CN113666970B - Preparation method of ruthenium metal complex - Google Patents

Preparation method of ruthenium metal complex Download PDF

Info

Publication number
CN113666970B
CN113666970B CN202110888426.5A CN202110888426A CN113666970B CN 113666970 B CN113666970 B CN 113666970B CN 202110888426 A CN202110888426 A CN 202110888426A CN 113666970 B CN113666970 B CN 113666970B
Authority
CN
China
Prior art keywords
reaction
hours
metal complex
solid
ruthenium metal
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202110888426.5A
Other languages
Chinese (zh)
Other versions
CN113666970A (en
Inventor
夏继波
肖会芝
杨钰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suzhou Youyi Landi Biotechnology Co ltd
Original Assignee
Suzhou Youyi Landi Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Suzhou Youyi Landi Biotechnology Co ltd filed Critical Suzhou Youyi Landi Biotechnology Co ltd
Priority to CN202110888426.5A priority Critical patent/CN113666970B/en
Publication of CN113666970A publication Critical patent/CN113666970A/en
Application granted granted Critical
Publication of CN113666970B publication Critical patent/CN113666970B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
    • C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
    • C07F15/0046—Ruthenium compounds
    • C07F15/0053—Ruthenium compounds without a metal-carbon linkage
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • C09B57/10—Metal complexes of organic compounds not being dyes in uncomplexed form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Electrochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

The invention discloses a preparation method of a ruthenium metal complex. In polyacrylamide gel electrophoresis, the ruthenium metal complex prepared by the method can be used for protein staining and can be used for new ruthenium metal complex for nucleic acid staining, and when the ruthenium metal complex is used for staining, the method is simple and convenient to operate, does not need decolorization, is economical, has high detection sensitivity, is compatible with mass spectrum, does not pollute the environment, and can be directly used for ultraviolet or blue light excitation observation.

