CN102924531A - Iridium-selenium polypyridine complex as well as preparation method and application thereof - Google Patents
Iridium-selenium polypyridine complex as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an iridium-selenium polypyridine complex as well as a preparation method and an application of the iridium-selenium polypyridine complex. The iridium-selenium polypyridine complex disclosed by the invention has the chemical formula of [Ir (N-C)2(phenSe)]+(N-C=ppy, dfppy, btp, 2-pq or dbq), is derived from selenium-5, 6-diamine-1, 10-ortho phenanthroline, has a good living cell mitochondria coloring characteristic, a strong cell membrane penetration capacity and low cytotoxicity and has a great application potential in the aspects of biolabeling and cell imaging.
Description
Technical field
The present invention relates to viable cell staining technique field, be specifically related to a series of new iridium-selenium multi-pyridine ligand, and its preparation method and application.
Background technology
Along with day by day deep to cell research of people, the biomedical optical image technologies such as Magnetic resonance imaging (MRI), PET (PET), single photon emission fault imaging (SPECT) and laser confocal fluorescence microscope technology also obtain development, and the exploitation of suitable bio-imaging reagent is a current study hotspot.At present, the commercial fluorescence dye major part that is applied to the cell imaging field is some organic molecules, such as PI, DAPI, EB, Hoechst, MitoTracker, JC-1 etc.Yet these organic molecules exist some shortcomings (V. Fernandez-Moreira, F. L. Thorp-Greenwood and M. P. Coogan.
Chem. Commun.2010,
46, 186-202): water-soluble relatively poor, water surrounding (in substratum) easily produce precipitation or with cytosis after at once separate out, affect Color; Have higher cytotoxicity, after dyestuff and the cytosis, cause the death of cell, impact is to the observation of cell standard state; Light stability is low, because being subjected in the air or the effect of promoting agent group (singlet oxygen etc.) in the medium, dyestuff is after the excitation wavelength irradiation, fluorescence constantly weakens, it is serious that light is drifted phenomenon, be unfavorable for that imaging stablizes (M. S. Lowry, W. R. Hudson, R. A. Pascal and S. Bernhard.
J. Am. Chem. Soc.2004,
126, 14129-14135); Stoke displacement between excitation spectrum and emmission spectrum is little, generally about tens nanometers, is unfavorable for distinguishing the self-quenching of endogenous fluorescence and reduction dyestuff itself.Based on some shortcomings that organic dye exists, people more turn to sight on the transition metal complex with good photoelectricity attribute.
Transition metal complex, particularly iridium multi-pyridine ligand are because its good optical physics and spectrochemical property have advantages of aspect the cell imaging reagent that it is unique.Compare with other transition metal complexes, the iridium multi-pyridine ligand has high cell migration rate, easily by Cell uptake.In addition, iridium metal complex seems more excellent at aspects such as luminosity and light stability, therefore receives more many concerns.The hyperfluorescenceCeng Yongminggaoyingguang of the common dependence of iridium metal complex itself is positioned certain position of cell, thereby becomes good cell dye.The Cell uptake of these title complexs and cell dyeing research thereof tentatively begin.The first iridium multi-pyridine ligand [Ir (dfpy) that is applied to cell imaging reported for work in 2008
2(bpy)] (PF
6) and [Ir (dfpy)
2(quqo)] (PF
6), wherein the introducing of F element has increased the lipotropy of title complex, makes it more easily pass through cytolemma.Laser co-focusing experiment shows that title complex is positioned tenuigenin, has the anti-photobleaching effect that is better than DAPI, be good tenuigenin dyestuff (M. Yu, Q. Zhao, L. Shi, F. Li, Z. Zhou, H. Yang, T. Yi and C. Huang,
Chem. Commun., 2008,2115-2117).The dipyridyl ring of part is modified the alkyl (C of different lengths
2H
6,-C
10H
21,-C
18H
37), can strengthen it to the nuclear membrane perviousness, show roughly the same Color.This class title complex all tends to the tenuigenin location, particularly examine Zhou Dingwei, this strong effectively locating effect is inferred the hydrophobic tissue that derives from long alkyl chain and examine week, such as interaction (the K. K. W. Lo between endoplasmic reticulum and the golgi body etc., P. K. Lee and J. S. Y. Lau
Organometallics, 2008,27,2998 – 3006).The complex of iridium that a class contains dipyridyl quinoxaline part is also studied by this working group, find that this class complex of iridium can enter nucleus, interact with DNA, nucleus is dyeed, this is the first nucleus fluorescent probe (K. Y. Zhang in this class complex of iridium, S. P. Y. Li, N. Zhu, L. W. S. Or, M. S. H. Cheung, Y. W. Lam, and K. K. W. Lo
Inorg. Chem., 2010,49,2530-2540).In general, all kinds of iridium metal complexes that are applied to the cell dyeing imaging of having reported at present, the overwhelming majority dyes and is positioned tenuigenin, and the iridium metal complex that is applied to the dyeing of viable cell plastosome has no report at present.
