CN115260261B - A method for preparing a fluorescent dye-modified deoxynucleoside solid phase carrier - Google Patents
A method for preparing a fluorescent dye-modified deoxynucleoside solid phase carrier Download PDFInfo
- Publication number
- CN115260261B CN115260261B CN202110479298.9A CN202110479298A CN115260261B CN 115260261 B CN115260261 B CN 115260261B CN 202110479298 A CN202110479298 A CN 202110479298A CN 115260261 B CN115260261 B CN 115260261B
- Authority
- CN
- China
- Prior art keywords
- compound
- organic solvent
- fluorescent dye
- solid phase
- trifluoroacetamide
- 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
Links
- 239000007790 solid phase Substances 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 title claims abstract description 13
- 238000006243 chemical reaction Methods 0.000 claims abstract description 40
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 32
- 239000003960 organic solvent Substances 0.000 claims abstract description 30
- 150000001875 compounds Chemical class 0.000 claims abstract description 27
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 15
- 229940126214 compound 3 Drugs 0.000 claims abstract description 14
- 229940125898 compound 5 Drugs 0.000 claims abstract description 14
- -1 diamine trifluoroacetamide derivative Chemical class 0.000 claims abstract description 14
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 13
- 229940125782 compound 2 Drugs 0.000 claims abstract description 13
- 150000002148 esters Chemical class 0.000 claims abstract description 12
- 239000000126 substance Substances 0.000 claims abstract description 11
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- 150000008064 anhydrides Chemical class 0.000 claims abstract description 7
- 150000007530 organic bases Chemical class 0.000 claims abstract description 6
- JBWYRBLDOOOJEU-UHFFFAOYSA-N 1-[chloro-(4-methoxyphenyl)-phenylmethyl]-4-methoxybenzene Chemical compound C1=CC(OC)=CC=C1C(Cl)(C=1C=CC(OC)=CC=1)C1=CC=CC=C1 JBWYRBLDOOOJEU-UHFFFAOYSA-N 0.000 claims abstract description 5
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 48
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 36
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 24
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 24
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 23
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 16
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 15
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 14
- 239000007787 solid Substances 0.000 claims description 9
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 6
- YWQZUENVQOQEHJ-PIXDULNESA-N (e)-3-[1-[(2r,4s,5r)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-2,4-dioxopyrimidin-5-yl]prop-2-enoic acid Chemical group C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(\C=C\C(O)=O)=C1 YWQZUENVQOQEHJ-PIXDULNESA-N 0.000 claims description 5
- NRKYWOKHZRQRJR-UHFFFAOYSA-N 2,2,2-trifluoroacetamide Chemical compound NC(=O)C(F)(F)F NRKYWOKHZRQRJR-UHFFFAOYSA-N 0.000 claims description 4
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 claims description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-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
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 4
- UAOMVDZJSHZZME-UHFFFAOYSA-N diisopropylamine Substances CC(C)NC(C)C UAOMVDZJSHZZME-UHFFFAOYSA-N 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
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 claims description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 3
- 150000004985 diamines Chemical class 0.000 claims description 3
- 229940014800 succinic anhydride Drugs 0.000 claims description 3
- GSNUFIFRDBKVIE-UHFFFAOYSA-N DMF Natural products CC1=CC=C(C)O1 GSNUFIFRDBKVIE-UHFFFAOYSA-N 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
- 125000004202 aminomethyl group Chemical group [H]N([H])C([H])([H])* 0.000 claims description 2
- VANNPISTIUFMLH-UHFFFAOYSA-N glutaric anhydride Chemical compound O=C1CCCC(=O)O1 VANNPISTIUFMLH-UHFFFAOYSA-N 0.000 claims description 2
- 125000004817 pentamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 claims description 2
- 229920005990 polystyrene resin Polymers 0.000 claims description 2
- 229910000028 potassium bicarbonate Inorganic materials 0.000 claims description 2
- 235000015497 potassium bicarbonate Nutrition 0.000 claims description 2
- 239000011736 potassium bicarbonate Substances 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
- 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
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 2
- ZFFMLCVRJBZUDZ-UHFFFAOYSA-N 2,3-dimethylbutane Chemical group CC(C)C(C)C ZFFMLCVRJBZUDZ-UHFFFAOYSA-N 0.000 claims 1
- 239000011324 bead Substances 0.000 claims 1
- 239000011521 glass Substances 0.000 claims 1
- 239000000843 powder Substances 0.000 claims 1