Description

Preparation method of ruthenium metal complex
The invention discloses a ruthenium metal complex and application thereof in polyacrylamide gel electrophoresis, wherein the application date is 2019, 4, 30 and 2019103644446, and the ruthenium metal complex is a part of a preparation method of a compound.
Technical Field
The invention belongs to the gel electrophoresis technology, and particularly relates to a ruthenium metal complex, a preparation method thereof and application thereof in polyacrylamide gel electrophoresis.
Background
The polyacrylamide gel is a compact reticular structure, and has a molecular sieve effect, a concentration effect and a charge effect. Polyacrylamide gel electrophoresis is a common electrophoresis technique using polyacrylamide gel as a support medium, and is widely used for separation and purification of proteins and nucleic acids.
In polyacrylamide gel electrophoresis, the staining reagent is mainly an organic reagent represented by coomassie brilliant blue and silver staining. Among them, coomassie brilliant blue staining is widely used for protein staining, but cannot be used for staining of nucleic acids. Even when coomassie blue stains proteins, there are a number of disadvantages, such as poor visualization of low abundance proteins, i.e. poor sensitivity, lengthy time consumption, and uncontrolled decolorization time.
In polyacrylamide gel electrophoresis, silver staining is not only used for protein but also for nucleic acid staining, and has higher sensitivity, but is often incompatible with downstream tests such as mass spectrometry or sequencing, and more importantly, a large amount of formaldehyde is required to be used as a reducing agent in the silver staining process, and the formaldehyde causes great harm to operators and also causes great pollution to the environment.
Disclosure of Invention
The invention discloses a novel ruthenium metal complex which can be used for protein dyeing and nucleic acid dyeing in polyacrylamide gel electrophoresis, and when used for dyeing, the method is simple and convenient to operate, does not need decolorization, saves materials, has high detection sensitivity, is compatible with mass spectrum, does not pollute the environment, and can be directly used for ultraviolet or blue light excitation observation.
The invention adopts the following technical scheme:
ruthenium metal complex with the chemical structural formula as follows:
wherein L is a connecting bridge, in particular-NH- (CH) 2 ) a -[O-(CH 2 ) b ] x -[O-(CH 2 ) c ] y -NH-, wherein a is 2 to 20, b and c are each independently 2 to 3, x is 0 or 1 to 20, y is 0 or 1, preferably a, b, c, x, y are integers; r is an anion selected from chloride, bromide, iodide or perchlorate ions.
The invention also discloses application of the ruthenium metal complex in preparing gel dye, preferably in preparing polyacrylamide gel dye; the invention also discloses application of the ruthenium metal complex in gel electrophoresis, preferably polyacrylamide gel electrophoresis; furthermore, the invention also discloses application of the ruthenium metal complex as a protein gel dye or a nucleic acid gel dye.
The product of the invention is improved in molecular structure, so that the dye can be detected by an ultraviolet excited instrument and can be observed by blue light; the method is not only suitable for protein staining in polyacrylamide gel electrophoresis, but also suitable for nucleic acid staining in polyacrylamide gel electrophoresis, and does not pollute the environment.
The invention also discloses a dye-bubble solution for polyacrylamide gel electrophoresis, which comprises a ruthenium metal complex. Preferably, the bubble dye solution for polyacrylamide gel electrophoresis is prepared from ruthenium metal complex solution and diH 2 O, methanol and acetic acid.
Preferably, the concentration of the ruthenium metal complex solution is 0.05 to 1wt%, and more preferably, the ruthenium metal complex solution is an aqueous ruthenium metal complex solution. Ruthenium metal complex solution, diH 2 The volume ratio of O, methanol and acetic acid is 1:58:28:13.
The invention also discloses a preparation method of the ruthenium metal complex, which comprises the following steps:
(1) Adding the phenanthroline into concentrated sulfuric acid, then dropwise adding fuming sulfuric acid, and then reacting to prepare an intermediate I;
(2) Reacting the intermediate I with phosphorus oxychloride to obtain a solid; then reacting the solid with a connecting bridge compound in the presence of an amine compound to prepare an intermediate II; the connecting bridge compound is NH 2 -(CH 2 ) a -[O-(CH 2 ) b ] x -[O-(CH 2 ) c ] y -NH-boc；
(3) Reacting bathophenanthroline with ruthenium trichloride in ethylene glycol, and then adding an intermediate II; continuing the reaction to prepare an intermediate III;
(4) Dropwise adding trifluoroacetic acid into the intermediate III, and reacting to prepare an intermediate IV;
(5) Reacting bathophenanthroline with ruthenium trichloride in ethylene glycol, then adding an intermediate I, and continuing the reaction to prepare an intermediate V;
(6) Reacting the intermediate V with phosphorus oxychloride to obtain a solid; then reacting the solid with intermediate iv in the presence of an amine compound; then adding a salt compound; and continuing the reaction to obtain the ruthenium metal complex.
The invention also discloses a polyacrylamide gel electrophoresis method, which comprises the following steps:
(1) Adding the phenanthroline into concentrated sulfuric acid, then dropwise adding fuming sulfuric acid, and then reacting to prepare an intermediate I;
(2) Reacting the intermediate I with phosphorus oxychloride to obtain a solid; then reacting the solid with a connecting bridge compound in the presence of an amine compound to prepare an intermediate II; the connecting bridge compound is NH 2 -(CH 2 ) a -[O-(CH 2 ) b ] x -[O-(CH 2 ) c ] y -NH-boc；
(3) Reacting bathophenanthroline with ruthenium trichloride in ethylene glycol, and then adding an intermediate II; continuing the reaction to prepare an intermediate III;
(4) Dropwise adding trifluoroacetic acid into the intermediate III, and reacting to prepare an intermediate IV;
(5) Reacting bathophenanthroline with ruthenium trichloride in ethylene glycol, then adding an intermediate I, and continuing the reaction to prepare an intermediate V;
(6) Reacting the intermediate V with phosphorus oxychloride to obtain a solid; then reacting the solid with intermediate iv in the presence of an amine compound; then adding a salt compound; continuing to react to obtain the ruthenium metal complex;
