CN102344464B - High water-solubility ruthenium metal complex singlet oxygen fluorescent probe, preparation method thereof, and application thereof - Google Patents
High water-solubility ruthenium metal complex singlet oxygen fluorescent probe, preparation method thereof, and application thereof Download PDFInfo
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Abstract
The invention discloses a high water-solubility ruthenium metal complex singlet oxygen fluorescent probe, a preparation method thereof, and an application thereof. According to the invention, a transition metal ruthenium is adopted as a central ion of the ruthenium complex; and anthracene nucleus derivatives with sodium (or potassium) sulfonate groups are adopted as ligands. The ruthenium complex has properties of a singlet oxygen fluorescent probe and a better water-solubility than existing anthracene nucleus derivative ligand ruthenium complexes. The ruthenium complex is suitable for quantitative or qualitative determinations of <1>O2 in neutral or basic system solutions.
Description
Technical field
The present invention relates to singlet oxygen in the aqueous solution (
1o
2) mensuration, be specifically related to a kind of preparation and application thereof of the ruthenium metal complex singlet oxygen fluorescent probe that contains anthracycline derivatives part.
Background technology
Singlet oxygen is a kind of unsettled existence form of oxygen molecule in high energy excited state, and its chemical property is very active, more unstable compared with ground state oxygen, is one of usually said active oxygen species.Content is seldom in normal human body cell for active oxygen species, in healthy human body, in equilibrium state, and the active oxygen producing in these bodies has important effect to vital movement, and they participate in diversified physiological activity in vivo, as the conduction of signal, neurotransmission, the adjusting of muscular tone, wriggling, the adjusting of hematoblastic polymerization and blood pressure height, immune control, learning and memory, the generation of energy, the merisis of cell rule etc.Once but this balance is broken, they just may produce injury effect to human body, cause film lipid peroxidation or degreasing, cause a series of physiological change such as film differential permeability is lost, ion exosmoses in a large number, metabolism disorder, when serious, cause necrocytosis.Singlet oxygen is as a kind of precious reagent in organic chemistry, at many photochemistry and optical-biological reaction, in the processes such as the phototransformation of photodegradation, pollutent, chemoluminescence, organism deterioration by oxidation or even photocarcinogenesis, all playing the part of very important role.In organic synthesis, singlet oxygen makes to introduce oxygen in highly three-dimensional single-minded organic compound and becomes very easy.In living things system, singlet oxygen has great impact to life system.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 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 body, can cause the disease relevant with oxidative damage, 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 generated singlet 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 density lipoprotein components by chemically generated singlet oxygen, J.Biol.Chem., 1993, 268, 18502.c) A.Gomes, E.Fernandes, Jos é L.F.C.Lima, Fluorescence probes used for detection of reactive oxygen species, J.Biochem.Biophys.Methods., 2005, 65, 45.].Singlet oxygen is in the hyperplasia of cell, differentiation, in the regulation and control such as apoptosis, also play an important role, be considered to a kind of new second messenger [(a) R.W.Redmond, I.E.Kochevar, Spatially resolved cellular responses to singlet oxygen, 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, and how, Photochem.Photobiol.Sci., 2002, 1, 1-21.].On the contrary, also can utilize the strong oxidizing property of singlet oxygen to kill the virocyte in body.Scientist has utilized this strong oxidizing property of singlet oxygen to treat tumour, experiment shows that malignant cell