CN102344464A - 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 that contains the ruthenium metal complexes singlet oxygen fluorescence 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, and its chemical property is very active, and is more unstable than ground state oxygen, is one of usually said active oxygen species.Active oxygen species content in normal human body cell seldom is in equilibrium state in the human body of health, and the active oxygen that produces in these bodies has important effect to vital movement; They participate in diversified physiological activity in vivo, like 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.In case but this balance broken, they just possibly produce injury effect to human body, cause peroxo-of film fat or degreasingization, cause a series of physiological change such as film differential permeability forfeiture, ion exosmose in a large number, metabolism disorder, cause necrocytosis when serious.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 like photodegradation, pollutent, all playing the part of crucial role.In organic synthesis, singlet oxygen makes that in highly three-dimensional single-minded organic cpds, introducing oxygen becomes very easy.In living things system, singlet oxygen has great influence to the 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, and it possibly cause the strong oxidizing property damage to body, thereby causes the generation of body lipid peroxidation, causes the damage of microbial film, arteriole, DNA, protein and cns; Accelerate the old and feeble and dead of body, therefore be considered to toxicity species important in the body, can cause and the oxidative damage diseases associated, like [(a) K.Briviba such as cataract, wet lung, mellitus, amyotrophy, nutritive deficiency, psychosis and tumour germinatings; 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 also plays an important role in regulation and control such as proliferation of cells, differentiation, apoptosis, is 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 intravital virocyte.Scientist has utilized this strong oxidizing property of singlet oxygen to treat tumour, experiment show that malignant cell obtains and the ability of sensitizing agent medicine that combines singlet oxygen significantly greater than the healthy tissues molecule, like this through after the illumination; The singlet oxygen that drug molecule produces is kill tumor cell optionally just, and this photodynamic therapy technology is with a wide range of applications in the diagnosis of tumour with in treating, and has been applied to down reproductive tract precancerous lesion, carcinoma in situ and late malignant tumour at present; Diagnosis and treatment [a) D.G.J.E.J.D.Dolmans, R.K.J.Fukumura, timeline:Photodynamic therapy for cancer like many positions such as carcinoma vulvae and ovarian cancers; 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, (methylene blue, MB) the photosensitization method produces with methylene blue
1O
2Come photodynamics diagnosis [L.C.Harber, A.S.Fleischer, R.L.Baer, Erythropoietic protoporphyria and photohemolysis.J.Am.Med.Assoc., 1964,189,191.] to blood plasma sterilization 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 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 is good is significant, and 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 the interior singlet oxygen of detection bodies exactly; Research for prevention, diagnosis and the pathology of some disease all has crucial 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 that being used to of having reported at present detected singlet oxygen mainly contains following several kinds:
(1) phosphorimetry.The principle of phosphorimetry is: be in the singlet oxygen of excited oxygen molecule, its energy is higher than the ground state oxygen molecule, when electronics returns ground state by the excited state transition, releases energy and phosphoresces with the form of light.The near infrared phosphorescence that it utilizes singlet oxygen self cancellation to produce at the 1268nm place detects, and this method is the most direct, and organism is not had infringement, and selectivity is high, is considered to detect the standard method of singlet oxygen.But sensitivity is low, detecting signal is 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.This method is to utilize 9,10-diphenylanthrancene (DPA) with
1O
2Characteristic reaction generate the variation that stable endoperoxide causes the DPA absorption spectrum, measure through the variation that detects the DPA absorption spectrum
1O
2, this method selectivity is good, and 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.].This method has been used to measure content [a) M.J.Steinbeck, A.U.Khan, the M.J.Karnovsky of the singlet oxygen in the pungency phagocytic cell; 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 are a kind of fluorescent chemicals (λ
Ex/ λ
Em=375/436), can optionally generate its endoperoxide with the singlet oxygen reaction, fluorescence own disappears.In recent years, utilizing fluorescent signal is that the organic fluorescence probe method that detects means comprises two types: (a) utilize
1O
2Fluoresceins probe molecule specificity reaction with having anthracene nucleus makes probe become hyperfluorescence property 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 detects in real time.(b) utilize transmission ofenergy between fluorescent probe molecule, excite probe molecule to send strong DF, 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, Quantum Electron., 2002,32,83.].This type probe mainly contains phthalocyanine pigment and tetraazoporphine verivate etc., behind the energy that receives singlet oxygen, can near 700nm, send fluorescence and be used for detecting.
