlorous oxidation capacity are the strongest, meanwhile, detect very crucial for the physiological and pathological effect of clearly explaining them to their highly selective.In nineteen ninety, scientist finds that nitrogen protoxide and the superoxide anion in organism can generate endogenic peroxynitrite (OONO by the addition reaction of nearly diffusion control first
-) [1].Further research discloses, and the cytotoxicity of nitrogen protoxide (NO) is to a great extent by OONO
-mediation [1-3].No matter be in direct mode or indirectly by a series of downstream reaction material, peroxynitrite can be oxidized various substrates, comprise transition metal center, halfcystine, methionine(Met), tryptophane and Histidine etc., or by making tyrosine nitration modified protein.Peroxynitrite can also be induced lipid peroxidation, attacks nucleoside base and the sugar-phosphoric acid ester skeleton [4-9] of DNA.These processes cause protein to lose function, membrane degradation and DNA damage.In the time that cell injury cannot initiatively be repaired, can there is apoptosis or necrosis.Associated now clearly set up [7] between peroxynitrite and various pathological states (as cardiovascular disorder, circulation shock, inflammation, cancer, apoplexy, defeated filling damage, nerve degenerative diseases and diabetes again).Therefore the peroxynitrite level that, accurately detects cell for medical diagnosis on disease and related pathologies physiological probe into significant.
Peroxynitrite can be nitrated free or protein in tyrosine, therefore, can measure OONO by the method for the immunohistology of cell nitrotyrosine
-level [10-11].But, this method defectiveness.First tyrosine can be by other mechanism by nitrated [12-15], and this can cause actual measured value higher.Meanwhile, the method cannot provide high-resolution space time information.Based on luminous, especially the technology based on fluorescence becomes the routine techniques means of biology, medical field gradually.The fluorescent probe of using in research is in early days as selectivity very poor [16-21] such as dihydro dichlorofluorescein (DHDCF) and diacetic acid esters derivative and dihydro rhodamines (DHR).Therefore, while understanding these datas, must obtain carefully, to avoid get the wrong sow by the ear [22].Chemiluminescence probe also has identical problem [23-25].A few class OONO are reported recently
-the agent of small molecules fluorescence imaging, comprise rhodamine B hydroazide[26], HPF/APF[27], NiSPY series [28], HKGreen series [29-31], tetramethyl ethylene ketone boric acid ester [32-33], BzSeO-Cy[34-35] and CyPSe[36].Wherein commercialization of HPF and APF., HKGreen series is compared with DHR with DHDCP, and selectivity increases, and in some biomedical researches, be applied [38-39].But, (comprise ClO for hyperoxia voltinism material
-and HO
.) highly selective remain problem.In addition, hyperchannel probe allows automatic calibration, and has avoided the complicacy [40-41] due to decolouring, inhomogeneous probe load and inhomogeneous excite etc.Obviously, be necessary to develop many signalling channels Small-molecule probe.
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Summary of the invention
The invention provides the fluorescent probe of a class novelty, to measure, to detect or to screen active nitrogen (RNS) or active oxygen (ROS), as
1o
2, O
2 -, NO, H
2o
2, OH, OCl
-, ONOO
-with alkyl peroxide free radical (ROO
.) etc.Described probe is especially to peroxynitrite (ONOO
-) and hypochlorous acid (ClO
-) there is highly selective, can provide responsive, proportional-type, multichannel fluorescent signal, and can be applicable in the experiment of cell micro-imaging.
Particularly, fluorescent probe of the present invention has the structure shown in general formula I:
In formula,
R
1, R
2, R
3, R
4, R
5, R
7, R
8and R
9independently be selected from separately: hydrogen, C1-C6 alkyl, aryl, cyano group, sulfonic group, sulfahydantoin, halogen, alkenyl, amide group, acyloxy, alkoxy carbonyl, alkoxyl group, alkylaryl, hydroxyalkyl, alkylthio, acyl group (for example formyl radical), amino, hydroxyl, sulfydryl, aryloxy (for example phenoxy group, benzyloxy), alkoxy aryl, alkynyl, formamyl, nitro, the alkyl (for example trifluoromethyl) that halogen replaces, low alkyl group sulfo-, low-grade alkyl amino, carbonyl, or alkylsulfonyl;
R
6oH, SH, NH
2, NHR or N(R)
2;
R is independently selected from H, alkyl, alkoxyl group, alkenyl, alkynyl and aryl.
In one embodiment, R
1, R
2, R
3and R
4independently be selected from separately: hydrogen, C1-C6 alkyl, acyl group, cyano group, sulfonic group; sulfahydantoin, halogen, alkenyl, amide group, acyloxy; alkoxy carbonyl, alkoxyl group, hydroxyalkyl, alkylthio, acyl group (for example formyl radical); amino, hydroxyl, sulfydryl, alkynyl; formamyl, nitro, the alkyl (for example trifluoromethyl) that halogen replaces, lower alkoxy; low alkyl group sulfo-, low-grade alkyl amino, carbonyl, or alkylsulfonyl.
In one embodiment, R
1, R
2and R
3independently be selected from separately the C1-C6 alkyl of H, C1-C6 alkyl, alkenyl, alkynyl, halogen, C1-C6 alkoxyl group, amino, hydroxyl, halogen replacement and the C1-C6 alkoxyl group that halogen replaces.
