CN103205135A - Photosensitive dye osmium complex and preparation method thereof, canceration early stage deoxyribonucleic acid (DNA) oxidative damage quick detection kit and detection method - Google Patents
Photosensitive dye osmium complex and preparation method thereof, canceration early stage deoxyribonucleic acid (DNA) oxidative damage quick detection kit and detection method Download PDFInfo
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Abstract
The invention provides a photosensitive dye osmium complex and a preparation method thereof, and a kit and a method for performing quick canceration early stage deoxyribonucleic acid (DNA) oxidative damage detection. The photosensitive dye osmium complex has the structural general formula of LL'L''Os-Y4, wherein Y is a halide ion, ClO<4->, BF<4->, PF<6-> or OTs<->; L and L' are independently selected from 2,2'-dipyridyl and a derivative thereof, phenanthroline and a derivative thereof and DPPZ; and L'' is a precursor with 2,2'-dipyridyl and 4,4'-dipyridyl. The kit at least comprises a main body compound 8-element cucurbit urils (a component A), the photosensitive dye osmium complex (a component B) and pure water or phosphate and borate buffering solution (a component D). By adopting the kit, even under the condition of no instrumental equipment, a user can individually and quickly detect 8-oxoG under visible light with eyes only by one drop of blood.
Description
Technical field
The present invention is specifically related to a kind of light-sensitive coloring agent osmium title complex and preparation method thereof, uses this light-sensitive coloring agent osmium title complex to carry out test kit and the detection method of the early stage DNA oxidative damage of canceration rapid detection.
Background technology
Cancer in case find to be in late period mostly, makes the doctor have no plan to make improvements, and has only early diagnosis just can prevent trouble before it happens.The by product of 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 easiest to be oxidized in four bases of DNA, its main oxidation products is 8-oxygen-7,8-dihydro deoxy-guanine (8-hydroxyl guanine, 8-oxoG), it is comparatively stable after it forms in vivo, be the important biomolecule marker of DNA oxidative damage, can reflect the DNA extent of damage to its monitoring, thereby early warning, prevention are carried out in canceration.Therefore, quick, single-minded, detect the research of the 8-oxoG method among the DNA reliably, have important theoretical and realistic meaning.
The conventional sense method of 8-oxoG mainly contains among the DNA at present: enzyme immunoassay, high performance liquid chromatography and electrochemistry/mass spectrometry, kapillary chromatography of gases-mass spectrometry, gel electrophoresis etc.But these methods do not possess specificity, and need earlier to DNA be hydrolyzed, pre-treatment such as enzymolysis, derivatize, hybridization, detect by special technology and instrument, analysis time is longer again, has limited their applying in 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) unique in can single-minded oxidation 8-oxoG(0.5 ± 0.1V vs Ag/AgCl) chemical reagent, it has obtained good application in the Electrochemical Detection of 8-oxoG at present, its principle is as follows.
But this electrochemical detection method needs earlier by chemical bond Os (bpy)
3 2+Probe molecule grafts 5 ' end of DNA segment, perhaps probe molecule and DNA respectively evenly is coated to electrode surface layer by layer and makes film, and duplicate packages is coated with till the uniformity coefficient of film and thickness reach requirement, operates quite loaded down with trivial detailsly, is not suitable for conventional sense.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of canceration early stage DNA oxidative damage quick detection kit and detection method are provided, be used for early stage special early warning, the prevention of canceration.In addition, another object of the present invention provides a kind of novel light-sensitive coloring agent osmium title complex and preparation method thereof.
For achieving the above object, the present invention adopts following technical scheme:
1. a light-sensitive coloring agent osmium title complex is characterized in that, this light-sensitive coloring agent osmium title complex has following general structure I:
LL’L’’Os-Y
4 （Ⅰ）
In the formula I, Y is halide-ions, ClO
4 -, BF
4 -, PF
6 -Or OTs
-, L, L ' are 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, has C
1-10The phenothiazinyl of alkyl or halogen,
L ' ' is following part:
Wherein, n is the integer of 1-10, R
3Be 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, has C
1-10The phenothiazinyl of alkyl or halogen, R
4Be C
1-10Alkyl.
