CN106008495A - Preparation of novel nucleic acid dye for polyacrylamide gel electrophoresis - Google Patents
Preparation of novel nucleic acid dye for polyacrylamide gel electrophoresis Download PDFInfo
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- CN106008495A CN106008495A CN201610412156.XA CN201610412156A CN106008495A CN 106008495 A CN106008495 A CN 106008495A CN 201610412156 A CN201610412156 A CN 201610412156A CN 106008495 A CN106008495 A CN 106008495A
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- reaction
- nucleic acid
- preparation
- polyacrylamide gel
- gel electrophoresis
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- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 34
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 34
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 34
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 239000000980 acid dye Substances 0.000 title abstract description 11
- 238000006243 chemical reaction Methods 0.000 claims abstract description 68
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims abstract description 57
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims abstract description 48
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims abstract description 24
- 150000001875 compounds Chemical class 0.000 claims abstract description 22
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000000975 dye Substances 0.000 claims abstract description 20
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 claims abstract description 20
- MUDSDYNRBDKLGK-UHFFFAOYSA-N 4-methylquinoline Chemical compound C1=CC=C2C(C)=CC=NC2=C1 MUDSDYNRBDKLGK-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229960003080 taurine Drugs 0.000 claims abstract description 10
- UTBVIMLZIRIFFR-UHFFFAOYSA-N 2-methylthio-1,3-benzothiazole Chemical compound C1=CC=C2SC(SC)=NC2=C1 UTBVIMLZIRIFFR-UHFFFAOYSA-N 0.000 claims abstract description 8
- VUQUOGPMUUJORT-UHFFFAOYSA-N methyl 4-methylbenzenesulfonate Chemical compound COS(=O)(=O)C1=CC=C(C)C=C1 VUQUOGPMUUJORT-UHFFFAOYSA-N 0.000 claims abstract description 8
- NVRVNSHHLPQGCU-UHFFFAOYSA-N 6-bromohexanoic acid Chemical compound OC(=O)CCCCCBr NVRVNSHHLPQGCU-UHFFFAOYSA-N 0.000 claims abstract description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 36
- FVAUCKIRQBBSSJ-UHFFFAOYSA-M sodium iodide Chemical compound [Na+].[I-] FVAUCKIRQBBSSJ-UHFFFAOYSA-M 0.000 claims description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 22
- 238000003756 stirring Methods 0.000 claims description 21
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 18
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 18
- 239000007787 solid Substances 0.000 claims description 15
- 239000002904 solvent Substances 0.000 claims description 14
- 235000009518 sodium iodide Nutrition 0.000 claims description 10
- 239000000376 reactant Substances 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 5
- 150000002148 esters Chemical class 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
- 229910021529 ammonia Inorganic materials 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 239000012295 chemical reaction liquid Substances 0.000 claims 1
- 150000002825 nitriles Chemical class 0.000 claims 1
- 238000010792 warming Methods 0.000 claims 1
- 239000007850 fluorescent dye Substances 0.000 abstract description 6
- 229920000642 polymer Polymers 0.000 abstract description 6
- 231100000086 high toxicity Toxicity 0.000 abstract description 3
- -1 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate Chemical compound 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 239000000499 gel Substances 0.000 description 20
- 239000000047 product Substances 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 229920002401 polyacrylamide Polymers 0.000 description 6
- 238000003384 imaging method Methods 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 239000011543 agarose gel Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000008595 infiltration Effects 0.000 description 3
- 238000001764 infiltration Methods 0.000 description 3
- 206010020751 Hypersensitivity Diseases 0.000 description 2
- 208000026935 allergic disease Diseases 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 2
- 230000009610 hypersensitivity Effects 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 1
- 229910052794 bromium Inorganic materials 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- TWYVVGMYFLAQMU-UHFFFAOYSA-N gelgreen Chemical compound [I-].[I-].C1=C(N(C)C)C=C2[N+](CCCCCC(=O)NCCCOCCOCCOCCCNC(=O)CCCCC[N+]3=C4C=C(C=CC4=CC4=CC=C(C=C43)N(C)C)N(C)C)=C(C=C(C=C3)N(C)C)C3=CC2=C1 TWYVVGMYFLAQMU-UHFFFAOYSA-N 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
- C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
- C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Biomedical Technology (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides preparation of a novel nucleic acid dye for polyacrylamide gel electrophoresis. Following reactions are sequentially conducted:, wherein is 4-methylquinoline, and is 6-bromohexanoic acid; , wherein is 2-(methylmercapto)benzothiazole, and is methyl p-toluenesulfonate; , wherein DMF is N,N-dimethyl formamide, Et3N is triethylamine, and HCl(conc.) is concentrated hydrochloric acid; , wherein is 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU), DIPEA is N, N-diisopropyl ethylamine, is taurine, and is a product compound. Improvement is conducted on a molecular structure, therefore, the nucleic acid dye can easily penetrate into a high-density high polymer, the high sensitivity and high stability of the nucleic acid dye are kept, and the technical problem that a fluorescent dye serving as a nucleic acid gel dye is difficult to penetrate into the high polymer and the problem that the nucleic acid dye generally has the high toxicity are solved.
Description
Technical field
The present invention relates to fluorescent dye field, be specifically related to a kind of novel nucleic acids for polyacrylamide gel electrophoresis and contaminate
The preparation of material.
Background technology
The dyestuff of the existing safety non-toxic being used in agarose gel, as biotium company of the U.S. produces
Gelgreen dyestuff is excessive due to molecular weight, it is impossible to apply in polyacrylamide gel electrophoresis.The reason caused is: polyacrylamide
Amine gel is close is the higher three-dimensional high polymer of dense degree.This highly dense polymer architecture so that separating difference
Magnitude range, variable concentrations scope nucleic acid molecules time, as nucleic acid gel dyestuff fluorescent dye particularly macromole very
Difficult infiltration is entered so that nucleic acid staining poor effect.
For the gel imaging instrument that nucleic acid electrophoresis is common, it is generally divided into two kinds of wave bands, i.e. ultraviolet and the gel of about 488nm
Laser scanner.Gel electrophoresis generally uses agarose gel and polyacrylamide gel two media.The latter's polyacrylamide
Gel is close is the high polymer that dense degree is the highest.This highly dense structure, the fluorescent dye as nucleic acid gel dyestuff is contaminating
It is difficult to infiltration during the nucleic acid molecules that color separates and enters so that nucleic acid staining poor effect.
Polyacrylamide gel electrophoresis is network structure, has molecular sieving effect, concentrates effect, charge effect, for little
The separating effect of sheet segment DNA (5bp-500bp) is best.Agarose gel about can distinguish the DNA fragmentation of difference 100bp, and it is differentiated
Though rate is lower than polyacrylamide gel, and when carrying out high-voltage power supply, polyacrylamide gel can only be used.The applicant exists
(Application No. 201410149438.6, invention and created name was the preparation of a kind of new nucleic acid dyestuff and answered in April, 2014
With) a patent application in, mention a kind of novel nucleic acid dye, this dyestuff is maintaining as excellent nucleic acid dye
Hypersensitivity, high stability, compared with detecting instrument compatibility in the case of, improve from molecular structure, such that it is able to very
Readily penetrate in the high polymer of this highly dense degree of polyacrylamide gel.But this dyestuff can be only used in
On ultraviolet gel scanner based on 254nm, it is impossible to compatible ultraviolet gel scanner based on 488nm.
Summary of the invention
For solving above-mentioned technical problem, we have proposed a kind of new nucleic acid dyestuff for polyacrylamide gel electrophoresis
Preparation, the present invention improves from molecular structure, such that it is able to penetrate into easily this highly dense degree height gather
In thing.
