CN103788135A - Ruthenium polypyridine complex and preparation method and application of [Ru(L)2(tppp)]<2+> thereof - Google Patents
Ruthenium polypyridine complex and preparation method and application of [Ru(L)2(tppp)]<2+> thereof Download PDFInfo
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Abstract
The invention discloses a ruthenium polypyridine complex and a preparation method and application of [Ru(L)2(tppp)]<2+> thereof. 2, 2'-bipyridine and 1,10'-phenanthroline are used as auxiliary ligands, and tppp is used as a main ligand (tppp=12-(2-thienyl)-pyrido[2', 3':5, 6]pyrazino[2,3-f][1,10]phenanthroline. The ruthenium complex disclosed by the invention has a recognition function, a combination function and a stabilization function on a telomere G-quadruplex. According to TO competition, the combining capacity between the complex and the G-quadruplex is stronger than that of double-stranded DNA, indicating that the complex has a recognition function; according to a TO competition experiment and a DNA melting temperature measurement experiment, the combining capacity and stabilizing capacity of the ruthenium complex a2 on the G-quadruplex are obviously stronger than those of a1.
Description
Technical field
The present invention relates to a kind of ruthenium (II) title complex and preparation method thereof and and the effect of telomeric dna between application.
Background technology
Along with social develop rapidly and progress, there is very large change in the mankind's living environment, and associated disease also increases day by day.Although when economy and development in science and technology, all kinds of transmissible diseases and infectious diseases are controlled gradually, malignant tumour has become one of common cause of current death.The whole world approximately has more than 1,010 ten thousand people to suffer from malignant tumour every year.Chemotherapy is the important means for the treatment of cancer, but the chemotherapeutics of use still exists the problems such as side effect is large, the resistance of tumour clinically, is the target that investigators struggle so seek cancer therapy drug efficient, low toxicity.
Telomere is a kind of special nucleoprotein complex body of eukaryotic cell end of chromosome, and the tumor-necrosis factor glycoproteins of the rich G (guanine base) being repeated by one section of 5-8 base series connection is feature.The activity inhibited of Telomerase in normal human tissue is reactivated in tumour cell, makes it can maintain the length of telomere, participates in pernicious diffusion.Therefore, the research of inhibition telomerase activation becomes cancer therapy drug action method.
Ruthenium (II) title complex is the very important hypotoxic cancer therapy drug of highly selective inhibition telomerase activation, potential of a class.
Summary of the invention
Object of the present invention is exactly to provide a kind of ruthenium (II) title complex that telomeric dna is had to recognition capability, binding ability and stabilizing power in order to overcome the defect that above-mentioned prior art exists, and study the effect of itself and telomeric dna, thereby provide theoretical foundation for the screening of cancer therapy drug.
Object of the present invention can be achieved through the following technical solutions:
1. the chemical formula of a ruthenium (II) multi-pyridine ligand is [Ru (bpy)
2tppp] (PF
6)
2or [Ru (phen)
2tppp] (PF
6)
2.It is characterized in that, with 2,2 '-dipyridyl, 1,10 '-phenanthroline is co-ligand; Take tppp as main part (tppp=12-(2-thienyl)-pyrido[2 ', 3 ': 5,6] pyrazino[2,3-f] [1,10] phenanthroline), so form following formula ruthenium (II) title complex:
Wherein L is 2,2 '-dipyridyl, 1,10 '-phenanthroline
According to described ruthenium (II) title complex of claim 1, it is characterized in that, wherein said compound has following formula structure:
According to the ruthenium of claim 1 (II) title complex, it is characterized in that, wherein said compound has following formula structure:
The preparation method of described ruthenium (II) title complex, is characterized in that, the method comprises the following steps:
(1) according to the synthetic bppp(bppp=12-bromo-pyrido[2 ' of document, 3 ': 5,6] pyrazino[2,3-f] [1,10] phenanthroline) obtain khaki color powder.
(3) step (2) is processed to the thick product and the 2-thienyl boric acid that obtain; be dissolved in (volume ratio of toluene and ethanol is 2:1) in toluene and ethanol; under argon shield; be warming up to 80-85 ℃; continuous heating to solid all dissolves; add sodium carbonate solution and tetra-triphenylphosphine palladium; at 80-85 ℃, continue to stir 12 hours, be cooled to room temperature, add distilled water wash sodium carbonate; using dichloromethane extraction; merge organic phase, add one night of anhydrous sodium sulfate drying, then suction filtration obtains filtrate; pressure reducing and steaming solvent, obtains blackish green powder.
