CN103205135B - Photosensitive dye osmium complex and preparation method thereof, carcinogenesis DNA oxidative damage quick detection kit and detection method - Google Patents
Photosensitive dye osmium complex and preparation method thereof, carcinogenesis DNA oxidative damage quick detection kit and detection method Download PDFInfo
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Abstract
The invention provides a kind of photosensitive dye osmium complex and preparation method thereof, use this photosensitive dye osmium complex to carry out test kit and the detection method of carcinogenesis DNA oxidative damage rapid detection.Photosensitive dye osmium complex has general structure: LL ' L ' ' Os-Y
4, wherein Y is halide-ions, ClO
4 -, BF
4 -, PF
6 -or OTs
-, L, L ' and be selected from 2 independently of one another, 2 '-dipyridyl and derivative, phenanthroline and derivative thereof or DPPZ, L ' ' for having 2, the part of 2 '-dipyridyl and 4,4'-Bipyridine structure.Test kit at least comprises: host compound eight yuan of melon rings (composition A), above-mentioned photosensitive dye osmium complex (composition B) and pure water or phosphoric acid salt, borate buffer solution (components D).Use this test kit, only need one to bleed sample, even if under the condition not by any plant and instrument, by means of naked eyes, under visible light illumination can single-minded, rapid detection 8-oxoG.
Description
Technical field
The present invention is specifically related to a kind of photosensitive dye osmium complex and preparation method thereof, uses this photosensitive dye osmium complex to carry out test kit and the detection method of carcinogenesis DNA oxidative damage rapid detection.
Background technology
Cancer, once find mostly to be in late period, makes doctor have no plan to make improvements, only has early diagnosis just can prevent trouble before it happens.The by product of the aerobic metabolisms such as chemical reaction, radiation and singlet oxygen, superoxide radical and hydroxyl radical free radical all can cause the oxidative damage of DNA, produces transgenation, aging and some degenerative disease of induced tumor, body cell.And guanine G is the most oxidized in four of DNA bases, its major oxidation product is 8-oxygen-7,8-dihydro deoxy-guanine (8-hydroxy guanine, 8-oxoG), it is comparatively stable after being formed in vivo, be the important biomolecule marker of DNA oxidative damage, the DNA extent of damage can be reflected to its monitoring, thus early warning, prevention are carried out to canceration.Therefore, quick, single-minded, reliably to detect the 8-oxoG method in DNA research, has very important theory and realistic meaning.
In current DNA, the common detection methods of 8-oxoG mainly contains: enzyme immunoassay, high performance liquid chromatography and electrochemistry/mass spectrometry, kapillary meteorological chromatograph-mass spectrometer coupling, gel electrophoresis etc.But these methods do not possess specificity, and the pre-treatment such as needs are first hydrolyzed to DNA, enzymolysis, derivatize, hybridization, detect by special technology and instrument, analysis time is longer again, limits their applying on routine physical examination and outpatient service chemical examination.
Terpyridyl osmium (Os (bpy)
3 3+, 0.63V vs Ag/AgCl) and be bibliographical information (R.C.Holmberg, M.T.Tierney, P.A.Ropp, E.E.Berg, M.W.Grinstaff, H.H.Thorp, Inorg.Chem.2003,42,6379; V.A.Szalai, M.J.Singer, H.H.Thorp, J.Am.Chem.Soc.2002,124,1625; P.A.Ropp, H.H.Thorp, Chem.Biol.1999,6,599; A.Mugweru, B.Wang, J.Rusling, Anal.Chem.2004,76,5557; L.Dennany, R.J.Forster, B.White, M.Smyth, J.F.Rusling, J.Am.Chem.Soc.2002,124,1625) uniquely can single-minded oxidation 8-oxoG(0.5 ± 0.1V vs Ag/AgCl in) chemical reagent, it obtains good application in the Electrochemical Detection of 8-oxoG at present, and its principle is as follows.
But this electrochemical detection method need first by chemical bond by Os (bpy)
3 2+probe molecule grafts 5 ' end of DNA segment, or by probe molecule and DNA layer by layer respectively even application make film to electrode surface, duplicate packages painting, until the uniformity coefficient of film and thickness reach requirement, operates quite loaded down with trivial details, is not suitable for conventional sense.
Summary of the invention
The object of the invention is for the deficiencies in the prior art, a kind of carcinogenesis DNA oxidative damage quick detection kit and detection method are provided, for special early warning, the prevention of carcinogenesis.In addition, another object of the present invention is to provide a kind of novel photosensitive dye osmium complex and preparation method thereof.
For achieving the above object, the present invention adopts following technical scheme:
1. a photosensitive dye osmium complex, is characterized in that, this photosensitive dye osmium complex has having structure formula I:
LL’L’’Os-Y
4（Ⅰ）
In formula I, Y is halide-ions, ClO
4 -, BF
4 -, PF
6 -or OTs
-, L, L ' and be selected from following part independently of one another:
Wherein, R
1and R
2be selected from H, C independently of one another
1-10alkyl, CHO, COOH, NH
2, NO
2, CN, OH, SH, C
1-6alkoxyl group, C
1-6alkylamino, C
1-6amide group, C
1-6haloalkyl, there is C
1-10the phenothiazinyl of alkyl or halogen,
L ' ' is following part:
Wherein, n is the integer of 1-10, R
3for H, C
1-10alkyl, CHO, COOH, NH
2, NO
2, CN, OH, SH, C
1-6alkoxyl group, C
1-6alkylamino, C
1-6amide group, C
1-6haloalkyl, there is C
1-10the phenothiazinyl of alkyl or halogen, R
4for C
1-10alkyl.
2, for the preparation of a method for above-mentioned photosensitive dye osmium complex, it is characterized in that, comprise the steps:
(1) preparation is with L, L ' be the osmium title complex intermediate of part
The ligand L of following compound, L ' and the metal osmium salt being negative ion with Y ' will be selected from independently of one another simultaneously or carry out coordination reaction successively, obtain the osmium title complex intermediate of general formula LL ' Os-Y ', temperature of reaction is 20-200 DEG C, reaction times is 1-24h, reaction solvent be selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and wherein arbitrarily two or more according to mixed solvent of arbitrary proportion composition
Wherein, R
1and R
2be selected from H, C independently of one another
1-10alkyl, CHO, COOH, NH
2, NO
2, CN, OH, SH, C
1-6alkoxyl group, C
1-6alkylamino, C
1-6amide group, C
1-6haloalkyl, there is C
1-10the phenothiazinyl of alkyl or halogen;
(2) intermediate 1 is prepared
By 4-methyl-4 '-R
3-dipyridyl and N-Lithiodiisopropylamide (LDA) react, temperature of reaction is-78-50 DEG C, reaction times is 0.5-8h, reaction solvent be selected from methylene dichloride, chloroform, tetrahydrofuran (THF), acetonitrile, DMF, DMSO and wherein arbitrarily two or more according to mixed solvent of arbitrary proportion composition, after reaction terminates, without separation, directly to the Br (CH adding n=1-10 in system
2)
nbr, obtains intermediate 1, and temperature of reaction is-78-100 DEG C, and the reaction times is 1-48h, reaction solvent be selected from methylene dichloride, chloroform, tetrahydrofuran (THF), acetonitrile, DMF, DMSO and wherein arbitrarily two or more according to mixed solvent of arbitrary proportion composition,
Wherein, R
3for H, C
1-10alkyl, CHO, COOH, NH
2, NO
2, CN, OH, SH, C
1-6alkoxyl group, C
1-6alkylamino, C
1-6amide group, C
1-6haloalkyl, there is C
1-10the phenothiazinyl of alkyl or halogen;
(3) intermediate 2 is prepared
Intermediate 1 is carried out coordination with osmium title complex intermediate LL ' Os-Y ', obtain intermediate 2, temperature of reaction is 20-200 DEG C, reaction times is 1-24h, reaction solvent be selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and wherein arbitrarily two or more according to mixed solvent of arbitrary proportion composition;
(4) intermediate 3 is prepared
By intermediate 2 under KI catalysis with 4,4 '-second bipyridine reacts, obtain intermediate 3, temperature of reaction is 20-200 DEG C, reaction times is 1-24h, reaction solvent be selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and wherein arbitrarily two or more according to mixed solvent of arbitrary proportion composition;
(5) preparation is with L, L ', L " be the osmium title complex of part
By intermediate 3 and C
1-10alkane iodide R
4i reacts, obtain general formula LL ' L " the osmium title complex of Os-Y '; temperature of reaction is 20-200 DEG C, and the reaction times is 1-24h, reaction solvent be selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and wherein arbitrarily two or more according to mixed solvent of arbitrary proportion composition;
(6) target product is prepared
By osmium title complex LL ' L " Os-Y ' carries out negative ion replacement(metathesis)reaction with containing the sodium salt of Y, sylvite or ammonium salt by molar ratio 1:1-10, obtains target product LL ' L ' ' Os-Y
4, described Y is halide-ions, ClO
4 -, BF
4 -, PF
6 -or OTs
-, temperature of reaction is 10-100 DEG C, and the reaction times is 5 minutes-2h, reaction solvent be selected from methyl alcohol, ethanol, ethylene glycol, acetone, water, DMF, DMSO and wherein arbitrarily two or more according to mixed solvent of arbitrary proportion composition; Or in step (3) ~ (5), carry out the above-mentioned negative ion replacement(metathesis)reaction of replacing Y ' with Y respectively, finally obtain target product LL ' L ' ' Os-Y
4.
3, a carcinogenesis DNA oxidative damage quick detection kit, is characterized in that, this test kit comprises A, B, D tri-kinds of compositions:
(1) composition A: host compound eight yuan of melon rings;
(2) composition B: as the above-mentioned photosensitive dye osmium complex of guest compound I;
(3) components D: pure water or phosphoric acid salt, borate buffer solution.
4, the test kit as described in above-mentioned 3, is characterized in that, this test kit also comprises further:
(4) composition C: as the fluorescent small molecule of guest compound II, this fluorescent small molecule has having structure general formula I I:
Wherein, R
5and R
6be selected from H, C independently of one another
1-10alkyl, CHO, COOH, NH
2, CN, OH, SH, C
1-6alkoxyl group, C
1-6alkylamino, C
1-6amide group, C
1-6haloalkyl, there is C
1-10the phenothiazinyl of alkyl or halogen.
5, a carcinogenesis DNA oxidative damage method for quick, is characterized in that, the method uses the test kit described in above-mentioned 3 to detect, and specifically comprises the steps:
(1) be first dissolved in components D by composition A, B according to the ratio of mol ratio 1-5:1, with the densitometer of composition B, be made into the solution that concentration is 10 μMs of-1mM, then get this appropriate solution and join in transparent vessel, rare gas element deoxygenation, seals stand-by;
(2) joined by testing sample in above-mentioned transparent vessel, after illumination 15-30 minute, visual color changes or detects absorption spectrum change with opticinstrument.
6, a carcinogenesis DNA oxidative damage method for quick, is characterized in that, the method uses the test kit described in above-mentioned 4 to detect, and specifically comprises the steps:
(1) first composition A, B, C are dissolved in components D according to the ratio of mol ratio 1-5:1:1, with the densitometer of composition B or C, be made into the solution that concentration is 10 μMs of-1mM, then get this appropriate solution and join in transparent vessel, rare gas element deoxygenation, seals stand-by;
(2) joined by testing sample in above-mentioned transparent vessel, after illumination 15-30 minute, visual color changes or detects ultravioletvisible absorption and/or fluorescence emission spectrum change with opticinstrument.
7, the detection method as described in above-mentioned 5 or 6, is characterized in that, the light source that described illumination adopts is the houselights that sunlight maybe can excite described photosensitive dye osmium complex.
Beneficial effect of the present invention is: develop a kind of efficient, quick, special, reliable 8-oxoG detection kit and 8-oxoG specificity method for quick.This detection method is easy and simple to handle, and operator do not need through professional training, only needs one to bleed sample, even if under the condition not by any plant and instrument, by means of naked eyes, single-minded, quick (about 15 minutes) can detect 8-oxoG under visible light illumination.When this technology is highly suitable for routine physical examination and outpatient service chemical examination, primary dcreening operation monitoring is carried out to carcinogenesis DNA oxidative damage.In addition, new compound photosensitive dye osmium complex LL ' L ' ' Os-Y of the present invention
4detect at 8-oxoG oxidative damage and there is in association area application prospect widely.
Accompanying drawing explanation
Fig. 1 is the changing conditions that when adding 8-oxoG sample in test kit of the present invention, ultraviolet-visible absorption spectroscopy produces with the prolongation of light application time.
Fig. 2 is ultraviolet-visible absorption spectroscopy when not adding 8-oxoG sample in test kit of the present invention.
Fig. 3 is the photo in kind (left: not add 8-oxoG sample, right: to add 8-oxoG sample) adding quartz cell colour-change before and after 8-oxoG sample in test kit of the present invention.
Fig. 4 is the changing conditions that when adding 8-oxoG sample in test kit of the present invention, fluorescence emission spectrum produces with the prolongation of light application time.
Fig. 5 is fluorescence emission spectrum when not adding 8-oxoG sample in test kit of the present invention.
Fig. 6 is the DPV(Differential pulse voltammetry of 8-oxoG) graphic representation.
Embodiment
< photosensitive dye osmium complex >
New compound photosensitive dye osmium complex of the present invention has having structure formula I:
LL’L’’Os-Y
4（Ⅰ）
In formula I, Y is halide-ions, ClO
4 -, BF
4 -, PF
6 -or OTs
-, L, L ' and be selected from following part independently of one another:
Wherein, R
1and R
2be selected from H, C independently of one another
1-10alkyl, CHO, COOH, NH
2, NO
2, CN, OH, SH, C
1-6alkoxyl group, C
1-6alkylamino, C
1-6amide group, C
1-6haloalkyl, there is C
1-10the phenothiazinyl of alkyl or halogen,
L ' ' is following part:
Wherein, n is the integer of 1-10, R
3for H, C
1-10alkyl, CHO, COOH, NH
2, NO
2, CN, OH, SH, C
1-6alkoxyl group, C
1-6alkylamino, C
1-6amide group, C
1-6haloalkyl, there is C
1-10the phenothiazinyl of alkyl or halogen, R
4for C
1-10alkyl.
Unless otherwise indicated, term used herein " alkyl " comprises straight chained alkyl and branched-chain alkyl.Such as, " C
1-6alkyl " comprise C
6alkyl, C
5alkyl, methyl, ethyl, n-propyl, sec.-propyl and the tertiary butyl.Similar rule is also applicable to other group used in this specification sheets.Term used herein " halogen " comprises fluorine, chlorine, bromine, iodine.
In a specific embodiment of the present invention, preferably, R
1and R
2be selected from H, C independently of one another
1-8alkyl, COOH, NH
2, OH or halogen; More preferably, R
1and R
2be selected from H, C independently of one another
1-6alkyl or COOH; Optimally, R
1and R
2be selected from H or methyl independently of one another.
In a specific embodiment of the present invention, preferred n is the integer of 1-8, and most preferably n is the integer of 3-7.
In a specific embodiment of the present invention, preferably, R
3for H, C
1-8alkyl, COOH, NH
2, OH or halogen; More preferably, R
3for H, C
1-6alkyl or COOH; Optimally, R
3for H or methyl.
In a specific embodiment of the present invention, preferably, R
4for C
1-6alkyl; More preferably, R
4for C
1-4alkyl; Optimally, R
4for methyl or ethyl.
In a specific embodiment of the present invention, the preferred halide-ions of Y.
In a specific embodiment of the present invention, preferably, R
5and R
6be selected from H, C independently of one another
1-8alkyl, COOH, NH
2, OH or halogen; More preferably, R
5and R
6be selected from H, C independently of one another
1-6alkyl, NH
2or OH; Optimally, R
5and R
6be selected from NH independently of one another
2or OH.
Optimally, photosensitive dye osmium complex of the present invention is selected from following compound:
The preparation method > of < photosensitive dye osmium complex
Method for the preparation of above-mentioned photosensitive dye osmium complex comprises the steps:
(1) preparation is with L, L ' be the osmium title complex intermediate of part
Ligand L, the L ' (R of following compound will be selected from independently of one another
1, R
2implication the same) with the metal osmium salt that is negative ion with Y ' simultaneously or carry out coordination reaction successively, obtain the osmium title complex intermediate of general formula LL ' Os-Y ', temperature of reaction is 20-200 DEG C, reaction times is 1-24h, reaction solvent be selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and wherein arbitrarily two or more according to mixed solvent of arbitrary proportion composition.
In above-mentioned reaction, L, L ' can be identical, also can be different.As L, L ' identical time, preferably by reaction with part simultaneously with metal osmium reactant salt, metal osmium salt: the mol ratio of part preferably controls in the scope of 1:1.8 ~ 3.As L, L ' not identical time, preferably by reaction ligand L, L ' respectively with metal osmium reactant salt, namely after metal osmium salt first reacts with ligand L or L ', then with ligand L ' or L react, metal osmium salt: the mol ratio of ligand L or L ' preferably controls in the scope of 1:0.8 ~ 2.
In addition, the metal osmium salt being negative ion with Y ' can be enumerated but be not limited to (NH
4)
2[OsCl
6], K
2osCl
6, OsCl
3deng.
In a preferred embodiment, temperature of reaction is 40-200 DEG C, and the reaction times is 1-20h, reaction solvent be selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF and wherein arbitrarily two or more according to mixed solvent of arbitrary proportion composition.
In one more preferably embodiment, temperature of reaction is 80-200 DEG C, and the reaction times is 1-10h, reaction solvent be selected from ethylene glycol, acetonitrile, water, DMF and wherein arbitrarily two or more according to mixed solvent of arbitrary proportion composition.
In a most preferred embodiment, temperature of reaction is 150-200 DEG C, and the reaction times is 1-5h, and reaction solvent is selected from ethylene glycol, DMF and mixed solvent thereof.
(2) intermediate 1 is prepared
By 4-methyl-4 '-R
3-dipyridyl (R
3implication the same) react with N-Lithiodiisopropylamide (LDA), temperature of reaction is-78-50 DEG C, reaction times is 0.5-8h, reaction solvent be selected from methylene dichloride, chloroform, tetrahydrofuran (THF), acetonitrile, DMF, DMSO and wherein arbitrarily two or more according to mixed solvent of arbitrary proportion composition, after reaction terminates, without separation, directly to the Br (CH adding n=1-10 in system
2)
nbr, obtains intermediate 1, and temperature of reaction is-78-100 DEG C, and the reaction times is 1-48h, reaction solvent be selected from methylene dichloride, chloroform, tetrahydrofuran (THF), acetonitrile, DMF, DMSO and wherein arbitrarily two or more according to mixed solvent of arbitrary proportion composition.
Above-mentioned 4-methyl-4 '-R
3in the reaction of-dipyridyl and N-Lithiodiisopropylamide, 4-methyl-4 '-R
3the mol ratio of-dipyridyl and N-Lithiodiisopropylamide (4-methyl-4 '-R
3-dipyridyl: N-Lithiodiisopropylamide) be preferably 1:0.8 ~ 3, be more preferably 1:0.8 ~ 2, be particularly preferably 1:0.9 ~ 1.5.Temperature of reaction is preferably-78 ~ 25 DEG C, is more preferably-78 ~ 0 DEG C.Reaction times is preferably 0.5 ~ 4h, is more preferably 1 ~ 2h.The preferred methylene dichloride of reaction solvent, chloroform, tetrahydrofuran (THF), acetonitrile, more preferably methylene dichloride, chloroform, tetrahydrofuran (THF), particularly preferably methylene dichloride, tetrahydrofuran (THF).
In addition, 4-methyl-4 '-R
3-dipyridyl and Br (CH
2)
nthe mol ratio of Br (4-methyl-4 '-R
3-dipyridyl: Br (CH
2)
nbr) be preferably 1:0.8 ~ 10, be more preferably 1:0.8 ~ 5, be particularly preferably 1:2 ~ 5.With Br (CH
2)
nin the reaction of Br, temperature of reaction is preferably-78 ~ 40 DEG C, is more preferably-78 ~ 30 DEG C, is particularly preferably 0 ~ 30 DEG C.Reaction times is preferably 1 ~ 12h, is more preferably 1 ~ 8h, is particularly preferably 1 ~ 4h.The preferred methylene dichloride of reaction solvent, chloroform, tetrahydrofuran (THF), acetonitrile, more preferably methylene dichloride, chloroform, tetrahydrofuran (THF), particularly preferably methylene dichloride, tetrahydrofuran (THF).
After product is preferably separated with the organic solvent extraction such as methylene dichloride, chloroform, thick product is further with separation and purifications such as silica gel column chromatographies.
(3) intermediate 2 is prepared
Intermediate 1 is carried out coordination with osmium title complex intermediate LL ' Os-Y ', obtain intermediate 2, temperature of reaction is 20-200 DEG C, reaction times is 1-24h, reaction solvent be selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and wherein arbitrarily two or more according to mixed solvent of arbitrary proportion composition.
In above-mentioned reaction, and the mol ratio of LL ' Os-Y ' and intermediate 1 (LL ' Os-Y ': intermediate 1) be preferably 1:1 ~ 10, be more preferably 1:1 ~ 8, be particularly preferably 1:1 ~ 5.Temperature of reaction is preferably 100 ~ 200 DEG C, is more preferably 150 ~ 200 DEG C.Reaction times is preferably 1 ~ 12h, is more preferably 1.5 ~ 5h.The preferred ethylene glycol of reaction solvent, acetonitrile, water, DMF, more preferably ethylene glycol, acetonitrile, water, particularly preferably ethylene glycol.
Product is preferably with separation and purifications such as silica gel column chromatographies, and the product obtained carries out Y ion exchange (implication of Y is the same) by the sodium salt, sylvite, ammonium salt etc. containing Y.
(4) intermediate 3 is prepared
By intermediate 2 under KI catalysis with 4,4 '-second bipyridine reacts, obtain intermediate 3, temperature of reaction is 20-200 DEG C, reaction times is 1-24h, reaction solvent be selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and wherein arbitrarily two or more according to mixed solvent of arbitrary proportion composition.
In above-mentioned reaction, intermediate 2 and 4, the mol ratio (intermediate 2:4,4 '-second bipyridine) of 4 '-second bipyridine is preferably 1:1 ~ 10, is more preferably 1:1 ~ 6, is particularly preferably 1:1 ~ 3.Temperature of reaction is preferably 50 ~ 150 DEG C, is more preferably 100 ~ 130 DEG C.Reaction times is preferably 3 ~ 15h, is more preferably 4 ~ 7h.Reaction solvent preferred alcohol, ethylene glycol, acetonitrile, water, DMF, more preferably ethylene glycol, acetonitrile, DMF, particularly preferably acetonitrile, DMF.
Product is preferably with separation and purifications such as silica gel column chromatographies, and the product obtained carries out Y ion exchange by the sodium salt, sylvite, ammonium salt etc. containing Y.
(5) preparation is with L, L ', L " be the osmium title complex of part
By intermediate 3 and C
1-10alkane iodide R
4i reacts, obtain general formula LL ' L " the osmium title complex of Os-Y '; temperature of reaction is 20-200 DEG C, and the reaction times is 1-24h, reaction solvent be selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and wherein arbitrarily two or more according to mixed solvent of arbitrary proportion composition.
In above-mentioned reaction, intermediate 3 and R
4mol ratio (the intermediate 3:R of I
4i) be preferably 1:1 ~ 10, be more preferably 1:2 ~ 8, be particularly preferably 1:4 ~ 6.Temperature of reaction is preferably 50 ~ 150 DEG C, is more preferably 80 ~ 130 DEG C.Reaction times is preferably 6 ~ 18h, is particularly preferably 10 ~ 15h.The preferred ethylene glycol of reaction solvent, acetonitrile, water, DMF, more preferably ethylene glycol, acetonitrile, DMF, particularly preferably acetonitrile, DMF.
Product is preferably with separation and purifications such as silica gel column chromatographies, and the product obtained carries out Y ion exchange by the sodium salt, sylvite, ammonium salt etc. containing Y.
(6) target product is prepared
Target product LL ' L ' ' Os-Y is obtained by two kinds of modes
4: (a) as described above, carries out Y displacement respectively in step (3) ~ (5), finally obtains target product LL ' L ' ' Os-Y
4; (b) only in step (5), the osmium title complex LL ' L to obtaining " Os-Y ' carries out Y displacement, obtains target product LL ' L ' ' Os-Y
4.
Each product generally carries out negative ion replacement(metathesis)reaction by molar ratio (each product: Y salt) 1:1-10 with containing the sodium salt of Y, sylvite or ammonium salt etc.Temperature of reaction is 10-100 DEG C, is preferably 20 ~ 80 DEG C, is more preferably 20 ~ 60 DEG C, is particularly preferably 20 ~ 40 DEG C.Reaction times is 5 minutes-2h, is preferably 5 minutes ~ 1.5h, is more preferably 8 minutes ~ 1.5h, is particularly preferably 10 minutes ~ 1h.Reaction solvent be selected from methyl alcohol, ethanol, ethylene glycol, acetone, water, DMF, DMSO and wherein arbitrarily two or more according to mixed solvent of arbitrary proportion composition, particular methanol, ethanol, ethylene glycol, acetone, water, more preferably methyl alcohol, acetone, water, particularly preferably methyl alcohol, acetone.
The photosensitive dye osmium complex of being synthesized by aforesaid method of the present invention, can adopt nuclear-magnetism, mass spectrum to determine its structure.
< carcinogenesis DNA oxidative damage quick detection kit >
This test kit comprises A, B, D tri-kinds of compositions, and wherein composition B is above-mentioned photosensitive dye osmium complex,
(1) composition A: host compound eight yuan of melon rings;
(2) composition B: as the photosensitive dye osmium complex of guest compound I;
(3) components D: pure water or phosphoric acid salt, borate buffer solution.
In addition, this test kit also preferably comprise further there is having structure general formula fluorescent small molecule as guest compound II (composition C):
Wherein, R
5and R
6be selected from H, C independently of one another
1-10alkyl, CHO, COOH, NH
2, CN, OH, SH, C
1-6alkoxyl group, C
1-6alkylamino, C
1-6amide group, C
1-6haloalkyl, there is C
1-10the phenothiazinyl of alkyl or halogen.
Optimally, this guest compound 2 is selected from following compound:
< carcinogenesis DNA oxidative damage method for quick >
The method uses mentioned reagent box to detect, and when test kit comprises A, B, D tri-kinds of compositions, detection method specifically comprises the steps:
(1) first composition A, B are dissolved in components D according to the ratio of mol ratio 1-5:1, with the densitometer of composition B, be made into the solution that concentration is 10 μMs of-1mM, then getting appropriate (such as 0.1-5mL) this solution joins in transparent vessel (such as quartz cell), rare gas element deoxygenation, seals stand-by;
(2) joined by testing sample in above-mentioned transparent vessel, after illumination 15-30 minute, visual color changes or detects ultraviolet-visible absorption spectroscopy change with opticinstrument.
When test kit comprises A, B, C, D tetra-kinds of compositions, detection method specifically comprises the steps:
(1) first composition A, B, C are dissolved in components D according to the ratio of mol ratio 1-5:1:1, with the densitometer of composition B or C, be made into the solution that concentration is 10 μMs of-1mM, then getting appropriate (such as 0.1-5mL) this solution joins in transparent vessel (such as quartz cell), rare gas element deoxygenation, seals stand-by;
(2) joined by testing sample in above-mentioned transparent vessel, after illumination 15-30 minute, visual color changes or detects ultraviolet-visible absorption spectroscopy and/or fluorescence emission spectrum change with opticinstrument.
The light source that illumination adopts comprises sunlight, houselights etc. and can excite arbitrarily composition B(photosensitive dye osmium complex) light source.Detection after illumination both can utilize direct visual perception, also can by optical detecting instruments such as arbitrary ultraviolet, fluorescence.
< design philosophy > of the present invention
Terpyridyl osmium is used to carry out 8-oxoG in the method for Electrochemical Detection, the single-minded oxygenant Os (bpy) of 8-oxoG
3 3+by electrochemistry by anodizing, in view of terpyridyl osmium is a kind of light-sensitive coloring agent, Os (bpy) can be excited by illumination completely
3 2+, make it transit to excited state
*os (bpy)
3 2+, excited state by electron transmission to electron acceptor(EA) methyl amethyst (MV
2+) after, obtain oxidation state Os (bpy)
3 3+and methyl amethyst free radical (MV
+), its principle is as follows.
But Os (bpy)
3 2+with MV
2+intermolecular reaction also exists slower, the inefficient defect of electron transmission speed, is unfavorable for practical application.If will both if following compound is (referred to as Os (bpy)
3 2+-MV
2+) shown in couple together like that (two parts namely in osmium part (L, L ') be 2,2 '-dipyridyl, and another one part (L ") has 2; 2 '-dipyridyl and 4; 4 '-dipyridyl structure), changed into intramolecular interaction, just can be realized Os (bpy) under illumination
3 3+to MV
2+efficiently single-minded oxidation, obtains oxidisability transition state Os (bpy)
3 3+-MV
+.
The present invention carries out photosensitive dye osmium complex structure design based on above-mentioned technological thought just.Through the research that the present inventor is deep in a large number, found that:
(1) as ligand L, L ', when being used in 2, when 2 '-dipyridyl ring introduces suitable substituent derivative, or ligand L, L ' one or both of adopt phenanthroline and derivative thereof or DPPZ(dipyrido [3 simultaneously, 2-a'2', 3'-c] phenazine) time, can obtain and only use dipyridyl quite even better oxidation effectiveness.Namely as ligand L, L ', following part (R can be selected from independently of one another
1, R
2implication as above).
Research shows, only adopts 2, and when 2 '-dipyridyl and derivative thereof are as part, the reactive force between the photosensitive dye osmium complex of acquisition and DNA double spirane structure is relatively weak; When adopting phenanthroline and derivative thereof as part, between the photosensitive dye osmium complex of acquisition and DNA double spirane structure, there is stronger interaction, and more weak with the reactive force of RNA; And when adopting DPPZ as part, DPPZ in photosensitive dye osmium complex can cuttage enter in DNA double spirane structure, be " the interacting in quasi-molecule " on DNA skeleton by photosensitive dye osmium complex and the intermolecular role transformation of 8-oxoG, thus the 8-oxoG more efficiently in O-DNA, obtain better detected result.In addition, although 2, the Detection results of 2 '-dipyridyl and derivative thereof is slightly poor, and its synthesis is relatively simple, and preparation cost is lower.
(2) as ligand L ", suitable substituting group can be introduced in 2,2 '-dipyridyl and 4,4'-Bipyridine structure, i.e. ligand L " optional from following part (R
3, R
4implication as above).
But, with Os (bpy)
3 2+-MV
2+for example, under the driving of potential difference, MV
+electronics certainly will be returned to rapidly Os (bpy)
3 3+, whole system is returned to again original state Os (bpy)
3 2+-MV
2+.Only has stable live in MV
+on electronics, make it not return fast, just can obtain single-minded oxidation 8-oxoG oxidation state Os (bpy)
3 3+.And eight yuan of melon rings (Cucurbit [8] uril, CB [8]) are to MV
2+, MV
+and the self-assembly performance of their derivative uniqueness can address these problems well, its principle is as follows.
By Os (bpy)
3 2+-MV
2+1:1 coordination compound is self-assembled in aqueous (referred to as Os with CB [8]
2+-MV
2+/ CB [8]), under light illumination, Os
2+-MV
2+(λ
max=480nm) absorb luminous energy and transit to excited state * Os
2+-MV
2+, excited state forms charge separation transition state Os by cyclic voltammetry method
3+-MV
+, CB [8] and MV
+self-assembly play certain stabilization by this charge separation transition state, thus delay the quick passback of electronics, make oxidisability transition state (Os
3+-MV
+)/CB [8] can stable existence in solution.After adding testing sample, if there is 8-oxoG in sample, (Os
3+-MV
+)/CB [8] will carry out specificity oxidation to it, generate reduction-state (Os
2+-MV
+)/CB [8], the now Os of two molecules
2+-MV
+will with CB [8] further self-assembly, form more stable free radical dimer self-assembly coordination compound (Os
2+-MV
+)
2/ CB [8], solution presents the dimeric characteristic absorbance (λ of this free radical
max=560nm), there is the remarkable color change of Glassless.Whole process can be schematically as follows.
The present invention just based on above-mentioned technological thought carry out carcinogenesis DNA oxidative damage quick detection kit design.Using eight yuan of melon rings as host compound, using photosensitive dye osmium complex of the present invention as guest compound (guest compound I), these compounds are dissolved in after in pure water or phosphoric acid salt, borate buffer solution, just can to carry out containing 8-oxoG sample efficient, quick, special, detect reliably.
In addition, in view of the change only having absorption spectrum in said process, restriction can be caused to detection to a certain extent.Consider that fluorescence detection method has simple and direct, efficient characteristic, can appropriate fluorescent small molecule is incorporated in system as 2,6-bisnaphthol (guest compound II), make itself and MV
2+under Charger transfer (charge transfer) effect, jointly enter into CB [8] cavity and carry out self-assembly.After free radical dimer is formed in CB [8] cavity, fluorescent small molecule by " exclusion " out, is dissociated in solution, is made the change simultaneously producing color and fluorescence in testing process from CB [8], thus give this system more powerful practicality, strengthen Detection results further.Its principle (fluorescent small molecule with 2,6-bisnaphthol for example) as follows.
These test kits of the application of the invention, can efficient, quick, special, reliably detect 8-oxoG, detect easy and simple to handle, and operator do not need through professional training, one is only needed to bleed sample, even if under the condition not by any plant and instrument, by means of naked eyes, single-minded, quick (about 15 minutes) 8-oxoG can be detected under visible light illumination, when being highly suitable for routine physical examination and outpatient service chemical examination, primary dcreening operation monitoring be carried out to carcinogenesis DNA oxidative damage.Certainly, the present invention also can carry out the detection of 8-oxoG by the opticinstrument such as UV detector, luminoscope.
Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, some simple deduction or replace can also be made, all should be considered as belonging to protection scope of the present invention.In addition, detecting as DNA oxidative damage is new compound photosensitive dye osmium complex LL ' L ' ' Os-Y of the present invention
4a kind of purposes; but can not assert that compound of the present invention only detects for DNA oxidative damage; for general technical staff of the technical field of the invention; under the consideration being used as the identical mechanism of action that DNA oxidative damage detects based on the compounds of this invention; some simple inferences can also be made; draw other application purpose of compound of the present invention, all should be considered as belonging to protection scope of the present invention.The features and advantages of the present invention will become apparent after with reference to accompanying drawing and embodiments of the invention.
Embodiment
The synthesis of embodiment 1Os-C3-MV
(1) Os (bpy)
2cl
2synthesis:
By (NH
4)
2[OsCl
6] (1.0g, 2.3mmol) and 2,2 '-dipyridyl (0.72g, 4.6mmol) is dissolved in 30ml ethylene glycol, under nitrogen protection magnetic agitation reflux 1h.After reaction mixture cool to room temperature, add 1M Na
2s
2o
4(60mL) in ice-water bath, 30min is kept.Filter, the atropurpureus throw out priority water obtained and ether fully wash, and vacuum-drying obtains black solid 0.92g, productive rate 70%.
(2) synthesis of intermediate 1:
4-methyl-2,2 '-second bipyridine (2.5g, 13.5mmol) is dissolved in dry THF(50mL) in, solution is placed in ice-water bath, by the LDA(2.0mL Diisopropylamine of brand-new, and 1.6M n-Butyl Lithium 8.1mL, 10mL THF) dropwise add, solution becomes black, continues to stir 1h, get 1,2-ethylene dibromide (50mmol, 4.3mL) adds rapidly, then ice-water bath is removed, continue under room temperature to stir 3h, add 100mL phosphoric acid buffer (pH=7.0) stopped reaction.Solution CH
2cl
2(3 × 50mL) extracts, and collects CH
2cl
2layer also with anhydrous magnesium sulfate drying, obtains pale yellow oil after evaporate to dryness.Thick product silica gel column chromatography is separated, collects the component of ethyl acetate-dichloromethane (1:9), obtain oily product 2.2g after evaporate to dryness, yield 61%.
1H NMR(CDCl
3,400MHz):δ2.16-2.23(m,2H,C
2H
2),2.82(t,2H,C
1H
2,J=7.2Hz),3.38(t,2H,C
3H
2,J=6.4Hz),7.11-7.12(m,1H,H
5),7.25-7.28(m,1H,H
5′),7.78(dt,1H,H4′, J=2.0,8.0Hz),8.26(s,1H,H
3),8.40(d,1H,H
3′,J=8.4Hz),8.56(d,1H,H
6,J=4.8Hz),8.66-8.67(m,1H,H
6′),API-MS,m/z:[M+H]
+=277.0.
(3) synthesis of intermediate 2:
By intermediate 1(0.56g, 2.02mmol) and Os (bpy)
2cl
2(0.29g, 0.51mmol) joins in 5ml ethylene glycol solvent, under nitrogen protection, and lucifuge reflux 2h.Be separated with silica gel column chromatography after cooling, developping agent is CH
3cOCH
3: H
2o:KNO
3saturated aqueous solution=20:1:1(V/V/V), collect blackish green component, evaporate to dryness developping agent, be dissolved in dry methyl alcohol, cross and filter insoluble saltpetre, then methyl alcohol is concentrated into about 5mL, then dropped to the NH that 10ml is saturated
4pF
6in the aqueous solution, under room temperature, react 0.5h, displacement NO
3 -gegenion, by the sedimentation and filtration of separating out, washing, obtains blackish green product 0.49g(and intermediate 2 after drying), yield 90%.
(4) synthesis of intermediate 3:
By intermediate 2(0.42g, 0.39mmol), 4,4 '-second bipyridine (1.22g, 0.78mmol) and KI(0.13g, 0.78mmol) be dissolved in the DMF of 10mL drying, under nitrogen lucifuge be heated to 115 DEG C reaction 5h.Carry out separation and purification with silica gel column chromatography after solvent evaporated, developping agent is CH
3cOCH
3: H
2o:KNO
3saturated aqueous solution=10:1:1(V/V/V), collect the blackish green component that polarity is larger, evaporate to dryness developping agent, be dissolved in dry methyl alcohol, cross and filter insoluble saltpetre, then methyl alcohol is concentrated into about 5mL, then dropped to the NH that 10ml is saturated
4pF
6in the aqueous solution, under room temperature, react 0.5h, displacement NO
3 -gegenion, by the sedimentation and filtration of separating out, washing, obtains blackish green product 0.32g(and intermediate 3 after drying), yield 64%.
(5) synthesis of Os-C3-MV:
By intermediate 3(0.30g, 0.23mmol), methyl iodide (1.63g, 1.15mmol) is dissolved in the acetonitrile of 10ml drying, lucifuge heated overnight at reflux under nitrogen.Carry out separation and purification with silica gel column chromatography after solvent evaporated, developping agent is CH
3cOCH
3: H
2o:KNO
3saturated aqueous solution=5:1:1(V/V/V), collect blackish green component, evaporate to dryness developping agent, be dissolved in dry methyl alcohol, cross and filter insoluble saltpetre, then methyl alcohol is concentrated into about 5mL, then dropped to the NH that 10ml is saturated
4pF
6in the aqueous solution, under room temperature, react 0.5h, displacement NO
3 -gegenion, by the sedimentation and filtration of separating out, washing, obtains blackish green target product 0.31g after drying, yield 92%.
1H NMR（400MHz,Acetone-d
6）:δ2.60-2.69（m,2H,-CH
2-）,3.16（t,2H,-CH
2-）,4.76（s,3H,N
+-CH
3）,5.06（t,2H,N
+-CH
2-）,7.41（d,1H,bpy′-H）,7.43-7.48（m,5H,bpy-H and bpy′-H）,7.83（d,1H,bpy′-H）,7.91-8.02（m,10H,bpy-Hand bpy′-H）,8.67（s,1H,bpy′-H）,8.72（d,1H,bpy′-H）,8.77-8.82（m,8H,bpy-H and MV
2+-H
β）,9.35-9.38（d,4H,MV
2+-H
α）.HRMS（ESI,m/z）:[(M-4PF
6 -）]
4+calculated for C
44H
40N
8Os218.0750,found218.0175.
The synthesis of embodiment 2Os-C4-MV
The synthesis of this compound can refer to the synthetic route of above-mentioned Os-C3-MV, and except utilizing dibromopropane to replace except ethylene dibromide, other is the same.
1H NMR（400MHz,Acetone-d
6）:δ1.92-1.99（m,2H,-CH
2-）,2.27-2.33（m,2H,-CH
2-）,3.03（t,2H,-CH
2-,J=7.8Hz）,4.76（s,3H,N
+-CH
3）,4.98（t,2H,N
+-CH
2-,J=7.4Hz）,7.37（d,1H,bpy′-H,J=5.6Hz）,7.45-7.48（m,5H,bpy-H and bpy′-H）,7.81（d,1H,bpy′-H,J=5.9Hz）,7.92-8.03（m,10H,bpy-H and bpy′-H）,8.66（s,1H,bpy′-H）,8.75（d,1H,bpy′-H,J=8.1Hz）,8.78-8.82（m,8H,bpy-H and MV
2+-H
β）,9.33（d,2H,MV
2+-H
α,J=6.6Hz）,9.37（d,2H,MV
2+-H
α,J=6.4Hz）.HRMS（ESI,m/z）:[(M-4PF
6 -）]
4+calculated for C
45H
42N
8Os221.5781,found221.5775.
The synthesis of embodiment 3Os-C7-MV
The synthesis of this compound is with reference to the synthetic route of above-mentioned Os-C3-MV, and except utilizing dibromo-hexane to replace except ethylene dibromide, other is the same.
1H NMR（400MHz,Acetone-d
6）:δ1.43-1.51（m,6H,-CH
2-）,1.70-1.74（m,2H,-CH
2-）,2.17-2.22（m,2H,-CH
2-）,2.91（t,2H,-CH
2-,J=7.5Hz）,4.76（s,3H,N
+-CH
3）,4.94（t,2H,N
+-CH
2-,J=7.2Hz）,7.36（d,1H,bpy′-H,J=5.6Hz）,7.45-7.49（m,5H,bpy-H and bpy′-H）,7.81（d,1H,bpy′-H,J=5.9Hz）,7.94-8.05（m,10H,bpy-H and bpy′-H）,8.67（s,1H,bpy′-H）,8.78（d,1H,bpy′-H,J=8.1Hz）,8.80-8.83（m,8H,bpy-H and MV
2+-H
β）,9.35（d,2H,MV
2+-H
α,J=7.2Hz）,9.39（d,2H,MV
2+-H
α,J=7.0Hz）.HRMS（ESI,m/z）:[(M-4PF
6 -）]
4+calculated forC
48H
48N
8Os232.0899,found232.0895.
Embodiment 4 ultraviolet-visible absorption spectroscopy change detection
Prepare Os-C3-MV, 8-hydroxy guanine respectively, (concentration is respectively 1.0 × 10 to the aqueous solution of 2,6-bisnaphthols
-3mol/L), CB [8] concentration of aqueous solution is made into 8.0 × 10
-5mol/L.Utilize above-mentioned mother liquor to prepare respectively and obtain 2mL solution 1(Os-C3-MV:2 to be measured, 6-bisnaphthol: CB [8]: 8-hydroxy guanine=mol ratio 1:1:1:1, Os-C3-MV final concentration is 0.05mM) and solution 2(Os-C3-MV:2 to be measured, 6-bisnaphthol: CB [8]=mol ratio 1:1:1, Os-C3-MV final concentration is 0.05mM), solution 1,2 to be measured is moved in homemade quartz cell respectively, supersound process passes into argon gas 1h after being about 20min, utilize the oxygen in bubbling argon method removing solution, rapidly quartz cell sealed membrane is sealed.The quartz cell that solution 1 to be measured is housed is placed on continuous illumination under 150W xenon lamp, respectively at 20min, 40min, 80min, 120min, 180min, its ultraviolet-visible absorption spectroscopy change (Ultraviolet Detector: Lambda35, Perkin Elmer company of the U.S.) is measured during 240min.For exclusive PCR, quartz cell is placed in the circulator bath of about 20 DEG C in During Illumination, prevents illumination from causing the temperature of whole quartz cell to raise.In addition, illumination do not carried out to the quartz cell that solution 2 to be measured is housed and directly measure the change of its ultraviolet-visible absorption spectroscopy.The detected result of solution 1,2 to be measured respectively as shown in Figure 1, 2.
Embodiment 5 colour-change is observed
Prepare solution 1 and 2 to be measured similarly to Example 4, after then carrying out deoxygenation, encapsulation process similarly to Example 4, take pictures with camera when quartz cell to be placed under xenon lamp illumination 20 minutes, compare 8-hydroxy guanine sample add before and after colour-change.Result as shown in Figure 3.
Embodiment 6 fluorescence emission spectrum change detection
The operation such as preparation, deoxygenation, encapsulation process of solution 1 and 2 to be measured is with embodiment 4.The quartz cell that solution 1 to be measured is housed is placed on continuous illumination under 150W xenon lamp, respectively at 30min, 60min, 90min, its fluorescence emission spectrum change (fluorescence detector: Perkin-Elmer Ls55 spectrophotofluorometer) is measured when 120min, 150min, 180min.For exclusive PCR, quartz cell is placed in the circulator bath of about 20 DEG C in During Illumination, prevents illumination from causing the temperature of whole quartz cell to raise.In addition, illumination do not carried out to the quartz cell that solution 2 to be measured is housed and directly measure the change of its fluorescence emission spectrum.The detected result of solution 1,2 to be measured respectively as shown in Figure 4,5.
The mensuration of embodiment 78-hydroxy guanine DPV curve
Preparation 8-hydroxy guanine concentration is 1.0 × 10
-2the aqueous solution of mol/L, utilize above-mentioned mother liquor to obtain solution to be measured (the 0.1M phosphate buffer soln of solvent: pH=7.5) that 2mL8-hydroxy guanine concentration is 0.25mM, this solution to be measured is moved in homemade quartz cell, passes into argon gas 30 minutes to remove the oxygen in solution.Adopt three-electrode system, working electrode is glassy carbon electrode (diameter 3mm), and reference electrode is saturated calomel electrode (saturated KCl solution), and supporting electrode is platinum filament, at room temperature measures its electrochemical properties.DPV graphic representation records on the CHI660D electrochemical workstation of Shanghai Chen Hua Instrument Ltd., and sweep velocity is 25mV.S
-1.
Claims (6)
1. prepare a method for photosensitive dye osmium complex, it is characterized in that, comprise the steps:
(1) preparation is with L, L ' be the osmium title complex intermediate of part
The ligand L of following compound, L ' and the metal osmium salt being negative ion with Y ' will be selected from independently of one another simultaneously or carry out coordination reaction successively, obtain the osmium title complex intermediate of general formula LL ' Os-Y ', temperature of reaction is 20-200 DEG C, reaction times is 1-24h, reaction solvent is selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and wherein two or more is according to the mixed solvent of arbitrary proportion composition arbitrarily, and the metal osmium salt being negative ion with Y ' is (NH
4)
2[OsCl
6], K
2osCl
6or OsCl
3;
(2) intermediate 1 is prepared
By 4-methyl-4 '-R
3-dipyridyl and N-Lithiodiisopropylamide LDA react, temperature of reaction is-78-50 DEG C, reaction times is 0.5-8h, reaction solvent be selected from methylene dichloride, chloroform, tetrahydrofuran (THF), acetonitrile, DMF, DMSO and wherein arbitrarily two or more according to mixed solvent of arbitrary proportion composition, after reaction terminates, without separation, directly to adding Br (CH in system
2)
nbr, obtains intermediate 1, and temperature of reaction is-78-100 DEG C, and the reaction times is 1-48h, reaction solvent be selected from methylene dichloride, chloroform, tetrahydrofuran (THF), acetonitrile, DMF, DMSO and wherein arbitrarily two or more according to mixed solvent of arbitrary proportion composition,
(3) intermediate 2 is prepared
Intermediate 1 is carried out coordination with osmium title complex intermediate LL ' Os-Y ', obtain intermediate 2, temperature of reaction is 20-200 DEG C, reaction times is 1-24h, reaction solvent be selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and wherein arbitrarily two or more according to mixed solvent of arbitrary proportion composition;
(4) intermediate 3 is prepared
By intermediate 2 under KI catalysis with 4,4 '-second bipyridine reacts, obtain intermediate 3, temperature of reaction is 20-200 DEG C, reaction times is 1-24h, reaction solvent be selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and wherein arbitrarily two or more according to mixed solvent of arbitrary proportion composition;
(5) preparation is with L, L ', L " be the osmium title complex of part
By intermediate 3 and C
1-10alkane iodide R
4i reacts, obtain general formula LL ' L " the osmium title complex of Os-Y '; temperature of reaction is 20-200 DEG C, and the reaction times is 1-24h, reaction solvent be selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and wherein arbitrarily two or more according to mixed solvent of arbitrary proportion composition;
(6) target product is prepared
By osmium title complex LL ' L " Os-Y ' carries out negative ion replacement(metathesis)reaction with containing the sodium salt of Y, sylvite or ammonium salt by molar ratio 1:1-10, obtains target product LL ' L " Os-Y
4, temperature of reaction is 10-100 DEG C, and the reaction times is 5 minutes-2h, reaction solvent be selected from methyl alcohol, ethanol, ethylene glycol, acetone, water, DMF, DMSO and wherein arbitrarily two or more according to mixed solvent of arbitrary proportion composition; Or in step (3) ~ (5), carry out the above-mentioned negative ion replacement(metathesis)reaction of replacing Y ' with Y respectively, finally obtain target product LL ' L " Os-Y
4;
Described photosensitive dye osmium complex has having structure formula I:
LL’L”Os-Y
4(Ⅰ)
In formula I, Y is halide-ions, ClO
4 -, BF
4 -, PF
6 -or OTs
-, L, L ' and be selected from following part independently of one another:
Wherein, R
1and R
2be selected from H, C independently of one another
1-10alkyl, CHO, COOH, NH
2, NO
2, CN, OH, SH, C
1-6alkoxyl group, C
1-6alkylamino, C
1-6amide group, C
1-6haloalkyl, there is C
1-10the phenothiazinyl of alkyl or halogen,
L " be following part:
Wherein, n is the integer of 1-10, R
3for H, C
1-10alkyl, CHO, COOH, NH
2, NO
2, CN, OH, SH, C
1-6alkoxyl group, C
1-6alkylamino, C
1-6amide group, C
1-6haloalkyl, there is C
1-10the phenothiazinyl of alkyl or halogen, R
4for C
1-10alkyl.
2. a carcinogenesis DNA oxidative damage quick detection kit, is characterized in that, this test kit comprises A, B, D tri-kinds of compositions:
(1) composition A: host compound eight yuan of melon rings;
(2) composition B: as the photosensitive dye osmium complex according to claim 1 of guest compound I;
(3) components D: pure water or phosphoric acid salt, borate buffer solution.
3. test kit as claimed in claim 2, it is characterized in that, this test kit also comprises further:
(4) composition C: as the fluorescent small molecule of guest compound II, this fluorescent small molecule has having structure general formula I I:
Wherein, R
5and R
6be selected from H, C independently of one another
1-10alkyl, CHO, COOH, NH
2, CN, OH, SH, C
1-6alkoxyl group, C
1-6alkylamino, C
1-6amide group, C
1-6haloalkyl, there is C
1-10the phenothiazinyl of alkyl or halogen.
4. a 8-hydroxy guanine method for quick, is characterized in that, the method uses the test kit described in claim 2 to detect, and specifically comprises the steps:
(1) first composition A, B are dissolved in components D according to the ratio of mol ratio 1-5:1, with the densitometer of composition B, be made into the solution that concentration is 10 μMs of-1mM, then get this appropriate solution and join in transparent vessel, rare gas element deoxygenation, seals stand-by;
(2) joined by testing sample in above-mentioned transparent vessel, after illumination 15-30 minute, visual color changes or detects absorption spectrum change with opticinstrument.
5. a 8-hydroxy guanine method for quick, is characterized in that, the method uses the test kit described in claim 3 to detect, and specifically comprises the steps:
(1) first composition A, B, C are dissolved in components D according to the ratio of mol ratio 1-5:1:1, with the densitometer of composition B or C, be made into the solution that concentration is 10 μMs of-1mM, then get this appropriate solution and join in transparent vessel, rare gas element deoxygenation, seals stand-by;
(2) joined by testing sample in above-mentioned transparent vessel, after illumination 15-30 minute, visual color changes or detects ultravioletvisible absorption and/or fluorescence emission spectrum change with opticinstrument.
6. the detection method as described in claim 4 or 5, is characterized in that, the light source that described illumination adopts is the houselights that sunlight maybe can excite described photosensitive dye osmium complex.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6262264B1 (en) * | 1998-06-01 | 2001-07-17 | Roche Diagnostics Corporation | Redox reversible imidazole osmium complex conjugates |
| CN101023502A (en) * | 2004-07-29 | 2007-08-22 | 洛桑聚合联合学院 | 2,2 -bipyridine ligand, sensitizing dye and dye sensitized solar cell |
| CN101379140A (en) * | 2006-02-08 | 2009-03-04 | 岛根县 | Photosensitizer dye |
| CN102124060A (en) * | 2008-06-19 | 2011-07-13 | 宇部兴产株式会社 | Dinuclear ruthenium complex dye, ruthenium-osmium complex dye, photoelectric conversion element using any one of the complex dyes, and photochemical cell |
-
2013
- 2013-04-25 CN CN201310148539.7A patent/CN103205135B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6262264B1 (en) * | 1998-06-01 | 2001-07-17 | Roche Diagnostics Corporation | Redox reversible imidazole osmium complex conjugates |
| CN101023502A (en) * | 2004-07-29 | 2007-08-22 | 洛桑聚合联合学院 | 2,2 -bipyridine ligand, sensitizing dye and dye sensitized solar cell |
| CN101379140A (en) * | 2006-02-08 | 2009-03-04 | 岛根县 | Photosensitizer dye |
| CN102124060A (en) * | 2008-06-19 | 2011-07-13 | 宇部兴产株式会社 | Dinuclear ruthenium complex dye, ruthenium-osmium complex dye, photoelectric conversion element using any one of the complex dyes, and photochemical cell |
Non-Patent Citations (3)
| Title |
|---|
| Generation by Electron Transfer of an Emitting State Not Observed by Photoexcitation in a Linked Ru(bpy)32+-Methyl Viologen;Xiaohong Xu等;《J. Am. Chem. Soc.》;19960417;第118卷(第15期);3656-3660 * |
| Site-selective electron transfer from purines to electrocatalysts: voltammetric detection of a biologically relevant deletion in hybridized DNA duplexe;Patricia A Ropp等;《Chemistry & Biology》;19990802;第6卷(第9期);599-605 * |
| The photoinduced long-lived charge-separated state of Ru(bpy)3–methylviologen with cucurbit[8]uril in aqueous solution;Shiguo Sun等;《Chem. Commun》;20060824(第40期);4195-4197 * |
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