Summary of the invention
The object of the invention is to following several: the preparation method and application that a series of iridium-selenium multi-pyridine ligand is provided.
The present invention is achieved through the following technical solutions above-mentioned purpose:
Invention provides a kind of iridium-selenium multi-pyridine ligand, chemical formula [Ir (N-C)
2(phenSe)]
2+(N-C=ppy, dfppy, btp, 2-pq or dbq), structural formula are as shown in the formula among I, II, III, IV and the V any one:
Invention provides the preparation method of above-mentioned iridium-selenium multi-pyridine ligand simultaneously, may further comprise the steps:
With chlorine bridging dimer [(N-C)
2Ir (μ-Cl)]
2(suc as formula VI) joins in the ethanol/methylene mixed solvent heating reflux reaction under the protection of inert gas with the many pyridine ligands of selenium.
Wherein N-C is 2-(2-pyridyl) thionaphthene (btp), 2-(2, the 4-difluorophenyl) pyridine (dfppy), dibenzo [f, h] quinoline Nuo Xilin (dbp), 2-phenylquinoline (2-pq) and 2-phenylpyridine (ppy), the respectively corresponding compound that obtains above-mentioned formula I-formula V.[(N-C)
2Ir (μ-Cl)]
2Can be synthetic by existing method, but concrete preparation method's reference literature S. Sprouse, K. A. King, P. J. Spellane, and R. J. Watts,
J. Am. Chem. Soc., 1984,
106, 6647-6653.
The many pyridine ligands of selenium wherein are suc as formula shown in the VII.The many pyridine ligands of this selenium are called for short phen-Se, and its chemical name is selenium-5,6-diamines-1,10-phenanthroline.The existing document of preparation method's reference of the many pyridine ligands of this selenium (Qian Li, Dongdong Sun, Yanhui Zhou, Du Liu, Qianling Zhang and Jie Liu,
Inorg.Chem. Commun., 2012,
20, 142) and method, its synthetic route has been carried out partly optimizing and revising, reduced synthesis step.Step is as follows: with 1,10-phenanthroline-5, the mixture heating up of 6-diketone, tin anhydride, ammonium acetate and Glacial acetic acid stirs, and is cooled to room temperature behind 6 h that reflux, and filters, and filtrate is poured in the frozen water, standing over night, suction filtration; Water and ether repeatedly wash respectively, vacuum-drying.Wherein 1,10-phenanthroline-5, the 6-diketone can prepare with the mature technology of common synthetic many pyridine ligands, as reference literature (M. Yamada, Y. Tanaka, Y. Yoshimato, S. Kuroda and I. Shimao,
Bull. Chem. Soc. Jpn., 1992,65,1006.) and method.The many pyridine ligands of selenium can be applicable to prepare viable cell plastosome dyestuff.
Ethanol/methylene mixed solvent described in the inventive method is ethanol/methylene (v/v=2:1).Reflux time is 6-10h.
Above-mentioned back flow reaction is cooled to room temperature after finishing, and filters, and adds NH in the filtrate
4PF
6The methyl alcohol saturated solution, revolve steaming, the precipitation of generation is dry after filtering, crosses neutral alumina column, with the ratio wash-out that methylene dichloride and acetone mixed solution increase gradually in polarity, obtains to dissolve the elutriant of pure products, is drying to obtain described iridium-selenium multi-pyridine ligand.