(4) in three-necked flask, add ruthenium trichloride (RuCl
33H
2o), a hydration lithium chloride, and with dipyridyl or with phenanthroline, than being 1:1:1, is dissolved in DMF(dimethyl formamide by amount of substance) in, under argon shield in 140 ℃ of reflux 8 hours.Be cooled to room temperature, add acetone, be stirred to bottom solid dispersed with glass stick, put refrigerator freezing and spend the night, suction filtration obtains atropurpureus crystallite cis-[RuL
2cl
2] 2H
2o(L=bpy, phen).
(5) take the thick product of step (4) and the thick product of step (3) is dissolved in 35mL ethylene glycol and 5mLH
2the mixed solvent of O, under argon shield, refluxes 6 hours in 120 ℃.After reaction finishes, be cooled to room temperature, add the dilution of 50mL water, suction filtration is removed unreacted part, obtains burgundy clear filtrate.In filtrate, add hexafluoro to close ammonium phosphate, stir, obtain scarlet precipitation, leave standstill after the night, suction filtration, water and ether washing leaching cake several respectively, vacuum-drying.
(6) step (5) is processed to the thick product silica gel column chromatography obtaining and separate, be then spin-dried for eluent, obtain powdery product and be product.
Eluent described in step (6) is mixed by the volume ratio of 1:3 and 1:5 respectively by toluene and acetonitrile.
The application of ruthenium (II) title complex, this title complex has recognition reaction, keying action and stabilization to telomere G-tetra-serobilas.
Telomere G-tetra-serobilas that described telomere G-tetra-serobilas are the mankind.
Ruthenium (II) title complex is competed telomere G-tetra-serobilas is had to fluorescent quenching phenomenon by TO, illustrates that described ruthenium (II) title complex can replace TO and be deposited in the position on DNA, with telomere G-tetra-serobila DNA, keying action occurs, and has the effect of identification G-tetra-serobilas.
The raising of the DNA melting temperature(Tm) determination experiment of ruthenium (II) title complex by PCR instrument to telomere G-tetra-serobila melting temperature(Tm)s, illustrates that title complex can stablize G-tetra-chain body structures.
Carried out hydrogen spectrum (
1h NMR) and electrospray ionization mass spectrum (ES-MS) sign.Utilize the means such as TO competition and PCR instrument to detect designed synthetic ruthenium (II) title complex to G-tetra-serobila recognition capabilities, binding ability and stabilizing power.
Under normal temperature, do not have fluorescence material can with the competitive assay of thiazole orange (TO) study itself and DNA key and character.The fluorescence intensity of TO own is very weak, but adds after nucleic acid, and its fluorescence intensity increases greatly.In the time adding small molecules, judge that by observing the process of fluorescent quenching small molecules and TO compete the size in conjunction with DNA ability.
In PCR instrument (BIORAD icycler (iQ5)), apply FRET technology, tested the melting temperature(Tm) T of ruthenium (II) title complex to fluorescent label DNA F22T (sequence (FAM-[(G3T2A) 3AG3]-TAMRA)
mthe change of value.Thereby reflect the difference of Different Complex to G-tetra-serobila DNA Thermodynamically stable effects.
Compared with prior art, ruthenium (II) title complex prepared by the present invention has stronger recognition capability to telomere G-tetra-serobilas, binding ability and stabilization, thus provide theoretical foundation for the screening of cancer therapy drug.