2, a kind of method for the preparation of above-mentioned light-sensitive coloring agent osmium title complex is characterized in that, comprises the steps:
(1) preparation is the osmium title complex intermediate of part with L, L '
To be selected from the ligand L, L ' of following compound independently of one another and be the metal osmium salt while of negative ion with Y ' or carry out coordination reaction successively, obtain the osmium title complex intermediate of general formula LL ' Os-Y ', temperature of reaction is 20-200 ℃, reaction times is 1-24h, reaction solvent is selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein
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, has C
1-10The phenothiazinyl of alkyl or halogen;
(2) preparation intermediate 1
With 4-methyl-4 '-R
3-dipyridyl and N-Lithiodiisopropylamide (LDA) reaction, temperature of reaction is-78-50 ℃, reaction times is 0.5-8h, reaction solvent is selected from methylene dichloride, chloroform, tetrahydrofuran (THF), acetonitrile, DMF, DMSO and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein, after reaction finishes, without separation, directly add the Br (CH of n=1-10 in the system
2)
nBr obtains intermediate 1, and temperature of reaction is-78-100 ℃, and the reaction times is 1-48h, and reaction solvent is selected from methylene dichloride, chloroform, tetrahydrofuran (THF), acetonitrile, DMF, DMSO and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein,
Wherein, R
3Be 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, has C
1-10The phenothiazinyl of alkyl or halogen;
(3) preparation intermediate 2
Intermediate 1 is carried out coordination with osmium title complex intermediate LL ' Os-Y ', obtain intermediate 2, temperature of reaction is 20-200 ℃, reaction times is 1-24h, and reaction solvent is selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein;
(4) preparation intermediate 3
With intermediate 2 under KI catalysis with 4, the reaction of 4 '-second bipyridine, obtain intermediate 3, temperature of reaction is 20-200 ℃, reaction times is 1-24h, and reaction solvent is selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein;
(5) preparation is with L, L ', L " be the osmium title complex of part
With intermediate 3 and C
1-10Alkane iodide R
4The I reaction, obtain general formula LL ' L " the osmium title complex of Os-Y '; temperature of reaction is 20-200 ℃, and the reaction times is 1-24h, and reaction solvent is selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein;
(6) preparation target product
With osmium title complex LL ' L " Os-Y ' carries out the negative ion replacement(metathesis)reaction with the sodium salt, sylvite or the ammonium salt that contain Y 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 ℃, and the reaction times is 5 minutes-2h, and reaction solvent is selected from methyl alcohol, ethanol, ethylene glycol, acetone, water, DMF, DMSO and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein; Perhaps in step (3)～(5), carry out the above-mentioned negative ion replacement(metathesis)reaction with Y displacement Y ' respectively, finally obtain target product LL ' L ' ' Os-Y
4
3, the early stage DNA oxidative damage of a kind of canceration quick detection kit is characterized in that, this test kit comprises A, B, three kinds of compositions of D:
(1) composition A: eight yuan of melon rings of host compound;
(2) composition B: as the above-mentioned light-sensitive coloring agent osmium title complex of guest compound I;
(3) components D: pure water or phosphoric acid salt, borate buffer solution.
As above-mentioned 3 described test kits, it is characterized in that 4, this test kit also further comprises:
(4) composition C: as the fluorescent small molecule of guest compound II, this fluorescent small molecule has following general structure II:
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, has C
1-10The phenothiazinyl of alkyl or halogen.
5, the early stage DNA oxidative damage of a kind of canceration method for quick is characterized in that, this method uses above-mentioned 3 described test kits to detect, and specifically comprises the steps:
(1) at first composition A, B are dissolved in the components D according to the ratio of mol ratio 1-5:1, with the densitometer of composition B, are made into the solution that concentration is 10 μ M-1mM, get this an amount of solution then and join in the transparent vessel, the rare gas element deoxygenation seals stand-by;
(2) testing sample is joined in the above-mentioned transparent vessel, after illumination 15-30 minute, visual inspection colour-change or detect absorption spectrum with opticinstrument and change.
6, the early stage DNA oxidative damage of a kind of canceration method for quick is characterized in that, this method uses above-mentioned 4 described test kits to detect, and specifically comprises the steps:
(1) at first composition A, B, C are dissolved in the 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 μ M-1mM, get this an amount of solution then and join in the transparent vessel, the rare gas element deoxygenation seals stand-by;
(2) testing sample is joined in the above-mentioned transparent vessel, after illumination 15-30 minute, visual inspection colour-change or detect with opticinstrument that UV, visible light absorbs and/or fluorescence emission spectrum changes.
As above-mentioned 5 or 6 described detection methods, it is characterized in that 7, the light source that described illumination is adopted is the houselights that sunlight maybe can excite described light-sensitive coloring agent osmium title complex.
Beneficial effect of the present invention is: developed 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 the sample of bleeding, even under the condition by any plant and instrument not, rely on naked eyes, can single-minded, quick (about 15 minutes) detect 8-oxoG under radiation of visible light.When this technology is highly suitable for routine physical examination and outpatient service chemical examination the early stage DNA oxidative damage of canceration is carried out the primary dcreening operation monitoring.In addition, new compound light-sensitive coloring agent osmium title complex LL ' L ' ' Os-Y of the present invention
4In the detection of 8-oxoG oxidative damage and association area, has application prospect very widely.
Description of drawings
Fig. 1 is the changing conditions that ultraviolet-visible absorption spectroscopy produces with the prolongation of light application time when adding the 8-oxoG sample in test kit of the present invention.
Fig. 2 is the ultraviolet-visible absorption spectroscopy when not adding the 8-oxoG sample in test kit of the present invention.
Fig. 3 is the photo in kind (left side: do not add the 8-oxoG sample, the right side: add the 8-oxoG sample) that adds 8-oxoG sample front and back quartz cell colour-change in test kit of the present invention.
Fig. 4 is the changing conditions that fluorescence emission spectrum produces with the prolongation of light application time when adding the 8-oxoG sample in test kit of the present invention.
Fig. 5 is the fluorescence emission spectrum when not adding the 8-oxoG sample in test kit of the present invention.
Fig. 6 is the DPV(Differential pulse voltammetry of 8-oxoG) graphic representation.
Embodiment
＜light-sensitive coloring agent osmium title complex 〉
New compound light-sensitive coloring agent osmium title complex of the present invention has following general structure I:
LL’L’’Os-Y
4 （Ⅰ）
In the formula I, Y is halide-ions, ClO
4 -, BF
4 -, PF
6 -Or OTs
-, L, L ' are 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, has C
1-10The phenothiazinyl of alkyl or halogen,
L ' ' is following part:
Wherein, n is the integer of 1-10, R
3Be 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, has C
1-10The phenothiazinyl of alkyl or halogen, R
4Be C
1-10Alkyl.