In one embodiment, R
1, R
2, R
3and R
4independently be selected from separately H, C1-C6 alkyl.
In one embodiment, R
5, R
7, R
8and R
9independently be selected from separately: hydrogen, C1-C6 alkyl, cyano group, sulfonic group; sulfahydantoin, halogen, alkenyl, amide group; acyloxy, alkoxy carbonyl, alkoxyl group, hydroxyalkyl; alkylthio, acyl group (for example formyl radical), amino, hydroxyl; sulfydryl, alkynyl, formamyl, nitro; the alkyl (for example trifluoromethyl) that halogen replaces, low alkyl group sulfo-, low-grade alkyl amino, carbonyl or alkylsulfonyl.
In one embodiment, R
5, R
7, R
8and R
9independently be selected from separately H, halogen and C1-C6 alkyl.
In one embodiment, R
9be selected from H, halogen and C1-C6 alkyl.
In one embodiment, R
6be selected from H, halogen, hydroxyl, amino, NHR and N (R)
2.In other embodiments, R
6be selected from hydroxyl, amino and NHR and N (R)
2.
In one embodiment, R is selected from C1-C6 alkyl.
In one embodiment, R
1, R
3, R
4, R
5, R
8for H, R
2for C1-C6 alkyl, R
6for hydroxyl, amino and N (R)
2, R
7and R
9independent is separately H and halogen, and R is C1-C6 alkyl.
In one embodiment, compound of Formula I of the present invention is following compound 1-5:
The present invention also comprises the compound of general formula I I:
In formula, R
1-R
8identical with the definition in formula I.
In a specific embodiment of formula II, R
1, R
2, R
3and R
4independently be selected from separately H, C1-C6 alkyl.
In a specific embodiment of formula II, R
5, R
7and R
8independently be selected from separately H, halogen and C1-C6 alkyl.
In a specific embodiment of formula II, R
6be selected from H, halogen and hydroxyl, amino.In other embodiments, R
6for hydroxyl.
In one embodiment, general formula I I compound is following compound 1a:
The present invention also comprises following compound 1b:
Described compound 1b can be used as fluorescence dye and uses, such as, for the common application scope of fluorescence dye, fluorescent mark, cell dyeing, cell imaging etc.
In the present invention, general formula I and II compound can be used for detection of active oxygen and/or active nitrogen.
Therefore, the invention provides active oxygen (ROS) in a kind of measure sample and the method for active nitrogen (RNS), it is characterized in that, said method comprising the steps of:
A) make general formula I of the present invention and/or II compound contact sample, to form fluorescent chemicals; With
B) measure the fluorescent characteristic of described fluorescent chemicals.
The invention provides a kind of active oxygen (ROS) in sample and high flux screening fluorescent method of active nitrogen (RNS) of detecting, wherein, described high throughput method comprises the following steps:
A) make general formula I of the present invention and/or II compound contact sample to form one or more fluorescent chemicalses; With
B) fluorescent characteristic of measuring described fluorescent chemicals is to determine active oxygen (ROS) in sample and the concentration of active nitrogen (RNS).
In detection method of the present invention, described active oxygen is selected from:
1o
2, O
2 -, ROO
.,
.oH, ClO-and H
2o
2; Described active nitrogen is selected from nitrogen protoxide, nitrogen peroxide, nitrous acid and peroxynitrite.
It is a kind of for measuring, detect or screen the fluorescent probe composition of active oxygen (ROS) and active nitrogen (RNS) that the present invention also provides, and it comprises general formula I of the present invention and/or II compound.
Brief description of the drawings
Fig. 1 shows fluorescence excitation and the emmission spectrum of compound 1, intermediate 1a and product 1b.
Fig. 2 shows in the phosphate buffered saline buffer with compound 1(50mM, pH7.4,25 μ M) detection OONO
-, ClO
-and HO
.fluorescence spectrum research.Wherein, A-1, B-1 and C-1 show respectively, and when the fluorescent emission at monitoring 620nm place, excitation spectrum is with the OONO of difference amount
-(A-1), ClO
-and HO (B-1)
.(C-1) variation; A-2, B-2 and C-2 are presented at respectively the intensity OONO of 355nm, 465nm and 575nm place fluorescence excitation spectrum
-(A-2), ClO
-and HO (B-2)
.(C-2) situation that equivalent changes; A-3, B-3 and C-3 show that respectively, while exciting with 550nm, fluorescence emission spectrum is with the OONO adding
-(A-3), ClO
-and HO (B-3)
.(C-3) changing conditions; A-4, B-4 and C-4 show that respectively the fluorescent emission intensity at 595nm place is with OONO
-(A-4), ClO
-and HO (B-4)
.(C-4) variation.
Fig. 3 shows the selectivity research of compound 1.Wherein, when A-1, B-1, C-1 and D-1 show respectively the fluorescent emission of monitoring 620nm place, excitation spectrum is with H
2o
2(A),
1o
2(B), NO (C) and O
2 .-variation (D); A-2, B-2, C-2 and D-2 show respectively and add H
2o
2(A),
1o
2(B), NO (C) and O
2 -(D) after, launch the excitation spectrum of gained in 550nm monitoring at solution.