Accompanying drawing explanation
Fig. 1 be the present invention obtain in potassium ion buffered soln, the TO of ruthenium (II) title complex a1 and G-tetra-serobila effects competition fluorescence spectrum figure;
Fig. 2 be the present invention obtain in sodium ion buffered soln, the TO of ruthenium (II) title complex a1 and G-tetra-serobila effects competition fluorescence spectrum figure;
Fig. 3 be the present invention obtain in potassium ion buffered soln, the TO of ruthenium (II) title complex a2 and G-tetra-serobila effects competition fluorescence spectrum figure;
Fig. 4 be the present invention obtain in sodium ion buffered soln, the TO of ruthenium (II) title complex a2 and G-tetra-serobila effects competition fluorescence spectrum figure;
Fig. 5 be the present invention obtain in potassium ion buffered soln, the TO competition replacement rate figure of two kinds of title complex a1 and a2 and G-tetra-serobila DNA effects;
Fig. 6 be the present invention obtain in sodium ion buffered soln, the TO competition replacement rate figure of two kinds of title complex a1 and a2 and G-tetra-serobila DNA effects;
Fig. 7 be the present invention obtain in potassium ion buffered soln, the TO of ruthenium (II) title complex a1 and double-stranded DNA effect competition fluorescence spectrum figure;
Fig. 8 be the present invention obtain in sodium ion buffered soln, the TO of ruthenium (II) title complex a1 and double-stranded DNA effect competition fluorescence spectrum figure;
Fig. 9 be the present invention obtain in potassium ion buffered soln, the TO of ruthenium (II) title complex a2 and double-stranded DNA effect competition fluorescence spectrum figure;
Figure 10 be the present invention obtain in sodium ion buffered soln, the TO of ruthenium (II) title complex a2 and double-stranded DNA effect competition fluorescence spectrum figure;
Figure 11 be the present invention obtain in potassium ion buffered soln, the melting temperature(Tm) experimental curve diagram of ruthenium (II) title complex a1 and G-tetra-serobila effects;
Figure 12 be the present invention obtain in sodium ion buffered soln, the melting temperature(Tm) experimental curve diagram of ruthenium (II) title complex a2 and G-tetra-serobila effects.
Figure 13 be the present invention obtain in potassium ion buffered soln, the melting temperature(Tm) experimental curve diagram of ruthenium (II) title complex a2 and G-tetra-serobila effects;
Figure 14 be the present invention obtain in sodium ion buffered soln, the melting temperature(Tm) experimental curve diagram of ruthenium (II) title complex a2 and G-tetra-serobila effects.
Embodiment
Below in conjunction with embodiment, the present invention is described in detail.
Embodiment mono-
New Ruthenium (II) title complex a1's is synthetic
1. according to the synthetic bppp(bppp=12-bromo-pyrido[2 ' of document [1], 3 ': 5,6] pyrazino[2,3-f] [1,10] phenanthroline) obtain khaki color powder.
2. take bppp(0.378g, 1mmoL), 2-thienyl boric acid (0.141g, 1.1mmoL), be dissolved in (volume ratio of toluene and ethanol is 2:1) in 40mL toluene and ethanol, under argon shield, be warming up to 80-85 ℃, continuous heating to solid all dissolves, add sodium carbonate solution (2M, 15mL) and tetra-triphenylphosphine palladium (0.116g, 0.1mmoL), at 80-85 ℃, continue to stir 12 hours, be cooled to room temperature, add the distilled water wash sodium carbonate (for washes clean washing three times) of 50mL, using dichloromethane extraction (extracting three times), merge organic phase, add one night of anhydrous sodium sulfate drying, then suction filtration obtains filtrate, pressure reducing and steaming solvent, obtain blackish green powder 0.268g.Productive rate: 70.3%.
3. in three-necked flask, add 1.56g(6mmol) ruthenium trichloride (RuCl
33H
2o); a 1.68g(28mmol) hydration lithium chloride; and 1.87g(12mmol) dipyridyl; be dissolved in 10ml DMF(dimethyl formamide) in; under argon shield, in 140 ℃ of reflux 8 hours, be cooled to room temperature, add 50ml acetone; be stirred to bottom solid with glass stick dispersed, put refrigerator freezing and spend the night.Suction filtration obtains atropurpureus solid.The alcohol heating reflux 0.5 hour that all solids is added to 150mL, is cooled to room temperature, and suction filtration obtains atropurpureus crystallite, is washed till closely colourless, dry with frozen water.Calculate productive rate approximately 65% with dipyridyl.
4. accurately take 0.4mmol cis-[Ru(bpy)
2cl
2] 2H
2o, 0.229g(0.6mmol) product of step 3, be dissolved in 35mL ethylene glycol and 5mL H
2the mixed solvent of O, under argon shield, refluxes 6 hours in 120 ℃.Reaction starts solution and is atropurpureus, gradually becomes red along with reaction is tending towards perfect solution color.After reaction finishes, be cooled to room temperature, add the dilution of 50mL water, suction filtration is removed unreacted part, obtains burgundy clear filtrate.In filtrate, add 2g hexafluoro to close ammonium phosphate, stir, obtain scarlet precipitation, leave standstill after the night, suction filtration, water and ether washing leaching cake several respectively, vacuum-drying.Crude product is crossed to 200～300 order neutral alumina columns to be separated; with acetonitrile/toluene (3:1; v/v) wash-out; red zone elutriant in the middle of collecting; being placed on stink cupboard with preservative film sealing makes solvent evaporates (product is less; revolve while steaming and conventionally can be attached to flask walls, cannot collect), can obtain approximately 0.2～0.3g of product.Productive rate approximately 55%.