(7) Adding a sample to be tested into polyacrylamide gel, and then performing electrophoresis test; after the electrophoresis test is finished, the polyacrylamide gel is dye blasted in a bubble dye solution, and finally ultraviolet excitation or blue light excitation detection is utilized to finish the polyacrylamide gel electrophoresis; the sample to be tested comprises protein or nucleic acid; the bubble dye solution comprises the ruthenium metal complex.
In the technical scheme, the polyacrylamide gel is prepared in the prior art or according to the prior art.
In the technical scheme, the mass fraction of fuming sulfuric acid is 20%, and concentrated sulfuric acid is a conventional commercial product; the amine compound is triethylamine; the salt compound contains an anion R, preferably a sodium salt compound such as sodium chloride, sodium iodide, sodium bromide or sodium perchlorate.
In the technical scheme, in the step (1), after the reaction is finished, cooling the reaction liquid to room temperature, adding the reaction liquid into diethyl ether, then centrifuging, adding the obtained solid into water, adjusting the solid to be neutral by alkali, spin-drying, and purifying by a column to obtain an intermediate I; preferably, the diethyl ether is precooled diethyl ether, sodium hydroxide is used for adjusting to be neutral, column purification is silica gel column purification, and the eluting agent is acetonitrile and water with the volume ratio of 90:10.
In the technical scheme, in the step (2), after the reaction of the intermediate I and phosphorus oxychloride is finished, cooling to room temperature, adding diethyl ether, and then centrifuging to obtain a solid; after the reaction of the solid and the connecting bridge compound is finished, spin-drying the reaction liquid, and purifying by a column to prepare an intermediate II; preferably, diethyl ether is added under ice water bath, column purification is silica gel column purification, and eluent is dichloromethane and methanol with the volume ratio of 95:5.
In the technical scheme, in the step (3), after the continuous reaction is finished, cooling to normal temperature, then dripping the reaction liquid into ethyl acetate, then pouring out supernatant, and purifying a residue column to prepare an intermediate III; preferably, the column purification is alumina column purification, and the eluent is acetonitrile and water with the volume ratio of 95:5.
In the technical scheme, in the step (4), after the reaction is finished, dichloromethane is added after the reaction solution is dried in a spinning way, and the intermediate IV is obtained after the reaction solution is dried in a spinning way again.
In the technical scheme, in the step (5), after the continuous reaction is finished, cooling to normal temperature, then dripping the reaction liquid into ethyl acetate, then pouring out supernatant, and purifying a residue column to prepare an intermediate V; preferably, the column purification is alumina column purification, and the eluent is acetonitrile and water with the volume ratio of 95:5.
In the technical scheme, in the step (6), after the reaction of the intermediate V and phosphorus oxychloride is finished, cooling to room temperature, adding diethyl ether, and centrifuging to obtain a solid; after the continuous reaction is finished, purifying the reaction liquid by a column and then spin-drying to obtain the ruthenium metal complex; preferably, diethyl ether is added under ice water bath, column purification is silica gel column purification, and eluent is dichloromethane and methanol with the volume ratio of 95:5.
In the above technical scheme, in the step (1), the bathophenanthroline is added into the concentrated sulfuric acid under the ice water bath, preferably, the bathophenanthroline is added into the concentrated sulfuric acid in batches under the ice water bath, and further preferably, the bathophenanthroline is added into the concentrated sulfuric acid in five batches under the ice water bath; the reaction was carried out at 90℃for 12 hours.
In the technical scheme, in the step (2), the reaction time of the intermediate I and phosphorus oxychloride is 4 hours, and the temperature is 100 ℃; the reaction time of the solid and the connecting bridge compound is 12h, and the temperature is normal temperature.
In the technical scheme, in the step (3), the reaction time of the bathophenanthroline and the ruthenium trichloride is 12 hours, and the temperature is 95 ℃; the reaction was continued for 6 hours at 120 ℃.
In the technical scheme, in the step (4), trifluoroacetic acid is added dropwise to the intermediate III in the presence of dichloromethane in an ice bath, and then the reaction is carried out for 12 hours to prepare the intermediate IV.
In the technical scheme, in the step (5), the reaction time of the bathophenanthroline and the ruthenium trichloride is 12 hours, and the temperature is 95 ℃; the reaction was continued for 6 hours at 120 ℃.
In the technical scheme, in the step (6), the reaction time of the intermediate V and phosphorus oxychloride is 4 hours, and the temperature is 100 ℃; the reaction time of the solid and the intermediate IV is 12 hours, and the temperature is normal temperature; the reaction is continued for 24 hours at normal temperature.
In the technical scheme, in the step (1), the dosage ratio of the bathophenanthroline to the concentrated sulfuric acid to the fuming sulfuric acid is 2.0g to 10mL to 1mL; in the step (2), the dosage ratio of the intermediate I to the phosphorus oxychloride to the amine compound to the connecting bridge compound is 0.2g to 4mL to 0.5g to 4mL; in the step (3), the mass ratio of the bathophenanthroline, the ruthenium trichloride and the intermediate II is 167:52:205; in the step (4), the dosage ratio of the intermediate III to the trifluoroacetic acid is 34mg to 1mL; in the step (5), the mass ratio of the bathophenanthroline to the ruthenium trichloride to the intermediate I is 167:52:124; in the step (6), the dosage ratio of the intermediate V to the phosphorus oxychloride to the intermediate IV to the amine compound to the salt compound is 35mg to 1mL to 41mg to 0.1mL to 0.1g.
Specifically, the preparation method of the ruthenium metal complex disclosed by the invention can be exemplified by the following steps:
(1) 10ml of concentrated sulfuric acid is added into a 100 mL reaction bottle, the mixture is cooled in an ice bath, 2.0g of bathophenanthroline is evenly added for 5 times, the mixture is stirred for 30min, 20wt% of fuming sulfuric acid is added dropwise, the mixture is heated to 90 ℃ for reaction for 12 hours, TLC (thin layer chromatography) tracking is finished, and a developing agent is acetonitrile, wherein the water ratio of acetonitrile is as follows: 15. after the reaction, cooling to room temperature, pouring into 200 mL pre-cooled diethyl ether, stirring for 1h, centrifugally collecting solid, dissolving in 20ml of water, adding sodium hydroxide to adjust pH to 7, spin-drying, purifying by a silica gel column, eluting with acetonitrile, wherein the water=90: 10, collecting and spin-drying to obtain a product 2.9 g intermediate I.