obtains and is greater than significantly healthy tissues molecule in conjunction with the ability of the sensitizing agent medicine of singlet oxygen, like this by after illumination, the singlet oxygen that drug molecule produces just can optionally kill tumour cell, this photodynamic therapy technology is with a wide range of applications in the diagnosis of tumour and treatment, be applied at present lower reproductive tract precancerous lesion, carcinoma in situ and late malignant tumour, as the diagnosis at many positions such as carcinoma vulvae and ovarian cancer and treatment [a) D.G.J.E.J.D.Dolmans, R.K.J.Fukumura, timeline:Photodynamic therapy for cancer, Nat.Rev.Cancer, 2003, 3, 380.b) A.P.Castano, P.M.Mroz, R.Hamblin, Photodynamic therapy and anti-tumour immunity.Nat.Rev.Cancer, 2006, 6, 535.c) N.Nishiyama, W.D.Jang, K.Kazunori, Supramolecular nanocarriers integrated with dendrimers encapsulating photosensitizers for effective photodynamic therapy and photochemical gene delivery.New J.Chem., 2007, 31, 1074.d) T.Oba, Photosensitizer nanoparticles for photodynamic therapy.Curr.Bioact.Comp., 2007, 3, 239.].Medically, by methylene blue (methylene blue, MB) photosensitization method, produce
1o
2carry out the photodynamics diagnosis [L.C.Harber, A.S.Fleischer, R.L.Baer, Erythropoietic protoporphyria and photohemolysis.J.Am.Med.Assoc., 1964,189,191.] to Plasma disinfection and tumour.This photodynamic therapy technology is with a wide range of applications in the diagnosis of tumour and treatment.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 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 singlet oxygen targeted cells system in real time, can be in good time the content of singlet oxygen in detection bodies exactly, prevention for some disease, the research of diagnosis and pathology has very important directive significance [K.Tanaka, T.Miura, N.Umezawa, Y.Urano, K.Kikuchi, T.Higuchi, T.Nagano, Rational design of fluorescein-based fluorescence probes, Mechanism-based design of a maximum fluorescence probe for singlet oxygen, J.Am.Chem.Soc., 2001, 123 (11), 2530.].The method for detection of singlet oxygen of having reported at present mainly contains following several:
(1) phosphorimetry.The principle of phosphorimetry is: the singlet oxygen in reactive oxygen species, its energy is higher than ground state oxygen molecule, when electronics returns ground state by excited state transition, with the form of light, releases energy and phosphoresces.The near infrared phosphorescence that it utilizes singlet oxygen self cancellation to produce at 1268nm place detects, and this method is the most direct, and to organism, without infringement, selectivity is high, is considered to detect the standard method of singlet oxygen.But sensitivity is low, detecting signal is weak, cannot be for very low concentrations
1o
2detection [K.Andersen, Z.Cao, P.R.Ogilby, L.Poulsen, I.Zebger, J.Phys.Chem.A.2002,106,8488.].
(2) Chemical Trapping absorption photometry.This method be utilize 9,10-diphenylanthrancene (DPA) with
1o
2characteristic reaction generate the variation that stable endoperoxide causes DPA absorption spectrum, by detecting the variation of DPA absorption spectrum, measure
1o
2this method selectivity is good, remolding sensitivity 1268nm phosphorescence is surveyed high [M.J.Steinbeck, A.U.Khan, M.J.Karnovsky, Extracellular production of singlet oxygen by stimulated macrophages quantified using 9,10-diphenylanthracene and perylene in a polystyrene film, J.Biol.Chem., 1993,268 (21), 15649.].The method has been used to measure the content [a) M.J.Steinbeck of the singlet oxygen in pungency phagocytic cell, A.U.Khan, M.J.Karnovsky, Intracellular singlet oxygen generation by phagocytosing neutrophils in response to particles coated with a chemical trap.J.Biol.Chem., 1992, 267, 13425.b) M.J.Steinbeck, A.U.Khan, M.J.Karnovsky, Extracellular production of singlet oxygen by stimulated macrophages quantified using 9, 10-diphenylanthracene and perylene in a polystyrene film.J.Biol.Chem., 1993, 268, 15649.].But this method is based on absorption spectrum, so sensitivity is still lower.