As a kind of specific embodiment, above-mentioned iridium-selenium multi-pyridine ligand can be made by following steps: synthesize [(N-C) first
2Ir (μ-Cl)]
2, joining in the ethanol/methylene (v/v=2:1) with many pyridine ligands of selenium phen-Se, the lower 65 ℃ of reflux 6h of argon shield are cooled to room temperature, filter NH
4PF
6The methyl alcohol saturated solution add in the filtrate, spin off part methyl alcohol, the precipitation of generation is dry through the suction filtration final vacuum, crosses neutral alumina column, is methylene dichloride and the drip washing of acetone mixed solution of 5:1 with volume ratio, vacuum-drying namely gets described iridium-selenium multi-pyridine ligand; Wherein N-C is 2-(2, the 4-difluorophenyl) pyridine (dfppy), 2-(2-pyridyl) thionaphthene (btp), dibenzo [f, h] quinoline Nuo Xilin (dbq), 2-phenylquinoline (2-pq) or 2-phenylpyridine (ppy).
Described methylene dichloride and acetone mixed solution are that volume ratio is that 1:2 is to methylene dichloride and the acetone mixed solution of 5:1.
Iridium of the present invention-selenium multi-pyridine ligand is applied to prepare viable cell plastosome dye reagent.
Show after deliberation above-mentioned 5 kinds of title complex [Ir (N-C) that contain phenanthroline diketone-selenium part of the present invention
2(phenSe)]
+(N-C=ppy, dfppy, btp, 2-pq or dbq) has strong fluorescence intensity and fluorescence lifetime under aqueous conditions.Thereby iridium-selenium multi-pyridine ligand can be used for preparing the cell mitochondrial dyestuff of viable cell.MTT and flow cytometry experimental result show that the cytotoxicity of title complex is low under the low consistency conditions, and be little to cell injury.
Compared with prior art, the present invention has following beneficial effect:
Iridium of the present invention-selenium multi-pyridine ligand simple and stable structure, selenium element and the combination of phenanthroline diketone have weakened title complex becomes hydrogen bond with water molecules ability in this iridium (II) title complex, greatly strengthened the fluorescent characteristic of title complex, simultaneously, as one of human essential elements, the introducing of selenium element has also strengthened the cell transmembrane ability of title complex.Under the condition that need not cell pretreatment (such as electroporation or solvent fixing etc.), this title complex can be successfully painted to the plastosome in the viable cell, and the characteristics with the cultivation time is short, dyeing is highly sensitive, the Premeabilisation of cells ability is strong, large stoke displacement, low cytotoxicity and fast light bleaching will have great application potential aspect biomarker and the cell imaging.
Description of drawings
Many pyridine ligands of Fig. 1 selenium molecular structural formula;
Fig. 2 iridium-selenium multi-pyridine ligand molecular structural formula;
The route of synthesis of the many pyridine ligands of Fig. 3 selenium;
Fig. 4 chlorine bridging dimer [(N-C)
2Ir (μ-Cl)]
2Molecular structural formula;
The route of synthesis of Fig. 5 iridium-selenium multi-pyridine ligand;
The fluorescence spectrum of Fig. 6 iridium-selenium multi-pyridine ligand, [Ir]=10 mM, (L=Phen-Se);
Fig. 7 HeLa cell is the title complex [Ir (ppy) 2 (phenSe)] of 10 μ M respectively
+(PF
6 -) (A), [Ir (dfppy) 2 (phenSe)]
+(PF
6 -) (B), [Ir (btp) 2 (phenSe)]
+(PF
6 -) (C), [Ir (2-pq) 2 (phenSe)]
+(PF
6 -) (D), [Ir (dbq) 2 (phenSe)]
+(PF
6 -) (E) cultivate 30 min, dye altogether rear cell imaging result with Mitotracker-Green (1 μ M).(a) fluorogram of Mitotracker-Green after painted to cell; (b) fluorogram of title complex after painted to cell; (c) bright field visible light cell color-patch map; (d) a, b, the stack of c; A, B, C, D and E in the bracket represent respectively this five kinds of title complexs.
Embodiment
Embodiment 1Part selenium-5,6-diamines-1, the preparation of 10-phenanthroline (phen-Se)
Part phen-Se synthetic can with reference to existing document (Qian Li, Dongdong Sun, Yanhui Zhou, Du Liu, Qianling Zhang and Jie Liu,
Inorg.Chem. Commun., 2012,
20, 142) and preparation, its synthetic route has been carried out partly optimizing and revising, reduced synthesis step, make its synthetic more simple and fast, productive rate is high.Only need through two steps, wherein the first step is the reaction of the technology maturation of common synthetic many pyridine ligands.
