CN115246866A - Synthetic method of Janus green B solid phase carrier - Google Patents
Synthetic method of Janus green B solid phase carrier Download PDFInfo
- Publication number
- CN115246866A CN115246866A CN202110453583.3A CN202110453583A CN115246866A CN 115246866 A CN115246866 A CN 115246866A CN 202110453583 A CN202110453583 A CN 202110453583A CN 115246866 A CN115246866 A CN 115246866A
- Authority
- CN
- China
- Prior art keywords
- janus green
- phase carrier
- solid phase
- reaction
- preparing
- 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.)
- Pending
Links
- XXACTDWGHQXLGW-UHFFFAOYSA-M Janus Green B chloride Chemical compound [Cl-].C12=CC(N(CC)CC)=CC=C2N=C2C=CC(\N=N\C=3C=CC(=CC=3)N(C)C)=CC2=[N+]1C1=CC=CC=C1 XXACTDWGHQXLGW-UHFFFAOYSA-M 0.000 title claims abstract description 52
- 239000007790 solid phase Substances 0.000 title claims abstract description 43
- 238000010189 synthetic method Methods 0.000 title description 4
- 238000002360 preparation method Methods 0.000 claims abstract description 7
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 69
- 238000006243 chemical reaction Methods 0.000 claims description 43
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 claims description 36
- 238000000034 method Methods 0.000 claims description 24
- 239000007787 solid Substances 0.000 claims description 24
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 20
- 238000010791 quenching Methods 0.000 claims description 20
- 230000000171 quenching effect Effects 0.000 claims description 20
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 18
- 235000010288 sodium nitrite Nutrition 0.000 claims description 18
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 claims description 15
- 239000003960 organic solvent Substances 0.000 claims description 14
- 239000012074 organic phase Substances 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 12
- HKOOXMFOFWEVGF-UHFFFAOYSA-N phenylhydrazine Chemical compound NNC1=CC=CC=C1 HKOOXMFOFWEVGF-UHFFFAOYSA-N 0.000 claims description 12
- 239000002904 solvent Substances 0.000 claims description 12
- MOVNSGGBTSIUGX-UHFFFAOYSA-N 2-n,2-n-diethyl-10-phenylphenazin-10-ium-2,8-diamine;chloride Chemical compound [Cl-].C12=CC(N(CC)CC)=CC=C2N=C2C=CC(N)=CC2=[N+]1C1=CC=CC=C1 MOVNSGGBTSIUGX-UHFFFAOYSA-N 0.000 claims description 11
- ALJHHTHBYJROOG-UHFFFAOYSA-N 7-(dimethylamino)phenothiazin-3-one Chemical compound C1=CC(=O)C=C2SC3=CC(N(C)C)=CC=C3N=C21 ALJHHTHBYJROOG-UHFFFAOYSA-N 0.000 claims description 10
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 9
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 claims description 7
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- 229940014800 succinic anhydride Drugs 0.000 claims description 7
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 6
- 229920005990 polystyrene resin Polymers 0.000 claims description 6
- 125000004202 aminomethyl group Chemical group [H]N([H])C([H])([H])* 0.000 claims description 5
- 239000011324 bead Substances 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 239000011521 glass Substances 0.000 claims description 5
- 150000007530 organic bases Chemical class 0.000 claims description 5
- 239000002994 raw material Substances 0.000 claims description 5
- 230000035484 reaction time Effects 0.000 claims description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- 150000008065 acid anhydrides Chemical class 0.000 claims description 4
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 4
- 150000008064 anhydrides Chemical class 0.000 claims description 3
- VANNPISTIUFMLH-UHFFFAOYSA-N glutaric anhydride Chemical compound O=C1CCCC(=O)O1 VANNPISTIUFMLH-UHFFFAOYSA-N 0.000 claims description 3
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 2
- 239000005695 Ammonium acetate Substances 0.000 claims description 2
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims description 2
- 229940043376 ammonium acetate Drugs 0.000 claims description 2
- 235000019257 ammonium acetate Nutrition 0.000 claims description 2
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 2
- 239000011736 potassium bicarbonate Substances 0.000 claims description 2
- 229910000028 potassium bicarbonate Inorganic materials 0.000 claims description 2
- 235000015497 potassium bicarbonate Nutrition 0.000 claims description 2