Unless otherwise indicated, term used herein " alkyl " comprises straight chained alkyl and branched-chain alkyl.For example, " C
1-6Alkyl " comprise C
6Alkyl, C
5Alkyl, methyl, ethyl, n-propyl, sec.-propyl and the tertiary butyl.Similarly rule also is applicable to other group that uses 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
3Be H, C
1-8Alkyl, COOH, NH
2, OH or halogen; More preferably, R
3Be H, C
1-6Alkyl or COOH; Optimally, R
3Be H or methyl.
In a specific embodiment of the present invention, preferably, R
4Be C
1-6Alkyl; More preferably, R
4Be C
1-4Alkyl; Optimally, R
4Be 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, light-sensitive coloring agent osmium title complex of the present invention is selected from following compound:
The preparation method of＜light-sensitive coloring agent osmium title complex 〉
Method for the preparation of above-mentioned light-sensitive coloring agent osmium title complex comprises the steps:
(1) preparation is the osmium title complex intermediate of part with L, L '
Ligand L, the L ' (R of following compound will be selected from independently of one another
1, R
2Implication the same) be metal osmium salt while of negative ion with Y ' or carry out coordination reaction successively, obtain the osmium title complex intermediate of general formula LL ' Os-Y ', temperature of reaction is 20-200 ℃, reaction times is 1-24h, and reaction solvent is selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein.
In the above-mentioned reaction, L, L ' can be identical, also can be different.As L, when L ' is identical, preferably will react with part while and metal osmium reactant salt, metal osmium salt: the mol ratio of part is preferably controlled in the scope of 1:1.8～3.As L, when L ' is inequality, preferably will react with ligand L, L ' respectively with metal osmium reactant salt, be after metal osmium salt reacts with ligand L or L ' earlier, again with ligand L ' or the L reaction, metal osmium salt: the mol ratio of ligand L or L ' is preferably controlled in the scope of 1:0.8～2.
In addition, be that the metal osmium salt of negative ion can be enumerated but is not limited to (NH with Y '
4)
2[OsCl
6], K
2OsCl
6, OsCl
3Deng.
In a preferred implementation, temperature of reaction is 40-200 ℃, and the reaction times is 1-20h, and reaction solvent is selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein.
Temperature of reaction is 80-200 ℃ more preferably in the embodiment at one, and the reaction times is 1-10h, and reaction solvent is selected from ethylene glycol, acetonitrile, water, DMF and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein.
In a most preferred embodiment, temperature of reaction is 150-200 ℃, and the reaction times is 1-5h, and reaction solvent is selected from ethylene glycol, DMF and mixed solvent thereof.
(2) preparation intermediate 1
With 4-methyl-4 '-R
3-dipyridyl (R
3Implication the same) with N-Lithiodiisopropylamide (LDA) reaction, temperature of reaction is-78-50 ℃, reaction times is 0.5-8h, reaction solvent is selected from methylene dichloride, chloroform, tetrahydrofuran (THF), acetonitrile, DMF, DMSO and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein, after reaction finishes, without separation, directly add the Br (CH of n=1-10 in the system
2)
nBr obtains intermediate 1, and temperature of reaction is-78-100 ℃, and the reaction times is 1-48h, and reaction solvent is selected from methylene dichloride, chloroform, tetrahydrofuran (THF), acetonitrile, DMF, DMSO and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein.
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, more preferably 1:0.8～2 are preferably 1:0.9～1.5 especially.Temperature of reaction is preferably-78～25 ℃, more preferably-78～0 ℃.Reaction times is preferably 0.5～4h, more preferably 1～2h.The preferred methylene dichloride of reaction solvent, chloroform, tetrahydrofuran (THF), acetonitrile, more preferably methylene dichloride, chloroform, tetrahydrofuran (THF), especially 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, more preferably 1:0.8～5 are preferably 1:2～5 especially.With Br (CH
2)
nIn the reaction of Br, temperature of reaction is preferably-78～40 ℃, more preferably-78～30 ℃, is preferably 0～30 ℃ especially.Reaction times is preferably 1～12h, and more preferably 1～8h is preferably 1～4h especially.The preferred methylene dichloride of reaction solvent, chloroform, tetrahydrofuran (THF), acetonitrile, more preferably methylene dichloride, chloroform, tetrahydrofuran (THF), especially preferably methylene dichloride, tetrahydrofuran (THF).
After product preferably separated with organic solvent extractions such as methylene dichloride, chloroforms, thick product was further refining with separation such as silica gel column chromatographies.
(3) preparation intermediate 2
Intermediate 1 is carried out coordination with osmium title complex intermediate LL ' Os-Y ', obtain intermediate 2, temperature of reaction is 20-200 ℃, reaction times is 1-24h, and reaction solvent is selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein.
In the above-mentioned reaction, and the mol ratio of LL ' Os-Y ' and intermediate 1 (LL ' Os-Y ': intermediate 1) be preferably 1:1～10, more preferably 1:1～8 are preferably 1:1～5 especially.Temperature of reaction is preferably 100～200 ℃, more preferably 150～200 ℃.Reaction times is preferably 1～12h, more preferably 1.5～5h.The preferred ethylene glycol of reaction solvent, acetonitrile, water, DMF, more preferably ethylene glycol, acetonitrile, water, especially preferably ethylene glycol.