(4) chemiluminescence probe method.This is one type of singlet oxygen fluorescence probe based on the photoinduction electron transport mechanism [X.H.Li, G.X.Zhang, H.M.Ma; D.Q.Zhang, J.Li, D.B.Zhu; J.Am.Chem.Soc.2004,126,11543.]; This type probe in detecting speed is fast and have very high sensitivity and good selectivity, but poorly water-soluble is unfavorable in the living things system
1O
2Mensuration.
(5) rare-earth fluorescent probe.The sharp group of Yuan Jing has prepared the series of rare earth fluorescent probe based on the long lifetime fluorescent characteristics of rare-earth fluorescent title complex, utilizes the time resolved fluorescence detection technique to measure singlet oxygen and has obtained effect [Yuan Jingli preferably; 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 type 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.].
We have reported singlet oxygen fluorescence probe [Y.J.Liu, K.Z.Wang, the Eur.J.Inorg.Chem. of a rhenium (I) title complex recently; 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 be 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, 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 further preparation have good water solubility, 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 good water solubility, excited by visible light, background fluorescence is low, selectivity good, highly sensitive novel
1O
2Fluorescent probe.
For realizing above-mentioned purpose, technical scheme of the present invention is following:
A kind of ruthenium complexe that contains the anthracycline derivatives part, its general structure are [RuA
mL
n] X
k, wherein: X is counter ion; L is the anthracycline derivatives part, and its structure is suc as formula shown in the I; A is the N^N assistant ligand; M=0 or 2, n=1 or 3, and m+n=3; K=1 or 2.
Among the formula I, R
1Be the straight or branched alkyl of hydrogen or C1-C10, R
2Be 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 the 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.Said alkyl is methyl, ethyl, propyl group, sec.-propyl, butyl, amyl group, hexyl for example; Said 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, said 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 an assistant ligand, and N^N assistant ligand commonly used is dipyridyl (bpy) and o-phenanthroline (phen) for example, but is not limited to this two kinds of parts.
Mostly above-mentioned counter ion X is negative univalent negatively charged ion, for example Cl
-, PF
6 -, ClO
4 -, NO
3 -, BF
4 -, CF
3SO
3 -X also can be negative dianion, for example SO
4 2-As X during for negative univalent anion, k=2; As X during for negative dianion, k=1.
Above-mentioned ruthenium complexe [RuA
mL
n] X
kThe preparation method following:
With hydrate ruthenium trichloride or RuA
2Cl
2In solvent, be heated to 100-140 ℃ with ligand L and carry out coordination reaction, as X ≠ Cl
-In time, also need to finish postcooling in coordination reaction, and add counter anion X stirring reaction certain hour, obtains thick product, through recrystallization or column chromatography (silica gel or aluminum oxide) separation and purification, obtains ruthenium complexe of the present invention again.
Coordination reaction normally under nitrogen protection in solvent back flow reaction 8 hours or longer time, for example more than the 24h; The coordination reaction solvent for use is terepthaloyl moietie, ethanol, methyl alcohol, N for example, dinethylformamide, acetonitrile etc.
Used ligand L can prepare through following method in the coordination reaction: under the nitrogen protection, part shown in the formula (1) and NaH (or KH) are carried out back flow reaction in organic solvent, be cooled to room temperature then, sulphonyl lactone or the MO shown in the adding formula (2)
3SC
iH
2iSlowly heat up again behind Br (i=1-10, M=Na or K) the stirring at room certain hour and carry out back flow reaction, separate out deposition, be cooled to suction filtration after the room temperature, the gained deposition is carried out recrystallization, obtain ligand L.The equation of reaction is following:
Formula (1) formula (2) L
Or
L
In the above-mentioned reaction formula, R
1Be 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 a central ion with the transition metal ruthenium; Anthracycline derivatives to have sodium sulfonate (or potassium) base is a part; 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 soln, utilize described title complex to catch in the system as fluorescent probe
1O
2, the fluorescence intensity of system is significantly strengthened, the fluorometry through excited by visible light is in can detection architecture
1O
2Concrete 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 through 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 said title complex or alkaline buffer solution, add a certain amount of solution to be measured, fluorescence intensity and absorbancy through excited by visible light mensuration system calculate its fluorescence quantum efficiency;
3. the amount of the singlet oxygen that the value of the fluorescence quantum efficiency that records according to step 2, the typical curve that obtains through step 1 are confirmed to contain in the solution to be measured.