(3) organic fluorescence probe method.Typical organic fluorescence probe 9,10-Dimethylanthracene (DMA), DMA is a kind of fluorescent chemicals (λ
ex/ λ
em=375/436), can optionally react with singlet oxygen and generate its endoperoxide, fluorescence own disappears.In recent years, utilizing fluorescent signal is that the organic fluorescence probe method that detects means comprises two classes: (a) utilize
1o
2react with the fluoresceins probe molecule specificity with anthracene nucleus, make probe become hyperfluorescenceZeng Yongminggaoyingguang molecule from original non-fluorescent molecule, thereby 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.].The method is short detection time, highly sensitive, but is not suitable for low ph environment and detects in real time.(b) utilize the transmission ofenergy between fluorescent probe molecule, excite probe molecule to send strong delayed fluorescence, and then for detection of
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, Quantum Electron., 2002,32,83.].This class probe mainly contains phthalocyanine pigment and porphyrazine derivative etc., after the energy that receives singlet oxygen, can near 700nm, send fluorescence for detection of.
(4) chemiluminescence probe method.This is the singlet oxygen fluorescence probe [X.H.Li of a class based on Photoinduced Electron transfer mechanism, G.X.Zhang, 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 good selectivity, but poorly water-soluble is unfavorable in living things system
1o
2mensuration.
(5) rare-earth fluorescent probe.The long lifetime fluorescent characteristics of Yuan Jing profit group based on rare-earth fluorescent title complex, prepared series of rare earth fluorescent probe, utilize time resolved fluorescence detection technique to measure singlet oxygen and obtained good effect [Yuan Jingli, Song Bo, Wang Guilan, 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, in ultraviolet region, is measured
1o
2time, living things system is had to damage [B.Song.G.L.Wang.M.Q.Tan.J.L.Yuan.New J.Chem.2005,29,1431.].
We have reported the singlet oxygen fluorescence probe [Y.J.Liu of a rhenium (I) title complex recently, K.Z.Wang, Eur.J.Inorg.Chem., 2008,5214.] and two China's application for a patent for invention [Wang Kezhi, Liu Yanju, Chen Chunhui, the preparation of ruthenium and iridium metal complex singlet oxygen fluorescent probe and application thereof, application number 200810240591.4; Wang Kezhi, Li Qi, Liu Yanju, Yin Hongju, the ruthenium complexe of Hdppz part is as the application of singlet oxygen fluorescence probe, application number 201110159371.0.].Though these title complexs can detect singlet oxygen under excited by visible light, while existing without singlet oxygen, this title complex shows larger background fluorescence, and detection sensitivity and selectivity water-soluble, singlet oxygen still need further improvement.Therefore further preparation has good water solubility, highly sensitive, selectivity good, applied widely, and the singlet oxygen fluorescence probe of excited by visible light has important practical significance.
Summary of the invention
The object of the invention is to develop good water solubility, excited by visible light, background fluorescence is low, selectivity good, highly sensitive novel
1o
2fluorescent probe.
For achieving the above object, technical scheme of the present invention is as follows:
A ruthenium complexe that contains anthracycline derivatives part, its general structure is [RuA
ml
n] X
k, wherein: X is counter ion; L is anthracycline derivatives part, and its structure is suc as formula shown in I; A is N^N assistant ligand; M=0 or 2, n=1 or 3, and m+n=3; K=1 or 2.
In formula I, R
1for the straight or branched alkyl of hydrogen or C1-C10, R
2for the straight or branched alkyl sodium sulfonate base of C1-C10 or the straight or branched alkylsulphonic acid potassium base of C1-C6.
R in above-mentioned ligand L
1be preferably the straight or branched alkyl of hydrogen or C1-C6, the straight or branched alkyl of hydrogen or C1-C4 more preferably, R
2be preferably straight or branched alkyl sodium sulfonate (or potassium) base of C1-C6, more preferably straight or branched alkyl sodium sulfonate (or potassium) base of C1-C4.Described alkyl is methyl, ethyl, propyl group, sec.-propyl, butyl, amyl group, hexyl for example; Described alkyl sodium sulfonate base for example-CH
2sO
3na ,-C
2h
4sO
3na ,-C
3h
6sO
3na ,-C
4h
8sO
3na ,-C
5h
10sO
3na and-C
6h
12sO
3na, described alkylsulphonic acid potassium base for example-CH
2sO
3k ,-C
2h
4sO
3k ,-C
3h
6sO
3k ,-C
4h
8sO
3k ,-C
5h
10sO
3k and-C
6h
12sO
3k.
