CN1986555A - Preparing process of Ru(II) polypyridine complex - Google Patents

Preparing process of Ru(II) polypyridine complex Download PDF

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CN1986555A
CN1986555A CN 200610125479 CN200610125479A CN1986555A CN 1986555 A CN1986555 A CN 1986555A CN 200610125479 CN200610125479 CN 200610125479 CN 200610125479 A CN200610125479 A CN 200610125479A CN 1986555 A CN1986555 A CN 1986555A
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bipy
dppx
add
phen
dppz
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何治柯
凌连生
黄珊
吉邢虎
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Wuhan University WHU
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Wuhan University WHU
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Abstract

The preparation process of Ru(II)-polypyridine complex includes the following steps: preparing 1,10-phenanthroline-5,6-diquinone, pyridine [3, 2-a:2', 3'-c] phenazine as ligand I, and 7,8-dimethyl pyridine [3, 2-a: 2', 3'-c] phenazine as ligand II, Ru(bipy)2Cl2.2H2O (bipy=2, 2'-dipyridyl) as precursor I and Ru(phen)2Cl2.2H2O as precursor II; adding certain amount of Ru(bipy)2Cl2.2H2O/Ru(phen)2Cl2.2H2O, ligand I/ligand II, and mixture liquid of methanol and water, heating reflux for certain time, heating to concentrate, adding water and boiling, freezing, adding sodium tetrafluoborate, filtering and separating out precipitate, re-crystallization in alcohol, and stoving under infrared lamp to obtain the Ru(II)-polypyridine complex. The Ru(II)-polypyridine complex is used as nucleic acid recognizing probe for the analysis and detection of nucleic acid, and has high stability, high sensitivity, high selectivity and other advantages.

Description

The preparation method of a kind of ruthenium (II) multi-pyridine ligand
Technical field
The invention belongs to the synthetic field of transition metal complex, more specifically relate to the preparation method of a kind of ruthenium (II) multi-pyridine ligand, this title complex is applicable to nucleic acid quantification, base mispairing and triple helical DNA detection.
Background technology
Ruthenium (II) multi-pyridine ligand Ru (bipy) 2(dppz) 2+And Ru (phen) 2(dppz) 2+In the aqueous solution, do not have fluorescence, combine back fluorescence with double-stranded DNA and can strengthen 10 4Doubly, therefore be called nucleic acid molecule " photoswitch " by image.They are a kind of new high-sensitive fluorescent probes, the not isomorphism type of nucleic acid and the content of different bases had very strong recognition capability, not only can distinguish DNA and RNA, b form dna, Z-DNA and A-DNA, poly (AT) and poly (GC), also can distinguish single stranded DNA and double-stranded DNA, also can be used for the hybridization analysis and the nucleic acid hybridization dynamics research of nucleic acid.Because ethidium bromide has the potential carcinogenic toxicity, triple strand dna to its fluorescence enhancement than double-stranded DNA a little less than, thereby ethidium bromide is restricted in triple strand dna detects.Nucleic acid molecule " photoswitch " class title complex not only can be used for the discriminance analysis of nucleic acid quantification analysis, oligonucleotide, nucleic acid hybridization dynamics research and triple strand dna detect, and have good stability, highly sensitive, advantages such as selectivity good, nontoxic, environmental protection, for novel diagnostic reagent research provides a kind of new technology.
Summary of the invention
The object of the present invention is to provide the preparation method of a kind of ruthenium (II) multi-pyridine ligand.At synthetic Ru (bipy) 2(dppz) 2+And Ru (phen) 2(dppz) 2+The basis on synthesized the title complex Ru (bipy) of two kinds of new rutheniums (II) in addition 2(dppx) 2+And Ru (phen) 2(dppx) 2+Their stable performances are used for the nucleic acid recognizing analysis and have easy, quick, sensitivity and advantages of environment protection.Successfully be used for nucleic acid quantification and detect base mispairing and the research of triple helical DNA detection.Ru (bipy) particularly 2(dppx) 2+And Ru (phen) 2(dppx) 2+, before this research, the researchist thinks that two methyl are arranged on the dppx part more, can not be used as nucleic acid molecule " photoswitch ", the applicant proves Ru (bipy) by long-term a large amount of research 2(dppx) 2+And Ru (phen) 2(dppx) 2+Can not only be used as nucleic acid molecule " photoswitch ", and detectability is low than corresponding dppz title complex, sensitivity is higher.