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 2
- 235000011181 potassium carbonates Nutrition 0.000 claims description 2
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 claims description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 2
- 235000017550 sodium carbonate Nutrition 0.000 claims description 2
- 235000011121 sodium hydroxide Nutrition 0.000 claims description 2
- ZAFNJMIOTHYJRJ-UHFFFAOYSA-N Diisopropyl ether Chemical compound CC(C)OC(C)C ZAFNJMIOTHYJRJ-UHFFFAOYSA-N 0.000 claims 1
- 238000003745 diagnosis Methods 0.000 abstract description 4
- 238000001308 synthesis method Methods 0.000 abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 21
- 238000005303 weighing Methods 0.000 description 16
- 230000001276 controlling effect Effects 0.000 description 15
- 239000007864 aqueous solution Substances 0.000 description 12
- 238000001914 filtration Methods 0.000 description 12
- 238000001704 evaporation Methods 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 108091034117 Oligonucleotide Proteins 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 229940125782 compound 2 Drugs 0.000 description 3
- 229940126214 compound 3 Drugs 0.000 description 3
- 238000002425 crystallisation Methods 0.000 description 3
- 230000008025 crystallization Effects 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 210000003470 mitochondria Anatomy 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 229960001701 chloroform Drugs 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- JFCQEDHGNNZCLN-UHFFFAOYSA-N glutaric acid Chemical compound OC(=O)CCCC(O)=O JFCQEDHGNNZCLN-UHFFFAOYSA-N 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 239000000987 azo dye Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 229940125904 compound 1 Drugs 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000012209 synthetic fiber Substances 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
- C07D241/36—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings condensed with carbocyclic rings or ring systems
- C07D241/38—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings condensed with carbocyclic rings or ring systems with only hydrogen or carbon atoms directly attached to the ring nitrogen atoms
- C07D241/46—Phenazines
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
The invention relates to a synthesis method of a Janus green B solid phase carrier, which can realize simple and efficient preparation of the Janus green B solid phase carrier and realize the purpose of efficient fluorescence diagnosis.
Description
Technical Field
The invention belongs to the field of biomolecule diagnosis, relates to a synthetic method of a probe quenching group, and particularly relates to a preparation method of a Janus green B solid phase carrier.
Background
Janus green B is a synthetic azo dye, and can be used in the printing and dyeing process of textile clothing, and used for dyeing and printing various natural and synthetic fibers. In biomedicine, janus green B is a living cell dye of specific mitochondria, which can make the mitochondria to present blue-green color, and cytoplasm to be colorless, and the distribution and the morphology of the mitochondria are observed under a high power microscope.
In molecular diagnosis, a quenching dye is that a fluorescent group is marked at the 5 'end and a quenching group is marked at the 3' end of an oligonucleotide probe, when the probe is complete, a fluorescence signal generated by a system exciting a donor is absorbed by the adjacent quenching group through the principle of fluorescence resonance transfer, so that the fluorescence signal of the donor cannot be detected at the moment. The fluorescent signal is only released when the two groups are separated by hybridization or nuclease hydrolysis, at which time the fluorescent signal is detected by the system and is measured analytically for diagnostic purposes.
The Jiannalv B is a good quenching dye, can be loaded on carriers such as controllable glass beads CPG and polystyrene resin PS to form solid phase carriers, the carriers can be directly used for a DNA automatic synthesizer, and the Jiannalv B is directly marked at the 3' end of the oligonucleotide to be used as a quenching group of a probe through a solid phase synthesis method.