Above-mentioned A is assistant ligand, and conventional N^N assistant ligand is dipyridyl (bpy) and o-phenanthroline (phen) for example, but is not limited to this two kinds of parts.
Above-mentioned counter ion X mostly is the negatively charged ion of negative monovalence, for example Cl
-, PF
6 -, ClO
4 -, NO
3 -, BF
4 -, CF
3sO
3 -.X can be also negative dianion, for example SO
4 2-.When X is negative univalent anion, k=2; When X is negative dianion, k=1.
Above-mentioned ruthenium complexe [RuA
ml
n] X
kpreparation method as follows:
By hydrate ruthenium trichloride or RuA
2cl
2in solvent, be heated to 100-140 ℃ with ligand L and carry out coordination reaction, as X ≠ Cl
-time also need be cooling after coordination reaction finishes, and add counter anion X stirring reaction certain hour, obtain thick product, then by recrystallization or column chromatography (silica gel or aluminum oxide) separation and purification, obtain ruthenium complexe of the present invention.
Coordination reaction normally under nitrogen protection in solvent back flow reaction 8 hours or longer time, for example, more than 24h; Coordination reaction solvent for use such as ethylene glycol, ethanol, methyl alcohol, DMF, acetonitrile etc.
In coordination reaction, ligand L used can be prepared by following method: under nitrogen protection, part shown in formula (1) and NaH (or KH) are carried out to back flow reaction in organic solvent, be then cooled to room temperature, add sulphonyl lactone or the MO shown in formula (2)
3sC
ih
2iafter Br (i=1-10, M=Na or K) stirring at room certain hour, slowly heat up and carry out back flow reaction again, separate out precipitation, be cooled to suction filtration after room temperature, gained precipitation is carried out to recrystallization, obtain ligand L.The equation of reaction is as follows:
Formula (1) formula (2) L
Or
L
In above-mentioned reaction formula, R
1for the straight or branched alkyl of hydrogen or C1-C10, be preferably the straight or branched alkyl of C1-C6, more preferably the straight or branched alkyl of C1-C4; I is 1~10 integer, is preferably 1~6 integer, more preferably 1~4 integer; M is Na or K.
Ruthenium complexe of the present invention is ion centered by transition metal ruthenium, the anthracycline derivatives of take with sodium sulfonate (or potassium) base is part, the performance with singlet oxygen fluorescence probe, and water-soluble better with respect to existing anthracycline derivatives part ruthenium complexe, be applicable in the aqueous solution of neutral and alkaline system
1o
2qualitatively or quantitatively determine.
The application process of this ruthenium complexe singlet oxygen fluorescence probe is: in neutrality or basic solution, utilize described title complex to catch in system as fluorescent probe
1o
2, the fluorescence intensity of system is significantly strengthened, the fluorometry by excited by visible light is in can detection system
1o
2.Concrete steps are:
1. in the 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, by excited by visible light, measure fluorescence intensity and the absorbancy of system, obtain corresponding fluorescence quantum efficiency, obtain fluorescence quantum efficiency with respect to the typical curve of singlet oxygen concentration;
2. in the neutrality containing described title complex or alkaline buffer solution, add a certain amount of solution to be measured, by excited by visible light, measure fluorescence intensity and the absorbancy of system, calculate its fluorescence quantum efficiency;
3. the value of the fluorescence quantum efficiency recording according to step 2, the typical curve obtaining by step 1 is determined the amount of the singlet oxygen containing in solution to be measured.