Product preferably separates refining with silica gel column chromatography etc., the sodium salt that the product that obtains can be by containing Y, sylvite, ammonium salt etc. carry out Y ion exchange (implication of Y is the same).
(4) preparation intermediate 3
With intermediate 2 under KI catalysis with 4, the reaction of 4 '-second bipyridine, obtain intermediate 3, temperature of reaction is 20-200 ℃, reaction times is 1-24h, and reaction solvent is selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein.
In the above-mentioned reaction, intermediate 2 and 4, the mol ratio of 4 '-second bipyridine (intermediate 2:4,4 '-second bipyridine) is preferably 1:1～10, and more preferably 1:1～6 are preferably 1:1～3 especially.Temperature of reaction is preferably 50～150 ℃, more preferably 100～130 ℃.Reaction times is preferably 3～15h, more preferably 4～7h.Reaction solvent preferred alcohol, ethylene glycol, acetonitrile, water, DMF, more preferably ethylene glycol, acetonitrile, DMF, especially preferably acetonitrile, DMF.
Product preferably separates refining with silica gel column chromatography etc., the sodium salt that the product that obtains can be by containing Y, sylvite, ammonium salt etc. carry out the Y ion exchange.
(5) preparation is with L, L ', L " be the osmium title complex of part
With intermediate 3 and C
1-10Alkane iodide R
4The I reaction, obtain general formula LL ' L " the osmium title complex of Os-Y '; temperature of reaction is 20-200 ℃, and the reaction times is 1-24h, and reaction solvent is selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein.
In the above-mentioned reaction, intermediate 3 and R
4The mol ratio of I (intermediate 3:R
4I) be preferably 1:1～10, more preferably 1:2～8 are preferably 1:4～6 especially.Temperature of reaction is preferably 50～150 ℃, more preferably 80～130 ℃.Reaction times is preferably 6～18h, is preferably 10～15h especially.The preferred ethylene glycol of reaction solvent, acetonitrile, water, DMF, more preferably ethylene glycol, acetonitrile, DMF, especially preferably acetonitrile, DMF.
Product preferably separates refining with silica gel column chromatography etc., the sodium salt that the product that obtains can be by containing Y, sylvite, ammonium salt etc. carry out the Y ion exchange.
(6) preparation target product
Can obtain target product LL ' L ' ' Os-Y by dual mode
4: (a) as described above, in step (3)～(5), carry out the Y displacement respectively, finally obtain target product LL ' L ' ' Os-Y
4(b) only in step (5), to the osmium title complex LL ' L that obtains " Os-Y ' carries out Y displacement, obtains target product LL ' L ' ' Os-Y
4
Generally (each product: Y salt) 1:1-10 carries out the negative ion replacement(metathesis)reaction to each product by molar ratio with sodium salt, sylvite or the ammonium salt etc. that contain Y.Temperature of reaction is 10-100 ℃, is preferably 20～80 ℃, more preferably 20～60 ℃, is preferably 20～40 ℃ especially.Reaction times is 5 minutes-2h, is preferably 5 minutes～1.5h, and more preferably 8 minutes～1.5h is preferably 10 minutes～1h especially.Reaction solvent is selected from methyl alcohol, ethanol, ethylene glycol, acetone, water, DMF, DMSO and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein, particular methanol, ethanol, ethylene glycol, acetone, water, more preferably methyl alcohol, acetone, water, special particular methanol, acetone.
By the synthetic light-sensitive coloring agent osmium title complex of aforesaid method of the present invention, can adopt nuclear-magnetism, mass spectrum to determine its structure.
The early stage DNA oxidative damage of＜canceration quick detection kit 〉
This test kit comprises A, B, three kinds of compositions of D, and wherein composition B is above-mentioned light-sensitive coloring agent osmium title complex,
(1) composition A: eight yuan of melon rings of host compound;
(2) composition B: as the light-sensitive coloring agent osmium title complex of guest compound I;
(3) components D: pure water or phosphoric acid salt, borate buffer solution.
In addition, this test kit preferably comprises further that also fluorescent small molecule with following general structure is 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, has C
1-10The phenothiazinyl of alkyl or halogen.
Optimally, this guest compound 2 is selected from following compound:
The early stage DNA oxidative damage of＜canceration method for quick 〉
This method uses the mentioned reagent box to detect, and when comprising A, B, three kinds of compositions of D in the test kit, detection method specifically comprises the steps:
(1) at first composition A, B are dissolved in the components D according to the ratio of mol ratio 1-5:1, densitometer with composition B, be made into the solution that concentration is 10 μ M-1mM, getting an amount of (for example 0.1-5mL) this solution then joins in the transparent vessel (for example quartz cell), the rare gas element deoxygenation seals stand-by;
(2) testing sample is joined in the above-mentioned transparent vessel, after illumination 15-30 minute, visual inspection colour-change or detect ultraviolet-visible absorption spectroscopy with opticinstrument and change.