Fluorescence quantum efficiency can calculate through 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, through 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 adopt the anthracycline derivatives have sodium sulfonate (or potassium) base be the ruthenium complexe of part as singlet oxygen fluorescence probe, its good water solubility and fluorescence own very a little less than, excited by visible light and with the singlet oxygen reaction after cause fluorescence intensity to strengthen significantly.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 the water-insoluble fluorescent probe and need add organic solvent and help dissolving and to the interference that mensuration is brought, be highly suitable in the living things system
1O
2Mensuration.
2, has selectivity preferably, with other active oxygen species (H
2O
2, OH, ONOO
-) effect fluorescent signal no change almost.
3, have higher singlet oxygen detection sensitivity, detect lower limit and can reach 1.3nM.
4, probe and
1O
2Effect back 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, more helps practical application.
Description of drawings
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) influence of ultraviolet-absorption 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) influence 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 the system between the singlet oxygen concentration, wherein: (a) be singlet oxygen concentration and the relation between the system fluorescence intensity in the 50mM of pH=7.10 phosphate buffer soln; (b) be singlet oxygen concentration and the relation between the system fluorescence intensity in the 0.1M of pH=10.5 carbonate buffer solution.
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 the system between the singlet oxygen concentration, wherein: (a) be singlet oxygen concentration and the relation between the system fluorescence quantum efficiency in the 50mM of pH=7.10 phosphate buffer soln; (b) be singlet oxygen concentration and the relation between the system fluorescence quantum efficiency in the 0.1M of pH=10.5 carbonate buffer solution.
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 active oxygen species (effect of [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 active oxygen species (effect of [ROS]=0.2mM).
Embodiment
Through embodiment the present invention is further specified below.
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:
The elementary operation process is following:
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 the nitrogen protection, take by weighing NaH (50% is scattered in the MO for 0.19g, 7.88mmol), it is inferior to give a baby a bath on the third day after its birth with the normal hexane that heavily steams, and add 20mL and heavily steam DMF, aip (1.2g, 3.03mmol), reflux three hours.Be cooled to room temperature, add 1, (557mg, 4.24mmol), stirring at room one hour slowly is warming up to 110 ℃ to 4-fourth sulphonyl lactone again, refluxes 24 hours, has the orange deposition to separate out midway.Be cooled to room temperature, suction filtration gets thick product.Thick product is used the suitable solvent recrystallization, gets 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 by weighing aip-C
4H
8-SO
3Na (183mg, 0.33mmol) and RuCl
33H
2(26.1mg 0.1mmol) is put in the 50mL round-bottomed flask O, under the nitrogen protection, adds methanol (20mL/5mL), 100 ℃ of backflow 60h.Be cooled to room temperature, filter, concentrate, get thick product.Thick product gets the red product of 62mg, productive rate: 32.7% through the neutral alumina column chromatography.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 in pure water, record of room temperature
3] Cl
2Solubleness is less than 3.66 * 10
-6Mol, [Ru (aip)
3] Cl
2Solubleness is less than 3.34 * 10
-6Mol, and [Ru (aip-C
4H
8-SO
3Na)
3] Cl
2Solubleness is greater than 1.96 * 10
-5Mol explains that sulfonic introducing solubleness increases at least 5 times.
The method of embodiment 2, title complex probe singlet oxygen in buffered soln and detection performance
1, with [Ru (aip-C
4H
8SO
3Na)
3] Cl
2Be example, introduce title complex of the present invention detects singlet oxygen in neutral and basic soln as fluorescent probe method and detection performance.
In the neutral system
1O
2Mensuration: at first in the phosphate buffer soln of the pH=7.10 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 is 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 fluorescence intensity of system strengthens gradually simultaneously.
In the alkaline system
1O
2Mensuration: containing 10mM Na
2MoO
4The carbonate buffer solution of pH=10.5 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 soln; M.Q.Tan, B.Song, G.L.Wang, J.L.Yuan, Free Radic.Biol.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 fluorescence intensity of system strengthens gradually simultaneously.
Title complex all has the quite good detecting performance to singlet oxygen in neutral and basic soln, referring to Fig. 1～Fig. 5, the singlet oxygen under the neutrallty condition is by H
2O
2/ NaClO system produces at the 50mM of pH=7.10 phosphate buffer soln; Singlet oxygen can be by H in the alkaline environment
2O
2/ Na
2The MoO system produces at the carbonate buffer solution of the 0.1M of pH=10.5.Measuring uses instrument to be 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 (by part internal electron shift π-π * transition produce) of anthracene nucleus part between 350nm-400nm constantly reduces, and as shown in Figure 1 (the arrow direction does among Fig. 1
1O
2The direction that concentration increases), prove anthracene nucleus be with
1O
2Reactive activity group, reaction have generated its endoperoxide (shown in the following reaction formula), have destroyed the structure of anthracene nucleus, cause its absorption peak strength to reduce.And acromion is (about 434nm; Shifting π-π * transition by the part internal electron at main part center produces) with metal about 460nm be not very greatly to the absorption peak absorbance variation of part charge transfer singlet state; And in the process that changes, tangible red shift and blue shift phenomenon do not appear in the absorption peak peak position and the absorption peak about 470nm of anthracene nucleus part.
Do not adding H
2O
2The time system fluorescence intensity very a little less than, along with
1O
2The increase of concentration, the fluorescence intensity at 600nm place strengthens gradually, and it is as shown in Figure 2 that (the arrow direction does among Fig. 2
1O
2Fluorescence intensity ratio is respectively 54.1 and 2.47 when the direction that concentration increases), having singlet oxygen and no singlet oxygen to exist in neutrality and the alkaline buffer solution.Title complex [Ru (aip-C
4H
8SO
3Na)
3] Cl
2Before the singlet oxygen effect, its metal shifts to energy takes place between the triplet state of part charge transfer triplet state and anthracene nucleus, causes fluorescence by cancellation; But after the singlet oxygen effect; Because the generation of endoperoxide, energy shifts and is blocked, and therefore sends stronger fluorescence.And in neutral system, (83.3 μ M～88.5mM) become good linear relationship, linearly dependent coefficient reaches 0.991 to fluorescence intensity level, shown in Fig. 3 (a) within the specific limits with the singlet oxygen concentration value.
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, through 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 (aip-C
4H
8SO
3Na)
3] Cl
2(5 * 10
-6M) as shown in Figure 4 in the quantum yield and the relation between the singlet oxygen concentration of the endoperoxide that generates under neutrality and the alkaline condition.Be not difficult to find, if with the curve among 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 the 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
-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 1.6nM and 1.3nM according to three times of calculating of background standard deviation, show that this title complex is right
1O
2Has very high sensitivity.
2, with title complex [Ru (aip-C
4H
8SO
3Na)
3] Cl
2The method that in solution, detects singlet oxygen is identical, and it is as shown in the table that the singlet oxygen of other title complexs that this paper mentions detects performance:
Claims (10)
1. ruthenium complexe that contains the anthracycline derivatives part, its general structure is [RuA
mL
n] X
k, wherein: X is counter ion; L is the anthracycline derivatives part, and its structure is suc as formula shown in the I; A is the N^N assistant ligand; M=0 or 2, n=1 or 3, and m+n=3; K=1 or 2;
Among the formula I, R
1Be the straight or branched alkyl of hydrogen or C1-C10, R
2Be the straight or branched alkyl sodium sulfonate base of C1-C10 or the straight or branched alkylsulphonic acid potassium base of C1-C22.
2. ruthenium complexe as claimed in claim 1 is characterized in that, said A is dipyridyl or o-phenanthroline.
3. ruthenium complexe as claimed in claim 1 is characterized in that said X is selected from Cl
-, PF
6 -, ClO
4 -, NO
3 -, BF
4 -, CF
3SO
3 -And SO
4 2-In a kind of.
4. ruthenium complexe as claimed in claim 1 is characterized in that, R among the formula I
1Be the straight or branched alkyl of hydrogen or C1-C6, R
2Be the straight or branched alkyl sodium sulfonate base of C1-C6 or the straight or branched alkylsulphonic acid potassium base of C1-C6.
5. ruthenium complexe as claimed in claim 4 is characterized in that, R among the formula I
1Be the straight or branched alkyl of hydrogen or C1-C4, R2 is the straight or branched alkyl sodium sulfonate base of C1-C4 or the straight or branched alkylsulphonic acid potassium base of C1-C4.
6. the preparation method of the arbitrary said ruthenium complexe of claim 1～5 is with hydrate ruthenium trichloride or RuA
2Cl
2In solvent, be heated to 100-140 ℃ with ligand L and carry out coordination reaction, as X ≠ Cl
-In time, also need to finish postcooling in coordination reaction, and add counter anion X stirring reaction certain hour, obtains thick product, through recrystallization or column chromatographic isolation and purification, obtains said ruthenium complexe then.
7. preparation method as claimed in claim 6; It is characterized in that, prepare ligand L: under the nitrogen protection, part shown in the formula (1) and NaH or KH are carried out back flow reaction in organic solvent according to following method; Be cooled to room temperature then, sulphonyl lactone or the MO shown in the adding formula (2)
3SC
iH
2iSlowly heat up again behind the Br stirring at room certain hour and carry out back flow reaction, separate out deposition, be cooled to suction filtration after the room temperature, the gained deposition is carried out recrystallization, obtain ligand L;
Formula (1) formula (2) L
Or
L
Wherein, R
1Straight or branched alkyl for hydrogen or C1-C10; I is 1～10 integer; M is Na or K.
8. the arbitrary said ruthenium complexe of claim 1～5 is as the purposes of singlet oxygen fluorescence probe.
9. purposes as claimed in claim 8 is characterized in that, utilizes said ruthenium complexe to measure the content of singlet oxygen in neutrality or the alkaline aqueous solution, may further comprise the steps:
1) in known neutrality that can effectively produce singlet oxygen or basic soln system, adds said ruthenium complexe; Under a series of singlet oxygen concentration conditions; Measure the fluorescence intensity and the absorbancy of system through 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 said ruthenium complexe or alkaline buffer solution, add a certain amount of solution to be measured, measure its fluorescence intensity and absorbancy, calculate fluorescence quantum efficiency through excited by visible light;
3) according to step 2) value of the fluorescence quantum efficiency that records, the typical curve that obtains through step 1) is confirmed the content of singlet oxygen in the solution to be measured.
10. purposes as claimed in claim 9 is characterized in that, said step 1) and 2) in fluorescence quantum efficiency calculate through 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 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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| CN105062463B (en) * | 2015-07-23 | 2017-11-03 | 北京师范大学 | The binuclear ruthenium of different alkyl chain lengths is used as the lysosome probe in cell |
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| CN105062463B (en) * | 2015-07-23 | 2017-11-03 | 北京师范大学 | The binuclear ruthenium of different alkyl chain lengths is used as the lysosome probe in cell |
| CN109251746A (en) * | 2018-11-23 | 2019-01-22 | 曲靖师范学院 | The preparation and its application of anthracene nucleus bridged binuclear ruthenium complex fluorescence probe |
| CN109251746B (en) * | 2018-11-23 | 2021-10-08 | 曲靖师范学院 | Preparation and application of anthracene ring bridged binuclear ruthenium complex fluorescent probe |
| CN111253398A (en) * | 2020-03-08 | 2020-06-09 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | Fluorescent compound for detecting nerve injury and application thereof |
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