Disclosure of Invention
The invention aims to provide a preparation method of a Janus green B solid phase carrier.
The technical problem to be solved by the invention is realized by the following technical scheme:
the synthesis method of the Janus green B solid phase carrier comprises the following steps:
(1) Obtaining O-DMTr-Janus green B by methylene violet 3RAX through reaction a;
(2) O-DMTr-Janus green B reacting to obtain Janus green B succinate;
(3) And (c) obtaining the Janus green B solid phase carrier through reaction c.
Wherein the reaction a is as follows:
preferably, the reaction temperature is-5 to 20 ℃;
preferably, in the reaction a process, firstly adding acid water into the raw material methylene violet, then dropwise adding sodium nitrite, reacting for a period of time, and then adding 4- (N-hydroxyethyl-N- (2-O- (4,4-dimethyl triphenylmethyl) ethyl)) amino-aniline solution to continue reacting to obtain O-DMTr-Janus green B;
preferably, the ratio of the sodium nitrite to the methylene violet is 0.5-1.5;
preferably, the pH is adjusted to 4-7 before adding the 4- (N-hydroxyethyl-N- (2-O- (4,4-dimethyltriphenylmethyl) ethyl)) amino-aniline solution;
preferably, the regulator for regulating the pH value can be one or a mixture of sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate and ammonium acetate;
preferably, the reaction time after adding the sodium nitrite is 1-3 hours, and the reaction time after adding the 4- (N-hydroxyethyl-N- (2-O- (4,4-dimethyl triphenylmethyl) ethyl)) amino-aniline solution is 1-24 hours;
preferably, after the reaction is finished, filtering is carried out, a solid is collected and crystallized by dichloromethane/methanol to obtain solid O-DMTr-Janus green B;
preferably, the sulfuric acid in the reaction a is 1-6N sulfuric acid, and can also be 1-6N hydrochloric acid;
preferably, the reaction a process is: weighing a certain amount of methylene violet 3RAX into a dry flask, adding acid water, controlling the temperature at-5-20 ℃, dropwise adding sodium nitrite, wherein the ratio of the sodium nitrite to the methylene violet is 0.5-1.5, the reaction time is 1-3 hours, then adjusting the pH value to 4-7 by using alkali, adding 4- (N-hydroxyethyl-N- (2-O- (4,4-dimethyl triphenylmethyl) ethyl)) amino-aniline solution, continuously reacting for 1-24 hours, filtering after the reaction is finished, collecting a solid, and purifying the solid to obtain a pure O-DMTr-Jiannalv B;
preferably, the purification method is crystallization or column chromatography, and the crystallization is dichloromethane/methanol crystallization.
Wherein the reaction b is:
preferably, in the reaction B process, dissolving the O-DMTr-Janus green B in a first organic solvent, and then sequentially adding acid anhydride and organic base for reaction;
the first organic solvent is any one of DMF, dichloromethane, trichloromethane, ethyl acetate, diethyl ether and acetonitrile;
the organic base is any one of triethylamine, pyridine, diisopropyl ethyl amine and diisopropyl ethyl amine;
the anhydride is any one of succinic anhydride, glutaric anhydride and diethanol anhydride;
preferably, the acid anhydride is succinic anhydride and the organic solvent is dichloromethane;
preferably, after the reaction is finished, water is added for quenching, extraction and demixing are carried out, an organic phase is collected, and reduced pressure distillation is carried out to remove impurities so as to obtain the Janus green B succinate.
Wherein the reaction c is:
preferably, in the reaction c, after the Jiannalv B succinate is added into a second organic solvent for dissolving, a condensing agent and p-nitrophenol are sequentially added, water is added for quenching after the reaction is completely carried out at room temperature, an organic phase is obtained by extraction, the solvent is removed under reduced pressure, and the residue is added into a solid phase carrier for reaction to obtain a Jiannalv B solid phase carrier;
preferably, the second organic solvent is any one of DMF, dichloromethane, trichloromethane, ethyl acetate, diethyl ether and acetonitrile.