When comprising A, B, four kinds of compositions of C, D in the test kit, detection method specifically comprises the steps:
(1) at first composition A, B, C are dissolved in the components D according to the ratio of mol ratio 1-5:1:1, densitometer with composition B or C, be made into the solution that concentration is 10 μ M-1mM, getting an amount of (for example 0.1-5mL) this solution then joins in the transparent vessel (for example quartz cell), the rare gas element deoxygenation seals stand-by;
(2) testing sample is joined in the above-mentioned transparent vessel, after illumination 15-30 minute, visual inspection colour-change or detect ultraviolet-visible absorption spectroscopy and/or fluorescence emission spectrum changes with opticinstrument.
The light source that illumination is adopted comprises that sunlight, houselights etc. can excite composition B(light-sensitive coloring agent osmium title complex arbitrarily) light source.Detection after the illumination both can utilize the naked eyes direct viewing, also can be by optical detecting instrument such as ultraviolet, fluorescence arbitrarily.
＜design philosophy of the present invention 〉
Use the terpyridyl osmium 8-oxoG to be carried out in the method for Electrochemical Detection the single-minded oxygenant Os (bpy) of 8-oxoG
3 3+By anodizing, in view of the terpyridyl osmium is a kind of light-sensitive coloring agent, can excite Os (bpy) by illumination fully by electrochemistry
3 2+, make it transit to excited state
*Os (bpy)
3 2+, excited state passes to electron acceptor(EA) methyl amethyst (MV with electronics
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 exists slower, the inefficient defective of electronics transmission speed, is unfavorable for practical application.If (brief note is: Os (bpy) as following compound with both
3 2+-MV
2+) shown in couple together like that (being that two parts (L, L ') in the osmium part are 2,2 '-dipyridyl, and another one part (L ") has 2; 2 '-dipyridyl and 4; 4 '-dipyridyl structure), change it into intramolecular interaction, just can realize Os under the illumination (bpy)
3 3+To MV
2+Efficient single-minded oxidation obtains oxidisability transition state Os (bpy)
3 3+-MV
+
The present invention just is being based on above-mentioned technological thought and is carrying out light-sensitive coloring agent osmium complex structure design.Through a large amount of deep discussion and the researchs of the present inventor, found that:
(1) as ligand L, L ', when using 2, when introducing suitable substituent derivative on 2 '-dipyridyl ring, perhaps ligand L, L ' one or both of adopt phenanthroline and derivative or DPPZ(dipyrido[3 simultaneously, 2-a'2', 3'-c] phenazine) time, can obtain and only use the suitable even better oxidation effectiveness of dipyridyl.Namely as ligand L, L ', can be selected from following part (R independently of one another
1, R
2Implication as above).
Studies show that, only adopt 2,2 '-dipyridyl and derivative thereof be during as part, the light-sensitive coloring agent osmium title complex of acquisition relative with the reactive force between the dna double spirane structure a little less than; When adopting phenanthroline and derivative thereof as part, have stronger interaction between the light-sensitive coloring agent osmium title complex of acquisition and the dna double spirane structure, and with the reactive force of RNA a little less than; And when adopting DPPZ as part, DPPZ in the light-sensitive coloring agent osmium title complex can enter in the dna double spirane structure in cuttage, be " interacting in the quasi-molecule " on the DNA skeleton with light-sensitive coloring agent osmium title complex and the intermolecular role transformation of 8-oxoG, thereby the 8-oxoG in the O-DNA obtains better detected result more efficiently.In addition, though 2, the detection effect of 2 '-dipyridyl and derivative thereof is slightly poor, and it is synthetic simple relatively, and preparation cost is lower.
(2) as ligand L ", can be 2,2 '-dipyridyl and 4 is introduced suitable substituting group, i.e. ligand L in 4 '-dipyridyl structure " optional from following part (R
3, R
4Implication as above).
Yet, with Os (bpy)
3 2+-MV
2+Be example, under the driving of potential difference, MV
+Electronics certainly will be returned rapidly and give Os (bpy)
3 3+, whole system is returned to original state Os (bpy) again
3 2+-MV
2+Has only the stable MV of living
+On electronics, it can not be returned fast, just can obtain the oxidation state Os (bpy) of single-minded oxidation 8-oxoG
3 3+And eight yuan of melon rings (Cucurbit[8] uril, CB[8]) to MV
2+, MV
+The self-assembly performance that reaches their derivative uniqueness can address these problems well, and its principle is as follows.
With Os (bpy)
3 2+-MV
2+With CB[8] (brief note is: Os to be self-assembled into the 1:1 coordination compound in the aqueous solution
2+-MV
2+/ CB[8]), under illumination, Os
2+-MV
2+(λ
Max=480nm) absorb luminous energy to transit to excited state * Os
2+-MV
2+, excited state forms charge separation transition state Os by the intramolecularly transfer transport
3+-MV
+, CB[8] and MV
+Self-assembly will play certain stabilization to this charge separation transition state, thereby 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 having 8-oxoG, (Os in the sample
3+-MV
+)/CB[8] will carry out the specificity oxidation to it, generate and go back ortho states (Os
2+-MV
+)/CB[8], this moment two molecules Os
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), the remarkable color change of Glassless takes place.Whole process can be schematically as follows.
The present invention just is being based on above-mentioned technological thought and is carrying out the early stage DNA oxidative damage of canceration quick detection kit design.With eight yuan of melon rings as host compound, with light-sensitive coloring agent osmium title complex of the present invention as guest compound (guest compound I), after being dissolved in these compounds in pure water or phosphoric acid salt, the borate buffer solution, just can carry out efficient, quick, special, reliable detection to containing the 8-oxoG sample.