1H-NMR(DMSO-d
6,TMS,400MHz):9.95(lH,d),9.64(2H,dd),9.02(IH,d),8.99(4H,dd),8.26(5H,m),8.15(2H,t),8.07(2H,m),7.95(1H,d),7.84(2H,d),7.78(2H,d),7.61(2H,t)7.40(3H,m)MALDI-MS?for?al:calcd.778.1[M]+,found779.1[M]
+.
Embodiment bis-
New Ruthenium (II) title complex a2's is synthetic
1. according to the synthetic bppp(bppp=12-bromo-pyrido[2 ' of document [1], 3 ': 5,6] pyrazino[2,3-f] [1,10] phenanthroline) obtain khaki color powder.
2. take bppp(0.378g, 1mmoL), 2-thienyl boric acid (0.141g, 1.1mmoL), be dissolved in (volume ratio of toluene and ethanol is 2:1) in 40mL toluene and ethanol, under argon shield, be warming up to 80-85 ℃, continuous heating to solid all dissolves, add sodium carbonate solution (2M, 15mL) and tetra-triphenylphosphine palladium (0.116g, 0.1mmoL), at 80-85 ℃, continue to stir 12 hours, be cooled to room temperature, add the distilled water wash sodium carbonate (for washes clean washing three times) of 50mL, using dichloromethane extraction (extracting three times), merge organic phase, add one night of anhydrous sodium sulfate drying, then suction filtration obtains filtrate, pressure reducing and steaming solvent, obtain blackish green powder 0.268g.Productive rate: 70.3%.
3. in three-necked flask, add 1.56g(6mmol) ruthenium chloride (RuCl
33H
2o); a 1.68g(28mmol) hydration lithium chloride; and 2.38g(12mmol) phenanthroline; be dissolved in 10ml DMF (dimethyl formamide); under argon shield, in 140 ℃ of reflux 8 hours, be cooled to room temperature, add 50ml acetone; be stirred to bottom solid with glass stick dispersed, put refrigerator freezing and spend the night.Suction filtration obtains atropurpureus solid.The alcohol heating reflux 0.5 hour that all solids is added to 150mL, is cooled to room temperature, and suction filtration obtains atropurpureus crystallite, is washed till closely colourless, dry with frozen water.Calculate productive rate approximately 65% with phenanthroline.
4. accurately take 0.4mmol cis-[Ru(phen)
2cl
2] 2H
2o, 0.229g(0.6mmol) product of step 3, be dissolved in 35mL ethylene glycol and 5mL H
2the mixed solvent of O, under argon shield, refluxes 6 hours in 120 ℃.Reaction starts solution and is atropurpureus, gradually becomes red along with reaction is tending towards perfect solution color.After reaction finishes, be cooled to room temperature, add the dilution of 50mL water, suction filtration is removed unreacted part, obtains burgundy clear filtrate.In filtrate, add 2g hexafluoro to close ammonium phosphate, stir, obtain scarlet precipitation, leave standstill after the night, suction filtration, water and ether washing leaching cake several respectively, vacuum-drying.Crude product is crossed to 200～300 order neutral alumina columns to be separated; with acetonitrile/toluene (5:1; v/v) wash-out; red zone elutriant in the middle of collecting; be placed on stink cupboard with preservative film sealing and make solvent evaporates (product is less, revolves while steaming and conventionally can be attached to flask walls, cannot collect); can obtain approximately 0.2～0.3g of product, productive rate approximately 50%.
1H-NMR(DMSO-d
6,TMS,400MHz):9.94(IH,d),9.60(2H,dd),9.01(lH,d),8.81(4H,t),8.42(4H,s),8.30(2H,d),8.23(3H,m),8.08(2H,d),7.94(3H,m),7.80(4H,m),7.37(1H,t)MALDI-MS?for?a2:calcd.826.1[M]+,found827.1[M]+.
Embodiment tri-
Tris-HCl damping fluid:
Buffer A: 10mM Tris, 100mM KCl, pH=7.0;
Buffer B: 10mM Tris, 100mM NaCl, pH=7.0;
General compound method: accurately take 0.303g Tris salt, 1.865g KCl/1.460g NaCl, dissolves completely with 60mL sterilizing triple distillation water, slowly regulate pH value to 7.0 with dilute hydrochloric acid, proceed to 250ml volumetric flask, with triple distillation water constant volume, mix rear for subsequent use.
The preparation of complex solution:
Accurately take 2～3mg title complex (depending on complex molecule amount, the ruthenium complexe expection concentration that the present embodiment is prepared is 200 μ M, volume is 10mL, preparation container is 15mL Corning centrifuge tube, so need the theoretical value weighing to be: molecular weight/1000 × 2mg), first dissolve with 50-100 μ L DMSO, then be settled to 10mL with pure water, obtain the title complex storing solution of 200 μ M.
The preparation of DNA solution and the mensuration of concentration:
(1) G-tetra-serobila DNA(22AG)
Compound method: 22AG the DNA(5 '-AGGGTTAGGGTTAGGGTTA GGG-3 ' that gets about 10OD, 22AG), with buffer A and the B dissolving of corresponding volume (seeing label), after sealing, be heated to 90 ℃ and keep 5 minutes with water-bath, slowly cool to and put into 4 ℃ of refrigerator and cooled after room temperature and hide more than 24 hours, for subsequent use.
(2) double-stranded DNA (Duplex-DNA)
Compound method: get respectively the single stranded DNA (5 '-TT CCC TTTT CCC TTT ATA TAT ATA TAT ATA TAT ATA TAT ATA TAT TT CCC TTTT CCC TT-3 ') of about 10OD and corresponding complementary single-stranded dna (3'-AA GGG AAAA GGG AAA TAT ATA TAT ATA TAT ATA TAT ATA TAT ATA AA GGG AAAA GGG AA-5'), with the damping fluid C dissolving of corresponding volume (seeing label), then dissolving single stranded DNA completely by two is blended in same centrifuge tube, after sealing, be heated to 90 ℃ and keep 5 minutes with water-bath, slowly cool to and put into 4 ℃ of refrigerator and cooled after room temperature and hide more than 24 hours, for subsequent use.
The TO competitive assay of ruthenium (II) title complex
Instrument: Hitachi FP7000 fluorophotometer
Instrument parameter: excitation wavelength: 480nm, slit width: 10nm, voltage: 500V
Sweep limit: 500-700nm
In potassium ion and sodium ion damping fluid, preparation is 2 μ M containing TO concentration respectively, the concentration of G-tetra-chain liquid solutions is 1 μ M, in sample pool, add 5 μ L concentration is ruthenium (II) title complex a1 and the a2 solution of 200 μ M at every turn, by liquid-transfering gun compressing repeatedly, mix, after 5 minutes, detect fluorescent quenching situation, so repeatedly, drip 13-15 time, shown in its result Fig. 1-4.
Result shows: ruthenium (II) title complex a1 and a2 have good quenching phenomenon to G-tetra-serobila DNA, illustrates that ruthenium (II) title complex a1 and a2 can replace the position that TO is combined with DNA, with DNA, keying action occurs.No matter known by Fig. 5-6 is that the binding ability of ruthenium (II) title complex a2 and G-tetra-serobilas is better than a2 under potassium ion and sodium ion condition.
In potassium ion and sodium ion damping fluid, preparation is 2 μ M containing TO concentration respectively, the concentration of double-stranded DNA solution is 1 μ M, in sample pool, add 5 μ L concentration is ruthenium (II) title complex a1 and the a2 solution of 200 μ M at every turn, by liquid-transfering gun compressing repeatedly, mix, after 5 minutes, detect fluorescent quenching situation, so repeatedly, drip 13-15 time, shown in its result Fig. 7-10.
Result shows: no matter be under potassium ion or sodium ion condition, ruthenium (II) title complex a1 and a2 have binding ability to double-stranded DNA, if but by the known binding ability of fluorogram 7-10 and G-tetra-serobila DNA, illustrate that this title complex has recognition capability to G-tetra-serobilas.
Ruthenium (II) title complex is for the stabilizing power research of telomere G-tetra-serobila DNA
Instrument: BIORAD icycler (iQ5) PCR instrument
The present invention's application FRET technology, has tested title complex a1 and the a2 melting temperature(Tm) T to fluorescent label DNA F22T (sequence (FAM-[(G3T2A) 3AG3]-TAMRA)
mthe change of value.Thereby reflect the difference of Different Complex to G-tetra-serobila DNA Thermodynamically stable effects.FRET experiment is carried out in PCR instrument, the KCl that use contains 100mM or the dipotassium hydrogen phosphate of NaCl or disodium hydrogen phosphate buffer solution (10mM, pH=7.0), add annealing to form the F22T (250nM) of G-tetra-serobilas, and the title complex of finite concentration ratio, jointly hatch 1 hour, carry out T
mvalue is measured as shown in Figure 11-14.
Result shows:
In the time there is no title complex (0.2 μ M G-tetra-serobilas, 100mM NaCl), the T of the F22T of G-tetra-serobilas
mbe worth less; Along with adding and the increase of concentration of title complex a1, the T of F22T
mvalue increases gradually.Finally adding title complex a1 concentration is that 3.5 μ M make G-tetra-serobila T
mvalue has raise 17 ℃; In the time that title complex a2 concentration is increased to 3.5 μ M, G-tetra-serobila T
mvalue has improved 21.5 ℃.Therefore, under sodium damping fluid, title complex has concentration dependent to the stabilization of G-tetra-serobilas, and the effect of stable G-tetra-serobilas of title complex a2 is greater than title complex a1.
In the time there is no title complex (0.2 μ M G-tetra-serobilas, 100mM KCl), the T of the F22T of G-tetra-serobilas
mbe worth less; Along with adding and the increase of concentration of title complex a1, the T of F22T
mvalue increases gradually.Finally adding title complex a1 concentration is that 3.5 μ M make G-tetra-serobila T
mvalue has raise 20.5 ℃; When title complex a2 concentration is increased to 3.5 μ M, G-tetra-serobila T
mvalue has improved 24.5 ℃.Therefore, under potassium damping fluid, title complex has concentration dependent to the stabilization of G-tetra-serobilas, and the effect of stable G-tetra-serobilas of title complex a2 is greater than title complex a1.
Reference:
[I]ShuoShi,Hai-LiangHuang,XingGao,JournalofInorganicBiochemistry121。
Claims (10)
1. ruthenium (II) multi-pyridine ligand, is characterized in that, its chemical formula is [Ru (bpy)
2tppp] (PF
6)
2or [Ru (phen)
2tppp] (PF
6)
2, with 2,2 '-dipyridyl, 1,10 '-phenanthroline is co-ligand; Take tppp as main part (tppp=12-(2-thienyl) – pyrido[2 ', 3 ': 5,6] pyrazino[2,3-f] [1,10] phenanthroline), so form following formula ruthenium (II) title complex:
Wherein L is 2,2 '-dipyridyl, 1,10 '-phenanthroline.
2. according to described ruthenium (II) title complex of claim 1, it is characterized in that, wherein said compound has following formula structure:
3. according to the ruthenium of claim 1 (II) title complex, it is characterized in that, wherein said compound has following formula structure:
4. a preparation method for ruthenium as claimed in claim 2 (II) title complex, is characterized in that, the method comprises the following steps:
(1) synthetic bppp(bppp=12-bromo-pyrido[2 ', 3 ': 5,6] pyrazino[2,3-f] [1,10] phenanthroline) obtain khaki color powder;
(2) step (1) is processed to the thick product and the 2-thienyl boric acid that obtain, be dissolved in toluene and ethanol, the volume ratio of toluene and ethanol is 2:1, under argon shield, be warming up to 80-85 ℃, continuous heating to solid all dissolves, and adds sodium carbonate solution and tetra-triphenylphosphine palladium, continues to stir 12 hours at 80-85 ℃, be cooled to room temperature, add distilled water wash sodium carbonate, then use dichloromethane extraction, merge organic phase, add one night of anhydrous sodium sulfate drying, then suction filtration obtains filtrate, and pressure reducing and steaming solvent obtains blackish green powder;
(3) in three-necked flask, add ruthenium trichloride (RuCl
33H
2o), one hydration lithium chloride, and dipyridyl,, be dissolved in dimethyl formamide than for 1:1:1 by amount of substance, under argon shield in 140 ℃ of reflux 8 hours, be cooled to room temperature, add acetone, be stirred to bottom solid with glass stick dispersed, put refrigerator freezing and spend the night, suction filtration obtains atropurpureus crystallite;
(4) take the thick product of step (3) and the thick product of step (2) is dissolved in ethylene glycol and H
2the mixed solvent of O, under argon shield, refluxes 6 hours in 120 ℃; after reaction finishes, be cooled to room temperature, add thin up, suction filtration is removed unreacted part; obtain burgundy clear filtrate; in filtrate, add hexafluoro to close ammonium phosphate, stir, obtain scarlet precipitation; leave standstill after the night; suction filtration, water and ether washing leaching cake several respectively, vacuum-drying.