Preferably, the condensing agent is any one of DCC, EDC, HOBt, HBTU, HATu and PyBop;
preferably, the reaction temperature is 0-30 ℃;
preferably, the solid phase carrier is any one of controllable glass bead powder CPG and aminomethyl polystyrene resin.
The technical advantages of the invention are as follows: the Janus green B solid phase carrier is simply and efficiently synthesized, and no synthetic method for the Janus green B solid phase carrier modified by the 3' end of the oligonucleotide is reported in China at present. The Janus green B solid phase carrier can be directly used for a DNA automatic synthesizer, is directly marked at the 3' end of oligonucleotide to be used as a quenching group, and achieves the aim of diagnosis by analyzing and measuring a fluorescence signal.
Drawings
FIG. 1 shows the synthesis procedure of Janus green B solid phase carrier.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more clear, the following description of the present application will be made in detail and completely with reference to the specific embodiments and the accompanying drawings. It should be understood that the described embodiments are only a few embodiments of the present application, not all embodiments, and are not intended to limit the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
In order to realize the purpose of preparing a Janus green B solid phase carrier, the application provides a synthesis method of the Janus green B solid phase carrier, which comprises the following steps:
(1) O-DMTr-Janna green B (Compound 2): weighing a certain amount of methylene violet 3RAX (compound 1) into a dry flask, adding acid water, controlling the temperature at-5-20 ℃, dropwise adding sodium nitrite, wherein the ratio of sodium nitrite to methylene violet is 0.5-1.5, reacting for 1-3 hours, then adjusting the pH value to 4-7 with alkali, adding 4- (N-hydroxyethyl-N- (2-O- (4,4-dimethyltriphenylmethyl) ethyl)) amino-aniline solution, continuing to react for 1-24 hours, filtering after the reaction is finished, collecting solid, and crystallizing the solid with dichloromethane/methanol to obtain a pure product O-DMTr-Janus green B (compound 2).
(2) Janana green B succinate (compound 3): weighing a certain amount of O-DMTr-Janus green B (compound 2) in a dry flask, adding a first organic solvent for dissolving, sequentially adding succinic anhydride and organic base, reacting at room temperature to be complete, adding water for quenching, extracting for layering, collecting an organic phase, and evaporating under reduced pressure to remove the solvent to obtain Janus green B succinate (compound 3), wherein the first organic solvent is preferably dichloromethane.
(3) Janana green B solid support (compound 4): weighing a certain amount of Janus green B succinate (compound 3), adding a second organic solvent into a dry flask for dissolving, then adding a condensing agent and p-nitrophenol, reacting at room temperature till the reaction is complete, adding water for quenching, extracting and layering, collecting an organic phase, evaporating under reduced pressure to remove the solvent, then dissolving the residue into the organic solvent, and adding a solid phase carrier, such as: controlling the temperature of the glass bead powder CPG to be 0-30 ℃, reacting until the reaction is finished, filtering, and collecting solid to obtain the Janus green B solid phase carrier CPG powder, wherein the solid can be specifically but not only limited to the Janus green B solid phase carrier CPG powder, and the second organic solvent is preferably dichloromethane.
In order to verify that the technical scheme has excellent technical effects, the application further provides the following embodiments:
example 1
Preparation of O-DMTr-Janna Green B
Weighing 10 g of methylene violet 3RAX, adding 20 ml of 3N HCl aqueous solution into a flask, controlling the temperature at-5 ℃, dropwise adding 2.16 g of sodium nitrite aqueous solution, controlling the ratio of sodium nitrite to methylene violet to be 1.2, reacting for 1 hour, then adjusting the pH value to 6 by using sodium hydroxide, adding 12.8 g of DMF solution of 4- (N-hydroxyethyl-N- (2-O- (4,4-dimethyltriphenylmethyl) ethyl)) amino-aniline, continuing to react for 2 hours, after the reaction is finished, filtering, collecting solid, and crystallizing the solid by using dichloromethane/methanol to obtain a pure product O-DMTr-jiana green B.