Fig. 4 shows the viable cell micro-imaging that uses compound 1 to carry out, and has shown respectively with (A-B) 20 μ M compd As, (C-D) 20 μ M compound 1 and 200 μ M ClO
-, (E-F) 20 μ M compound 1 and 200 μ M SIN-1 and the differing and fluorescence microscopy image of human glioma cells that (G-H) 5 μ M product 1b process.Scale is 50 μ m.
Fig. 5 shows in the phosphate buffered saline buffer of compound 1a(50mM, pH7.4,25 μ M) in the time that 550nm excites, fluorescence emission spectrum is with added OONO
-the variation (left side) of equivalent, and the fluorescent emission intensity at 595nm place is with OONO
-the variation (right side) of concentration.
Fig. 6 has shown in the phosphate buffered saline buffer of compound 4(50mM, pH7.4,25 μ M) add respectively 15 equivalent OONO in solution
-or 5 equivalent ClO
-after the fluorescent emission curve that obtains.Excitation wavelength is 550nm.
Embodiment
In the application, " alkyl " is often referred to side chain or straight chain saturation alkane base.In some embodiments, alkyl contains 1-10 carbon atom, and in a preferred embodiment, alkyl contains 1-6 carbon atom, and in preferred embodiment, alkyl has 1-4 carbon atom.The example of alkyl has methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, the tertiary butyl, n-heptyl, n-hexyl, n-octyl and positive decyl." low alkyl group " is often referred to the alkyl of individual, preferred 1-4 the carbon atom of 1-6.
" alkoxyl group " refers to the oxygen base being replaced by alkyl, conventionally contains 1-6 carbon atom, preferably contains 1-4 carbon atom.
In the application, " alkenyl " ordinary representation has the univalence hydrocarbyl of at least one two key, conventionally contains 2-8 carbon atom, preferably contains 2-6 carbon atom, can be straight or branched.The example of alkenyl includes but not limited to vinyl, propenyl, pseudoallyl, butenyl, isobutenyl, hexenyl etc.
In the application, " alkynyl " explains the univalence hydrocarbyl with at least one triple bond conventionally, conventionally contains 2-8 carbon atom, preferably contains 2-6 carbon atom, can be straight or branched.The example of alkenyl comprises ethynyl, proyl, isopropyl alkynyl, butynyl, isobutyl alkynyl, hexin base etc.
In the application, " aryl " refers to optional carbocyclic ring or the heteroaromatic group replacing.In some embodiments, aryl is included in monocycle or the bicyclic groups that loop section contains 6-14 carbon atom, as the naphthyl of the xenyl of the phenyl of phenyl, xenyl, naphthyl, replacement, replacement or replacement.In other embodiments, aryl is the phenyl of phenyl or replacement.
" aryloxy " refers to the oxygen base being replaced by aryl.The example of aryloxy includes but not limited to phenoxy group etc.
" aralkyl " refers to the alkyl being replaced by aryl.Some non-limitative examples of aralkyl comprise benzyl and styroyl.
" acyl group " refer to formula-C (=O) H ,-C (=O)-alkyl ,-C (=O)-aryl or-monoradical of C (=O)-aralkyl.
" halogen " refers to fluorine, chlorine, bromine and iodine.
R of the present invention
1-R
9each group in definition is optionally replaced by 1-3 substituting group.These substituent non-limitative examples comprise: halogen, alkyl, alkenyl, alkynyl, aryl, hydroxyl, alkoxyl group, amino, nitro, cyano group, amido, carboxyl, alkylsulfonyl, sulphonamide, haloalkyl are (for example; trifluoromethyl), cycloalkyl (for example, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl), amino, O-low alkyl group, O-aryl, aralkyl etc.
" active oxygen " or ROS represent the ion with oxidisability, free radical and the non-free radical containing aerobic.Some non-limitative examples of active oxygen comprise
1o
2, O
2 -, ROO
.,
.oH, ClO
-and H
2o
2.
" active nitrogen " or RNS represent the ion with oxidisability, free radical and the non-free radical that contain nitrogen.Some non-limitative examples of active nitrogen comprise nitrogen protoxide (NO
.), nitrogen peroxide (NO
2 .), nitrite (NO
2 -) and peroxynitrite (ONOO
-).
" sulfonic group " refer to-SO
3h group.
" sulfahydantoin " refer to-SO
2nRR ' group, wherein R, R ' can be alkyl.
" amide group " refer to-C (O)-NR ' R " group, wherein R, R ' can be alkyl.
" acyloxy " refer to-C (O)-OR-group, wherein R can be alkyl.
The invention provides can be used as fluorescent probe with measure, detect or screening active nitrogen (RNS) if peroxynitrite or active oxygen (ROS) are as hypochlorous general formula I and/or II compound.
In some embodiments, general formula I of the present invention and/or II compound can be used for special detection, measurement or screening peroxynitrite or hypochlorous acid and other ROS, RNS selectively.In further embodiment, general formula I of the present invention and/or II compound can exist other active oxygens and/or active nitrogen as
1o
2, O
2 -, NO, H
2o
2,
.oH, ClO
-, ONOO
-during with alkyl peroxide free radical (ROO), react with peroxynitrite or hypochlorous acid selectively.