In addition, in view of the variation of having only absorption spectrum in the said process, can cause restriction to detection to a certain extent.Consider that fluorescence detection method has simple and direct, characteristic efficiently, appropriate fluorescent small molecule can be incorporated in the system as 2,6-bisnaphthol (guest compound II), make itself and MV
2+Under charge transfer (charge transfer) effect, entering into CB[8 jointly] cavity carries out self-assembly.When the free radical dimer at CB[8] form in the cavity after, fluorescent small molecule is from CB[8] " being squeezed " come out, free in solution, make the change that produces color and fluorescence in the testing process simultaneously, thereby give this system more powerful practicality, further strengthen and detect effect.Its principle (fluorescent small molecule is example with 2,6-bisnaphthol) as follows.
These test kits of the application of the invention, can detect 8-oxoG efficient, quick, special, reliably, detect easy and simple to handle, and operator do not need through professional training, only need the sample of bleeding, even under the condition by any plant and instrument not, rely on naked eyes, under radiation of visible light, can single-minded, quick (about 15 minutes) detect 8-oxoG, when being highly suitable for routine physical examination and outpatient service chemical examination the early stage DNA oxidative damage of canceration be carried out the primary dcreening operation monitoring.Certainly, the present invention also can carry out the detection of 8-oxoG by opticinstruments such as uv-spectrophotometric instrument, luminoscopes.
Above content be in conjunction with concrete preferred implementation to further describing that the present invention does, can not assert that concrete enforcement of the present invention is confined to these explanations.For the general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, can also make some simple deduction or replace, all should be considered as belonging to protection scope of the present invention.In addition, detecting as the DNA oxidative damage is new compound light-sensitive coloring agent osmium title complex LL ' L ' ' Os-Y of the present invention
4A kind of purposes; but can not assert that compound of the present invention only is used for the DNA oxidative damage and detects; for the general technical staff of the technical field of the invention; under the consideration of the same function mechanism that detects as the DNA oxidative damage based on The compounds of this invention; can also make some simple inferences; 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 with reference to the accompanying drawings with after the embodiments of the invention.
Embodiment
Embodiment 1Os-C3-MV's is synthetic
(1) Os (bpy)
2Cl
2Synthetic:
With (NH
4)
2[OsCl
6] (1.0g, 2.3mmol) with 2, (0.72g 4.6mmol) is dissolved in the 30ml ethylene glycol 2 '-dipyridyl, at nitrogen protection lower magnetic force stirring heating backflow 1h.Behind the reaction mixture cool to room temperature, add 1M Na
2S
2O
4(60mL) in ice-water bath, keep 30min.Filter, the atropurpureus throw out priority water and the ether that obtain fully wash, and vacuum-drying gets black solid 0.92g, productive rate 70%.
(2) intermediate 1 is synthetic:
With 4-methyl-2,2 '-second bipyridine (2.5g 13.5mmol) is dissolved in dry THF(50mL) in, solution places ice-water bath, with the LDA(2.0mL Diisopropylamine of new system, and 1.6M n-Butyl Lithium 8.1mL, 10mL THF) dropwise add, solution becomes black, continues to stir 1h, get 1, (50mmol 4.3mL) adds rapidly the 2-ethylene dibromide, then ice-water bath is removed, continue to stir 3h under the room temperature, add 100mL phosphoric acid buffer (pH=7.0) stopped reaction.Solution CH
2Cl
2(CH is collected in 3 * 50mL) extractions
2Cl
2Layer is also used anhydrous magnesium sulfate drying, obtains faint yellow oily thing behind the evaporate to dryness.Thick product is separated with silica gel column chromatography, collect the component of ethyl acetate-methylene dichloride (1:9), obtain oily product 2.2g behind the 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) intermediate 2 is synthetic:
With intermediate 1(0.56g, 2.02mmol) with Os (bpy)
2Cl
2(0.29g 0.51mmol) joins in the 5ml ethylene glycol solvent, under the nitrogen protection, and lucifuge reflux 2h.Separate with silica gel column chromatography the cooling back, and developping agent is CH
3COCH
3: H
2O:KNO
3Saturated aqueous solution=20:1:1(V/V/V), collect blackish green component, the evaporate to dryness developping agent is dissolved in it in dry methyl alcohol, removes by filter insoluble saltpetre, methyl alcohol is concentrated into about 5mL again, then it is dropped to the saturated NH of 10ml
4PF
6In the aqueous solution, react 0.5h under the room temperature, displacement NO
3 -Gegenion, with the sedimentation and filtration of separating out, washing, getting blackish green product 0.49g(after the drying is intermediate 2), yield 90%.
(4) intermediate 3 is synthetic:
With intermediate 2(0.42g, 0.39mmol), 4, (1.22g 0.78mmol) and KI(0.13g, 0.78mmol) is dissolved among the DMF of 10mL drying 4 '-second bipyridine, and lucifuge is heated to 115 ℃ of reaction 5h under the nitrogen.Carry out separation and purification with silica gel column chromatography after the solvent evaporated, developping agent is CH
3COCH
3: H
2O:KNO
3Saturated aqueous solution=10:1:1(V/V/V), collect the bigger blackish green component of polarity, the evaporate to dryness developping agent is dissolved in it in dry methyl alcohol, removes by filter insoluble saltpetre, methyl alcohol is concentrated into about 5mL again, then it is dropped to the saturated NH of 10ml
4PF
6In the aqueous solution, react 0.5h under the room temperature, displacement NO
3 -Gegenion, with the sedimentation and filtration of separating out, washing, getting blackish green product 0.32g(after the drying is intermediate 3), yield 64%.