Example 2
Preparation of Janus green B succinate
Weighing 7 g of O-DMTr-Jianna green B into a dry flask, adding 100 ml of dichloromethane for dissolving, sequentially adding 1.2 g of succinic anhydride and 2.3 ml of triethylamine, reacting at room temperature until the reaction is complete, adding 5% NaHCO 3 Quenching the aqueous solution, extracting and layering, collecting an organic phase, and evaporating the solvent under reduced pressure to obtain the Janus green B succinate.
Example 3
Preparation of Janus green B solid phase carrier
Weighing 7 g of Janus green B succinate, adding 100 ml of dichloromethane into a dry flask for dissolving, then sequentially adding 1.8 g of Dicyclohexylimine (DCC) and 1 g of p-nitrophenol, reacting at room temperature until the reaction is complete, adding water for quenching, extracting and layering, collecting an organic phase, evaporating the solvent under reduced pressure, dissolving the residue into 100 ml of dichloromethane, adding 30 g of controllable glass bead powder CPG, controlling the temperature to be 30 ℃, reacting until the reaction is completed, filtering, and collecting a solid to obtain Janus green B solid phase carrier CPG powder.
Example 4
Method for preparing Janus green B solid phase carrier by taking methylene violet 3RAX as raw material
(1) Weighing 10 g of methylene violet 3RAX, adding 20 ml of 6N sulfuric acid aqueous solution into a flask, controlling the temperature at 20 ℃, dropwise adding 2.16 g of sodium nitrite aqueous solution, controlling the ratio of sodium nitrite to methylene violet to be 0.5, reacting for 3 hours, then adjusting the pH value to 4 by using sodium hydroxide, adding 12.8 g of DMF (dimethyl formamide) solution of 4- (N-hydroxyethyl-N- (2-O- (4,4-dimethyl triphenylmethyl) ethyl)) amino-aniline, continuing to react for 12 hours, filtering after the reaction is finished, collecting solid, and crystallizing the solid by using dichloromethane/methanol to obtain a pure product O-DMTr-Jianna green B.
(2) Weighing 10 g of O-DMTr-Jianna green B into a dry flask, adding 130 ml of dichloromethane for dissolving, sequentially adding 1.5 g of glutaric anhydride and 2.8 ml of triethylamine, reacting at room temperature until the reaction is complete, adding 5% NaHCO 3 Quenching the aqueous solution, extracting and layering, collecting an organic phase, and evaporating the solvent under reduced pressure to obtain the Jiannalv B glutarate.
(3) Weighing 7 g of Janus green B glutarate, adding 100 ml of dichloromethane into a dry flask for dissolving, then sequentially adding 1.8 g of EDC and 1 g of p-nitrophenol, reacting at room temperature till completion, adding water for quenching, extracting and layering, collecting an organic phase, evaporating under reduced pressure to remove a solvent, then dissolving a residue into 100 ml of dichloromethane, adding 30 g of aminomethyl polystyrene resin, controlling the temperature to 15 ℃, reacting till completion, filtering, and collecting a solid to obtain the Janus green B solid phase carrier.
Example 5
Method for preparing Janus green B solid phase carrier by taking methylene violet 3RAX as raw material
(1) Weighing 10 g of methylene violet 3RAX, adding 20 ml of 2N hydrochloric acid aqueous solution into a flask, controlling the temperature at 5 ℃, dropwise adding 2.16 g of sodium nitrite aqueous solution, controlling the ratio of sodium nitrite to methylene violet to be 1.5, reacting for 2 hours, then adjusting the pH value to 7 by using sodium hydroxide, adding 12.8 g of DMF (dimethyl formamide) solution of 4- (N-hydroxyethyl-N- (2-O- (4,4-dimethyl triphenylmethyl) ethyl)) amino-aniline, continuing to react for 18 hours, after the reaction is finished, filtering, collecting solid, and crystallizing the solid by using dichloromethane/methanol to obtain a pure product O-DMTr-Jianna green B.