The present invention also comprises the fluorescent probe composition that comprises general formula I of the present invention and/or II compound.In some embodiments, fluorescent probe composition described herein also comprises the conventional all kinds of SOLVENTS in this area, acid, alkali, damping fluid or its combination.
The present invention also provides for directly or indirectly measuring ROS in chemistry or biological sample (as the cell or tissue of microorganism or animal) and/or RNS(as peroxynitrite and/or hypochlorous acid) reagent composition.Described reagent composition comprises fluorescent probe described herein, for example, compound described in general formula I and/or II.In some embodiments, reagent composition also comprises the conventional all kinds of SOLVENTS in this area, acid, alkali, damping fluid or its combination.The concentration of the compound described in composition formula of I and/or II can freely be selected according to practical situation, common concentration range at 1 μ M within the scope of 100 μ M.
The present invention also provides especially peroxynitrite of the RNS(in measure sample) or especially hypochlorous acid of ROS() method.In some embodiments, the method comprises the following steps: (a) make fluorescent probe contact sample described herein to form fluorescent chemicals; (b) measure the fluorescent characteristic of described fluorescent chemicals.In some embodiments, fluorescent characteristic is to adopt methods described herein or any method well known by persons skilled in the art to measure.
The present invention also provides the especially peroxynitrite of RNS(detecting in sample) or ROS(especially hypochlorous acid) high flux screening fluorescent method.In some embodiments, described high flux screening fluorescence comprises the following steps: (a) make fluorescent probe contact sample described herein to form one or more fluorescent chemicalses; (b) measure the fluorescent characteristic of described fluorescent chemicals.
Can implement above-mentioned detection or measuring method with composition of the present invention.Conventionally, in the compounds of this invention contacting with sample or composition, the concentration of the compounds of this invention can freely be selected according to practical situation, common concentration range at 1 μ M within the scope of 100 μ M.
Fluorescent characteristic can adopt methods described herein or any method well known by persons skilled in the art to measure.For example, fluoroscopic examination can be implemented by spectrophotofluorometer or fluorescent microscope.Described fluorescent characteristic can comprise the ratio of the fluorescence intensity between fluorescence intensity or several characteristic wavelength of a certain characteristic wavelength.Can pass through this fluorescent characteristic, judge in solution which kind of reaction has occurred, in judgement sample, have thus which kind of ROS and/or RNS, and/or its concentration.
The sample that is applicable to the inventive method can be various chemical examples or biological sample.Biological sample can be the biological sample from microorganism, animal or plant cell or tissue.Described biological sample is generally vitro samples.
The oxidation mechanism of compound of Formula I of the present invention carries out in two steps.Taking compound 1 and 4 as example, the first step oxidation is to be first oxidized phenol or the aniline ring that cloud density is larger, loses after two electronics, and the amino on coumarin ring removes to carry out nucleophilic attack, form intermediate product, active oxygen reoxidizes intermediate state and obtains final product structure.This detection mechanism is fully verified, the results are shown in Figure 1.R in mutual-through type I
1, R
2, R
3and R
4the structural modification of position can not affect the detection performance of this compounds to different oxygenants.To R
5, R
6, R
7, R
8and R
9modification can make the redox potential of this phenyl ring change, obviously, such structural modification can make this compounds have different selectivity to different oxygenants.
Provide as follows the concrete preparation flow of compound 1 and 1a, the synthetic route of compound 2-5 and other compound and compound 1 are similar.
The compound that compound in general formula I I can be oxidized in corresponding general formula I by different oxygenants obtains.
Below the mode with specific embodiment is described to the present invention.Should be understood that these specific embodiments are only illustrative, the present invention is not limited to these specific embodiments.The all ingredients, the reaction conditions etc. that in embodiment, use except as otherwise noted, otherwise are conventional reagent and the reaction conditions using in this area.
Embodiment 1: the preparation of compound 1
The meta-methoxy phenol (0.45mmol) of the fluoro-4-methylcoumarin of 6-nitro-7-100mg (0.45mmol) and 56mg and the salt of wormwood (0.45mmol) of 62mg join in the acetonitrile of 20ml, return stirring spends the night, then suction filtration, filtrate decompression is spin-dried for to solvent, product separates by chromatographic column, elutriant petrol ether/ethyl acetate mixed solvent (2:1, V/V), obtain yellow solid 82mg, yield 60%.
Get the compound 210mg(0.64mmol that step obtains) in reaction flask, add the 5%Pd/C of catalytic amount, methyl alcohol is made solvent, put into hydrogenating reduction device and react 6h, reaction finishes rear suction filtration, and filtrate decompression is spin-dried for to solvent, very short chromatographic column elimination Pd/C for product, then obtain 181mg faint yellow solid with methylene dichloride recrystallization, productive rate 95%.
Get 180mg reducing compound (0.61mmol), make solvent with dry methylene dichloride, then under ice bath, add 0.5ml BBr
3(0.75mmol), remove ice bath, stirring at normal temperature 6h, reaction finishes rear with methyl alcohol cancellation reaction, then extracts three times with a large amount of methylene dichloride and water, merge organic phase, organic phase anhydrous magnesium sulfate drying, elimination magnesium sulfate, decompression is spin-dried for solvent, obtain the milky crystalline compounds 1 of 158mg, productive rate 92% by methylene dichloride and recrystallizing methanol.