(2) Into a 25 mL reaction flask was charged 0.2g of intermediate I, 4ml phosphorus oxychloride POCl 3 Heated to 100℃and reacted for 4 hours. TLC traces with dichloromethane: methanol=90: 10. after the reaction, cooling to room temperature, adding 25 ml diethyl ether under ice bath cooling, precipitating solid, stirring for 30min, centrifuging, pouring out supernatant, and vacuum drying the solid for 1 hour. The solid was stirred with 5 ml DMF, cooled in an ice bath, and 0.5g of tert-butyl (3- (2- (3-aminopropoxy) ethoxy) propyl) carbamate, 0.5. 0.5 ml triethylamine TEA, was added and reacted at room temperature for 12 hours. The reaction solution was spin-dried and purified on silica gel, eluting with dichloromethane, methanol=95: and 5, collecting and spin-drying to obtain a product 60 mg intermediate II.
(3) 167 mg red phenanthroline, 52 mg ruthenium trichloride RuCl are added into a 25 mL reaction bottle 3 5. 5 ml ethylene glycol, heated to 95℃and stirred for reaction for 12 hours. TLC trace, developing solvent acetonitrile, water=90: 10. after the reaction, the temperature is reduced to normal temperature, 205 and mg intermediate II are added, heated to 120 ℃ and stirred for reaction for 6 hours. TLC trace, developing solvent acetonitrile, water=90: 10. after the reaction was completed, the temperature was lowered to room temperature, the reaction solution was added dropwise to 50ml of ethyl acetate, after stirring for 30min, the supernatant was decanted, and the remaining solid alumina was purified by column chromatography, eluting with acetonitrile, water=95: 5, collecting and spin-drying to obtain a product 34mgAn intermediate III.
(4) To a 25 mL reaction flask was added 34mg of intermediate III, 5 mL of dichloromethane, and 1ml of trifluoroacetic acid TFA dropwise under ice-bath, followed by stirring for 12 hours. TLC traces with acetonitrile: water=90: 10. after the reaction is finished, spin-drying, adding dichloromethane 5 ml, stirring for 30min, spin-drying, and collecting to obtain 32 mg intermediate IV.
(5) 167 and mg red phenanthroline, 52 and mg ruthenium trichloride and 5 and ml glycol are added into a 25 and mL reaction bottle, heated to 95 ℃ and stirred for reaction for 12 hours. TLC trace, developing solvent acetonitrile, water=90: 10. after the reaction is finished, the temperature is reduced to normal temperature, 124 mg intermediate I is added, the temperature is heated to 120 ℃ and the reaction is stirred for 6 hours. TLC trace, developing solvent acetonitrile, water=90: 10. after the reaction was completed, the temperature was lowered to room temperature, the reaction solution was added dropwise to 50ml of ethyl acetate, after stirring for 30min, the supernatant was decanted, and the remaining solid alumina was purified by column chromatography, eluting with acetonitrile, water=95: and 5, collecting and spin-drying to obtain the product 35mg intermediate V.
(6) In a 25 mL reaction flask was charged 35mg of intermediate V, 1ml of phosphorus oxychloride, and heated to 100deg.C for 4 hours. TLC traces with dichloromethane: methanol=90: 10. after the reaction, cooling to room temperature, adding 5 ml diethyl ether under ice bath cooling, precipitating solid, stirring for 30min, centrifuging, pouring out supernatant, and vacuum drying the solid for 1 hour. The solid was stirred with 1ml DMF, cooled in an ice bath, and 41. 41mg intermediate IV, 0.1ml triethylamine was added and reacted at room temperature for 12 hours. The reaction mixture was dried by spin-drying, 1ml of pure water and 0.1g of sodium iodide were added, followed by stirring for 24 hours. Purifying silica gel by column, eluting with dichloromethane, wherein methanol=95: 5, collecting the spin-dried product 60 mg, wherein when a=y=3, b=x=2, c=3, y=1, r=i - 。
In the polyacrylamide gel electrophoresis method disclosed by the invention, a sample to be tested is prepared according to the test requirement in the prior art; the bubble dye solution comprises the ruthenium metal complex solution and diH 2 O, methanol, acetic acid, preferably 0.05-1 wt% of ruthenium metal complex solution, and diH 2 The volume ratio of O, methanol and acetic acid is 1:58:28:13. The prior art discloses a method for SDS-PAGE (polyacrylamide gel electrophoresis)) The dyes of (a) are basically coomassie brilliant blue, lumitein protein gel dye (a fluorescent dye designed by the expert of Biotechnology company in the United states for SDS-PAGE experimental protein detection), thermo Scientific Krypton protein staining reagent and Krypton protein staining reagent; compared with the existing disclosed protein gel dye, the ruthenium metal complex disclosed by the invention has the advantages that the dyeing process is extremely simple, the high-sensitivity, rapid dyeing, simplicity, easiness in use and good compatibility are integrated, the ruthenium metal complex is a protein gel dye for detecting protein in SDS-PAGE and 2D gel, the unique structure can ensure that the time and the cost are saved more than those of the conventional fluorescent dye, the fluorescent dyeing technology is greatly improved, and the problem of protein quantitative error caused by the difference of protein dyeing can be avoided while the sensitivity equal to or higher than that of other fluorescent dyes is provided; the dye can provide extremely high signal intensity for various proteins and has a linear range of three to four orders of magnitude, so that very small amounts of protein can be detected.
The existing protein staining reagent, such as Lumitein protein gel dye of Biotium, can only be detected by an ultraviolet imager or a laser fluorescence scanner due to the characteristic of the molecular structure, can only be used for protein staining in polyacrylamide gel electrophoresis, and cannot be widely used for nucleic acid staining in the polyacrylamide gel electrophoresis. Similarly, the Thermo Scientific Krypton protein staining reagent can only be detected by an instrument with excitation light of 520nm, and the dye is not applicable to staining of polyacrylamide gel electrophoresis nucleic acid. In polyacrylamide gel electrophoresis, silver staining is not only used for protein but also for nucleic acid staining, and has higher sensitivity, but is often incompatible with downstream tests such as mass spectrometry or sequencing, and more importantly, a large amount of formaldehyde is required to be used as a reducing agent in the silver staining process, and the formaldehyde causes great harm to operators and also causes great pollution to the environment.