(1) 1,10-phenanthroline-5, the 6-diketone
Can reference literature (M. Yamada, Y. Tanaka, Y. Yoshimato, S. Kuroda and I. Shimao,
Bull. Chem. Soc. Jpn., 1992,65,1006) and the method preparation.Under the ice bath cooling, in the three neck round-bottomed flasks that 4.0 g o-phenanthrolines and 4.0 g Potassium Bromides are housed, drip 40 cm
3The vitriol oil and 20 cm
3The mixed solution of concentrated nitric acid, simultaneously induction stirring.After dropwising, remove ice bath, be warming up to 85 ℃ of reactions 3 hours.After reaction finished, stopped heating also removed prolong, allowed the bromine ease go.Cooled orange-yellow reaction soln is poured 200 cm into fully
3In the frozen water, with the sodium hydroxide solution of 10 M pH=7 that neutralize carefully, there is a large amount of yellow mercury oxides to produce.With this suspension liquid with 4 ' 100 cm
3Chloroform extraction.Merge organic phase, use 50 cm
3Spend the night with anhydrous sodium sulfate drying again after the water washing.Boil off chloroform after the filtration, the solid product ethyl alcohol recrystallization gets orange-yellow needle-like crystal, and fusing point is 258 ℃.
(2) selenium-5,6-diamines-1,10-phenanthroline
With 1,10-phenanthroline-5,6-diketone (2.1 g, 10 mmol), tin anhydride (2.1g, 19 mmol), ammonium acetate (7.7 g, 100 mmol) stirs with the mixture heating up of 100 mL Glacial acetic acid, is cooled to room temperature behind 6 h that reflux, filter, filtrate is poured in the 600 mL frozen water, leave standstill after the night, if precipitate less, available ammoniacal liquor is regulated pH to neutral, suction filtration.Water and ether repeatedly wash respectively, and vacuum-drying gets yellowish brown solid 2.3 g, i.e. selenium-5,6-diamines-1,10-phenanthroline.Productive rate: 79.6%.ES-MS?(CH
3OH):?m/z?287.2[M+1]
+，595.3[M-Na
+]
2+
Embodiment 2Title complex [Ir (ppy)
2(phenSe)]
+(PF
6)
-, [Ir (dfppy)
2(phenSe)]
+(PF
6)
-, [Ir (btp)
2(phenSe)]
+(PF
6)
-, [Ir (2-pq)
2(phenSe)]
+(PF
6)
-, [Ir (dbq)
2(phenSe)]
+(PF
6)
-Synthetic method:
(1) [(dfppy)
2Ir (μ-Cl)]
2Synthetic
This compound synthetic can with reference to existing document (T. Peng, Y. Yang, Y. Liu, D. Ma, Z. Hou and Y. Wang,
Chem. Commun., 2011,47,3150) and preparation.Take by weighing IrCl
30.33 g(is 1.1 mmol approximately), 2-(2,4 difluorobenzene base) pyridine (dfppy) 0.501 g(2.62 mmol), in the mixed solution of 40 mL ethylene glycol ethyl ethers and water (V1:V2=3:1), the lower 120 ℃ of backflows of argon shield approximately 24 hours.After being chilled to room temperature, add suitable quantity of water, suction filtration is collected solid precipitation, uses normal hexane, and ether repeatedly washs, and oven dry obtains yellow-green colour powder, productive rate 53.5%.
(2) [(ppy)
2Ir (μ-Cl)]
2Synthetic
Synthetic method is same [(dfppy)
2Ir (μ-Cl)]
2, ppy (0.426 g, 2.75 mmol) is replaced dfpy, other steps are identical.Productive rate 67.7%.
(3) [(btp)
2Ir (μ-Cl)]
2Synthetic
Synthetic method is same [(ppy)
2Ir (μ-Cl)]
2Synthetic, btp (0.580 g, 2.75 mmol) is replaced dfppy, other steps are identical.Productive rate 45.6%.
(4) [(2-pq)
2Ir (μ-Cl)]
2Synthetic
Synthetic method is same [(ppy)
2Ir (μ-Cl)]
2Synthetic, 2-pq (0.564 g, 2.75 mmol) is replaced dfppy, other steps are identical.Productive rate 65.4%.