The Analysis and Identification data of compound 1:
1H?NMR(400MHz,DMSO)δ(ppm)7.18(t,J=8.0Hz,1H),7.08(s,1H),6.71(s,1H),6.56(dd,J
1=8.0Hz,J
2=1.6Hz,1H),6.46(dd,J
1=8.0Hz,J
2=2.0Hz,1H),6.41(t,J
1=2.0Hz,J
2=1.6Hz,1H),6.24(d,J=1.2Hz,1H),3.81(s,3H),2.50(s,3H);
13C?NMR(100MHz,DMSO)δ(ppm)160.7,159.9,157.5,153.2,146.8,145.4,137.5,130.9,116.3,111.6,109.2,108.9,106.4,105.9,18.7;EI-HRMS(m/z)283.0842;[M+H]+(MW?calcd=283.0845)。
Embodiment 2: the preparation of compound 2
The 4-chloro-3-hydroxyl p-methyl benzenesulfonic acid ester (2.91mmol) of the fluoro-4-methylcoumarin of 6-nitro-7-650mg (2.91mmol) and 870mg and the salt of wormwood (3.00mmol) of 415mg join in the acetonitrile of 40ml, return stirring spends the night, then suction filtration, filtrate decompression is spin-dried for to solvent, product separates by chromatographic column, elutriant petrol ether/ethyl acetate mixed solvent (15:4, V/V), obtain yellow solid 320mg, yield 22%.
Get the compound 200mg(0.40mmol that step obtains) in reaction flask, add the 5%Pd/C of catalytic amount, methyl alcohol is made solvent, put into hydrogenating reduction device and react 6h, reaction finishes rear suction filtration, and filtrate decompression is spin-dried for to solvent, very short chromatographic column elimination Pd/C for product, then obtain 157mg faint yellow solid by recrystallization from ethyl acetate/petroleum ether, productive rate 83%.
Get 100mg compound (0.21mmol) and 11mg(0.27mmol) sodium hydroxide, make solvent with dehydrated alcohol, then back flow reaction 6h, reaction finishes rear use and adds a small amount of saturated NaHCO
3solution neutralization reaction liquid, then adds column chromatography silica gel decompression to be spin-dried for solvent, carries out column chromatography obtain 52mg yellow solid compound 2, productive rate 77% by dichloromethane/ethyl acetate (15:1, V/V).
The data of compound 2:
1h NMR (400MHz, DMSO) δ (ppm) 10.38 (s, 1H), 7.32 (d, J=8.8Hz, 1H), 7.09 (s, 1H), 6.84 (s, 1H), 6.60 (d, J=2.8Hz, 1H), 6.50 (dd, J
1=8.8Hz, J
2=2.8Hz, 1H), 6.27 (d, J=0.8Hz, 1H), 5.17 (s, 2H), 2.37 (s, 3H);
13cNMR (100MHz, DMSO) δ (ppm) 160.7,156.2,154.5,153.2,145.7,145.2,137.8,130.9,116.9,114.8,113.4,110.0,109.5,107.4,106.4,18.7; ESI-HRMS (m/z) 318.0526; [M+H]+(MW calcd=318.0533).
Embodiment 3: the preparation of compound 3
The meta-methoxy phenol (0.45mmol) of the fluoro-4-methylcoumarin of 6-nitro-7-100mg (0.45mmol) and 56mg and the salt of wormwood (0.45mmol) of 62mg join in the acetonitrile of 20ml, return stirring spends the night, then suction filtration, filtrate decompression is spin-dried for to solvent, product separates by chromatographic column, elutriant petrol ether/ethyl acetate mixed solvent (2:1, V/V), obtain yellow solid 82mg, yield 60%.
Get the compound 210mg(0.64mmol that step obtains) in reaction flask, add the 5%Pd/C of catalytic amount, methyl alcohol is made solvent, put into hydrogenating reduction device and react 6h, reaction finishes rear suction filtration, and filtrate decompression is spin-dried for to solvent, very short chromatographic column elimination Pd/C for product, then obtain 181mg faint yellow solid compound 3, productive rate 95% with methylene dichloride recrystallization.
The data of compound 3:
1h NMR (400MHz, CDCl
3) δ (ppm) 7.28 (t, J=10.4Hz, 1H), 7.02 (s; 1H), 6.77 (d, J=10.4Hz, 1H); 6.75 (s, 1H), 6.66 (d, J=10.4Hz; 1H), 6.64 (s, 1H); 6.19 (s, 1H), 3.81 (s; 3H), 2.39 (s, 3H);
13c NMR (100MHz, CDCl
3) δ (ppm) 159.1,156.9,154.9,154.4,151.5,145.1,142.1,135.6,123.2,114.4,114.2,112.2,108.9,105.0,56.6,18.6; EI-HRMS (m/z) 297.1003[M+H]+(MW calcd=297.1001).
Embodiment 4: the preparation of compound 4
The m-nitrophenol (2.24mmol) of the fluoro-4-methylcoumarin of 6-nitro-7-500mg (2.24mmol) and 311mg and the salt of wormwood (2.25mmol) of 310mg join in the acetonitrile of 20ml, return stirring spends the night, then suction filtration, filtrate decompression is spin-dried for to solvent, product separates by chromatographic column, eluent ethylacetate mixed solvent, obtains yellow solid 440mg, yield 57%.