Drawings
FIG. 1 is a staining chart of the prior art Lumitein (A) and the prior art Coomassie blue (B);
FIG. 2 is a chart showing the staining of the ruthenium metal complex (A) of the present invention, the conventional Lumitein (B);
FIG. 3 is a graph showing the staining of nucleic acids in a PAGE gel stained with ruthenium metal complexes according to the present invention and detected under UV and blue light, respectively.
Detailed Description
Example 1
100 10mL of concentrated sulfuric acid is added into a mL reaction bottle, the mixture is cooled in ice bath, 2.0g of bathophenanthroline is evenly added for 5 times, the mixture is stirred for 30min, 1mL of 20wt% fuming sulfuric acid is added dropwise, the mixture is heated to 90 ℃ for reaction for 12 hours, TLC (thin layer chromatography) is tracked until the reaction is finished, and a developing agent is acetonitrile, water=85: 15. cooling to room temperature after the reaction is finished, pouring the mixture into 200 mL ice water bath pre-cooled diethyl ether, stirring for 1h, centrifugally collecting solids, dissolving the solids in 20ml of water, adding sodium hydroxide to adjust the pH to 7, spin drying, purifying by a silica gel column, and eluting with acetonitrile, wherein the water=90: 10, collecting and spin-drying to obtain a product 2.9 g intermediate I.
Into a 25 mL reaction flask was charged 0.2g of intermediate I, 4ml phosphorus oxychloride POCl 3 Heated to 100℃and reacted for 4 hours. TLC traces with dichloromethane: methanol=90: 10. after the reaction, cooling to room temperature, adding 25 ml diethyl ether under ice bath cooling, precipitating solid, stirring for 30min, centrifuging, pouring out supernatant, and vacuum drying the solid for 1 hour. The solid was stirred with 5 ml DMF, cooled in an ice bath, and 0.5g of tert-butyl (3- (2- (3-aminopropoxy) ethoxy) propyl) carbamate, 0.5. 0.5 ml triethylamine TEA, was added and reacted at room temperature for 12 hours. The reaction solution was spin-dried and purified on silica gel, eluting with dichloromethane, methanol=95: and 5, collecting and spin-drying to obtain a product 60 mg intermediate II.
25 167, mg red phenanthroline, 52, mg ruthenium trichloride RuCl are added into a mL reaction bottle 3 5. 5 ml ethylene glycol, heated to 95℃and stirred for reaction for 12 hours. TLC trace, developing solvent acetonitrile, water=90: 10. after the reaction, the temperature is reduced to normal temperature, 205 and mg intermediate II are added, heated to 120 ℃ and stirred for reaction for 6 hours. TLC trace, developing solvent acetonitrile, water=90: 10. after the reaction was completed, the temperature was lowered to room temperature, the reaction solution was added dropwise to 50ml of ethyl acetate, after stirring for 30min, the supernatant was decanted, and the remaining solid alumina was purified by column chromatography, eluting with acetonitrile, water=95: and 5, collecting and spin-drying to obtain the product 34mg intermediate III.
25 To the mL reaction flask was added 34mg intermediate III, 5 mL dichloromethane, and 1mL trifluoroacetic acid TFA dropwise under ice bath, followed by stirring for 12 hours. TLC traces with acetonitrile: water=90: 10. after the reaction is finished, spin-drying, adding dichloromethane 5 ml, stirring for 30min, spin-drying, and collecting to obtain 32 mg intermediate IV.
25 167 and mg red phenanthroline, 52 and mg ruthenium trichloride and 5 mL glycol are added into a mL reaction bottle, heated to 95 ℃ and stirred for reaction for 12 hours. TLC trace, developing solvent acetonitrile, water=90: 10. after the reaction is finished, the temperature is reduced to normal temperature, 124 mg intermediate I is added, the temperature is heated to 120 ℃ and the reaction is stirred for 6 hours. TLC trace, developing solvent acetonitrile, water=90: 10. after the reaction was completed, the temperature was lowered to room temperature, the reaction solution was added dropwise to 50ml of ethyl acetate, after stirring for 30min, the supernatant was decanted, and the remaining solid alumina was purified by column chromatography, eluting with acetonitrile, water=95: and 5, collecting and spin-drying to obtain the product 35mg intermediate V.
In a 25 mL reaction flask was charged 35mg of intermediate V, 1ml of phosphorus oxychloride, and heated to 100deg.C for 4 hours. TLC traces with dichloromethane: methanol=90: 10. after the reaction, cooling to room temperature, adding 5 ml diethyl ether under ice bath cooling, precipitating solid, stirring for 30min, centrifuging, pouring out supernatant, and vacuum drying the solid for 1 hour. The solid was stirred with 1ml DMF, cooled in an ice bath, and 41. 41mg intermediate IV, 0.1ml triethylamine was added and reacted at room temperature for 12 hours. The reaction mixture was dried by spin-drying, 1ml of pure water and 0.1g of sodium iodide were added, followed by stirring for 24 hours. Purifying silica gel by column, eluting with dichloromethane, wherein methanol=95: 5, collecting the spin-dried product 60 mg, wherein when a=y=3, b=x=2, c=3, y=1, R=I - 。
Example two
Compared with the existing similar products, the fluorescent dye disclosed by the invention has the advantages of simplicity and convenience in operation, no need of decolorization, saving, high detection sensitivity, compatibility of mass spectrum, no pollution to the environment and the like, can be detected on an ultraviolet excitation instrument and a blue light excitation instrument, and well solves the defect that a large gel cannot be observed by an ultraviolet gel imager; not only can dye the protein of SDS-PAGE gel, but also is applicable to the dyeing of polyacrylamide gel electrophoresis nucleic acid.
The method for performing polyacrylamide gel electrophoresis by using the ruthenium metal complex prepared in the first embodiment comprises the following steps of adding a sample to be detected into polyacrylamide gel, and then performing electrophoresis test; after the electrophoresis test is finished, the polyacrylamide gel is dye blasted in a bubble dye solution, and finally ultraviolet excitation or blue light excitation detection is utilized to finish the polyacrylamide gel electrophoresis; the bubble dye solution comprises the ruthenium metal complex prepared in the first embodiment.
The specific protein polyacrylamide gel electrophoresis operation is as follows:
(1) gel concentration: preparing 10% SDS-PAGE separating gel and 5% SDS-PAGE upper gel, wherein the specific raw materials are all existing products