(5) [(dbq)
2Ir (μ-Cl)]
2Synthetic
Synthetic method is same [(ppy)
2Ir (μ-Cl)]
2Synthetic, dbq (0.506 g, 2.2 mmol) is replaced dfppy, other steps are identical.Productive rate 50.6%.
(6) [Ir (dfppy)
2(phenSe)]
+(PF
6)
-Synthetic
Will be [(dfppy)
2Ir (μ-Cl)]
2(0.244 g, 0.2 mmol) and phenSe (0.114 g, 0.4 mmol) are dissolved in the 48ml ethanol/methylene (v/v=2:1), under the argon atmosphere 65
oC reacts 6h, is cooled to room temperature, filters ammonium hexafluorophosphate (NH
4PF
6) the methyl alcohol saturated solution add in the filtrate, spin off part methyl alcohol, separate out a large amount of yellow mercury oxides, filter vacuum-drying.Thick product dissolves with a small amount of methylene dichloride, packing into approximately, 15 cm neutral aluminas (200 order) post separates, with methylene dichloride/acetone mixed solvent (increasing gradually solvent polarity) drip washing, collect the component under 5:1 methylene dichloride-acetone drip washing, the pressure reducing and steaming solvent, get yellow powdery solid, productive rate 49.8%.ES-MS?(CH
3OH):?m/z?857.1?[M-PF
6 -]
+.?
1H?NMR?(300?MHz,?d6-DMSO)?δ?9.21?(d,?J?=?7.6?Hz,?1H),?8.27?(t,?J?=?7.4?Hz,?2H),?8.10?–?7.93?(m,?2H),?7.67?(d,?J?=?5.8?Hz,?1H),?7.10?(t,?J?=?6.6?Hz,?1H),?7.05?–?6.91?(m,?1H),?5.66?(dd,?J?=?8.4,?2.2?Hz,?1H).
(7) [Ir (ppy)
2(phenSe)]
+(PF
6)
-Synthetic
Synthetic method is with [Ir (dfppy)
2(phenSe)]
+(PF
6)
-Synthetic.Will be [(ppy)
2Ir (μ-Cl)]
2(0.215 g, 0.2 mmol) substitutes [(dfppy)
2Ir (μ-Cl)]
2Get orange-yellow powdery solid, productive rate 48.4%.ES-MS?(CH
3OH):?m/z?785.2?[M-PF
6 -]
+.?
1H?NMR?(300?MHz,?Acetonitrile)?δ?9.17?(d,?J?=?6.8?Hz,?1H),?8.29?(d,?J?=?5.2?Hz,?1H),?8.06?(d,?J?=?8.1?Hz,?1H),?7.90?–?7.75?(m,?3H),?7.57?(d,?J?=?5.8?Hz,?1H),?7.07?(t,?J?=?7.5?Hz,?1H),?6.99?–?6.85?(m,?2H),?6.35?(d,?J?=?7.5?Hz,?1H)。
(8) [Ir (btp)
2(phenSe)]
+(PF
6)
-Synthetic
Synthetic method is with [Ir (dfppy)
2(phenSe)]
+(PF
6)
-Synthetic.Will be [(btp)
2Ir (μ-Cl)]
2(0.259 g, 0.2 mmol) substitutes [(dfppy)
2Ir (μ-Cl)]
2Red powdery solid, productive rate 54.9%.ES-MS?(CH
3OH):?m/z?899.1?[M-PF
6 -]
+.?
1H?NMR?(300?MHz,d6-?DMSO)?δ?9.24?–?9.17?(m,?1H),?8.21?–?8.12?(m,?1H),?8.04?(dd,?J?=?8.1,?5.3?Hz,?1H),?8.00?–?7.86?(m,?3H),?7.66?(d,?J?=?5.8?Hz,?1H),?7.23?(t,?J?=?7.6?Hz,?1H),?6.98?–?6.84?(m,?2H),?5.97?(d,?J?=?8.0?Hz,?1H).
(9) [Ir (2-pq)
2(phenSe)]
+(PF
6)
-Synthetic
Synthetic method is with [Ir (dfppy)
2(phenSe)]
+(PF
6)
-Synthetic.Will be [(2-pq)
2Ir (μ-Cl)]
2(0.255 g, 0.2 mmol) substitutes [(dfppy)
2Ir (μ-Cl)]
2Orange red powdery solid, productive rate 50.4%.Dissolve a small amount of title complex fully with methylene dichloride, add a small amount of toluene, make solvent slowly volatilization in air, obtain complex monocrystal.ES-MS?(CH
3OH):?m/z?887.2?[M-PF
6 -]
+.?
1H?NMR?(300?MHz,?Acetonitrile)?δ?8.98?(dd,?J?=?8.1,?1.4?Hz,?1H),?8.55?(dd,?J?=?5.3,?1.3?Hz,?1H),?8.39?–?8.29?(m,?2H),?8.18?(d,?J?=?7.8?Hz,?1H),?7.85?(dd,?J?=?8.1,?5.3?Hz,?1H),?7.71?(d,?J?=?7.2?Hz,?1H),?7.26?–?7.16?(m,?3H),?6.91?–?6.79?(m,?2H),?6.60?(d,?J?=?7.6?Hz,?1H).
(10) [Ir (dbq)
2(phenSe)]
+(PF
6)
-Synthetic
Synthetic method is with [Ir (dfppy)
2(phenSe)]
+(PF
6)
-Synthetic.Will be [(dbq)
2Ir (μ-Cl)]
2(0.275 g, 0.2 mmol) substitutes [(dfppy)
2Ir (μ-Cl)]
2Get orange-yellow powdery solid, productive rate 31.5%.Dissolve a small amount of title complex fully with methylene dichloride, add a small amount of toluene, make solvent slowly volatilization in air, obtain complex monocrystal.ES-MS?(CH
3OH):?m/z?938.1?[M-PF
6 -]
+.?
1H?NMR?(300?MHz,?d6-DMSO)?δ?9.20?(d,?J?=?8.2?Hz,?1H),?9.14?(d,?J?=?8.0?Hz,?1H),?8.82?(d,?J?=?8.2?Hz,?1H),?8.75?(d,?J?=?3.1?Hz,?1H),?8.35?(d,?J?=?8.2?Hz,?1H),?8.26?–?8.13?(m,?2H),?8.03?–?7.90?(m,?2H),?7.90?–?7.80?(m,?1H),?7.34?(t,?J?=?7.7?Hz,?1H),?6.52?(d,?J?=?7.2?Hz,?1H).
The complex fluorescent spectral detection the results are shown in Table 1 and Fig. 6.
Embodiment 3The laser co-focusing experiment of complex of iridium
Cell cultures: the Hela cell is cultivated in containing the DMEM substratum of 10% foetal calf serum, cell (5 ' l0
8/ L) be seeded at the bottom of the special-purpose glass of Laser Scanning Confocal Microscope in the culture dish culture dish diameter 35 mm, cover-glass thickness 0.085 ~ 0.13 mm wherein, ware center micro-pore diameter 10 mm, 5% CO
2Under 95% air conditions, 37 ℃ of cultivations, adherent growth 24 hours.
Laser Scanning Confocal Microscope-cell imaging: Hela cell and title complex { 5 μ M, [Ir (ppy) 2 (phenSe)]
+(PF
6 -) (A), [Ir (dfppy) 2 (phenSe)]
+(PF
6 -) (B), [Ir (btp) 2 (phenSe)]
+(PF
6 -) (C), [Ir (2-pq) 2 (phenSe)]
+(PF
6 -) (D), [Ir (dbq) 2 (phenSe)]
+(PF
6 -) (E) cultivation 30min, again with plastosome green fluorescence dyestuff Mitotracker-Green(50 nM) cultivate the regular hour, the sucking-off nutrient solution, then with PBS damping fluid washing 3 ~ 4 times, imaging on Leica TCS SP5 laser scanning co-focusing microscope, use 63 '/1.4 oily mirrors, as excitation light source, collect the fluorescence in 560 ~ 630nm scope with 405 nm light.Experimental result sees that (MitoTracker-Green excites with emission wavelength and is respectively 490,516 nm Fig. 7, and complex of iridium excites, emission wavelength is respectively 458,600 nm, scale: 50 mm).Plastosome green fluorescence dyestuff Mitotracker-Green wherein can not affected by mitochondrial membrane potential to be positioned the plastosome in the viable cell, from Fig. 6 experimental result as seen, a figure is that 490 nm wavelength excite lower MitoTracker-Green dyestuff to be positioned image in the Hela cell mitochondrial, b figure is that 458 nm wavelength excite lower title complex to be positioned image in the Hela cell, c figure is the Hela cell imaging under the light field, d figure is all image coverage diagrams, can see that a figure and b figure luminous position overlap, illustrate that title complex also is positioned the Hela cell mitochondrial, the good painted characteristic of viable cell plastosome is arranged.
Embodiment 4MTT vitro cytotoxicity analysis experiment
Select human cervical carcinoma cell (Hela), human liver cancer cell (HepG-2), the tumour cell such as lung carcinoma cell (A549) and people's normal breast epithelial cell (MCF-10A) carry out this research.Well-grown cell is become single cell suspension with 0.25% tryptic digestion, adopt blood counting chamber to carry out viable count, adjusting viable cell concentrations is 5 ' 10
4/ mL is inoculated in 96 well culture plates, and every hole 160 mL cultivated after 24 hours, added respectively the medicine of different concns again, placed 37 ℃, were containing 5% CO
2Incubator in hatched 48 hours, in finishing front 4 hours adding MTT 20 mL/holes, abandoning supernatant after 4 hours, add DMSO 100 mL/hole, vibrated about 10 minutes, and placed multi-functional microplate reader, for avoiding the inhalation effects of iridium multi-pyridine ligand itself, measure 570 nm and the 607 nm wavelength OD of place values, calculate survival rate by following formula:
The average OD value of the average OD value/control wells of survival rate %=medicine feeding hole ' 100%
Experimental result sees Table 2.The MTT experimental result shows that the cell survival rate of title complex is high under the low consistency conditions, and cytotoxicity is low, and is little to cell injury.
Table 2 mtt assay is analyzed 5 mM title complexs to HeLa, HepG-2, A549, the cytotoxicity of MCF-10A cell
The experiment of the laser co-focusing of complex of iridium and this title complex of MTT vitro cytotoxicity analysis description of test have the good painted characteristic of viable cell plastosome, stronger cytolemma penetrance and lower cytotoxicity, will have great application potential aspect biomarker and the cell imaging.
Claims (8)
1. iridium-selenium multi-pyridine ligand is characterized in that structural formula is as shown in the formula any one of I, II, III, IV and V:
。
2. the preparation method of iridium as claimed in claim 1-selenium multi-pyridine ligand is characterized in that may further comprise the steps:
With chlorine bridging dimer [(N-C)
2Ir (μ-Cl)]
2Join in the ethanol/methylene mixed solvent heating reflux reaction under the protection of inert gas with the many pyridine ligands of selenium;
N-C wherein is 2-(2, the 4-difluorophenyl) pyridine (dfppy), 2-(2-pyridyl) thionaphthene (btp), dibenzo [f, h] quinoline Nuo Xilin (dbp), 2-phenylquinoline (2-pq) or 2-phenylpyridine (ppy).
3. wherein many pyridine ligands of selenium structural formula is as follows
。
4. the preparation method of iridium as claimed in claim 2-selenium multi-pyridine ligand is characterized in that: described ethanol/methylene mixed solvent is ethanol/methylene (v/v=2:1).
5. the preparation method of iridium as claimed in claim 2-selenium multi-pyridine ligand, it is characterized in that: described reflux time is 6-10h.
6. the preparation method of iridium as claimed in claim 2-selenium multi-pyridine ligand is characterized in that: after the reaction end, be cooled to room temperature, filter, add NH in the filtrate
4PF
6The methyl alcohol saturated solution, revolve steaming, the precipitation of generation is dry after filtering, crosses neutral alumina column, with the ratio wash-out that methylene dichloride and acetone mixed solution increase gradually in polarity, obtains to dissolve the elutriant of pure products, is drying to obtain described iridium-selenium multi-pyridine ligand.
7. the preparation method of iridium as claimed in claim 2-selenium multi-pyridine ligand, it is characterized in that: described methylene dichloride and acetone mixed solution are that volume ratio is methylene dichloride and the acetone mixed solution that 1:2-5:1 arrives.
8. the as claimed in claim 1 application of iridium-selenium multi-pyridine ligand in preparation viable cell plastosome dye reagent.
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