For reaching above-mentioned purpose, technical scheme is as follows:
The preparation of a kind of new nucleic acid dyestuff for polyacrylamide gel electrophoresis, is divided into following four-step reaction and obtains
:
One, first step reaction
1.1 reaction equation
Wherein:
For 4-methylquinoline
For 6-bromocaproic acid
For intermediate compound I
Two, second step reaction
2.1 reaction equation
Wherein:
For 2-(methyl mercapto) benzothiazole
For methyl tosylate
For intermediate II
Three, three-step reaction
3.1 reaction equation
Wherein:
For intermediate compound I
For intermediate II
DMF is N,N-dimethylformamide
Et3N is triethylamine
NaI is sodium iodide
HCl (conc.) is concentrated hydrochloric acid
For intermediate III
Four, four-step reaction
4.1 reaction equation
Wherein:
For intermediate III
For 2-succinimido-1,1,3,3-tetramethylurea Tetrafluoroboric acid ester (TSTU)
DMF is N, N-dimethyl methyl
Amide
DIPEA is N, N-diisopropylethylamine
For taurine
Et3N is triethylamine
H2O is water
For product compound.
Preferably, the operation of described 1.1 steps is as follows: add 4-methylquinoline and 6-bromine in 100mL two-neck bottle
Caproic acid, with rubber stopper sealing post-heating 130 DEG C reaction 3h, then raises temperature to 150 DEG C of reaction 12h；TLC follows the tracks of reaction, developing solvent:
15%MeOH/CH2Cl2；The reactant liquor of heat is poured in 150mL acetonitrile, stirring, adds 100mL ethyl acetate, has solid to separate out, mistake
Filter；Gained solid adds 150mL ethyl acetate, stirs 2h；Filtering, solid is dried to obtain intermediate compound I.
Preferably, the operation of described 2.1 steps is as follows: add 2-in equipped with churned mechanically 250mL reaction bulb
(methyl mercapto) benzothiazole and methyl tosylate, heat 60 DEG C of reactions overnight, then raises temperature to 120 DEG C of reaction 2h；TLC with
Track has reacted, developing solvent: isopropanol: acetone: water: acetic acid: ammonia=35:25:25:14:8；Add ethyl acetate 100mL,
Back flow reaction 1h, cooled and filtered, solid is dried to obtain intermediate II.
Preferably, the operation of described 3.1 steps is as follows: adds intermediate compound I and DMF in 100mL round-bottomed flask, drips
Add Et3N, stirring at normal temperature 15min；In 20min, be added portionwise into intermediate II, after stirring at normal temperature reaction overnight；TLC with
Track has reacted, developing solvent: 20%MeOH/CH2Cl2；Reactant liquor is poured in 150mL water, stirring, adds NaI, is subsequently added dense
Hydrochloric acid, stirs 2h；Filtering, solid is dried to obtain intermediate III.
Preferably, the operation of described 4.1 steps is as follows: in 50mL eggplant-shape bottle, add intermediate III, DMF and
DIPEA, stirs 0.5h under ice bath, is dividedly in some parts TSTU, continues to react under condition of ice bath 1h；TLC follows the tracks of reaction and completes, and launches
Agent: MeOH:DCM=1:5；Preparation 0.6g taurine/20mL H2The solution of O, and be placed in ice bath cooling down 0.5h, with
Backward taurine solution add triethylamine and is slowly added dropwise into above reactant liquor, being stirred overnight reaction.TLC follows the tracks of and has reacted
Become, developing solvent: MeOH:DCM=1:10；Filtering, wash (20mL × 3), lyophilizing obtains product compound.
By technique scheme, the present invention not only maintain the hypersensitivity as excellent nucleic acid dye, high stability,
And solve the fluorescent dye as nucleic acid gel dyestuff and be difficult to permeate so that the technology of nucleic acid staining poor effect is difficult
Topic.Product can be used in the ultraviolet gel imaging instrument with SYBR Green optical filter or have the gel of excited by visible light device
Imager, such as the gel scanner based on Dark Reader camera system or 488nm laser instrument.Additionally this product also solves
The high toxicity problem that usual nucleic acid dye is had.Thus reached novel in design, operation and rationally and applied effective
Purpose.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
In having technology to describe, the required accompanying drawing used is briefly described, it should be apparent that, the accompanying drawing in describing below is only this
Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, it is also possible to
Other accompanying drawing is obtained according to these accompanying drawings.
Fig. 1 is the system of a kind of new nucleic acid dyestuff for polyacrylamide gel electrophoresis disclosed in the embodiment of the present invention
Mass spectrum time standby.
Detailed description of the invention
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Describe, it is clear that described embodiment is only a part of embodiment of the present invention rather than whole embodiments wholely.Based on
Embodiment in the present invention, it is every other that those of ordinary skill in the art are obtained under not making creative work premise
Embodiment, broadly falls into the scope of protection of the invention.
Below in conjunction with embodiment and detailed description of the invention, the present invention is further detailed explanation.
Embodiment.
As it is shown in figure 1, a kind of new nucleic acid dyestuff for polyacrylamide gel electrophoresis, it is characterised in that it is divided into
Following four-step reaction obtains:
One, first step reaction
1.1 reaction equation
Wherein:
For 4-methylquinoline
For 6-bromocaproic acid
For intermediate compound I
1.2 operation
In 100mL two-neck bottle, add 4-methylquinoline and 6-bromocaproic acid, seal post-heating 130 DEG C reaction with rubber stopper
3h, then raises temperature to 150 DEG C of reaction 12h.TLC follows the tracks of reaction, developing solvent: 15%MeOH/CH2Cl2.The reactant liquor of heat is poured into
In 150mL acetonitrile, stirring, add 100mL ethyl acetate, have solid to separate out, filter.Gained solid adds 150mL acetic acid second
Ester, stirs 2h.Filtering, solid is dried to obtain intermediate compound I.
The experimental data that first step reaction obtains is:
4-methylquinoline inventory: 6.8mL
6-bromocaproic acid inventory: 10g
The yield of intermediate compound I is: 12.4g theoretical value: 13.24g
Two, second step reaction
2.1 reaction equation
Wherein:
For 2-(methyl mercapto) benzothiazole
For methyl tosylate
For intermediate II
2.2 operation
2-(methyl mercapto) benzothiazole and methyl tosylate is added in equipped with churned mechanically 250mL reaction bulb,
Heat 60 DEG C of reactions overnight, then raise temperature to 120 DEG C of reaction 2h.TLC follows the tracks of reaction and completes, developing solvent: isopropanol: acetone: water:
Acetic acid: ammonia=35:25:25:14:8.Add ethyl acetate 100mL, back flow reaction 1h, cooled and filtered, solid be dried in
Mesosome II.
The experimental data that second step reaction obtains is:
2-(methyl mercapto) benzothiazole inventory: 19.8g
P-methyl benzenesulfonic acid methyl ester inventory: 24.4g
The yield of intermediate II is: 38.54g theoretical value: 40.43g
Three, three-step reaction
3.1 reaction equation
Wherein:
For intermediate compound I
For intermediate II
DMF is N,N-dimethylformamide
Et3N is triethylamine
NaI is sodium iodide
HCl (conc.) is concentrated hydrochloric acid
For intermediate III
3.2 operation
In 100mL round-bottomed flask, add intermediate compound I and DMF, be added dropwise to Et3N, stirring at normal temperature 15min.In 20min
Be added portionwise into intermediate II, after stirring at normal temperature reaction overnight.TLC follows the tracks of reaction and completes, developing solvent: 20%MeOH/
CH2Cl2.Reactant liquor is poured in 150mL water, stirring, adds NaI, is subsequently added concentrated hydrochloric acid, stirs 2h.Filtering, solid is dried
Intermediate III.
The experimental data that three-step reaction obtains is:
Intermediate compound I inventory: 7.13g
Intermediate II inventory: 7.72g
DMF inventory: 50mL
Triethylamine inventory: 5.85mL
Sodium iodide inventory: 3.15g
Concentrated hydrochloric acid inventory: 3.5mL
Intermediate III yield: 9.28g theoretical value: 11.19g
Four, four-step reaction
4.1 reaction equation
Wherein:
For intermediate III
For 2-succinimido-1,1,3,3-tetramethylurea Tetrafluoroboric acid ester (TSTU)
DMF is N,N-dimethylformamide
DIPEA is N, N-diisopropylethylamine
For taurine
Et3N is triethylamine
H2O is water
For product compound
4.2 operation
In 50mL eggplant-shape bottle, add intermediate III, DMF and DIPEA, under ice bath, stir 0.5h, be dividedly in some parts
TSTU, continues to react under condition of ice bath 1h.TLC follows the tracks of reaction and completes, developing solvent: MeOH:DCM=1:5.Preparation 0.6g cattle sulphur
Acid/20mL H2The solution of O, and be placed in ice bath cooling down 0.5h, with backward taurine solution adding triethylamine and delaying
Slowly it is added dropwise to above reactant liquor, is stirred overnight reaction.TLC follows the tracks of reaction and completes, developing solvent: MeOH:DCM=1:10.Filter,
Washing (20mL × 3), lyophilizing obtains product compound.
The experimental data that four-step reaction obtains is:
Intermediate III inventory: 1.6g
TSTU inventory: 1g
DIPEA inventory: 1mL
DMF inventory: 20mL
Taurine inventory: 0.6g
Water inventory: 20mL
Triethylamine inventory: 1mL
Product compound yield: 1.5g, theoretical value: 1.92g
By above-mentioned operation process, the product that the present invention obtains, not only maintain the height as excellent nucleic acid dye
Sensitivity, high stability, and solve the fluorescent dye as nucleic acid gel dyestuff and be difficult to infiltration and enter so that nucleic acid staining
The technical barrier of poor effect.Meanwhile, product can be used in the ultraviolet gel imaging instrument with SYBR Green optical filter or
There is the gel imaging instrument of excited by visible light device, as the gel based on Dark Reader camera system or 488nm laser instrument is swept
Retouch instrument.Additionally this product also solves the high toxicity problem that usual nucleic acid dye is had.Thus reached novel in design, reaction
Operation rationally and applies effective purpose.
Above-described is only the preferred embodiment of the present invention, it is noted that for those of ordinary skill in the art
For, without departing from the concept of the premise of the invention, it is also possible to make some deformation and improvement, these broadly fall into the present invention
Protection domain.
Claims (5)
1. the preparation for the new nucleic acid dyestuff of polyacrylamide gel electrophoresis, it is characterised in that be divided into following four
Step reaction obtains:
One, first step reaction
1.1 reaction equation
Wherein:
For 4-methylquinoline
For 6-bromocaproic acid
For intermediate compound I
Two, second step reaction
2.1 reaction equation
Wherein:
For 2-(methyl mercapto) benzothiazole
For methyl tosylate
For intermediate II
Three, three-step reaction
3.1 reaction equation
Its
In:For intermediate compound I
For intermediate II
DMF is N,N-dimethylformamide
Et3N is triethylamine
NaI is sodium iodide
HCl (conc.) is concentrated hydrochloric acid
For intermediate III
Four, four-step reaction
4.1 reaction equation
Wherein:
For intermediateFor 2-succinimido-1,1,3,
3-tetramethylurea Tetrafluoroboric acid ester DMF is N,N-dimethylformamide
DIPEA is N, N-diisopropylethylamineFor taurine
Et3N is triethylamine
H2O is water
For product compound.
The preparation of a kind of new nucleic acid dyestuff for polyacrylamide gel electrophoresis the most according to claim 1, it is special
Levying and be, the operation of described 1.1 steps is as follows:
In 100mL two-neck bottle, add 4-methylquinoline and 6-bromocaproic acid, react 3h with rubber stopper sealing post-heating 130 DEG C, with
After be warming up to 150 DEG C reaction 12h；TLC follows the tracks of reaction, developing solvent: 15%MeOH/CH2Cl2；The reactant liquor of heat pours 150mL second into
In nitrile, stirring, add 100mL ethyl acetate, have solid to separate out, filter；Gained solid adds 150mL ethyl acetate, stirring
2h；Filtering, solid is dried to obtain intermediate compound I.
3., according to claim, the preparation of a kind of new nucleic acid dyestuff for polyacrylamide gel electrophoresis described in 1, it is special
Levying and be, the operation of described 2.1 steps is as follows:
2-(methyl mercapto) benzothiazole and methyl tosylate, heating is added in equipped with churned mechanically 250mL reaction bulb
60 DEG C of reactions overnight, then raise temperature to 120 DEG C of reaction 2h；TLC follows the tracks of reaction and completes, developing solvent: isopropanol: acetone: water: second
Acid: ammonia=35:25:25:14:8；Adding ethyl acetate 100mL, back flow reaction 1h, cooled and filtered, solid is dried middle
Body II.
The preparation of a kind of new nucleic acid dyestuff for polyacrylamide gel electrophoresis the most according to claim 1, it is special
Levying and be, the operation of described 3.1 steps is as follows:
In 100mL round-bottomed flask, add intermediate compound I and DMF, be added dropwise to Et3N, stirring at normal temperature 15min；Add portionwise in 20min
Enter intermediate II, after stirring at normal temperature reaction overnight；TLC follows the tracks of reaction and completes, developing solvent: 20%MeOH/CH2Cl2；Reaction
Liquid is poured in 150mL water, stirring, adds NaI, is subsequently added concentrated hydrochloric acid, stirs 2h；Filtering, solid is dried to obtain intermediate III.
The preparation of a kind of new nucleic acid dyestuff for polyacrylamide gel electrophoresis the most according to claim 1, it is special
Levying and be, the operation of described 4.1 steps is as follows:
In 50mL eggplant-shape bottle, add intermediate III, DMF and DIPEA, under ice bath, stir 0.5h, be dividedly in some parts TSTU, continue
1h is reacted under continuous condition of ice bath；TLC follows the tracks of reaction and completes, developing solvent: MeOH:DCM=1:5；Preparation 0.6g taurine/20mL
H2The solution of O, and be placed in ice bath cool down 0.5h, with in backward taurine solution add triethylamine and be slowly added dropwise into
Above reactant liquor, is stirred overnight reaction.TLC follows the tracks of reaction and completes, developing solvent: MeOH:DCM=1:10；Filter, washing
(20mL × 3), lyophilizing obtains product compound.
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| CN110128481A (en) * | 2019-04-30 | 2019-08-16 | 苏州宇恒生物科技有限公司 | Ruthenium metal complex and its application in polyacrylamide gel electrophoresis |
| CN111363377A (en) * | 2020-03-13 | 2020-07-03 | 苏州宇恒生物科技有限公司 | Nucleic acid dye and preparation method and application thereof |
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| CN110128481A (en) * | 2019-04-30 | 2019-08-16 | 苏州宇恒生物科技有限公司 | Ruthenium metal complex and its application in polyacrylamide gel electrophoresis |
| CN111363377A (en) * | 2020-03-13 | 2020-07-03 | 苏州宇恒生物科技有限公司 | Nucleic acid dye and preparation method and application thereof |
| WO2021179394A1 (en) * | 2020-03-13 | 2021-09-16 | 苏州优逸兰迪生物科技有限公司 | Nucleic acid dye, preparation method therefor and use thereof |
| CN111363377B (en) * | 2020-03-13 | 2021-11-30 | 苏州优逸兰迪生物科技有限公司 | Nucleic acid dye and preparation method and application thereof |
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