a. Firstly preparing lower glue (separating glue), adding a glue preparation plate, immediately adding 95% ethanol liquid seal, enabling glue planes to be on the same horizontal line, and solidifying for half an hour, wherein the formula is shown in the following table:
b. adding upper layer glue (concentrated glue), pouring ethanol in the glue plate, adding upper layer glue until the plate is full of liquid, inserting comb (smooth side of comb is close to thick side of the glue plate), and solidifying for 30min-1 hr, wherein the formula is shown in the following table:
(2) loading: loading sequence: 1. after the rainbow markers 5ul 2. MKP002 markers are diluted twice in sequence, loading samples, 5ul each hole; manufacturer EZbiolab, product number and name: MKP002 Unstained Low Range Protein Marker (14.4-97.4 KD)); the loading amount is 1000ng/500ng/250ng/125ng/62.5ng/31.25 ng/15.625 ng/-7 ng respectively;
(3) electrophoresis: firstly, the voltage is 80V, electrophoresis is carried out for 30min, and after the electrophoresis is finished, the voltage is adjusted to 120V, and the electrophoresis is carried out for 1h;
(4) taking out the gel after electrophoresis, washing with tap water, prying the glass plate by using a small plate, and taking out the gel;
(5) preparing a bubble dye solution as shown in the Table, wherein the aqueous solution of Brightein is 0.5wt% (mass concentration) of the aqueous solution of the ruthenium metal complex prepared in example one
Putting the gel taken out into a bubble-dyeing liquid to be bubble-dyed for 90 minutes; then directly used for ultraviolet observation without decolorization;
(6) gel observation and photographing under an ultraviolet lamp are carried out, and a ruthenium metal complex staining chart is obtained.
Changing the Brightein aqueous solution in the step (5) into the existing coomassie blue solution, changing the bubble dyeing time to 6 hours, decoloring for 19 hours after bubble dyeing, and then using the decolored water solution for ultraviolet observation, wherein the rest is unchanged, so as to obtain a coomassie blue dyeing chart.
And (3) replacing the Brightein aqueous solution in the step (5) with the existing Lumitein solution of Biotium, changing the bubble dyeing time to 2 hours, decoloring for 2 hours after bubble dyeing, and then using the decolored product for ultraviolet observation, wherein the rest is unchanged, so as to obtain a Lumitein dyeing chart.
FIG. 1 is a staining chart of Lumitein (A) of existing Biotechnology, existing Coomassie blue (B), imaging Lumitein-stained gel using UV equipped with EtBr filter (UVP), and simultaneously imaging Coomassie brilliant blue-stained gel using white light converter (UVP), in which the sample concentrations are, in order from left to right: 1000ng/500ng/250ng/125ng/62.5ng/31.25 ng/15.625 ng/-7 ng. It can be seen that both the sensitivity and staining time of Lumitein are superior to coomassie blue.
FIG. 2 is a staining chart of ruthenium metal complex (A) of the invention, prior Lumitein (B), using UV equipped with EtBr filter (UVP) to image stained gel, in which sample concentrations from left to right are in order: 50ng/20ng/12.5ng/6.25ng/3.125ng/1.5625ng/0.78125 ng/-0.3 ng, the product is more sensitive and clearer than Lumitein of Biotechnology, and the sensitivity is higher than that of Lumitein.
As can be seen by combining the two drawings, the product is more sensitive and clearer than coomassie blue staining, the time is far faster than coomassie staining, the time is changed into 90 minutes from tens of hours, the sensitivity is 1000 times of that of coomassie staining, and the customer can not miss any possible target protein due to the high sensitivity.
Example III
The specific protein polyacrylamide gel electrophoresis operation is as follows:
(1) gel concentration: preparing 10% SDS-PAGE separating gel and 5% SDS-PAGE upper gel, wherein the specific raw materials are all existing products
a. Firstly preparing lower glue (separating glue), adding a glue preparation plate, immediately adding 95% ethanol liquid seal, enabling glue planes to be on the same horizontal line, and solidifying for half an hour, wherein the formula is shown in the following table:
b. adding upper layer glue (concentrated glue), pouring ethanol in the glue plate, adding upper layer glue until the plate is full of liquid, inserting comb (smooth side of comb is close to thick side of the glue plate), and solidifying for 30min-1 hr, wherein the formula is shown in the following table:
(2) loading: several 100bp markers are commercially available, 5ul per well; specifically, I. 10 uL 100bp Ladder (manufacturer: CWBio; product number: CW 0636); 10 uL 100bp Ladder (manufacturer: CWBio; product number: CW 0636); III. 10 uL 100bp Ladder DNA Marker (manufacturer: bioMed; product number: DM 112), IV. 10 uL 100bp DNA Ladder (manufacturer: trans; product number: BM 301), V. 10 uL 100bp DNA Ladder (manufacturer: trans; product number: BM 301);
(3) electrophoresis: the voltage is 120V, and electrophoresis is carried out for 1h;
(4) taking out the gel after electrophoresis, washing with tap water, prying the glass plate by using a small plate, and taking out the gel;
(5) preparing a soaking dye solution, soaking and dyeing the taken gel for 30min as shown in the table; the aqueous ruthenium metal complex solution prepared in example one wherein the aqueous Brightein solution is 0.5wt% (mass concentration)
(6) The gel after the bubble dyeing is used for observation and photographing under an ultraviolet exciter or a blue light exciter without decoloring.
FIG. 3 is a graph showing the staining of nucleic acids in a PAGE gel with ruthenium metal complexes according to the present invention, and the staining of nucleic acids in the gel was detected by UV and blue light, respectively, wherein the UV and blue light are both front and back sides of the same gel, and the samples from left to right in the two graphs are I. 10 uL 100bp Ladder (manufacturer: CWBio; product number: CW 0636); 10 uL 100bp Ladder (manufacturer: CWBio; product number: CW 0636); III. 10 uL 100bp Ladder DNA Marker (manufacturer: bioMed; product number: DM 112), IV. 10 uL 100bp DNA Ladder (manufacturer: trans; product number: BM 301), V. 10 uL 100bp DNA Ladder (manufacturer: trans; product number: BM 301); experimental results show that Brightein can well dye nucleic acid in a PAGE gel, the dyed strips are bright, and can be observed under an ultraviolet exciter and a blue light exciter, so that the defect that the large gel cannot be observed by an ultraviolet gel imager is well overcome; the existing Biotium Lumitein, thermo Scientific Krypton, cannot be used for nucleic acid staining.