Get the compound 140mg(0.41mmol that step obtains) in reaction flask, add the 5%Pd/C of catalytic amount, methyl alcohol is made solvent, put into hydrogenating reduction device and react 6h, reaction finishes rear suction filtration, and filtrate decompression is spin-dried for to solvent, very short chromatographic column elimination Pd/C for product, then obtain 102mg faint yellow solid compound 4, productive rate 88% with ethyl acetate and sherwood oil recrystallization.
The data of compound 4:
1h-NMR (400MHz, CDCl
3) δ (ppm) 7.16 (t, J=8.0Hz, 1H), 6.95 (s, 1H), 6.80 (s, 1H), 6.52 (dd, J
1=8.0Hz, J
2=1.6Hz, 1H), 6.45 (dd, J
1=8.0Hz, J
2=1.6Hz, 1H), 6.39 (s, 1H), 6.20 (s, 1H), 2.39 (s, 3H);
13c NMR (100MHz, CDCl
3) δ (ppm) 161.4,156.5,151.9,148.3,148.2,147.2,134.6,130.7,115.6,113.3,111.4,109.1,108.9,105.9,105.7,18.8; EI-HRMS (m/z) 282.1004[M+H]+(MW calcd=282.1004).
Embodiment 5: the preparation of compound 5
The 3-diethylin phenol (0.90mmol) of the fluoro-4-methylcoumarin of 6-nitro-7-200mg (0.90mmol) and 148mg and the salt of wormwood (0.90mmol) of 116mg join in the acetonitrile of 20ml, return stirring spends the night, then suction filtration, filtrate decompression is spin-dried for to solvent, product separates by chromatographic column, eluent ethylacetate mixed solvent, obtains yellow solid 180mg, yield 55%.
Get the compound 100mg(0.27mmol that step obtains) in reaction flask, add the 5%Pd/C of catalytic amount, methyl alcohol is made solvent, put into hydrogenating reduction device and react 6h, reaction finishes rear suction filtration, and filtrate decompression is spin-dried for to solvent, very short chromatographic column elimination Pd/C for product, then obtain 78mg faint yellow solid compound 5, productive rate 85% with ethyl acetate and sherwood oil recrystallization.
The data of compound 5:
1h-NMR (400MHz, CDCl
3) δ (ppm) 7.18 (t, J=8.0Hz, 1H), 6.92 (s, 1H); 6.74 (s, 1H), 6.51 (d, J=7.6Hz, 1H); 6.38 (s, 1H), 6.28 (d, J=7.6Hz, 1H); 6.14 (s, 1H), 3.97 (s, 2H); 3.33 (q, J=6.4,4H), 2.36 (s; 3H), 1.16 (t, J=6.4,6H); ESI-MS (m/z) 339.5; [M+H]+(MWcalcd=339.2).
Embodiment 6: the preparation of compound 1a and 1b
Get compound 1(0.6mmol) and be dissolved in 20mL methylene dichloride, then slowly dropping is low adds chlorine bleach liquor, runs out of until thin-layer chromatography is determined raw material.Then add 1 Glacial acetic acid and solution is spin-dried for.Separate and obtain compound 1a(106mg, orange solids by column chromatography (methylene dichloride: methyl alcohol=200:1)) and 1b(10 milligram, brown solid).
Compound 1a's
1h NMR (400MHz, CDCl
3): δ 8.02 (s, 1H), 7.43 (d, J=9.6Hz, 1H), 7.24 (s; 1H), 8.87 (dd, J=9.6,1.6Hz, 1H), 6.38 (d; J=1.6Hz, 1H), 6.34 (s, 1H), 2.51 (s, 3H);
13c NMR (100MHz, CDCl
3): δ: 186.2,159.4,155.8,151.5,148.8,148.7,146.2,135.2,134.9,130.2,126.6,118.2,114.9,108.2,104.4,18.9; C
16h
11nO
4eI-MS (m/z) [M-e
-]
+, calculated value 281.3; Actual value 281.1.
Compound 1b's
1h NMR (400MHz, CDCl
3): δ 7.64 (d, J=8.8Hz, 1H), 7.28 (s, 1H), 6.88 (dd, J=8.8,2.0Hz, 1H), 6.76 (s, 1H), 6.31 (s, 1H), 5.96 (d, J=2.0Hz, 1H), 2.51 (s, 3H) .EI-HRMS279.0525[M]
+(MW calculated value=279.2470).
Embodiment 7: utilize compound 1 to OONO
-carry out fluoroscopic examination and selectivity research thereof
The maximum excitation wavelength of compound 1 is at 350nm place, and this is the typical emission wavelength of tonka bean camphor.The emission peak of compound 1 is at 525nm(Fig. 1, A).Oxidation intermediate 1a absorbs at 479nm place, and in 585nm place transmitting (Fig. 1, B).Compared with compound 1, the fluorescence excitation of intermediate 1a has the red shift of 124nm, and transmitting has the red shift of 63nm.Final oxidation products 1b excites and launches further red shift to 576nm and 595nm(Fig. 1, C).Because the excitation maximum of compound 1,1a and 1b fully separates mutually, and simultaneously they to be transmitted in about 600nm place overlapping, therefore can excite near the fluorescence experiments of (355nm, 475nm and 575nm) single transmit (600nm) to monitor this three kinds of materials by one three simultaneously.In following spectral investigation, the inventor has selected the emissive porwer at monitoring 620nm place, to obtain the complete excitation band of product 1b.Or, optionally under the existence of probe 1 and intermediate 1a, excite product 1b, because the maximum excitation wavelength of product 1b is longer.