(5) Os-C3-MV's is synthetic:
With intermediate 3(0.30g, 0.23mmol), (1.63g 1.15mmol) is dissolved in the acetonitrile of 10ml drying methyl iodide, and the lucifuge reflux is spent the night under the nitrogen.Carry out separation and purification with silica gel column chromatography after the solvent evaporated, developping agent is CH
3COCH
3: H
2O:KNO
3Saturated aqueous solution=5:1:1(V/V/V), collect blackish green component, the evaporate to dryness developping agent is dissolved in it in dry methyl alcohol, removes by filter insoluble saltpetre, methyl alcohol is concentrated into about 5mL again, then it is dropped to the saturated NH of 10ml
4PF
6In the aqueous solution, react 0.5h under the room temperature, displacement NO
3 -Gegenion, with the sedimentation and filtration of separating out, washing gets blackish green target product 0.31g, yield 92% after the drying.
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.
Embodiment 2Os-C4-MV's is synthetic
The synthetic of this compound can be with reference to the synthetic route of above-mentioned Os-C3-MV, and except utilizing dibromopropane to replace the 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.
Embodiment 3Os-C7-MV's is synthetic
The synthetic route of the above-mentioned Os-C3-MV of synthetic reference of this compound, except utilizing dibromo-hexane to replace the 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 spectroscopies change to be measured
Prepare Os-C3-MV, 8-hydroxyl guanine, 2 respectively, (concentration is respectively 1.0 * 10 to the aqueous solution of 6-bisnaphthol
-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-hydroxyl guanine=mol ratio 1:1:1:1, the 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, the Os-C3-MV final concentration is 0.05mM), solution 1 to be measured, 2 is moved into respectively in the homemade quartz cell, feed argon gas 1h behind the about 20min of supersound process, utilize the argon gas Bubbling method to remove oxygen in the solution, rapidly with quartz cell with sealing film phonograph seal.The quartz cell that solution 1 to be measured is housed is placed on continuous illumination under the 150W xenon lamp, respectively at 20min, 40min, 80min, 120min, 180min, measure its ultraviolet-visible absorption spectroscopy during 240min and change (Ultraviolet Detector: Lambda35, U.S. Perkin Elmer company).Disturb for getting rid of, in the illumination process, quartz cell placed the circulator bath about 20 ℃, prevent that illumination from causing that the temperature of whole quartz cell raises.In addition, the quartz cell that solution 2 to be measured is housed is not carried out illumination and directly measure its ultraviolet-visible absorption spectroscopy variation.Solution 1 to be measured, 2 detected result are respectively as shown in Figure 1, 2.
Embodiment 5 colour-change are observed
Prepare solution 1 to be measured and 2 similarly to Example 4, carry out deoxygenation, encapsulation process similarly to Example 4 then after, quartz cell is placed under the xenon lamp illumination takes pictures with camera in the time of 20 minutes, the colour-change before and after relatively 8-hydroxyl guanine sample adds.The result as shown in Figure 3.
Embodiment 6 fluorescence emission spectrums change to be measured
Operations such as solution 1 to be measured and 2 preparation, deoxygenation, encapsulation process are with embodiment 4.The quartz cell that solution 1 to be measured is housed is placed on continuous illumination under the 150W xenon lamp, respectively at 30min, 60min, 90min, 120min, 150min measures its fluorescence emission spectrum and changes (fluorescence detector: Perkin-Elmer Ls55 spectrophotofluorometer) during 180min.Disturb for getting rid of, in the illumination process, quartz cell placed the circulator bath about 20 ℃, prevent that illumination from causing that the temperature of whole quartz cell raises.In addition, the quartz cell that solution 2 to be measured is housed is not carried out illumination and directly measure its fluorescence emission spectrum variation.Solution 1 to be measured, 2 detected result are respectively shown in Fig. 4,5.
The mensuration of embodiment 78-hydroxyl guanine DPV curve
Preparation 8-hydroxyl guanine concentration is 1.0 * 10
-2The aqueous solution of mol/L, utilize above-mentioned mother liquor to obtain 2mL8-hydroxyl guanine concentration and be the solution to be measured (the 0.1M phosphate buffer soln of solvent: pH=7.5) of 0.25mM, should be moved in the homemade quartz cell by solution to be measured, feed argon gas 30 minutes to remove the oxygen in the 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.The DPV graphic representation records at the CHI660D electrochemical workstation of Shanghai occasion China Instr Ltd., and sweep velocity is 25mV.S
-1
Claims (7)
1. a light-sensitive coloring agent osmium title complex is characterized in that, this light-sensitive coloring agent osmium title complex has following general structure I:
LL’L’’Os-Y
4 （Ⅰ）
In the formula I, Y is halide-ions, ClO
4 -, BF
4 -, PF
6 -Or OTs
-, L, L ' are 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, has C
1-10The phenothiazinyl of alkyl or halogen,
L ' ' is following part:
Wherein, n is the integer of 1-10, R
3Be 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, has C
1-10The phenothiazinyl of alkyl or halogen, R
4Be C
1-10Alkyl.
2. the method for the preparation of the described light-sensitive coloring agent osmium of claim 1 title complex is characterized in that, comprises the steps:
(1) preparation is the osmium title complex intermediate of part with L, L '
To be selected from the ligand L, L ' of following compound independently of one another and be the metal osmium salt while of negative ion with Y ' or carry out coordination reaction successively, obtain the osmium title complex intermediate of general formula LL ' Os-Y ', temperature of reaction is 20-200 ℃, reaction times is 1-24h, reaction solvent is selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein
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, has C
1-10The phenothiazinyl of alkyl or halogen;
(2) preparation intermediate 1
With 4-methyl-4 '-R
3-dipyridyl and N-Lithiodiisopropylamide LDA reaction, temperature of reaction is-78-50 ℃, reaction times is 0.5-8h, reaction solvent is selected from methylene dichloride, chloroform, tetrahydrofuran (THF), acetonitrile, DMF, DMSO and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein, after reaction finishes, without separation, directly add the Br (CH of n=1-10 in the system
2)
nBr obtains intermediate 1, and temperature of reaction is-78-100 ℃, and the reaction times is 1-48h, and reaction solvent is selected from methylene dichloride, chloroform, tetrahydrofuran (THF), acetonitrile, DMF, DMSO and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein,
Wherein, R
3Be 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, has C
1-10The phenothiazinyl of alkyl or halogen;
(3) preparation intermediate 2
Intermediate 1 is carried out coordination with osmium title complex intermediate LL ' Os-Y ', obtain intermediate 2, temperature of reaction is 20-200 ℃, reaction times is 1-24h, and reaction solvent is selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein;
(4) preparation intermediate 3
With intermediate 2 under KI catalysis with 4, the reaction of 4 '-second bipyridine, obtain intermediate 3, temperature of reaction is 20-200 ℃, reaction times is 1-24h, and reaction solvent is selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein;
(5) preparation is with L, L ', L " be the osmium title complex of part
With intermediate 3 and C
1-10Alkane iodide R
4The I reaction, obtain general formula LL ' L " the osmium title complex of Os-Y '; temperature of reaction is 20-200 ℃, and the reaction times is 1-24h, and reaction solvent is selected from methyl alcohol, ethanol, ethylene glycol, acetonitrile, water, DMF, DMSO and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein;
(6) preparation target product
With osmium title complex LL ' L " Os-Y ' carries out the negative ion replacement(metathesis)reaction with the sodium salt, sylvite or the ammonium salt that contain Y 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 ℃, and the reaction times is 5 minutes-2h, and reaction solvent is selected from methyl alcohol, ethanol, ethylene glycol, acetone, water, DMF, DMSO and two or more mixed solvent of forming according to arbitrary proportion arbitrarily wherein; Perhaps in step (3)～(5), carry out the above-mentioned negative ion replacement(metathesis)reaction with Y displacement Y ' respectively, finally obtain target product LL ' L ' ' Os-Y
4
3. the early stage DNA oxidative damage of a canceration quick detection kit is characterized in that, this test kit comprises A, B, three kinds of compositions of D:
(1) composition A: eight yuan of melon rings of host compound;
(2) composition B: as the described light-sensitive coloring agent osmium of the claim 1 of guest compound I title complex;
(3) components D: pure water or phosphoric acid salt, borate buffer solution.
4. test kit as claimed in claim 3 is characterized in that, this test kit also further comprises:
(4) composition C: as the fluorescent small molecule of guest compound II, this fluorescent small molecule has following general structure II:
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, has C
1-10The phenothiazinyl of alkyl or halogen.
5. the early stage DNA oxidative damage of a canceration method for quick is characterized in that, this method right to use requires 3 described test kits to detect, and specifically comprises the steps:
(1) at first composition A, B are dissolved in the components D according to the ratio of mol ratio 1-5:1, with the densitometer of composition B, are made into the solution that concentration is 10 μ M-1mM, get this an amount of solution then and join in the transparent vessel, the rare gas element deoxygenation seals stand-by;
(2) testing sample is joined in the above-mentioned transparent vessel, after illumination 15-30 minute, visual inspection colour-change or detect absorption spectrum with opticinstrument and change.
6. the early stage DNA oxidative damage of a canceration method for quick is characterized in that, this method right to use requires 4 described test kits to detect, and specifically comprises the steps:
(1) at first composition A, B, C are dissolved in the 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 μ M-1mM, get this an amount of solution then and join in the transparent vessel, the rare gas element deoxygenation seals stand-by;
(2) testing sample is joined in the above-mentioned transparent vessel, after illumination 15-30 minute, visual inspection colour-change or detect with opticinstrument that UV, visible light absorbs and/or fluorescence emission spectrum changes.
7. as claim 5 or 6 described detection methods, it is characterized in that the light source that described illumination is adopted is the houselights that sunlight maybe can excite described light-sensitive coloring agent osmium title complex.
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|---|---|---|---|---|
| CN109097460A (en) * | 2018-08-30 | 2018-12-28 | 青岛大学 | A kind of detection method of the nitrogenous base of oxidative modification |
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| CN114835634A (en) * | 2022-05-20 | 2022-08-02 | 贵州大学 | Preparation and application of supramolecular fluorescent probe capable of detecting o-nitrophenol in water |
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