Claims (4)

1. A method for preparing a ruthenium metal complex, comprising the steps of:
(1) Adding the phenanthroline into concentrated sulfuric acid, then dropwise adding fuming sulfuric acid, and then reacting to prepare an intermediate I;
(2) Reacting the intermediate I with phosphorus oxychloride to obtain a solid; then reacting the solid with a connecting bridge compound in the presence of an amine compound to prepare an intermediate II; the connecting bridge compound is NH 2 -(CH 2 ) a -[O-(CH 2 ) b ] x -[O-(CH 2 ) c ] y -NH-boc；
(3) Reacting bathophenanthroline with ruthenium trichloride in ethylene glycol, and then adding an intermediate II; continuing the reaction to prepare an intermediate III;
(4) Dropwise adding trifluoroacetic acid into the intermediate III, and reacting to prepare an intermediate IV;
(5) Reacting bathophenanthroline with ruthenium trichloride in ethylene glycol, then adding an intermediate I, and continuing the reaction to prepare an intermediate V;
(6) Reacting the intermediate V with phosphorus oxychloride to obtain a solid; then reacting the solid with intermediate iv in the presence of an amine compound; then adding a salt compound; continuing to react to obtain the ruthenium metal complex;
the chemical structural formula of the ruthenium metal complex is as follows:
;
wherein L is a connecting bridge, in particular-NH- (CH) 2 ) a -[O-(CH 2 ) b ] x -[O-(CH 2 ) c ] y -NH-, wherein a is 2 to 20, b and c are each independently 2 to 3, x is 0 or 1 to 20, y is 0 or 1; r is an anion selected from chlorine, bromine, iodine or perchlorate ions;
in the step (1), red phenanthroline is added into concentrated sulfuric acid under ice water bath; the reaction is carried out for 12 hours at 90 ℃;
in the step (2), the reaction time of the intermediate I and phosphorus oxychloride is 4 hours, and the temperature is 100 ℃; the reaction time of the solid and the connecting bridge compound is 12 hours, and the temperature is normal temperature;
in the step (3), the reaction time of the erythrophenanthroline and the ruthenium trichloride is 12 hours, and the temperature is 95 ℃; the reaction is continued for 6 hours at 120 ℃;
in the step (4), trifluoroacetic acid is added into the intermediate III dropwise in the presence of dichloromethane in an ice bath, and then the mixture is reacted for 12 hours to prepare an intermediate IV;
in the step (5), the reaction time of the erythrophenanthroline and the ruthenium trichloride is 12 hours, and the temperature is 95 ℃; the reaction is continued for 6 hours at 120 ℃;
in the step (6), the reaction time of the intermediate V and phosphorus oxychloride is 4 hours, and the temperature is 100 ℃; the reaction time of the solid and the intermediate IV is 12 hours, and the temperature is normal temperature; continuing the reaction for 24 hours at normal temperature;
in the step (1), the dosage ratio of the bathophenanthroline to the concentrated sulfuric acid to the fuming sulfuric acid is 2.0g to 10mL to 1mL; in the step (2), the dosage ratio of the intermediate I to the phosphorus oxychloride to the amine compound to the connecting bridge compound is 0.2g to 4mL to 0.5g to 4mL; in the step (3), the mass ratio of the bathophenanthroline, the ruthenium trichloride and the intermediate II is 167:52:205; in the step (4), the dosage ratio of the intermediate III to the trifluoroacetic acid is 34mg to 1mL; in the step (5), the mass ratio of the bathophenanthroline to the ruthenium trichloride to the intermediate I is 167:52:124; in the step (6), the dosage ratio of the intermediate V to the phosphorus oxychloride to the intermediate IV to the amine compound to the salt compound is 35mg to 1mL to 41mg to 0.1mL to 0.1g.
2. The method for preparing the ruthenium metal complex according to claim 1, wherein the mass fraction of fuming sulfuric acid is 20%, and the concentrated sulfuric acid is a conventional commercial product; the amine compound is triethylamine; the salt compound is a sodium salt compound.
3. The method for producing ruthenium metal complex according to claim 1, wherein in the step (1), after the reaction is completed, the reaction solution is cooled to room temperature, then added into diethyl ether, then subjected to centrifugal treatment, and the obtained solid is added into water, then adjusted to be neutral with alkali, and then spin-dried, and then subjected to column purification to obtain an intermediate I;
in the step (2), after the reaction of the intermediate I and phosphorus oxychloride is finished, cooling to room temperature, adding diethyl ether, and centrifuging to obtain a solid; after the reaction of the solid and the connecting bridge compound is finished, spin-drying the reaction liquid, and purifying by a column to prepare an intermediate II;
in the step (3), after the continuous reaction is finished, cooling to normal temperature, then dripping the reaction liquid into ethyl acetate, then pouring out supernatant, and purifying a residue column to prepare an intermediate III;
in the step (4), after the reaction is finished, adding dichloromethane after spin-drying the reaction solution, and spin-drying again to obtain an intermediate IV;
in the step (5), after the continuous reaction is finished, cooling to normal temperature, then dripping the reaction liquid into ethyl acetate, then pouring out supernatant, and purifying a residual column to prepare an intermediate V;
in the step (6), after the reaction of the intermediate V and phosphorus oxychloride is finished, cooling to room temperature, adding diethyl ether, and centrifuging to obtain a solid; after the continuous reaction is finished, the reaction liquid is purified by a column and then dried by spin to obtain the ruthenium metal complex.
4. The method for preparing ruthenium metal complex according to claim 3, wherein in the step (1), diethyl ether is precooled diethyl ether, sodium hydroxide is used for adjusting to be neutral, column purification is silica gel column purification, and eluent is acetonitrile and water in a volume ratio of 90:10;
in the step (2), diethyl ether is added under ice water bath, column purification is silica gel column purification, and eluent is dichloromethane and methanol with the volume ratio of 95:5;
in the step (3), the column purification is alumina column purification, and the eluent is acetonitrile and water with the volume ratio of 95:5;
in the step (5), the column purification is alumina column purification, and the eluent is acetonitrile and water with the volume ratio of 95:5;
in the step (6), diethyl ether is added under ice water bath, column purification is silica gel column purification, and eluent is dichloromethane and methanol with the volume ratio of 95:5.
CN202110888426.5A 2019-04-30 2019-04-30 Preparation method of ruthenium metal complex Active CN113666970B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110888426.5A CN113666970B (en) 2019-04-30 2019-04-30 Preparation method of ruthenium metal complex