(2) Weighing 7 g of O-DMTr-Jianna green B into a dry flask, adding 130 ml of DMF for dissolving, sequentially adding 1.2 g of succinic anhydride and 2.3 ml of triethylamine, reacting at room temperature till completion, adding 5% NaHCO3 aqueous solution for quenching, extracting for layering, collecting an organic phase, and evaporating under reduced pressure to remove the solvent to obtain Jianna green B succinate.
(3) Weighing 7 g of Janus green B succinate, adding 100 ml of dichloromethane into a dry flask for dissolving, then sequentially adding 1.8 g of DCC and 1 g of p-nitrophenol, reacting at room temperature till the reaction is complete, adding water for quenching, extracting and layering, collecting an organic phase, evaporating under reduced pressure to remove a solvent, then dissolving a residue into 100 ml of dichloromethane, adding 30 g of aminomethyl polystyrene resin, controlling the temperature to be 30 ℃, reacting till the reaction is completed, filtering, and collecting a solid to obtain the Janus green B solid phase carrier.
Example 6
Method for preparing Janus green B solid phase carrier by taking methylene violet 3RAX as raw material
(1) Weighing 10 g of methylene violet 3RAX, adding 20 ml of 1N hydrochloric acid aqueous solution into a flask, controlling the temperature at 15 ℃, dropwise adding 2.16 g of sodium nitrite aqueous solution, controlling the ratio of sodium nitrite to methylene violet to be 1.5, reacting for 3 hours, then adjusting the pH value to 5 by using sodium hydroxide, adding 12.8 g of DMF (dimethyl formamide) solution of 4- (N-hydroxyethyl-N- (2-O- (4,4-dimethyl triphenylmethyl) ethyl)) amino-aniline, continuing to react for 24 hours, after the reaction is finished, filtering, collecting solid, and crystallizing the solid by using dichloromethane/methanol to obtain a pure product O-DMTr-Jianna green B.
(2) Weighing 7 g of O-DMTr-Jianna green B into a dry flask, adding 130 ml of DMF for dissolving, sequentially adding 1.2 g of succinic anhydride and 2.3 ml of triethylamine, reacting at room temperature until the reaction is complete, adding 5% NaHCO 3 Quenching the aqueous solution, extracting and layering, collecting an organic phase, and evaporating the solvent under reduced pressure to obtain the Janus green B succinate.
(3) Weighing 7 g of Janus green B succinate, adding 100 ml of dichloromethane into a dry flask for dissolving, then sequentially adding 1.8 g of DCC and 1 g of p-nitrophenol, reacting at room temperature till the reaction is complete, adding water for quenching, extracting and layering, collecting an organic phase, evaporating the solvent under reduced pressure, then dissolving the residue into 100 ml of dichloromethane, adding 30 g of aminomethyl polystyrene resin, controlling the temperature to be 20 ℃, reacting till the reaction is completed, filtering, and collecting a solid to obtain the Janus green B solid phase carrier.
Although the description is given in terms of embodiments, not every embodiment includes only a single embodiment, and such description is for clarity only, and those skilled in the art will recognize that the embodiments described herein may be combined as a whole to form other embodiments as would be understood by those skilled in the art.
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.
Claims (15)
1. The preparation method of the Janus green B solid phase carrier is characterized by comprising the following steps: (1) Obtaining O-DMTr-Janus green B by methylene violet 3RAX through reaction a; (2) O-DMTr-Janus green B reacting to obtain Janus green B succinate; (3) And reacting c to obtain the Janus green B solid phase carrier.