(5) step (4) is processed to the thick product silica gel column chromatography obtaining and separate, be then spin-dried for eluent, obtain powdery product and be product.
5. a preparation method for ruthenium as claimed in claim 3 (II) title complex, is characterized in that, the method comprises the following steps:
(1) synthetic bppp(bppp=12-bromo-pyrido[2 ', 3 ': 5,6] pyrazino[2,3-f] [1,10] phenanthroline) obtain khaki color powder;
(2) step (1) is processed to the thick product and the 2-thienyl boric acid that obtain, being dissolved in the volume ratio of toluene and ethanol in toluene and ethanol is 2:1, under argon shield, be warming up to 80-85 ℃, continuous heating to solid all dissolves, add sodium carbonate solution and tetra-triphenylphosphine palladium, at 80-85 ℃, continue to stir 12 hours, be cooled to room temperature, add distilled water wash sodium carbonate, use again dichloromethane extraction, merge organic phase, add one night of anhydrous sodium sulfate drying, then suction filtration obtains filtrate, pressure reducing and steaming solvent, obtains blackish green powder;
(3) in three-necked flask, add ruthenium trichloride (RuCl
33H
2o), a hydration lithium chloride, and phenanthroline,, be dissolved in dimethyl formamide than for 1:1:1 by amount of substance, under argon shield in 140 ℃ of reflux 8 hours.Be cooled to room temperature, add acetone, be stirred to bottom solid dispersed with glass stick, put refrigerator freezing and spend the night, suction filtration obtains atropurpureus crystallite;
(4) take the thick product of step (3) and the thick product of step (2) is dissolved in ethylene glycol and H
2the mixed solvent of O, under argon shield, refluxes 6 hours in 120 ℃, after reaction finishes, be cooled to room temperature, add the dilution of 50mL water, suction filtration is removed unreacted part, obtain burgundy clear filtrate, in filtrate, add hexafluoro to close ammonium phosphate, stir, obtain scarlet precipitation, leave standstill after the night, suction filtration, water and ether washing leaching cake several respectively, vacuum-drying;
(5) step (4) is processed to the thick product silica gel column chromatography obtaining and separate, be then spin-dried for eluent, obtain powdery product and be product.
6. a ruthenium as claimed in claim 1 (II) title complex application, is characterized in that, this title complex has recognition reaction to telomere G-tetra-serobilas, keying action and stabilization.
7. the application of ruthenium according to claim 6 (II) title complex, is characterized in that, the telomeric DNA sequence that described telomere G-tetra-serobila sequences are the mankind.
8. the application of ruthenium according to claim 6 (II) title complex, it is characterized in that, the application of ruthenium (II) title complex can be competed telomere G-tetra-serobilas are had to fluorescent quenching phenomenon by TO, illustrate that described ruthenium (II) title complex can replace TO and be deposited in the position on DNA, with telomere G-tetra-serobila DNA, keying action occurs.
9. the application of ruthenium according to claim 6 (II) title complex, it is characterized in that, the application of ruthenium (II) title complex, competing known and G-tetra-serobila effects by TO is that quenching of fluorescence is greater than and double-stranded DNA effect, illustrates that ruthenium (II) title complex has recognition reaction to G-tetra-serobila DNA.
10. the application of ruthenium according to claim 6 (II) title complex, it is characterized in that, the known ruthenium of thermally-stabilised experiment (II) title complex by PCR instrument is improved the melting temperature(Tm) of G-tetra-serobilas, illustrates that described ruthenium (II) title complex all can make the structure of G-tetra-serobilas more stable.
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| CN113336797B (en) * | 2021-04-06 | 2022-09-27 | 江西科技师范大学 | Ruthenium polypyridine complex with triphenylphosphine structure and preparation method and application thereof |
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