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910364444.6A CN110128481B (en) 2019-04-30 2019-04-30 Ruthenium metal complex and application thereof in polyacrylamide gel electrophoresis
CN202110888426.5A CN113666970B (en) 2019-04-30 2019-04-30 Preparation method of ruthenium metal complex

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
CN201910364444.6A Division CN110128481B (en) 2019-04-30 2019-04-30 Ruthenium metal complex and application thereof in polyacrylamide gel electrophoresis

Publications (2)

Publication Number Publication Date
CN113666970A CN113666970A (en) 2021-11-19
CN113666970B true CN113666970B (en) 2024-01-12

Family

ID=67575942

Family Applications (2)

Application Number Title Priority Date Filing Date
CN202110888426.5A Active CN113666970B (en) 2019-04-30 2019-04-30 Preparation method of ruthenium metal complex
CN201910364444.6A Active CN110128481B (en) 2019-04-30 2019-04-30 Ruthenium metal complex and application thereof in polyacrylamide gel electrophoresis

Family Applications After (1)

Application Number Title Priority Date Filing Date
CN201910364444.6A Active CN110128481B (en) 2019-04-30 2019-04-30 Ruthenium metal complex and application thereof in polyacrylamide gel electrophoresis

Country Status (1)

Country Link
CN (2) CN113666970B (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1788395A1 (en) * 1998-10-27 2007-05-23 Molecular Probes, Inc. Luminescent protein stains containing transition metal complexes
CN103709202A (en) * 2013-12-26 2014-04-09 广东药学院 Ruthenium (II) complex, preparation method thereof, and application of complex as cell fluorescent dye
CN107417736A (en) * 2017-07-26 2017-12-01 广东医科大学 A kind of ruthenium complex and its synthetic method and application

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000025139A1 (en) * 1998-10-27 2000-05-04 Molecular Probes, Inc. Luminescent protein stains containing transition metal complexes
US9193746B2 (en) * 2006-12-07 2015-11-24 Biotium, Inc. Luminescent metal complexes and associated technology
CN103333211B (en) * 2013-06-05 2016-08-10 大连理工大学 One class dual wavelength transmitting, double Heteronu clear metal complex and its preparation method and application
US10203333B2 (en) * 2013-06-18 2019-02-12 Rubipy Scientific, Inc. Electronically neutral metal complexes as biological labels
IL229525A0 (en) * 2013-11-20 2014-01-30 Yeda Res & Dev Metal-based tris-bipyridyl complexes and uses thereof in electrochromic applications
US9505794B2 (en) * 2014-10-08 2016-11-29 Council Of Scientific And Industrial Research Ruthenium (II) complexes, preparation and uses thereof
CN106008495A (en) * 2016-06-13 2016-10-12 苏州宇恒生物科技有限公司 Preparation of novel nucleic acid dye for polyacrylamide gel electrophoresis
CN106188149B (en) * 2016-07-05 2019-03-22 上海师范大学 A kind of near-infrared metal iridium complex and its preparation method and application
CN109206454B (en) * 2018-04-26 2021-02-05 青岛农业大学 G-quadruplex selective probe based on binuclear iridium (III) complex

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1788395A1 (en) * 1998-10-27 2007-05-23 Molecular Probes, Inc. Luminescent protein stains containing transition metal complexes
CN103709202A (en) * 2013-12-26 2014-04-09 广东药学院 Ruthenium (II) complex, preparation method thereof, and application of complex as cell fluorescent dye
CN107417736A (en) * 2017-07-26 2017-12-01 广东医科大学 A kind of ruthenium complex and its synthetic method and application

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Ruthenium(II) multi carboxylic acid complexes: chemistry and application in dye sensitized solar cells;Hashem Shahroosvand et al.;《Dalton Transactions》;第43卷;第5158–5167页 *
杯[4]芳烃修饰的钌(II)配合物的合成与性质;黄秋颖 等;《无机化学学报》;第34卷(第4期);第217-229页 *

Also Published As

Publication number Publication date
CN113666970A (en) 2021-11-19
CN110128481B (en) 2021-08-06
CN110128481A (en) 2019-08-16

Similar Documents

Publication Publication Date Title
AP165A (en) Fluorescent nucleus acid dyes.
US6713622B1 (en) 4,7-dichlororhodamine dye labeled polynucleotides
CN101602762B (en) Asymmetric cyanine compound, preparation method and application thereof
US9193746B2 (en) Luminescent metal complexes and associated technology
CN106147753B (en) Thiazole orange styrene compound as G-quadruplex nucleic acid fluorescent probe
CN101723874B (en) Cyanine compound and application thereof in dyeing biological samples
EP0500779A4 (en)
CN110143966B (en) Spiropyran-naphthalimide derivative and synthesis method and application thereof
CN107721922B (en) Quinoline biological thiol fluorescent probe and preparation and application thereof
CN106883637B (en) Seven methine cyanine dyes of indoles and preparation method and application of uracil
CN105440725A (en) Asymmetric cyanine fluorescent dye, composition and application in biological sample dyeing
JPH06321908A (en) Optical sensor for measuring cation
CN113666970B (en) Preparation method of ruthenium metal complex
EP1947453B1 (en) Method for analyzing protein
CN106940376A (en) Protein detection
CN105001666B (en) A kind of asymmetric near-infrared squaraine dye and preparation method and application
WO1999038919A1 (en) Fluorescent dye
CN108586448A (en) A kind of quinoline salt type compound and its application
JP2016193897A (en) Ph dependent fluorescence compound
CN114634488B (en) Succinimide esters and methods of making, treating and detecting same
CN111334076A (en) High-brightness and high-light-stability cell nucleus fluorescent probe
US11952518B2 (en) Synthesis and application of fluorescent dye with phenanthridine and benzothiazole conjugated
CN113686943B (en) Multifunctional nucleic acid dye and preparation method and application thereof
CN112898228B (en) Mixed assembly based mixed aggregate FRET probes and their use in detecting mitochondrial autophagy
JPH08198873A (en) Compound-selective fluorescent labeled reagent having sulfhydryl group

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant