CN101475597B - Preparation and use of ruthenium and iridium metal complex singlet oxygen fluorescent probe - Google Patents

Preparation and use of ruthenium and iridium metal complex singlet oxygen fluorescent probe Download PDF

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CN101475597B
CN101475597B CN2008102405914A CN200810240591A CN101475597B CN 101475597 B CN101475597 B CN 101475597B CN 2008102405914 A CN2008102405914 A CN 2008102405914A CN 200810240591 A CN200810240591 A CN 200810240591A CN 101475597 B CN101475597 B CN 101475597B
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singlet oxygen
title complex
fluorescence
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CN101475597A (en
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王科志
刘艳菊
陈春晖
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Beijing Normal University
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Abstract

The present invention discloses the preparation and use of ruthenium and iridium complex singlet oxygen fluorescent probe. The complexes use transition metal ruthenium or iridium as central ions, anthracene derivatives as ligands and undergo greater changes in fluorescence performance before and after the reaction with singlet oxygen in a solution. According to the changes of a fluorescence signal of the complexes, detection of the singlet oxygen in the solution can be realized. When used in the detection the singlet oxygen, the complexes have the advantages of visible light excitation, good selectivity, high flexibility, simple detection means, etc. The complexes are suitable for detection of the singlet oxygen in a neutral or alkaline system.

Description

The preparation of ruthenium and iridium metal complex singlet oxygen fluorescent probe and application thereof
Technical field
The present invention relates to singlet oxygen in the solution ( 1O 2) mensuration, specifically a class contains the preparation and the application thereof of the ruthenium and the iridium metal complex singlet oxygen fluorescent probe of anthracycline derivatives part.
Background technology
Singlet oxygen is a kind of unsettled existence form that oxygen molecule is in high energy excited state, singlet oxygen is as a kind of precious reagent in the organic chemistry, at many photochemistry and optical-biological reaction, in the processes such as phototransformation, chemoluminescence, organism deterioration by oxidation or even photocarcinogenesis as photodegradation, pollutent, all playing the part of crucial role.In organic synthesis, singlet oxygen makes that introducing oxygen in highly three-dimensional single-minded organic compound becomes very easy.In living things system, singlet oxygen has great influence to the life system, and the physiology oxygenizement of singlet more and more receives investigator's concern.Singlet oxygen plays an important role in cell injury and apoptosis, it may cause the strong oxidizing property damage to body, thereby cause the generation of body lipid peroxidation, cause microbial film, arteriole, DNA, the damage of protein and central nervous system, accelerate the old and feeble and dead of body, therefore be considered to important toxicity species in the body, cause and the oxidative damage diseases associated, as cataract, pulmonary edema, diabetes, amyotrophy, nutritive deficiency, [(a) K.Briviba such as psychosis and tumour germinating, L.O.Klotz, H.Sies, Toxic and signaling effects of photochemically or chemically generatedsinglet oxygen in biological systems, Biol.Chem., 1997,378,1259.b) J.R.Wagner, P.A.Motchnik, R.Stocker, H.Sies, B.N.Ames, The oxidation of blood plasma and low densitylipoprotein components by chemically generated singlet oxygen, J.Biol.Chem., 1993,268,18502.].Singlet oxygen is in proliferation of cells, differentiation, also play an important role in the regulation and control such as apoptosis, be considered to a kind of new second messenger [(a) R.W.Redmond, I.E.Kochevar, Spatially resolved cellular responses to singletoxygen, Photochem.Photobiol., 2006,82 (5), 1178-1186. (b) N.L.Oleinick, R.L.Morris, I.Belichenko, The role of apoptosis in response to photodynamic therapy:what, where, why, andhow, Photochem.Photobiol.Sci., 2002,1,1-21.].On the contrary, people also can utilize the strong oxidizing property matter of singlet oxygen to remove to kill malignant cell or tissue, reach the purpose of curing cancer.Experiment finds, when malignant cell obtains and in conjunction with the ability of singlet oxygen sensitizers medicine during significantly greater than healthy tissues, after illumination, the singlet oxygen that drug molecule produces is kill tumor cell optionally just.Medically the singlet oxygen that produces with the photosensitive method of methylene blue comes the photodynamics diagnosis to blood plasma sterilization and tumour, and this photodynamic therapy technology is with a wide range of applications in the diagnosis of tumour with in treating.Singlet oxygen and human health and disease are closely related, are the focuses of current life science and chemical science crossing research.
Because singlet oxygen has consequence like this in photochemistry and photo bio process, 1O 2Detection receives much attention, particularly in the living things system 1O 2Detection more and more cause the concern of researcher.The small molecules fluorescent probe of the singlet oxygen that research and development highly sensitive and selectivity are good is significant, it can provide the important space distributed intelligence of biomacromolecule in the singlet oxygen targeted cells system in real time, can be in good time the content of singlet oxygen in the detection bodies exactly, prevention for some disease, the research of diagnosis and pathology all has crucial directive significance [K.Tanaka, T.Miura, N.Umezawa, Y.Urano, K.Kikuchi, T.Higuchi, T.Nagano, Rational design of fluorescein-based fluorescenceprobes, Mechanism-based design of a maximum fluorescence probe for singlet oxygen, J.Am.Chem.Soc., 2001,123 (11), 2530.].The method that being used to of having reported at present detected singlet oxygen mainly contains following several: (1) phosphorimetry, detect the standard method of singlet oxygen, the near infrared phosphorescence that utilizes singlet oxygen self cancellation to produce at the 1268nm place detects, this method does not have infringement to organism, the selectivity height, but sensitivity is low, detecting signal weak, can't be used for very lower concentration 1O 2Detection [K.Andersen, Z.Cao, P.R.Ogilby, L.Poulsen, I.Zebger, J.Phys.Chem.A.2002,106,8488.].(2) chemistry is caught absorption photometry, utilizes 9,10-diphenylanthrancene (DPA) with 1O 2Characteristic reaction generate the variation that stable endoperoxide causes the DPA absorption spectrum, measure by the variation that detects the DPA absorption spectrum 1O 2This method selectivity is good, though sensitivity is surveyed much higher [M.J.Steinbeck than 1268am phosphorescence, A.U.Khan, M.J.Karnovsky, Extracellular production of singlet oxygen by stimulatedmacrophages quantified using 9,10-diphenylanthracene and perylene in a polystyrene film, J.Biol.Chem., 1993,268 (21), 15649.], but this method is based on absorption spectrum, so sensitivity is still lower.(3) organic fluorescence probe method, utilizing fluorescent signal is that the organic fluorescence probe method that detects means comprises two classes: (a) utilize 1O 2Fluoresceins probe molecule specificity reaction with having anthracene nucleus makes probe become the hyperfluorescence molecule by original non-fluorescent molecule, thereby is used for 1O 2Detection (N.Umezawa, K.Tanaka, Y.Urano, K.Kikuchi, T.Higuchi, T.Nagano, Angew.Chem.Int.Ed.Engl.1999,38,2899; K.Tanaka, T.Miura, N.Umezawa, Y.Urano, K.Kikuchi, T.Higuchi, T.Nagano, J.Am.Chem.Soc.2001,123,2530.).This method is short, highly sensitive detection time, but is not suitable for low ph environment and detection in real time.(b) utilize transmission ofenergy between fluorescent probe molecule, excite probe molecule to send strong delayed fluorescence, and then be used for detecting 1O 2(A.A.Krasnovskii, C.Schweitzer, H.Leismann, C.Tanielian, E.A.Luk ' yanets, Quantum Electron., 2000,30,445; A.A.Krasnovskii, M.E.Bashtanov, N.N.Drozdova, O.A.Yuzhakova, E.A.Luk ' yanets, QuantumElectron., 2002,32,83.).This class probe mainly contains phthalocyanine pigment and porphyrazine derivative etc., can send fluorescence and be used for detecting behind the energy that receives singlet oxygen near 700nm.(4) chemiluminescence probe method, this is singlet oxygen fluorescence probe (X.H.Li, the G.X.Zhang of a class based on the photoinduction electron transport mechanism, H.M.Ma, D.Q.Zhang, J.Li, D.B.Zhu, J.Am.Chem.Soc.2004,126,11543.), this class probe in detecting speed is fast and have very high sensitivity and a good selectivity, but poorly water-soluble is unfavorable for living things system 1O 2Mensuration.(5) rare-earth fluorescent probe, the sharp group of Yuan Jing is based on the long lifetime fluorescent characteristics of rare-earth fluorescent title complex, prepared the series of rare earth fluorescent probe, utilize the time resolved fluorescence detection technique to measure singlet oxygen and obtained effect (Yuan Jingli, Song Bo, Wang Guilan preferably, Tan Mingqian, a kind of singlet oxygen fluorescence probe and application thereof based on europium complex, Chinese invention patent, application number 200510130851.9; Yuan Jingli, Song Bo, Wang Guilan, a kind of singlet oxygen europium coordination compound fluorescent probe and application thereof, Chinese invention patent, application number: 200510045768.1; Yuan Jingli, Song Bo, Wang Guilan, a kind of terbium coordination compound singlet oxygen fluorescent probe and application thereof, Chinese invention patent, application number 200510045767.7), the excitation wavelength of this class title complex is in the ultraviolet region, measures 1O 2The time, living things system there is damage (B.Song.G.L.Wang.M.Q.Tan.J.L.Yuan.New J.Chem.2005,29,1431.).Reported the singlet oxygen fluorescence probe (Y.J.Liu, K.Z.Wang, Eur.J.Inorg.Chem., 2008,5214.) of a rhenium (I) title complex recently.Though this title complex can detect singlet oxygen under excited by visible light, this title complex does not show bigger background fluorescence when having singlet oxygen and existing, and detection sensitivity and selectivity water-soluble, singlet oxygen still need further to improve.Therefore preparation have highly sensitive, selectivity good, and is applied widely, the singlet oxygen fluorescence probe of excited by visible light has important practical significance.
Summary of the invention
The objective of the invention is to develop excited by visible light, background fluorescence is low, and selectivity is good, and highly sensitive is novel 1O 2Fluorescent probe.
Technical scheme of the present invention is as follows:
A kind of ruthenium or complex of iridium that contains the anthracycline derivatives part, its general structure is [MA mL n] X k, wherein: M is ruthenium (Ru) or iridium (Ir); X is a counter anion; L is the anthracycline derivatives part, and its structural formula is suc as formula shown in the I:
Figure G2008102405914D00031
R in the formula 1Straight or branched alkyl for hydrogen or C1～C22; R 2Straight or branched alkyl for hydrogen or C1～C22; When M was Ir, A was C^N part (for example 2-phenylpyridine (ppy) or 7,8-benzoquinoline), m=2, n=1, k=1, i.e. (C^N) 2Ir (L) X; When M was Ru, A was N^N part (for example dipyridyl (bpy) or an o-phenanthroline), m=0 or 2, n=1 or 3, and m+n=3, k=2, i.e. Ru (L) 3X and (N^N) 2Ru (L) X 2
Figure G2008102405914D00041
Ppy 7,8-benzoquinoline bpy o-phenanthroline
Above-mentioned R 1And R 2The straight or branched alkyl of preferred hydrogen or C1～C6, described alkyl is methyl, ethyl, propyl group, sec.-propyl, butyl, amyl group, hexyl for example.Above-mentioned counter anion mostly is negative univalent ion, for example Cl -, ClO 4 -, PF 6 -, NO 3 -, CF 3SO 3 -, BF 4 -
The object lesson of title complex of the present invention is for example:
When M is an iridium, A is ppy, and L is 2-(9-anthryl)-1H-imidazoles [4,5-f] [1,10] phenanthroline (aip) or 2-(9-anthryl)-1-ethyl-1H-imidazoles [4,5-f] [1,10] phenanthroline (aeip), m=2, n=1, X=PF 6 -The time, k=1, the structure of this title complex is as shown in the formula shown in 1a or the 1b:
Figure G2008102405914D00042
When M is a ruthenium, A is bpy, and L is aeip, m=2, n=1, X=Cl -The time, k=2, the structure of this title complex is as shown in the formula shown in the 2a:
When M is a ruthenium, L is aip or aeip, m=0, n=3, X=PF 6 -The time, k=2, the structure of this title complex is as shown in the formula shown in 2b or the 2c:
Figure G2008102405914D00051
Above-mentioned complex of iridium (C^N) 2Ir (L) X can prepare by following method:
1) with C^N part and IrCl 33H 2O is heated to 120-160 ℃ of reaction and obtains dichloro bridge title complex (C^N) in solvent 4Ir 2Cl 2
2) dichloro bridge title complex (C^N) 4Ir 2Cl 2In solvent, be heated to 50-80 ℃ with ligand L and carry out coordination reaction, as X ≠ Cl -The time, also need cool off the back and further add counter anion X stirring reaction certain hour;
3) column chromatographic isolation and purification obtains described complex of iridium.
Above-mentioned steps 1) normally with reactant under nitrogen protection in solvent back flow reaction 18-30 hour; solvent for use is the mixing solutions (volume ratio of ethylene glycol monoethyl ether and water was good with 3: 1) of ethylene glycol monoethyl ether and water for example; reaction times was the best with 24 hours, the dichloro bridge title complex (C^N) that obtains 4Ir 2Cl 2Thick product is standby after ether and water washing again.
Above-mentioned steps 2) normally under nitrogen protection in solvent backflow 4-12 hour; solvent for use is for example in the mixing solutions of methylene dichloride and methyl alcohol (two kinds of solvent volume are good than with 2: 1); reaction times was generally 8 hours, was chilled to add the salt that contains counter ion after the room temperature and carry out stirring reaction.
Above-mentioned steps 3) column chromatography generally adopts silica gel or alumina column chromatography.
Above-mentioned ruthenium complexe (N^N) mRu (L) nX 2The preparation method be: at first with (N^N) mRuCl (3-m/2)In solvent, be heated to 80-180 ℃ with ligand L and carry out coordination reaction, as X ≠ Cl -In time, also need be reacted postcooling and be added counter anion stirring certain hour, uses column chromatography the purification of purifying or recrystallization at last and obtains described ruthenium complexe.
(N^N) mRuCl (3-m/2)With ligand L normally under nitrogen protection in solvent back flow reaction 4-12 hour, solvent for use is ethylene glycol, ethanol, DMF, acetonitrile etc. for example, and the reaction times was generally 8 hours, after reaction finishes, as X ≠ Cl -In time, also need be cooled off the back and added counter anion X stirring reaction certain hour, and thick product is purified through column chromatography (silica gel or alumina column) or recrystallization again.
Of the present invention is central ion with transition metal ruthenium or iridium, is that the title complex of part has the performance of singlet oxygen fluorescence probe with the anthracycline derivatives, is applicable in neutral and the alkaline system 1O 2Qualitative and/or quantitative assay.
This Ru or Ir are that the application process of the singlet oxygen fluorescence probe of central ion is: in neutrality or basic solution, utilize described title complex to catch in the system as fluorescent probe 1O 2And generate the endoperoxide of described title complex with the singlet oxygen effect, and the fluorescence intensity of system significantly strengthens, and the fluorometry by excited by visible light is in can detection architecture 1O 2
Concrete steps are:
1. in known neutrality that can effectively produce singlet oxygen or alkaline system, add title complex of the present invention, under a series of singlet oxygen concentration conditions, measure the fluorescence intensity and the absorbancy of system by excited by visible light, obtain corresponding fluorescence quantum efficiency, obtain the typical curve of fluorescence quantum efficiency with respect to singlet oxygen concentration;
2. add a certain amount of solution to be measured in neutrality that contains described title complex or alkaline buffer solution, fluorescence intensity and absorbancy by excited by visible light mensuration system calculate its fluorescence quantum efficiency;
3. the value of the fluorescence quantum efficiency that records according to step 2, the typical curve that obtains by step 1 are determined the amount of the singlet oxygen that contains in the solution to be measured.
Fluorescence quantum efficiency can calculate by following formula in the above-mentioned steps 1 and 2:
φ s＝φ std(A std/A s)(I s/I std)(η s/η std) 2
Wherein, subscript s and std represent system to be measured and reference material respectively, and φ is a fluorescence quantum efficiency, and A is the absorbancy of excitation wave strong point, and I is an emitted fluorescence intensity, and η is the refractive index of solution.With [Ru (bpy) 3] 2+Be reference material, the fluorescence quantum efficiency of its aqueous solution is φ Std=0.028, at A Std, I Std, η StdAnd η sUnder the known situation, by measuring the absorbance A of system to be measured in the excitation wave strong point sWith fluorescence intensity I s, can calculate its fluorescence quantum efficiency φ s
The present invention a little less than a series of background fluorescences have been synthesized in design on the basis of the singlet oxygen detection method of having reported, excited by visible light, and generate its endoperoxide fluorescence intensity with singlet oxygen reaction back and strengthen the big singlet oxygen fluorescence probe of amplitude.
Fluorescent probe of the present invention has following advantage:
1. be applicable in neutral and the alkaline system 1O 2Mensuration.
2. have higher singlet oxygen detection sensitivity, the lowest detection lower limit can reach 9.9nM.
3. has selectivity preferably, other active oxygen species (H 2O 2, OH, ONOO -) cause that system fluorescent signal enhanced amplitude is very little.
With 1O 2Effect back fluorescence intensity enhancement factor is big, reaches as high as 65 times, and quantum yield strengthens can reach 100 times.
Description of drawings
Fig. 1 has illustrated that singlet oxygen is to title complex Ru (aeip) in neutral and alkaline system 3(PF 6) 2(9.6897 * 10 -6M) influence of absorption spectrum, wherein: (a) be the variation of the absorption spectrum in the 50mM of pH=7.0 phosphate buffer soln; (b) be the variation of the absorption spectrum in the 0.1M of pH=9.95 carbonate buffer solution.
Fig. 2 has illustrated that singlet oxygen is to title complex Ru (aeip) in neutral and alkaline system 3(PF 6) 2(9.6897 * 10 -6M) influence of fluorescence spectrum, wherein: (a) be the variation of the fluorescence spectrum in the 50mM of pH=7.0 phosphate buffer soln; (b) be the variation of the fluorescence spectrum in the 0.1M of pH=9.95 carbonate buffer solution.
Fig. 3 has illustrated the title complex Ru (aeip) in neutral and alkaline system 3(PF 6) 2(9.6897 * 10 -6M) relation in fluorescence intensity and the system between the singlet oxygen concentration, wherein: (a) be singlet oxygen concentration in the 50mM of pH=7.0 phosphate buffer soln and the relation between the system fluorescence intensity; (b) be singlet oxygen concentration in the 0.1M of pH=9.95 carbonate buffer solution and the relation between the system fluorescence intensity.
Fig. 4 has illustrated the title complex Ru (aeip) in neutral and alkaline system 3(PF 6) 2(9.6897 * 10 -6M) relation between fluorescence quantum efficiency and the singlet oxygen concentration, wherein: be in the 50mM of pH=7.0 phosphate buffer soln (a); (b) be in the 0.1M of pH=9.95 carbonate buffer solution.
Fig. 5 illustrated neutral and alkaline system in title complex Ru (aeip) 3(PF 6) 2(9.6897 * 10 -6M) with active oxygen species (effect of [ROS]=30mM), wherein: be in the 50mM of pH=7.0 phosphate buffer soln (a); (b) be in the 0.1M of pH=9.95 carbonate buffer solution.
Embodiment
The present invention is further described below by embodiment.
Embodiment one, part aeip's is synthetic:
Under nitrogen protection; with the mineral oil among the anhydrous n-hexane flush away NaH 52mg (content about 50%), aip (358.2mg, 0.9mmol) [M.Mariappan; B.G.Maiya; Eur.J.Inorg.Chem.2005,2164-2173.], 10ml DMF; 100 ℃ of heating; reacted 1 hour, and stopped heats cold, add monobromethane 24.6mg (0.22mmol) to room temperature; continuation was 100～110 ℃ of reactions 24 hours; be chilled to room temperature, filter, will be dissolved in methylene dichloride behind the filtrate evaporate to dryness; refilter and remove insolubles; thick product silica gel column chromatography, (20: 1, v/v) drip washing obtained product to dichloromethane methanol.Productive rate 40%.Nmr analysis: 1H NMR (δ H, ppm, 500MHz, d 6-DMSO): 9.11～9.15 (m, 2H); 8.89～8.97 (m, 3H); 8.29 (d, J=8.5Hz, 2H); 7.63～7.85 (m, 2H); 7.62 (t, J=6.7Hz, 2H); 7.49～7.54 (m, 4H); 4.28～4.32 (m, 2H); 1.18 (t, J=7Hz, 3H). ultimate analysis: C 29H 20N 40.25H 2O: calculated value: C, 81.25; H, 4.82; N, 13.06. measured value: C, 81.41; H, 4.54; N, 12.08.
Embodiment two, (ppy) 2Ir (aip) PF 6, (ppy) 2Ir (aeip) PF 6Synthetic:
Under nitrogen protection, with IrCl 3.3H 2(0.75mmol, 116.4mg) (0.3mmol, 97.68mg), three water of 10ml ethylene glycol monoethyl ether and 3ml join in the there-necked flask O, are heated to back flow reaction 24 hours with 2-phenylpyridine (Hppy).Be cooled to room temperature, filter and obtain solid, water, ether are washed successively, obtain yellow solid product.It is iridium dichloro endo compound (ppy) 2Ir (μ-Cl) 2Ir (ppy) 2Dichloro endo compound (0.05mmol) and part (aip or aeip) (0.1mmol), methylene dichloride 12ml and methyl alcohol 6ml reacted 6 hours under nitrogen protection, were chilled to room temperature and added 5 times of normal NH 4PF 6Stirred 2 hours, underpressure distillation removes and desolvates, and solid dissolves with methylene dichloride, removes by filter insolubles, and methylene dichloride is removed in the filtrate decompression distillation, gained solid silica gel column chromatography, and (1: 80, v/v) drip washing obtained product to the acetone methylene dichloride.Productive rate is respectively: 68%; 65%.
(ppy) 2Ir (aip) PF 6Nmr analysis: 1H NMR (δ H, ppm, 500MHz, d 6-DMSO): 8.98 (s, 2H); 8.31 (d, J=8.26Hz, 4H); 8.21 (s, 2H); 8.11 (t, J=3.01Hz, 2H); 7.99 (t, J=7.69Hz, 4H); 7.81 (d, J=8.67Hz, 2H); 7.61 (m, 6H); 7.06 (m, 6H); 6.35 (d, J=7.44Hz, 2H). ultimate analysis: C 49H 32N 6PF 6IrC 4H 14O 2Calculated value: C, 56.03; H, 4.08; N, 7.40. measured value: C, 56.03; H, 4.104; N, 7.384. ground substance assistant laser desorption ionization flight time mass spectrum: m/Z=897, ([M-PF 6 -] +)
(Ppy) 2Ir (aeip) PF 6Nmr analysis: 1H NMR (δ H, ppm, 500MHz, d 6-DMSO): 9.23 (t, J=9.4Hz, 2H); 9.02 (s, 1H), 8.32 (m, 6H); 8.11 (dd, J 1=16Hz, J 2=2.5Hz, 2H); 7.96 (dd, J 1=8Hz, J 2=8Hz, 4H); 7.52 (m, 8H); 7.08 (t, 4H); 6.99 (d, J=7.5Hz, 2H); 6.34 (t, 2H); 4.37 (m, 2H); 1.20 (t, 3H). ground substance assistant laser desorption ionization flight time mass spectrum: m/Z=926.2, (M+H +). ultimate analysis: C 51H 36N 6PF 6Ir calculated value: C, 57.24; H, 3.39; N 7.85. measured value: C, 56.98; H, 3.33; N, 7.61.
Embodiment three, Ru (bpy) 2(aeip) Cl 2Synthetic:
Under nitrogen protection, with aeip (84.9mg, 0.2mmol), Ru (bpy) 2Cl 22H 2(104.1mg, 0.2mmol), the 10ml ethylene glycol solution was 140 ℃ of reactions 6 hours for O.Be cooled to room temperature, underpressure distillation removes and desolvates, and the solid methanol dissolving adds dioxane then and separates out oily matter, and inclining solvent, the oily matter dissolve with ethanol, and alumina column chromatography, (20: 1, v/v) drip washing obtained product to dichloromethane methanol, productive rate: 45%.Nmr analysis: 1H NMR (δ H, ppm, 500MHz, d 6-DMSO): 9.12 (d, J=8Hz, 2H); 8.93 (m, 5H); 8.67 (t, 2H); 8.25 (s, 2H); 8.16 (m, 4H); 7.95 (m, 2H); 7.89 (d, J=5.2Hz, 2H); 7.69 (m, 4H); 7.63 (d, J=6.4Hz, 2H); 7.55 (d, J=7.2Hz, 2H); 7.47 (d, J=4Hz, 4H) .4.38 (m, 2H); 1.20 (t, 3H). ultimate analysis: C 49H 36N 8RuC L2C 2H 6O 24H 2OC 4H 8O 23CH 3OH calculated value: C, 56.77; H, 5.75; N, 9.13. measured value: C, 56.41; H, 5.69; N, 9.78.
Embodiment four, Ru (aip) 3(PF 6) 2And Ru (aeip) 3(PF 6) 2Synthetic:
(1) Ru (aip) 3(PF 6) 2Synthetic
RuCl 3.3H 2(0.1mmol, 0.0261g) (0.33mmol 0.132g) joins in the 30mL ethylene glycol O, and nitrogen protection refluxed 8 hours down, was cooled to room temperature, added 10 times of normal NH with part aip 4PF 6Stirred 2 hours, separate out a large amount of red precipitates, filter and obtain red solid, ether is washed, solid is removed insolubles with second eyeball dissolution filter, solution spreads recrystallization with ether, separates out the solid silicone column chromatography, methanol acetone (3: 1, v/v) drip washing obtains product, obtains red solid 74mg after the vacuum-drying.Productive rate: 46.8%. 1H NMR (δ H, ppm, 500MHz, d 6-DMSO): 8.97 (d, 6H); 8.66 (dd, 12H); 7.69 (12H); 7.51 (t, 6H); 7.39 (t, 6H). ultimate analysis: C 81H 48F 12N 12P 2Ru10H 2O10CH 3CN6CH 3OH (FW=2363), calculated value: C, 54.38; N, 13.04; H, 5.20. observed value: C, 54.50; N, 13.03; H, 5.02. ground substance assistant laser desorption ionization flight time mass spectrum: m/z=1289 ([M-2PF 6 --H +] +).Infrared spectra (KBr compressing tablet, cm -1): 3443.3 (w); 1635.7 (s); 843.3 (s).
(2) Ru (aeip) 3(PF 6) 2Synthetic
RuCl 3.3H 2(0.2mmol, 0.0522g) (0.66mmol 0.280g) joins in the 30mL ethylene glycol O, and nitrogen protection refluxed 8 hours down, was cooled to room temperature, added 10 times of excessive NH with part aeip 4PF 6, stir 2h, filter and obtain red precipitate.DMF-ether diffusion recrystallization is separated out the solid silicone column chromatography, dichloromethane methanol (10: 1, v/v) drip washing obtains thick product, (30: 1, v/v) drip washing obtained product to products obtained therefrom recycle silicon plastic column chromatography dichloromethane methanol, obtains red solid 62mg after the vacuum-drying.Productive rate: 21.3%. 1H NMR (δ H, ppm, 500MHz, d 6-DMSO): 9.17 (d, 6H); 9.03 (s, 3H); 8.27 (12H); 7.95 (6H); 7.64 (18H); 7.39 (t, 6H); 4.41 (6H); 1.25 (t, 9H). ultimate analysis C 87H 60N 12F 12P 2Ru4H 2O: calculated value: C, 60.17; H, 3.95; N, 9.68. observed value: C, 60.18; H, 4.375; N, 10.38. ground substance assistant laser desorption ionization flight time mass spectrum: m/z=1374 ([M-2PF 6 --H] +).Infrared spectra (KBr compressing tablet, cm -1): 3443.5 (w), 3044.4 (s), 2977.8 (s), 2918.5 (s), 1660.5 (s), 841.0 (s).
Embodiment five
With Ru (aeip) 3(PF 6) 2Be example, introduce title complex of the present invention and in neutral and basic solution, detect the method for singlet oxygen and detect performance as fluorescent probe.
In the neutral system 1O 2Mensuration: at first in the phosphate buffer soln of the pH=7.0 that contains 10mM NaOCl, add title complex, in this system, add H then 2O 2, H 2O 2/ NaOCl system singlet oxygen productive rate in neutral solution almost be 100% (A.M.Held, D.J.Halko, J.K.Hurst, J.Am.Chem.Soc.1978,100,5732-5740.).Along with H 2O 2Adding, constantly produce in the system 1O 2, produced 1O 2With the title complex effect, the absorption of the anthracene nucleus in the title complex between 350nm-400nm constantly reduces, prove anthracene nucleus be with 1O 2The reactive activity group, the system fluorescence intensity strengthens gradually simultaneously.
In the alkaline system 1O 2Mensuration: containing 10mM Na 2MoO 4The carbonate buffer solution of pH=9-11 in add title complex, in system, add H then 2O 2, H 2O 2/ Na 2MoO 4System can effectively produce singlet oxygen (K.Tanaka, T.Miura, N.Umezawa, Y.Urano, K.Kikuchi, T.Higuchi, T.Nagano, J.Am.Chem.Soc., 2001,123,2530-2536 in basic solution; M.Q.Tan, B.Song, G.L.Wang, J.L.Yuan, Free Radic.Bio1.Med., 2006,40,1644-1653.).Along with H 2O 2Adding, system constantly produces 1O 2, produced 1O 2With the title complex effect, the absorption of the anthracene nucleus in the title complex between 350nm-400nm constantly reduces, prove anthracene nucleus be with 1O 2The reactive activity group, the system fluorescence intensity strengthens gradually simultaneously.
Title complex all has the quite good detecting performance to singlet oxygen in neutral and basic solution, referring to Fig. 1～Fig. 4, the singlet oxygen under the neutrallty condition is by H 2O 2/ NaClO system produces at the 50mM of pH=7.0 phosphate buffer soln; Singlet oxygen can be by H in the alkaline environment 2O 2/ Na 2MoO 4System produces at the carbonate buffer solution of the 0.1M of pH=9.95.Measuring with instrument is GBC Cintra 10e ultraviolet-visible spectrophotometer and Cary Eclipse spectrophotofluorometer.In neutral (alkalescence) buffered soln, add H 2O 2System promptly can produce 1O 2, along with 1O 2With the effect of title complex, the absorption between 350nm-400nm of anthracene nucleus part constantly reduces, as shown in Figure 1, prove anthracene nucleus be with 1O 2The reactive activity group.Do not adding H 2O 2The time system almost do not have fluorescent emission, along with 1O 2The increase of concentration, the fluorescent emission that ties up to the 602nm place at 464nm optical excitation lower body strengthens gradually, and (the arrow direction is among Fig. 2 as shown in Figure 2 1O 2The direction that concentration increases), fluorescence intensity ratio reaches 65 and 27 respectively when having singlet oxygen and no singlet oxygen to exist in the neutral and alkaline solution, and the quantum yield increase reaches 100 and 37 respectively.The fluorescence of system strengthen be since title complex with 1O 2Influence the emission characteristic of fluor thereby effect generates its endoperoxide, send stronger fluorescence thereby changed its luminescent properties.Under the 464nm optical excitation system 602nm place fluorescent emission intensity with 1O 2Relation between the concentration as shown in Figure 3, wherein in the alkaline system 1O 2Concentration 4.8 μ M to the fluorescence intensity of system between the 9.46mM with 1O 2Good linear relationship is arranged between the concentration, and linearly dependent coefficient reaches 0.99943.Calculate the fluorescence quantum efficiency of this system according to following formula:
φ s＝φ std(A std/A s)(I s/I std)(η s/η std) 2
In the following formula, subscript s and std represent system to be measured and reference material respectively, and φ is a quantum yield, and A is the absorbancy of excitation wave strong point, and I is a fluorescent emission intensity, and η is the refractive index of solution.With [Ru (bpy) 3] 2+Be reference material, the fluorescence quantum efficiency of its aqueous solution is φ Std=0.028, at A Std, I Std, η StdAnd η sUnder the known situation, by measuring the absorbance A of system to be measured in the excitation wave strong point sWith fluorescent emission intensity I s, can calculate its quantum yield φ sTitle complex Ru (aeip) 3(PF 6) 2(9.6897 * 10 -6M) as shown in Figure 4 in the quantum yield of the endoperoxide that generates under neutrality and the alkaline condition and the relation between the singlet oxygen concentration.With the curve among Fig. 4 as typical curve, can be with Ru (aeip) 3(PF 6) 2Be probe singlet oxygen concentration in the arbitrary unknown system of mensuration under neutral and alkaline condition.
In the effect experiment of title complex and active oxygen species, title complex and H 2O 2, OH, ONOO -Isoreactivity oxygen species do time spent system fluorescence intensity change very little, and title complex with 1O 2The fluorescence of effect back system strengthens greatly, and as shown in Figure 5, this shows that title complex is right 1O 2Has good selectivity.Right under neutral and alkaline condition 1O 2The detection minimum concentration be respectively 18nM and 27.8nM according to three times of calculating of background standard deviation, show that this title complex is right 1O 2Has very high sensitivity.
Embodiment six
With title complex Ru (aeip) 3(PF 6) 2The method that detects singlet oxygen in solution is identical, and it is as shown in the table that the singlet oxygen of all the other title complexs detects performance:

Claims (7)

1. a ruthenium or complex of iridium that contains the anthracycline derivatives part, its general structure is [MA mL n] X k, wherein: M is Ru or Ir; X is a counter anion; L is the anthracycline derivatives part, and its structural formula is suc as formula shown in the I:
R among the formula I 1And R 2Independent is the straight or branched alkyl of hydrogen or C1～C6; When M was Ir, A was the C^N part, and described C^N part is the 2-phenylpyridine, m=2, n=1, k=1; When M is Ru, m=0, n=3, k=2; Described counter anion is selected from Cl for negative monovalent ion -, ClO 4 -, PF 6 -, NO 3 -, CF 3SO 3 -And BF 4 -In a kind of.
2. title complex as claimed in claim 1 is one of following title complex 1a, 1b, 2b and 2c:
Figure FSB00000570589900012
。
3. complex of iridium (C^N) 2The preparation method of Ir (L) X comprises the following steps:
1) with C^N part and IrCl 33H 2O is heated to 120-160 ℃ of reaction and obtains dichloro bridge title complex (C^N) in solvent 4Ir 2Cl 2, wherein said C^N part is the 2-phenylpyridine;
2) dichloro bridge title complex (C^N) 4Ir 2Cl 2In solvent, be heated to 50-80 ℃ with ligand L and carry out coordination reaction, as X ≠ Cl -The time, also need cool off the back and add counter anion X stirring certain hour, wherein said counter anion X is selected from Cl for negative monovalent ion -, ClO 4 -, PF 6 -, NO 3 -, CF 3SO 3 -And BF 4 -In a kind of; L is the anthracycline derivatives part, and its structural formula is suc as formula shown in the I:
Figure FSB00000570589900021
R among the formula I 1And R 2Independent is the straight or branched alkyl of hydrogen or C1～C6;
3) column chromatographic isolation and purification obtains described complex of iridium.
4. ruthenium complexe Ru (L) 3X 2The preparation method, with RuCl 3In solvent, be heated to 80-180 ℃ with ligand L and carry out coordination reaction, as X ≠ Cl -In time, need be cooled off the back and added counter anion X and stir certain hour, uses column chromatography purifying or recrystallization at last and purifies and obtain described ruthenium complexe, and wherein said counter anion X is selected from Cl for negative monovalent ion -, ClO 4 -, PF 6 -, NO 3 -, CF 3SO 3 -And BF 4 -In a kind of; L is the anthracycline derivatives part, and its structural formula is suc as formula shown in the I:
Figure FSB00000570589900022
R among the formula I 1And R 2Independent is the straight or branched alkyl of hydrogen or C1～C6.
5. the described title complex of claim 1 is as the purposes of singlet oxygen fluorescence probe.
6. purposes as claimed in claim 5 is characterized in that, utilizes the content of the singlet oxygen in described complexes measuring neutrality or the alkaline solution to be measured, may further comprise the steps:
1) in known neutrality that can effectively produce singlet oxygen or alkaline system, adds the described title complex of claim 1, under a series of singlet oxygen concentration conditions, measure the fluorescence intensity and the absorbancy of system by excited by visible light, obtain corresponding fluorescence quantum efficiency, obtain the typical curve of fluorescence quantum efficiency with respect to singlet oxygen concentration;
2) in neutrality that contains described title complex or alkaline buffer solution, add a certain amount of solution to be measured, measure its fluorescence intensity and absorbancy, obtain its fluorescence quantum efficiency by excited by visible light;
3) according to step 2) value of the fluorescence quantum efficiency that records and the typical curve that step 1) obtains, determine the content of singlet oxygen in the solution to be measured.
7. purposes as claimed in claim 6 is characterized in that: fluorescence quantum efficiency can calculate by following formula described step 1) and 2):
φ s＝φ std(A std/A s)(I s/I std)(η s/η std) 2
Wherein, subscript s and std represent system to be measured and reference material respectively, and φ is a fluorescence quantum efficiency, and A is the absorbancy of excitation wave strong point, and I is an emitted fluorescence intensity, and η is the refractive index of solution.
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