In above-mentioned steps 1 and 2, fluorescence quantum efficiency can calculate by following formula:
φ
s=φ
std(A
std/A
s)(I
s/I
std)(η
s/η
std)
2
Wherein, subscript s and std represent respectively system to be measured and reference material, and φ is fluorescence quantum efficiency, and A is the absorbancy of excitation wave strong point, and I is the fluorescence intensity of transmitting, the refractive index that η is solution.With [Ru (bpy)
3]
2+for reference material, the fluorescence quantum efficiency of its aqueous solution is φ
std=0.028, at A
std, I
std, η
stdand η
sin known situation, by measuring system to be measured in the absorbance A of excitation wave strong point
swith fluorescence intensity I
s, can calculate its fluorescence quantum efficiency φ
s.
The present invention adopts ruthenium complexe that the anthracycline derivatives with sodium sulfonate (or potassium) base is part as singlet oxygen fluorescence probe, its good water solubility and fluorescence own is very weak, excited by visible light and react with singlet oxygen after cause fluorescence intensity significantly to strengthen.Its advantage mainly contains:
1, good water solubility, this ruthenium complexe probe is soluble in water, can in various buffered soln, use, and has overcome water-insoluble fluorescent probe and need to add organic solvent to help to dissolve and to measuring the interference bringing, be highly suitable in living things system
1o
2mensuration.
2, there is good selectivity, with other active oxygen species (H
2o
2, OH, ONOO
-) effect fluorescent signal almost unchanged.
3, there is higher singlet oxygen detection sensitivity, detect lower limit and can reach 1.3nM.
4, probe and
1o
2after effect, fluorescence intensity enhancement factor can reach 54.1 times.
5, excited by visible light, has changed the disadvantage of singlet oxygen probe in the past, is more conducive to practical application.
Accompanying drawing explanation
Fig. 1 has shown that singlet oxygen is to title complex [Ru (aip-C in neutral and alkaline system
4h
8-SO
3na)
3] Cl
2(5 * 10
-6m) impact of UV-Vis spectrum spectrum, wherein: (a) be the variation of the uv-visible absorption spectra in the 50mM of pH=7.10 phosphate buffer soln; (b) be the variation of the uv-visible absorption spectra in the 0.1M of pH=10.5 carbonate buffer solution.
Fig. 2 has shown that singlet oxygen is to title complex [Ru (aip-C in neutral and alkaline system
4h
8-SO
3na)
3] Cl
2(5 * 10
-6m) impact of fluorescence emission spectrum, wherein: (a) be the variation of the fluorescence emission spectrum in the 50mM of pH=7.10 phosphate buffer soln; (b) be the variation of the fluorescence emission spectrum in the 0.1M of pH=10.5 carbonate buffer solution.
Fig. 3 has shown the title complex [Ru (aip-C in neutral and alkaline system
4h
8-SO
3na)
3] Cl
2(5 * 10
-6m) relation in fluorescence intensity and system between singlet oxygen concentration, wherein: (a) be singlet oxygen concentration in the 50mM of pH=7.10 phosphate buffer soln and the relation between system fluorescence intensity; (b) be singlet oxygen concentration in the 0.1M of pH=10.5 carbonate buffer solution and the relation between system fluorescence intensity.
Fig. 4 has shown the title complex [Ru (aip-C in neutral and alkaline system
4h
8-SO
3na)
3] Cl
2(5 * 10
-6m) relation in fluorescence quantum efficiency and system between singlet oxygen concentration, wherein: (a) be singlet oxygen concentration in the 50mM of pH=7.10 phosphate buffer soln and the relation between system fluorescence quantum efficiency; (b) be singlet oxygen concentration in the 0.1M of pH=10.5 carbonate buffer solution and the relation between system fluorescence quantum efficiency.
Fig. 5 has shown title complex [Ru (aip-C in the 50mM of neutral pH=7.10 phosphate buffer soln
4h
8-SO
3na)
3] Cl
2(5 * 10
-7m) with the effect of active oxygen species ([ROS]=0.2mM).