In the 50mM of the pH7.4 that contains 2.5%DMF phosphate buffered saline buffer, study the selectivity of compound 1 to various oxygenants.As shown in the fluorescence excitation spectrum of the transmitting gained in 620nm monitoring (seeing A-1 and the A-2 of accompanying drawing 2), add OONO
-after, compound 1 consumes fast, and produces intermediate 1a, as shown in the excitation intensity at 350nm and 467nm place.Product 1b at the signal of 575nm also along with OONO
-add and increase; But signal increases not obvious when lower equivalent.Work as OONO
-equivalent exceedes at 15 o'clock, and the signal of product starts sharply to raise, and finally reaches stable.Meanwhile, intermediate signal declines.This experiment is clear has proved that compound 1 is at OONO
-hyperchannel ability in detection.Also the emission scan value only exciting by 550nm is monitored the formation (A-3 of Fig. 2 and A-4) of product.The fluorescent signal of observing 595nm place is to strengthen gradually zero background.Fluorescence intensification factor is very high, and detection sensitivity is high.It should be noted OONO
-pass through H
+or CO
2catalysis is by way of being rearranged into rapidly NO
3 -(t
1/2～1.9s) [7].In addition, product 1b can effectively be excited by 550nm, and provides 595nm transmitting.This makes it can mate widely used TRITC filter group in imaging research, and this fluorophore has the emission bandpass of the logical and 600 ± 20nm of the excitation band of 555 ± 10nm.
In parallel research, the inventor finds ClO
-also can inducing compounds 1 be oxidized to intermediate 1a(Fig. 2, B).But compound 1 can not further be oxidized intermediate and become product 1b, observe product signal because there is no.Add the ClO of about 10 equivalents
-cause the probe signals completely dissolve at 350nm place, the strength of signal of the intermediate at 467nm place is corresponding reaches stable.Additionally add ClO
-produce unconspicuous spectrum change.ClO
-and OONO
-high oxidative capacity be all known.In the time of exploitation fluorescent probe, how to obtain high selectivity for the two is a very large challenge.Above-mentionedly experiment showed, that compound 1 is effectively to distinguish OONO
-and ClO
-fluorescent probe.
Expection is used compound 1 to detect OONO
-time, HO
.can produce and disturb, because found HO
.while being exposed to some baseline fluorescence probe as APF, HPF, HKgreen series (these probes also contain the aromatic portion that is rich in electronics), produce high fluorescent signal.But, what is interesting is, add the Fe (ClO up to 30 equivalents
4)
2solution is to containing excessive H
2o
2compound 1 solution in time, probe signals intensity only has 30% decline (accompanying drawing 2, C).Only find to exist a small amount of intermediate 1a and product 1b.With OONO
-the signal of oxidation is compared, and their strength of signal can be ignored.Trial adds more Fe (ClO
4)
2in this solution, result has produced Fe (OH)
2precipitation, this causes strong scattering of light.Consider maturity and the HO of Fenton chemistry
.strong oxidizing property, the HO producing in inventor's expectation system
.very likely by unpredictalbe by way of and non-oxide this probe (compound 1) this by way of being consumed.
The inventor has also detected other common active oxygen and reactive nitrogen species under the same conditions, comprises hydrogen peroxide (H
2o
2), single oxygen (
1o
2), peroxylradicals negatively charged ion (O
2 -) and nitrogen protoxide (NO), to assess their potential interference effect (Fig. 3).Add in a large number H
2o
2(about 17,500 equivalents),
1o
2(about 50 equivalents) and NO (about 10 equivalents) do not produce diacritic spectrum change in three passages of probe (compound 1), intermediate and product.By ClO
-stoste is added to and contains excessive H
2o
2probe solution in produce
1o
2.In the solution of air saturation, NO is oxidized to N
2o
3, the latter can be by aniline nitrosylation, and nitric oxide production many fluorescent probes (comprise DAF
43and NO
550 44) utilize this reaction.But the nucleophilicity compared with aniline of the amino in compound 1 reduces, this is because the cloud density of the tonka bean camphor core being connected with this amino is lower.Therefore, N
2o
3hydrolysis relatively faster.Because NO is at H
2the lower (～1.9mM of solubleness in O
7), the NO of higher dosage do not detected.Add excessive O
2 -(approximately Isosorbide-5-Nitrae 00 equivalent, is KO
2the form of solid) in probe solution, vigorous stirring, oxygen is overflowed and is stopped latter 5 minutes, has measured the fluorescence of this solution.The pickup electrode the earth that found that probe reduces, and intermediate or product passage all do not increase.This may be the oxidation that probe does not occur, and fluorescence is by due to other machine-processed quencher.This also with report consistent [27,45] in many documents, wherein do not find from O for phenol or the anilino probe of many para-orientation of oxidizing substance
2 -interference.