To achieve these goals, the present invention adopts following technical measures:
Its step is as follows:
(a) in the 250mL flask, add 15g o-phenanthroline and 8.0g Sodium Bromide successively, add the 20-150mL vitriol oil and 10-100mL concentrated nitric acid while stirring, reflux (temperature is controlled at 100-200 ℃) 10-200min, be cooled to room temperature (10-30 ℃) after, neutralize with 30% sodium hydroxide solution, the filtrate filtered dichloromethane extraction, heating (temperature is controlled at 100-200 ℃) extraction liquid is used recrystallizing methanol behind the evaporate to dryness, obtain 1,10-phenanthroline-5, the 6-diquinone; (b) in the 250mL flask, add 0.5g 1 respectively, 10-phenanthroline-5,6-diquinone and O-Phenylene Diamine (or 4,5-dimethyl-O-Phenylene Diamine), add 20-200mL ethanol then, heating (temperature is controlled at 100-200 ℃) backflow 5-60min, be cooled to room temperature (10-30 ℃) after, add 5-50ml water and then produce yellow creaming, filter the mixed solution (1: 10-10: 1 of back with the second alcohol and water, v/v) recrystallization, the yellow needle-like crystal that obtains, oven dry obtains part dppz, dppx under infrared lamp; (c) in the 100mL flask, add 2.5g RuCl respectively 32H 2O, 3.0g 2 ', 2 '-dipyridyl, 3.0g LiCl and 17mLDMF.Heating (temperature is controlled at 100-200 ℃) backflow 2-20h, after being cooled to room temperature (10-30 ℃), add 20-200mL acetone, keep 0 ℃ and spend the night, filter and obtain red-purple filtrate and sap green solid, it is inferior that the solid water is given a baby a bath on the third day after its birth, be light green until filtrate, it is inferior to give a baby a bath on the third day after its birth with the 5-50mL ether again, with the infrared lamp oven dry, obtains Ru (bipy) 2Cl 22H 2The O solid.Adopt o-phenanthroline to replace 2 ', 2 '-dipyridyl, by experimental procedure (c) preparation Ru (phen) 2Cl 22H 2O; (d) difference 0.88gRu (phen) in the flask of 250mL 2Cl 22H 2O and 0.45gdppz, add first alcohol and water (v: v=1: 10-10: mixed solution 50-200mL 1), heating (temperature is controlled at 100-200 ℃) backflow 1-10h, heating (temperature is controlled at 100-200 ℃) is concentrated to 5-50%, add 20-100mL water, boil (temperature is controlled at 100-200 ℃) 5-60min.In refrigerator, place 3-20h, add 10% Tetrafluoroboric acid sodium solution again, produce red precipitate, filter, solid ethyl alcohol recrystallization three times, the infrared lamp oven dry obtains red powder shape product Ru (phen) 2(dppz) (BF 4) 22H 2O.Replace dppz with dppx, prepare red powder shape product Ru (phen) by experimental procedure (d) 2(dppx) (BF 4) 22H 2O is with Ru (bipy) 2Cl 22H 2O replaces the Ru (phen) in the step (d) 2Cl 22H 2O is by experimental procedure (e) preparation Ru (bipy) 2(dppz) (BF 4) 21.5H 2O replaces dppz in the step (d) with dppx again, can prepare Ru (bipy) 2(dppx) (BF 4) 22H 2O.
Ruthenium (II) the multi-pyridine ligand stable in properties of the present invention preparation can be used as nucleic acid recognizing probe, is used for the analyzing and testing of nucleic acid, has easy, quick, sensitivity and advantages of environment protection.Below with Ru (bipy) 2(dppx) (BF 4) 22H 2O is an example, introduces the analytical performance and the analyzing and testing of above-mentioned ruthenium (II) multi-pyridine ligand and uses.Ru (bipy) 2(dppx) (BF 4) 22H 2The nuclear magnetic resonance spectrum HNMR of O as shown in Figure 1,1HNMR (acetonitrile-d3, Fig. 1) δ 9.57 (2H, dd), δ 8.63 (4H, m), δ 8.20 (4H), δ 8.08 (2H, m), δ 7.90 (4H, m), and δ 7.80 (2H, dd), δ 7.53 (2H, m), δ 7.35 (2H, m), and δ 2.57 (6H, s).Molecular formula C 40H 34N 8O 2F 8The calculated value of constituent content is respectively and is C, 51.4203 among the Ru; N, 11.9928; H, 3.6679, and measured value is respectively C, 51.125; N, 11.606; H, 3.995, calculated value conforms to measured value.The fragment peak of fast atom bombardment mass spectroscopy(FABMS) (FABMS) is as shown in Figure 2: [M 2++ BF 4 -] +811, M +724, [M-bipy] +568, [M-dppx-H] +Or[M-2bipy] +412, M 2+362, [M-dppx-bipy-H] +257.Ru (bipy) 2(dppx) 2+The fluorescence spectrum figure of-aqueous dna as shown in Figure 3.A is for adding the preceding Ru (bipy) of DNA 2(dppx) 2+Excite and emmission spectrum figure, as seen, Ru (bipy) 2(dppx) 2+The aqueous solution do not have fluorescent emission, the Raman scattering peak a little less than in the of is only arranged at 535nm wavelength place; B is Ru (bipy) behind the adding DNA 2(dppx) 2+-DNA excites and emmission spectrum figure, and as seen, this moment, the maximum excitation peak of system appeared at 467nm, and the Raman scattering peak at 535nm place slightly increases, and at very strong fluorescence emission peak of 595nm generation, promptly add DNA after, Ru (bipy) 2(dppx) 2+Fluorescence strengthens greatly.Ru (bipy) 2(dppx) 2+-aqueous dna uv-vis spectra is seen Fig. 4.A is for adding the preceding Ru (bipy) of DNA 2(dppx) 2+The ultraviolet-visible absorption spectroscopy figure of the aqueous solution, b are Ru (bipy) behind the adding DNA 2(dppx) 2+The ultraviolet-visible spectrogram of-DNA.As seen from Figure 4, Ru (bipy) 2(dppx) 2+Three absorption peaks are arranged, lay respectively at: 449.0nm, 379.2nm and 284.4nm place; After adding DNA, the absorption peak strength at 449.0nm, 379.2nm and 284.4nm place has reduced by 13.0%, 33.4% and 27.78% respectively, and the red shift of the absorption peak at 379.2nm and 284.4nm place difference is to 383.2nm and 287.2nm place simultaneously.Because strong hypochromic effect and red shift phenomenon usually are accompanied by the insertion bonding action between small molecules and the DNA base pair, this experimental phenomena has disclosed Ru (bipy) 2(dppx) 2+And the insertion effect between the DNA.Fig. 5 is [Fe (CN) 6] 4-To Ru (bipy) 2(dppx) 2+The quenching of fluorescence graphic representation of-DNA mixture.The curve that Fig. 5 orbicular spot forms is [Fe (CN) 6] 4-To Ru (phen) 3 2+The quenching of fluorescence graphic representation of-DNA mixture, the curve that cardinal points forms is [Fe (CN) 6] 4-To Ru (bipy) 2(dppx) 2+The quenching of fluorescence figure of-DNA mixture, as shown in Figure 5: [Fe (CN) 6] 4-To Ru (bipy) 2(dppx) 2+The fluorescence of-DNA mixture does not almost have quencher, therefore available [Fe (CN) 6] 4-Distinguish insertion bonding and groove bonding action between Ru (II) title complex and the DNA.As shown in Figure 5, along with [Fe (CN) 6] 4-The increase of concentration, Ru (phen) 3 2+The I of-DNA 0/ I value also increases gradually, this variation tendency and Ru (phen) 3 2+Relevant with the groove bonding mode of DNA; And Ru (bipy) 2(dppx) 2+The I of-DNA 0It is 1.0 constant that/I value remains at, and shows Ru (bipy) 2(dppx) 2+Be inserted into the double-spiral structure of DNA, duplex DNA can be protected Ru (bipy) well 2(dppx) 2+Fluorescence not by quencher, further confirmed Ru (bipy) 2(dppx) 2+And the insertion bonding action between the DNA.In addition, adopt the mode that increases ionic strength from DNA, to discharge the molecule of groove bonding, but be difficult to make the molecule that the insertion bonding action takes place to discharge.Therefore, change ionic strength and can be used as the another means of inserting bonding and groove bonding of distinguishing.NaCl is a kind of strong electrolyte, investigates NaCl to Ru (phen) 3 2+-DNA and Ru (bipy) 2(dppx) 2+(Fig. 6, the curve that round dot forms is that NaCl is to Ru (phen) in the fluorescence intensity influence of-DNA 3 2+The influence of-DNA fluorescence intensity, the curve that cardinal points forms then is that NaCl is to Ru (bipy) 2(dppx) 2+The influence of-DNA fluorescence intensity).Discovery is along with the increase of NaCl concentration, Ru (phen) 3 2+The I of-DNA 0/ I value also increases gradually, and Ru (bipy) 2(dppx) 2+The I of-DNA 0It is 1.0 constant that/I value remains at, and this result has further confirmed Ru (bipy) 2(dppx) 2+And the insertion bonding action between the DNA.DNA sex change experiment (Fig. 7) has confirmed that also the dna double spirane structure is to Ru (bipy) 2(dppx) 2+The provide protection of fluorescence.After the DNA thermally denature, the dna double spiral untwists, and becomes single stranded DNA, fluorescence intensity (curve b) and Ru (bipy) 2(dppx) 2+The aqueous solution (curve fluorescence intensity a) is very close, and Ru (bipy) before the DNA thermally denature 2(dppx) 2+The fluorescence of-DNA very strong (curve c).In the detection of DNA, investigated the interference (table 1) of concurrents such as protein, base, RNA and metal ion, the result shows: the above-mentioned concurrent of higher concentration does not almost disturb the detection of DNA.Utilize this method to detect calf thymus DNA, salmon fish sperm DNA and catfish fish sperm DNA, detectability is respectively 0.75ng/mL, 0.66ng/mL and 1.49ng/mL (table 2), and the result is satisfactory.Result of study also proves Ru (bipy) 2(dppx) 2+And Ru (phen) 2(dppx) 2+Can not only be used as nucleic acid molecule " photoswitch ", and detectability is low than corresponding dppz title complex, sensitivity higher (table 3).
It deposited the interference of material when table 1 detected DNA.
Concurrent Concentration (* 10 -5mol/L) The rate of recovery
A G U T BSA a RNA Na +(Cl -) Mg 2+(SO 4 2-) Ca 2+(Cl -) HPO 4 2-(K +) 1.35 1.65 1.0 1.9 4.0 b 8.0 b 100 5 5 10 104.3 101.9 99.0 102.0 104.2 105.0 99.6 99.2 98.5 102.0
● a: bovine serum albumin; B: concentration unit is μ g/mL
● experiment condition: Calfthymus DNA 0.4 μ g/mL, Ru (bipy) 2(dppx) 2+0.4 μ mol/L
Table 2 detects detectability, linearity range and the precision of different DNA.
Nucleic acid Equation of linear regression (c, μ g/mL) Detectability (3 σ, ng/mL) Relation conefficient Precision 0.4ng/mL
Calfthymus DNA Salmon sperm DNA Herring sperm DNA I＝0.133+237.6c I＝1.37+271.1c I＝2.76+119.7c 0.75 0.66 1.49 0.9992 0.9996 0.9998 0.45 0.37 0.65
● I is a relative intensity of fluorescence
The comparison of four kinds of rutheniums of table 3 (II) multi-pyridine ligand analytical performance
Title complex Calfthymus DNA detectability (ng/ml) poly(AT) 10Sensitivity (poly(GC) 10Sensitivity
Ru(bipy) 2(dppz) 2+Ru(phen) 2(dppz) 2+Ru(bipy) 2(dppx) 2+Ru(phen) 2(dppx) 2+ 3.3 2.0 0.75 0.30 6.6×10 8 8.7×10 8 2.3×10 9 5.2×10 9 4.5×10 8 6.0×10 8 1.3×10 9 3.4×10 9
Description of drawings
Fig. 1 is Ru (bipy) 2(dppx) (BF 4) 22H 2O's 1The HNMR collection of illustrative plates.
Fig. 2 is Ru (bipy) 2(dppx) 2+Mass spectrum.
Fig. 3 is Ru (bipy) 2(dppx) 2+Fluorescence spectrum figure in the-aqueous dna.
Fig. 4 is Ru (bipy) 2(dppx) 2+Ultraviolet-visible spectrogram in the-aqueous dna.
Fig. 5 is [Fe (CN) 6] 4-Quenching of fluorescence to Ru (II) title complex-DNA mixture.
Fig. 6 is the quenching of fluorescence of NaCl to Ru (II) title complex-DNA mixture.
Fig. 7 is that denatured DNA is to Ru (bipy) 2(dppx) 2+The influence of fluorescence intensity.
Embodiment
Embodiment 1: the preparation of ruthenium (II) multi-pyridine ligand:
1. in the 250mL flask, add 15g o-phenanthroline and 8.0g Sodium Bromide successively, add the 90mL vitriol oil and 45mL concentrated nitric acid while stirring, the heating (temperature is controlled at 120 ℃ or 150 ℃) backflow 50min, be cooled to 20 or 25 ℃ after, neutralize with 0% sodium hydroxide, the filtrate filtered dichloromethane extraction, extraction liquid is heated to 100 ℃, uses recrystallizing methanol behind the evaporate to dryness, get final product 1,10-phenanthroline-5,6-diquinone product;
2. in the 250mL flask, add 0.5g 1 respectively, 10-phenanthroline-5,6-diquinone and O-Phenylene Diamine add 100mL ethanol then, heating (temperature is controlled at 100 ℃ or 110 ℃) backflow 20min, after being cooled to 15 or 20 ℃, add 10ml water and then produce yellow creaming, and the back mixed solution with the second alcohol and water of filtration (3: 1, v/v) recrystallization, the yellow needle-like crystal that obtains, oven dry promptly gets part dppz under infrared lamp;
3. in the 100mL flask, add 2.5g RuCl respectively 32H 2O, 3.8g o-phenanthroline, 3.0gLiCl and 17mL DMF.Heating (temperature is controlled at 130 ℃ or 160 ℃) backflow 9h, after being cooled to 15 or 20 ℃, add 80mL acetone, keeping 0 ℃ spends the night, filtration obtains red-purple filtrate and sap green solid, and it is inferior that the solid water is given a baby a bath on the third day after its birth, and is light green until filtrate, it is inferior to give a baby a bath on the third day after its birth with the 10mL ether again, promptly gets Ru (phen) with the infrared lamp oven dry 2Cl 22H 2O;
4. in the flask of 250mL, distinguish 0.88g Ru (phen) 2Cl 22H 2O and 0.45g dppz, adding first alcohol and water (v: v=1: mixed solution 180mL 2), heating (temperature is controlled at 100 ℃ or 110 ℃) backflow 5.5h, heating (temperature is controlled at 100 ℃ or 110 ℃) is concentrated to 10%, add 50mL water, boil heating (temperature is controlled at 100 ℃) 10min.Place 12h in refrigerator, add 10% Tetrafluoroboric acid sodium solution again, produce red precipitate, filter, solid ethyl alcohol recrystallization three times with the infrared lamp oven dry, obtain red powder shape product Ru (phen) 2(dppz) (BF 4) 22H 2O.
Embodiment 2: the preparation of ruthenium (II) multi-pyridine ligand:
1. in the 250mL flask, add 15g o-phenanthroline and 8.0g Sodium Bromide successively, add the 90mL vitriol oil and 45mL concentrated nitric acid while stirring, the heating (temperature is controlled at 120 ℃ or 150 ℃) backflow 50min, be cooled to 20 or 25 ℃ after, neutralize with 30% sodium hydroxide, the filtrate filtered dichloromethane extraction, extraction liquid is heated to 100 ℃, uses recrystallizing methanol behind the evaporate to dryness, get final product 1,10-phenanthroline-5,6-diquinone product;
2. in the 250mL flask, add 0.5g 1 respectively, 10-phenanthroline-5,6-diquinone and 4,5-dimethyl-O-Phenylene Diamine, add 100mL ethanol then, heating (temperature is controlled at 110 ℃ or 130 ℃) backflow 20min, be cooled to 15 or 20 ℃ after, add 10ml water and then produce yellow creaming, the mixed solution of the back usefulness of filtration second alcohol and water (3: 1, v/v) recrystallization, the yellow needle-like crystal that obtains, oven dry promptly gets part dppx under infrared lamp;
3. in the 100mL flask, add 2.5g RuCl respectively 32H 2O, 3.8g o-phenanthroline, 3.0g LiCl and 17mL DMF.Heating (temperature is controlled at 120 ℃ or 150 ℃) backflow 9h, after being cooled to 15 or 20 ℃, add 80mL acetone, keeping 0 ℃ spends the night, filtration obtains red-purple filtrate and sap green solid, and it is inferior that the solid water is given a baby a bath on the third day after its birth, and is light green until filtrate, it is inferior to give a baby a bath on the third day after its birth with the 10mL ether again, promptly gets Ru (phen) with the infrared lamp oven dry 2Cl 22H 2O;
4. in the flask of 250mL, distinguish 0.88g Ru (phen) 2Cl 22H 2O and 0.51g dppx, adding first alcohol and water (v: v=1: mixed solution 180mL 2), heating (temperature is controlled at 100 ℃ or 110 ℃) backflow 5.5h, heating (temperature is controlled at 100 ℃ or 110 ℃) is concentrated to 10%, add 50mL water, boil (temperature is controlled at 100 ℃) 10min.Place 12h in refrigerator, add 10% Tetrafluoroboric acid sodium solution again, produce red precipitate, filter, solid ethyl alcohol recrystallization three times with the infrared lamp oven dry, obtain red powder shape product Ru (phen) 2(dppx) (BF 4) 22H 2O.
Embodiment 3: the preparation of ruthenium (II) multi-pyridine ligand:
1. in the 250mL flask, add 15g o-phenanthroline and 8.0g Sodium Bromide successively, add the 90mL vitriol oil and 45mL concentrated nitric acid while stirring, the heating (temperature is controlled at 120 ℃ or 150 ℃) backflow 50min, be cooled to 20 ℃ or 25 ℃ after, neutralize with 30% sodium hydroxide, the filtrate filtered dichloromethane extraction, extraction liquid is heated to 100 ℃, uses recrystallizing methanol behind the evaporate to dryness, get final product 1,10-phenanthroline-5,6-diquinone product;
2. in the 250mL flask, add 0.5g 1 respectively, 10-phenanthroline-5,6-diquinone and O-Phenylene Diamine add 100mL ethanol then, heating (temperature is controlled at 100 ℃ or 110 ℃) backflow 20min, after being cooled to 15 ℃ or 20 ℃, add 10ml water and then produce yellow creaming, and the back mixed solution with the second alcohol and water of filtration (3: 1, v/v) recrystallization, the yellow needle-like crystal that obtains, oven dry promptly gets part dppz under infrared lamp;
3. in the 100mL flask, add 2.5g RuCl respectively 32H 2O, 3.0g 2 ', 2 '-dipyridyl, 3.0gLiCl and 17mLDMF.Heating (temperature is controlled at 120 ℃ or 150 ℃) backflow 9h, after being cooled to 15 ℃ or 20 ℃, add 80mL acetone, keeping 0 ℃ spends the night, filtration obtains red-purple filtrate and sap green solid, and it is inferior that the solid water is given a baby a bath on the third day after its birth, and is light green until filtrate, it is inferior to give a baby a bath on the third day after its birth with the 10mL ether again, promptly gets Ru (bipy) with the infrared lamp oven dry 2Cl 22H 2The O solid;
4. in the flask of 250mL, distinguish 0.8g Ru (bipy) 2Cl 22H 2O and 0.45g dppz, adding first alcohol and water (v: v=1: mixed solution 180mL 2), heating (temperature is controlled at 100 ℃ or 110 ℃) backflow 5.5h, heating (temperature is controlled at 100 ℃ or 110 ℃) is concentrated to 10%, add 50mL water, boil (temperature is controlled at 100 ℃) 10min.Place 12h in refrigerator, add 10% Tetrafluoroboric acid sodium solution again, produce red precipitate, filter, solid ethyl alcohol recrystallization three times with the infrared lamp oven dry, obtain red powder shape product Ru (bipy) 2(dppz) (BF 4) 21.5H 2O.
Embodiment 4: the preparation of ruthenium (II) multi-pyridine ligand:
1. in the 250mL flask, add 15g o-phenanthroline and 8.0g Sodium Bromide successively, add the 90mL vitriol oil and 45mL concentrated nitric acid while stirring, the heating (temperature is controlled at 120 ℃ or 150 ℃) backflow 50min, be cooled to 20 ℃ or 25 ℃ after, neutralize with 30% sodium hydroxide, the filtrate filtered dichloromethane extraction, extraction liquid is heated to 100 ℃, uses recrystallizing methanol behind the evaporate to dryness, get final product 1,10-phenanthroline-5,6-diquinone product;
2. in the 250mL flask, add 0.5g 1 respectively, 10-phenanthroline-5,6-diquinone and 4,5-dimethyl-O-Phenylene Diamine, add 100mL ethanol then, heating (temperature is controlled at 110 ℃ or 130 ℃) backflow 20min, be cooled to 15 ℃ or 20 ℃ after, add 5-50ml water and then produce yellow creaming, the mixed solution of the back usefulness of filtration second alcohol and water (3: 1, v/v) recrystallization, the yellow needle-like crystal that obtains, oven dry promptly gets part dppx under infrared lamp;
3. in the 100mL flask, add 2.5g RuCl respectively 32H 2O, 3.0g 2 ', 2 '-dipyridyl, 3.0gLiCl and 17mLDMF.Heating (temperature is controlled at 130 ℃ or 160 ℃) backflow 9h, after being cooled to 15 ℃ or 20 ℃, add 80mL acetone, keeping 0 ℃ spends the night, filtration obtains red-purple filtrate and sap green solid, and it is inferior that the solid water is given a baby a bath on the third day after its birth, and is light green until filtrate, it is inferior to give a baby a bath on the third day after its birth with the 10mL ether again, promptly gets Ru (bipy) with the infrared lamp oven dry 2Cl 22H 2The O solid;
4. in the flask of 250mL, distinguish 0.88g Ru (bipy) 2Cl 22H 2O and 0.51g dppx, adding first alcohol and water (v: v=1: mixed solution 180mL 2), heating (temperature is controlled at 100 ℃ or 110 ℃) backflow 5.5h, heating (temperature is controlled at 100 ℃ or 110 ℃) is concentrated to 10%, add 50mL water, boil (temperature is controlled at 100 ℃) 10min.Place 12h in refrigerator, add 10% Tetrafluoroboric acid sodium solution again, produce red precipitate, filter, solid ethyl alcohol recrystallization three times with the infrared lamp oven dry, obtain red powder shape product Ru (bipy) 2(dppx) (BF 4) 22H 2O.
Embodiment 5: ruthenium (II) multi-pyridine ligand Ru (bipy) 2(dppx) 2+Be used for the nucleic acid recognizing analysis:
1. get 1mL 4 μ mol/L Ru (bipy) 2(dppx) 2+With dna solution in the 10mL colorimetric cylinder, add 1.0mL 0.1mol/L Tris damping fluid (pH=9.3), be settled to 10mL.Measure fluorescence intensity with fluorescence spectrophotometer, to measure the amount of DNA.In the detection of DNA, investigated the interference (seeing Table 1) of concurrents such as protein, base, RNA and metal ion, the result shows: the above-mentioned concurrent of higher concentration does not almost disturb the detection of DNA.Utilize this method to detect calf thymus DNA, salmon fish sperm DNA and catfish fish sperm DNA, detectability is respectively 0.75ng/mL, 0.66ng/mL and 1.49ng/mL (seeing Table 2), and the result is satisfactory.
Ru (bipy) 2(dppx) 2+When carrying out DNA detection, have sensitivity, simple, advantages such as Stokes shift is big, immunity from interference is strong, asepsis environment-protecting as probe.

Claims (5)

1. the preparation method of a ruthenium (II) multi-pyridine ligand, it comprises the following steps:
(a) in the 250mL flask, add 15g o-phenanthroline and 8.0g Sodium Bromide successively, add the 20-150mL vitriol oil and 10-100mL concentrated nitric acid while stirring, reflux 10-200min, temperature is controlled at 100-200 ℃, the cooling back neutralizes with 30% sodium hydroxide, temperature is controlled at 10-30 ℃, filters the rear filtrate dichloromethane extraction, the heating extraction liquid, use recrystallizing methanol behind the evaporate to dryness, get 1,10-phenanthroline-5,6-diquinone product;
(b) in the 250mL flask, add 0.5g1 respectively, 10-phenanthroline-5,6-diquinone and O-Phenylene Diamine/or 4,5-dimethyl-O-Phenylene Diamine adds 20-200mL ethanol, reflux 5-60min then, the cooling back adds 5-50ml water and then produces yellow creaming, filter the mixed solution 1 of back with the second alcohol and water: 10-10: the 1v/v recrystallization, obtain yellow needle-like crystal, under infrared lamp, dry part dppz, dppx;
(c) in the 100mL flask, add 2.5g RuCl respectively 32H 2O, 3.0g 2 ', 2 '-dipyridyl or 3.8g o-phenanthroline, 3.0g LiCl and 17mL DMF, reflux 2-20h, the cooling back adds 20-200mL acetone, and 0 ℃ is spent the night, and filters and obtains red-purple filtrate and sap green solid, it is inferior that the solid water is given a baby a bath on the third day after its birth, be light green until filtrate, give a baby a bath on the third day after its birth time with the 5-50mL ether again, with infrared lamp dry Ru (bipy) 2Cl 22H 2O and Ru (phen) 2Cl 22H 2The O solid;
(d) difference 0.88g Ru (phen) in the flask of 250mL 2Cl 22H 2O and 0.45g dppz add first alcohol and water 1: the mixed solution 50-200mL of 10-10: 1v/v, reflux 1-10h, heating is concentrated to 5-50%, add 20-100mL water, boil 5-60min, in refrigerator, place 3-20h, add 10% Tetrafluoroboric acid sodium solution again, produce red precipitate, filter, solid ethyl alcohol recrystallization three times, the infrared lamp oven dry obtains red powder shape product Ru (phen) 2(dppz) (BF 4) 22H 2O.
2, the preparation method of a kind of ruthenium according to claim 1 (II) multi-pyridine ligand, it is characterized in that: adopt 1 among the above-mentioned steps b, 10-phenanthroline-5,6-diquinone and O-Phenylene Diamine/4,5-dimethyl-O-Phenylene Diamine is a reactant, preparation dppz, dppx part.
3, the preparation method of a kind of ruthenium according to claim 1 (II) multi-pyridine ligand is characterized in that: described dppx replaces the dppz of d step, makes Ru (phen) by above-mentioned d step 2(dppx) (BF 4) 22H 2O.
4, the preparation method of a kind of ruthenium according to claim 1 (II) multi-pyridine ligand is characterized in that: described Ru (bipy) 2Cl 22H 2O replaces the Ru (phen) of d step 2Cl 22H 2O makes Ru (bipy) by the d step 2(dppz) (BF 4) 22H 2O.
5, the preparation method of a kind of ruthenium according to claim 1 (II) multi-pyridine ligand is characterized in that: described Ru (bipy) 2Cl 22H 2O replaces Ru (phen) 2Cl 22H 2O replaces dppz with dppx, makes Ru (bipy) by the d step 2(dppx) (BF 4) 22H 2O.
CN 200610125479 2006-12-15 2006-12-15 Preparing process of Ru(II) polypyridine complex Pending CN1986555A (en)

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CN102464676A (en) * 2010-11-05 2012-05-23 同济大学 Ruthenium (II)-polypyridine complex, and preparation method and application thereof
CN102590494A (en) * 2012-01-11 2012-07-18 南京工业大学 Molecular probe for detecting single-stranded and/or double-stranded deoxyribonucleic acid (DNA) and application of molecular probe
CN102675348A (en) * 2012-03-13 2012-09-19 中山大学 Selenium polypyridine ligand, ruthenium-selenium polypyridine complex, and preparation methods and applications of selenium polypyridine ligand and ruthenium-selenium polypyridine complex
CN103509059A (en) * 2012-12-31 2014-01-15 中山大学 Cyclometalated ruthenium complex, and preparation method and application thereof
CN103709203A (en) * 2014-01-02 2014-04-09 中山大学 Dinuclear ruthenium complex as well as preparation method and application thereof as living cell fluorescent dye
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Publication number Priority date Publication date Assignee Title
CN102464676A (en) * 2010-11-05 2012-05-23 同济大学 Ruthenium (II)-polypyridine complex, and preparation method and application thereof
CN102464676B (en) * 2010-11-05 2014-03-05 同济大学 Ruthenium (II)-polypyridine complex, and preparation method and application thereof
CN102590494A (en) * 2012-01-11 2012-07-18 南京工业大学 Molecular probe for detecting single-stranded and/or double-stranded deoxyribonucleic acid (DNA) and application of molecular probe
CN102590494B (en) * 2012-01-11 2014-04-09 南京工业大学 Molecular probe for detecting single-stranded and/or double-stranded deoxyribonucleic acid (DNA) and application of molecular probe
CN102675348A (en) * 2012-03-13 2012-09-19 中山大学 Selenium polypyridine ligand, ruthenium-selenium polypyridine complex, and preparation methods and applications of selenium polypyridine ligand and ruthenium-selenium polypyridine complex
CN103509059A (en) * 2012-12-31 2014-01-15 中山大学 Cyclometalated ruthenium complex, and preparation method and application thereof
CN103509059B (en) * 2012-12-31 2016-05-04 中山大学 A kind of Cyclometalated ruthenium complex and its preparation method and application
CN103709203A (en) * 2014-01-02 2014-04-09 中山大学 Dinuclear ruthenium complex as well as preparation method and application thereof as living cell fluorescent dye
CN103709203B (en) * 2014-01-02 2016-05-04 中山大学 One class double-core ruthenium complex and preparation method thereof and as the application of living cells fluorescent dye
CN106496281A (en) * 2016-10-20 2017-03-15 南方医科大学 A kind of many pyridine ruthenium metal complexs and its production and use

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