2. The method of preparing a Janus green B solid support according to claim 1, wherein the reaction a is:
3. the method for preparing the Janus green B solid-phase carrier as claimed in claim 2, wherein in the reaction a, acid water is added into the methylene violet as the raw material, sodium nitrite is added dropwise to react for a period of time, and then 4- (N-hydroxyethyl-N- (2-O- (4,4-dimethyltriphenylmethyl) ethyl)) amino-aniline solution is added to continue the reaction to obtain O-DMTr-Janus green B.
4. The method for preparing the Janus green B solid phase carrier as claimed in claim 3, wherein the ratio of the sodium nitrite to the methylene violet is 0.5-1.5.
5. The method of claim 3, wherein the pH is adjusted to 4-7 before adding the 4- (N-hydroxyethyl-N- (2-O- (4,4-dimethyltriphenylmethyl) ethyl)) amino-aniline solution.
6. The method for preparing Janus green B solid phase carrier as claimed in claim 5, wherein the pH regulator is one or more of sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate and ammonium acetate.
7. The method for preparing Janus green B solid phase carrier as claimed in claim 3, wherein the reaction time after adding sodium nitrite is 1-3 hours, and the reaction time after adding 4- (N-hydroxyethyl-N- (2-O- (4,4-dimethyltriphenylmethyl) ethyl)) amino-aniline solution is 1-24 hours.
8. The method for preparing Janus green B solid phase carrier as claimed in claim 3, wherein the sulfuric acid in reaction a is any one of 1-6N sulfuric acid or 1-6N hydrochloric acid.
9. The method of preparing a Janus green B solid support of claim 1, wherein reaction B is:
10. the method of claim 9, wherein the reaction comprises dissolving O-DMTr-jiannalv B in a first organic solvent, and sequentially adding an acid anhydride and an organic base to the solution to react.
11. The method for preparing Janus green B solid phase carrier of claim 10, wherein the first organic solvent is any one of DMF, dichloromethane, chloroform, ethyl acetate, diethyl ether and acetonitrile, the organic base is any one of triethylamine, pyridine, diisopropyl ether and diisopropyl ethylamine, and the acid anhydride is any one of succinic anhydride, glutaric anhydride and diethanol anhydride.
12. The method of preparing a Janus green B solid support of claim 1, wherein reaction c is:
13. the method for preparing the Janus green B solid phase carrier as defined in claim 12, wherein the reaction process comprises: adding Jiannalv B succinate into a second organic solvent for dissolving, sequentially adding a condensing agent and p-nitrophenol, reacting at room temperature completely, adding water for quenching, extracting to obtain an organic phase, removing the solvent under reduced pressure, dissolving the residue in a third organic solvent, adding a solid phase carrier, and reacting to obtain the Jiannalv B solid phase carrier.
14. The method for preparing Janus green B solid phase carrier of claim 13, wherein the condensing agent is any one of DCC, EDC, HOBt, HBTU, HATu, pyBop.
15. The method for preparing Janus green B solid phase carrier as claimed in claim 13, wherein the solid phase carrier is any one of controllable glass bead powder CPG and aminomethyl polystyrene resin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110453583.3A CN115246866A (en) | 2021-04-26 | 2021-04-26 | Synthetic method of Janus green B solid phase carrier |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110453583.3A CN115246866A (en) | 2021-04-26 | 2021-04-26 | Synthetic method of Janus green B solid phase carrier |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115246866A true CN115246866A (en) | 2022-10-28 |
Family
ID=83697405
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110453583.3A Pending CN115246866A (en) | 2021-04-26 | 2021-04-26 | Synthetic method of Janus green B solid phase carrier |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115246866A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050272088A1 (en) * | 2000-05-09 | 2005-12-08 | Biosearch Technologies, Inc. | Dark quenchers for donor-acceptor energy transfer |
| CN102618064A (en) * | 2012-02-28 | 2012-08-01 | 武汉工程大学 | Synthesizing process of Janus green B |
| CN102617492A (en) * | 2012-02-28 | 2012-08-01 | 武汉工程大学 | Process for synthesizing Janus green B |
-
2021
- 2021-04-26 CN CN202110453583.3A patent/CN115246866A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050272088A1 (en) * | 2000-05-09 | 2005-12-08 | Biosearch Technologies, Inc. | Dark quenchers for donor-acceptor energy transfer |
| CN102618064A (en) * | 2012-02-28 | 2012-08-01 | 武汉工程大学 | Synthesizing process of Janus green B |
| CN102617492A (en) * | 2012-02-28 | 2012-08-01 | 武汉工程大学 | Process for synthesizing Janus green B |
Non-Patent Citations (3)
| Title |
|---|
| KE YANG，等: "DNA Photocleavage and Binding Modes of Methylene Violet 3RAX and its Derivatives: Effect of Functional Groups", 《AUST. J. CHEM.》, vol. 70, no. 7, 20 February 2017 (2017-02-20), pages 830 - 836 * |
| 姚思，等: "健那绿B的合成及其中放流程的设计", 《武汉工程大学学报》, vol. 38, no. 3, 30 June 2016 (2016-06-30), pages 240 - 243 * |
| 梯泽，等: "《当代有机反应和合成操作》", vol. 1, 30 September 2008, 华东理工大学出版社, pages: 571 - 572 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107021984A (en) | A kind of Preparation Method And Their Intermediate of TAF nucleoside derivates | |
| CN109928937B (en) | Water-soluble column [ n ] arene and synthesis method thereof | |
| CN114014848B (en) | RNA fluorescent probe and preparation method and application thereof | |
| CN115246866A (en) | Synthetic method of Janus green B solid phase carrier | |
| CN108251516B (en) | DNA single molecule sequencing method and sequencing system | |
| CN102070635B (en) | Preparation method for ganciclovir valine ester derivative | |
| CN116854749B (en) | Method for synthesizing nucleotide intermediate | |
| CN113234113B (en) | Method for constructing 1, 2-cis-2-nitro-glucoside and galactose glucoside | |
| CN106831826B (en) | A kind of method that eldisine is prepared from catharanthus roseus | |
| CN106636241B (en) | Method for preparing esmollin intermediate by enzyme method | |
| CN108440319B (en) | Method for preparing aminolevulinic acid hydrochloride | |
| CN108117587B (en) | Photosensitive peptide nucleic acid monomer and synthesis method thereof | |
| CN102140124B (en) | Novel synthesis process of capecitabine | |
| CN101870717A (en) | Oligonucleotide direct condensation method reagent and application thereof | |
| US7329515B2 (en) | Solid support for the synthesis of 3′-amino oligonucleotides | |
| CN116283505A (en) | Method for synthesizing phloroglucinol from tri-acetylated triaminobenzoic acid | |
| CN109651224B (en) | Fluorescent protein cross-linking agent and preparation method thereof | |
| CN114349800B (en) | Ferrocene derivative and synthetic method and application thereof | |
| CN115894303B (en) | Preparation method of (3-amino bicyclo [1.1.1] pentane-1-yl) carbamic acid tert-butyl ester and intermediate thereof | |
| CN112368292A (en) | Phosphoramidite compound, preparation method and application thereof | |
| CN111909197B (en) | Preparation method of triphosphate compound and deoxynucleotide | |
| JP7345156B2 (en) | A method for causing a change in 19F chemical shift through double strand formation | |
| WO2020102980A1 (en) | Preparation method for nucleotide for sequencing | |
| CN116284190A (en) | Pseudo-uridine intermediate, and preparation method and application thereof | |
| CN112010852A (en) | Compound for inhibiting PCa cell transfer and application |
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 |