Fig. 6 has shown title complex [Ru (aip-C in the 0.1M of pH=10.5 carbonate alkalescence buffered soln
4h
8-SO
3na)
3] Cl
2(5 * 10
-7m) with the effect of active oxygen species ([ROS]=0.2mM).
Embodiment
Below by embodiment, the present invention is further described.
Embodiment 1, part aip-C
4h
8-SO
3na and title complex [Ru (aip-C
4h
8-SO
3na)
3] Cl
2synthetic.
Synthetic route is as follows:
Elementary operation process is as follows:
1, part aip-C
4h
8-SO
3na's is synthetic.
Aip is according to document [M.Mariappan, B.G.Maiya, Effects of anthracene and pyrene units on the interactions of novel polypyridylruthenium (II) mixed-ligand complexes with DNA.Eur.J.Inorg.Chem.2005,2164.] synthetic.
Under nitrogen protection, take NaH (50% is scattered in mineral oil for 0.19g, 7.88mmol), with the normal hexane heavily steaming, wash three times, add 20mL heavily to steam DMF, aip (1.2g, 3.03mmol), reflux three hours.Be cooled to room temperature, add Isosorbide-5-Nitrae-Ding sulphonyl lactone (557mg, 4.24mmol), stirring at room one hour, be more slowly warming up to 110 ℃, and reflux 24 hours, there is orange Precipitation midway.Be cooled to room temperature, suction filtration, obtains thick product.Thick product suitable solvent recrystallization, obtains yellowish pink solid.Product 560mg, productive rate: 33.4%.Hydrogen nuclear magnetic resonance spectrum (δ
h, ppm, 400MHz, D
2o-d
6): 8.97 (s, 1H), 8.80 (s, 1H), 8.27 (3H), 7.64 (3H), 7.22 (s, 1H), 7.04 (2H), 6.84 (4H), 3.78 (2H), 1.96 (2H), 1.41 (2H), 1.06 (2H).Ultimate analysis: C
30h
21n
4naO
3s3.5CH
3oH (FW=652), calculated value C, 61.65; H, 5.36; N, 8.59.Observed value C, 61.38; H, 4.95; N, 9.03.Ground substance assistant laser desorption ionization flight time mass spectrum: m/z=533 ([M-Na
+]
-), m/z=556 ([M+H
+]
+).
2, title complex [Ru (aip-C
4h
8-SO
3na)
3] Cl
2synthetic.
Take aip-C
4h
8-SO
3na (183mg, 0.33mmol) and RuCl
33H
2o (26.1mg, 0.1mmol) is put in 50mL round-bottomed flask, under nitrogen protection, adds methanol/water (20mL/5mL), 100 ℃ of backflow 60h.Be cooled to room temperature, filter, concentrated, obtain thick product.Thick product, through neutral alumina column chromatography, obtains the red product of 62mg, productive rate: 32.7%.Hydrogen nuclear magnetic resonance spectrum (δ
h, ppm, 400MHz, DMSO-d
6): 9.16 (d, 6H), 9.02 (s, 3H), 8.30 (m, 11H), 7.94 (m, 6H), 7.63 (m, 17H), 7.04 (2H), 4.43 (m, 3H), 4.28 (m, 3H), 1.91 (m, 10H), 1.81 (m, 6H), 1.29 (m, 6H).Ground substance assistant laser desorption ionization flight time mass spectrum: m/z=884 ([M-2Cl
-]
2+), m/z=1837 ([M+H
+]
+).
3, title complex [Ru (aip-C
4h
8-SO
3na)
3] Cl
2water-soluble detection.
28 ℃ of [Ru (aeip) that record in pure water of room temperature
3] Cl
2solubleness is less than 3.66 * 10
-6mol/L, [Ru (aip)
3] Cl
2solubleness is less than 3.34 * 10
-6mol/L, and [Ru (aip-C
4h
8-SO
3na)
3] Cl
2solubleness is greater than 1.96 * 10
-5mol/L, illustrates that sulfonic introducing solubleness increases at least 5 times.
The method of embodiment 2, Complex probe singlet oxygen in buffered soln and detection performance
1, with [Ru (aip-C
4h
8sO
3na)
3] Cl
2for example, introduce title complex of the present invention and as fluorescent probe, in neutral and basic solution, detect the method for singlet oxygen and detect performance.
In neutral system
1o
2mensuration: first in the phosphate buffer soln of the pH=7.10 that contains 10mM NaOCl, add title complex, then in this system, add H
2o
2, H
2o
2/ NaOCl system singlet oxygen productive rate in neutral solution is almost 100%[A.M.Held, D.J.Halko, J.K.Hurst, J.Am.Chem.Soc.1978,100,5732-5740.].Along with H
2o
2add, in system, constantly produce
1o
2, produce
1o
2with title complex effect, the absorption of the anthracene nucleus in title complex between 350nm-400nm constantly reduces, prove anthracene nucleus be with
1o
2the active group of reaction, the fluorescence intensity of system strengthens gradually simultaneously.
In alkaline system
1o
2mensuration: containing 10mM Na
2moO
4the carbonate buffer solution of pH=10.5 in add title complex, then in system, add H
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.Biol.Med., 2006,40,1644-1653.].Along with H
2o
2add, system constantly produces
1o
2, produce
1o
2with title complex effect, the absorption of the anthracene nucleus in title complex between 350nm-400nm constantly reduces, prove anthracene nucleus be with
1o
2the active group of reaction, the fluorescence intensity of system strengthens gradually simultaneously.
Title complex all has good detection performance to singlet oxygen in neutral and basic solution, and referring to Fig. 1~Fig. 5, the singlet oxygen under neutrallty condition is by H
2o
2/ NaClO system produces at the 50mM of pH=7.10 phosphate buffer soln; In alkaline environment, singlet oxygen can be by H
2o
2/ Na
2moO system produces at the carbonate buffer solution of the 0.1M of pH=10.5.Measuring is GBC Cintra 10e ultraviolet-visible spectrophotometer and Cary Eclipse spectrophotofluorometer with instrument.
In neutral (alkalescence) buffered soln, add H
2o
2system can produce
1o
2, along with
1o
2with the effect of title complex, the absorption (by part internal electron shift π-π * transition produce) of anthracene nucleus part between 350nm-400nm constantly reduces, and (in Fig. 1, arrow direction is as shown in Figure 1
1o
2the direction that concentration increases), prove anthracene nucleus be with
1o
2the active group of reaction, reaction has generated its endoperoxide (shown in following reaction formula), has destroyed the structure of anthracene nucleus, causes its absorption peak strength to reduce.And acromion (434nm left and right, by the part internal electron at main part center, shifting π-π * transition produces) and the metal of 460nm left and right to the absorption peak absorbance variation of part charge transfer singlet state, be not very greatly, and in the process changing, there is not obvious red shift and blue-shifted phenomenon in the absorption peak of the absorption peak peak position of anthracene nucleus part and 470nm left and right.
Do not adding H
2o
2time system fluorescence intensity very weak, along with
1o
2the increase of concentration, the fluorescence intensity at 600nm place strengthens gradually, and (in Fig. 2, arrow direction is as shown in Figure 2
1o
2when the direction that concentration increases), having singlet oxygen and existing without singlet oxygen in neutrality and alkaline buffer solution, fluorescence intensity ratio is respectively 54.1 and 2.47.Title complex [Ru (aip-C
4h
8sO
3na)
3] Cl
2before singlet oxygen effect, its metal shifts to there is energy between part charge transfer triplet state and the triplet state of anthracene nucleus, causes fluorescence to be quenched, but after singlet oxygen effect, due to the generation of endoperoxide, energy shifts and is blocked, and therefore sends stronger fluorescence.And in neutral system, fluorescence intensity level and singlet oxygen concentration value within the specific limits (83.3 μ M~88.5mM) become good linear relationship, linearly dependent coefficient reaches 0.991, as shown in Fig. 3 (a).
According to following formula, calculate the fluorescence quantum efficiency of this system:
φ
s=φ
std(A
std/A
s)(I
s/I
std)(η
s/η
std)
2
In above formula, subscript s and std represent respectively system to be measured and reference material, and φ is quantum yield, and A is the absorbancy of excitation wave strong point, and I is fluorescent emission intensity, the refractive index that η is solution.With [Ru (bpy)
3]
2+for reference material, the fluorescence quantum efficiency of its aqueous solution is φ
std=0.028, at A
std, I
std, η
stdand η
sin known situation, by measuring system to be measured in the absorbance A of excitation wave strong point
swith fluorescent emission intensity I
s, can calculate its quantum yield φ
s.Title complex [Ru (aip-C
4h
8sO
3na)
3] Cl
2(5 * 10
-6relation between the quantum yield of the endoperoxide M) generating under neutrality and alkaline condition and singlet oxygen concentration as shown in Figure 4.Be not difficult to find, if using the curve in Fig. 4 as typical curve, can be with [Ru (aip-C
4h
8sO
3na)
3] Cl
2for probe is measured the singlet oxygen concentration in unknown system under neutral and alkaline condition.
In the effect experiment of title complex and active oxygen species, title complex and H
2o
2, OH, ONOO
-it is very little that isoreactivity oxygen species are made used time system fluorescence intensity change, and title complex with
1o
2after effect, the fluorescence of system strengthens greatly, and as shown in Figure 5, this shows title complex pair
1o
2there is good selectivity.
Right under neutral and alkaline condition
1o
2detection minimum concentration, according to three of background standard deviation times of calculating, be respectively 1.6nM and 1.3nM, show this title complex pair
1o
2there is very high sensitivity.
2, with title complex [Ru (aip-C
4h
8sO
3na)
3] Cl
2the method that detects singlet oxygen in solution is identical, and it is as shown in the table that the singlet oxygen of other title complexs mentioned in this article detects performance:
Claims (3)
1. a ruthenium complexe that contains anthracycline derivatives part, its general structure is [RuA
ml
n] X
k, wherein: X is counter ion, and described counter ion are selected from Cl
-, PF
6 -, ClO
4 -, NO
3 -, BF
4 -, CF
3sO
3 -, SO
4 2-, when X is Cl
-, PF
6 -, ClO
4 -, NO
3 -, BF
4 -, CF
3sO
3 -time, k=2; When X is SO
4 2-time, k=1; L is anthracycline derivatives part, and its structure is suc as formula shown in I; A is N^N assistant ligand; M=0, n=3;
In formula I, R
1for the straight or branched alkyl of hydrogen or C1-C4, R
2for the straight or branched alkyl sodium sulfonate base of C1-C4 or the straight or branched alkylsulphonic acid potassium base of C1-C4.
2. the preparation method of ruthenium complexe described in claim 1, hydrate ruthenium trichloride and ligand L are heated to 100-140 ℃ in solvent and carry out coordination reaction, also need be cooling after coordination reaction finishes when X is not Cl-, and add the salt stirring reaction certain hour that contains counter anion X, obtain thick product, then by recrystallization or column chromatographic isolation and purification, obtain described ruthenium complexe.
3. preparation method as claimed in claim 2; it is characterized in that, described ligand L is prepared according to following method: under nitrogen protection, part shown in formula (1) and NaH or KH are carried out to back flow reaction in organic solvent; then be cooled to room temperature, add sulphonyl lactone or the MO shown in formula (2)
3sC
ih
2iafter Br stirring at room certain hour, slowly heat up and carry out back flow reaction again, separate out precipitation, be cooled to suction filtration after room temperature, gained precipitation is carried out to recrystallization, obtain ligand L;
Or
Wherein, R
1straight or branched alkyl for hydrogen or C1-C4; I is 1~4 integer; M is Na or K.
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