Embodiment 8: cell research
This experiment end user glioma cell line U87 studies compound 1 in external imaging applications and detects OONO
-ability.Wherein, OONO
-by commercially available OONO
-donor SIN-1(3-morpholine-Si get ketoimine, 3-morpholinosydnonimine[46]) original position generation.Itself does not produce active substance this cell strain, does not exert an influence to detecting, and is therefore suitable for biocompatibility and detection selectivity for assessing compound 1.
Be the biochemical Institute of Cell Biology of U87(by human glioma cells, Chinese Shanghai) cultivate and containing high glucose (Invitrogen, Carlsbad, USA), be supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen) and 1% penicillin/streptomycin (Sigma-Aldrich, St.Louis, USA) DMEM substratum in.Cell is maintained to 37 DEG C, 5%CO
2wetting atmosphere in.Spend IONS OF H
2o dilutes commercially available NaClO solution, preparation ClO
-mother liquor.In DMF, prepare compound 1(100mM) and SIN-1(10mM) mother liquor.Compound 1 (20M) is loaded in cell, hatches 15 minutes.Then use Hank balanced salt solution (D-Hanks) to clean cell, then by ClO
-be added in described cell with SIN-1 mother liquor (up to 200M).Hatch after 12 hours for 37 DEG C, with being housed, standard TRITC filter group (Olympus) closes the eyepiece (CF160 of 20X (0.5 numerical aperture), Nikon) inverted fluorescence microscope (Ti-S, Nikon) obtains fluoroscopic image.
Found that, compound 1 mother liquor is added to after cell culture, the rapid permeate through cell membranes of compound 1 enters cell.As expected, when for TRITC filter composition as time, compound 1(20 μ M) do not produce any background signal (accompanying drawing 4, B).This is very favorable for imaging applications, because the fluorescent signal based on zero background provides the highest contrast gradient, and can and clearly catch by sensitivity.And during MTT detects, compound 1 does not show obvious cytotoxicity in this level yet in 16 hours.Add ClO
-(200 μ M) do not produce any detectable signal (Fig. 4, D) in 12 hours.On the contrary, add SIN-1 (200 μ M) in the cell of compound 1 of par, to produce obvious fluorescence and strengthen (Fig. 4, F) to having added.Finally, loaded product 1b(5 μ M) cell also can be by clearly imaging (Fig. 4, H), this prove through SIN-1 process cell in formed product 1b.These experiment showed, in experiment in vitro that compound 1 is at specificity OONO
-in imaging, there is great application prospect.And dyestuff 1b can be for fluorescent mark, cell dyeing and imaging.
Expection, in the case of using the suitable filter group of the suitable spectral response curve with compound 1 and product 1b, can realize the multi channel imaging of compound 1.
Embodiment 9: utilize compound 1a to OONO
-carry out fluoroscopic examination
In the 50mM of the pH7.4 that contains 2.5%DMF phosphate buffered saline buffer, study compound 1a to OONO
-reactivity.Excite with 550nm, observe fluorescent emission with OONO
-change in concentration (seeing accompanying drawing 5).When lower equivalent, signal increases not obvious.Work as OONO
-equivalent exceedes at 125 o'clock, and the signal of product starts sharply to raise, and finally reaches stable.
Embodiment 10: utilize compound 4 to OONO
-carry out fluoroscopic examination
In the 50mM of the pH7.4 that contains 2.5%DMF phosphate buffered saline buffer, study compound 4 and added respectively 15 equivalent OONO
-with 5 equivalent ClO
-reactivity.Excite with 550nm, observe fluorescent emission with OONO
-with ClO
-change (seeing accompanying drawing 6).Result demonstration, the two can be oxidized probe 4, and generates fluorescence dye.
Claims (8)
1. there is the compound of general formula I or II structure:
In formula,
R
1, R
3, R
4, and R
5independent is separately hydrogen;
R
2be selected from C1-C6 alkyl;
R
6be selected from: OH, NH
2, NHR and N (R)
2;
R
7, R
8and R
9independently be selected from separately: H and halogen; With
R is independently selected from H and C1-C6 alkyl.
2. compound as claimed in claim 1, is characterized in that, in described general formula I or II, and R
2be selected from C1-C4 alkyl.
3. compound as claimed in claim 1 or 2, is characterized in that, in described general formula I or II, and R
7and R
8independently be selected from separately hydrogen.
4. compound as claimed in claim 1 or 2, is characterized in that, in described general formula I, and R
9be selected from H.
5. compound as claimed in claim 1, is characterized in that, in described general formula I, and R
1, R
3, R
4, R
5, R
8for H, R
2for C1-C6 alkyl, R
6for hydroxyl or amino, R
7and R
9independent is separately H;
In described general formula I I, R
1, R
3, R
4, R
5, R
8for H, R
2for C1-C6 alkyl, R
6for hydroxyl or amino, R
7for H or halogen.
6. compound as claimed in claim 1, is characterized in that, described compound is selected from following compound 1-5 and 1a:
7. a detection composition, it comprises the compound described in any one in claim 1-6.
8. the peroxynitrite in measure sample and/or a hypochlorous method, is characterized in that, said method comprising the steps of:
A) make in claim 1-6 described in any one composition contact sample described in general formula I and/or II compound or claim 7, to form fluorescent chemicals; With
B) measure the fluorescent characteristic of described fluorescent chemicals, thereby according to peroxynitrite and/or hypochlorous acid in described fluorescent characteristic working sample.
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