- 238000003745 diagnosis Methods 0.000 abstract description 5
- 230000006872 improvement Effects 0.000 description 16
- 239000002904 solvent Substances 0.000 description 12
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 238000010791 quenching Methods 0.000 description 9
- 239000012074 organic phase Substances 0.000 description 8
- 230000000171 quenching effect Effects 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 238000004090 dissolution Methods 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000004440 column chromatography Methods 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 238000001704 evaporation Methods 0.000 description 6
- 238000000605 extraction Methods 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 230000008020 evaporation Effects 0.000 description 4
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 229940125904 compound 1 Drugs 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 108020003215 DNA Probes Proteins 0.000 description 2
- 239000003298 DNA probe Substances 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- NAQMVNRVTILPCV-UHFFFAOYSA-N hexane-1,6-diamine Chemical compound NCCCCCCN NAQMVNRVTILPCV-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 150000008065 acid anhydrides Chemical class 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 229940043279 diisopropylamine Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 150000008300 phosphoramidites Chemical class 0.000 description 1
- ZWLUXSQADUDCSB-UHFFFAOYSA-N phthalaldehyde Chemical compound O=CC1=CC=CC=C1C=O ZWLUXSQADUDCSB-UHFFFAOYSA-N 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 239000001022 rhodamine dye Chemical class 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical class [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
- C07H19/073—Pyrimidine radicals with 2-deoxyribosyl as the saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- 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
- C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
- C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
-
- 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
-
- 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
- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
-
- 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
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Materials Engineering (AREA)
- Saccharide Compounds (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention relates to a preparation method of a fluorescent dye modified deoxynucleoside solid phase carrier, which comprises the steps of dissolving deoxynucleoside in a first organic solvent, adding 4,4' -dimethoxy triphenylchloromethane to obtain a compound 2, dissolving the compound 2 in a second organic solvent, adding a first condensing agent and a diamine trifluoroacetamide derivative to obtain a compound 3, dissolving the compound 3 in a methanol solution, adding an alkaline substance to react to obtain a compound 4, dissolving the compound 4 in a third organic solvent, adding fluorescent dye activated ester to obtain a compound 5, dissolving the compound 5 in a fourth organic solvent, sequentially adding anhydride and organic base to obtain a compound 6, then dissolving the compound 6 in a fifth organic solvent, sequentially adding a second condensing agent and a solid phase carrier, and separating the fluorescent dye modified deoxynucleoside solid phase carrier after reaction. The method is simple and convenient, has high yield and realizes the purpose of high-efficiency fluorescent diagnosis.
Description
Technical Field
The invention belongs to the field of biological molecular diagnosis, relates to a method for synthesizing a fluorescent group in a fluorescent probe, and in particular relates to a method for preparing a fluorescent dye modified deoxynucleoside solid phase carrier.
Background
The fluorescent probe uses fluorescent substances as indicators, and the fluorescent substances generate fluorescence under excitation of a certain wavelength, and the detected substances are quantitatively or qualitatively analyzed by detecting the generated fluorescence. Compared with other detection methods, the fluorescent probe has the advantages of high sensitivity, high selectivity, quick response, good repeatability, simple operation, low cost and the like in the aspect of biomedical detection, so that the fluorescent probe is widely applied to the aspects of molecular diagnosis, gene detection, antibody immunoassay and the like.
The fluorescent probe consists of three parts, namely a recognition group, a fluorescent group and a connector, wherein the recognition group determines the selectivity and the specificity of the probe, the fluorescent group determines the sensitivity of the probe, and the connector part plays a role of a connecting hinge in the probe. The fluorescent dye exists in the fluorescent probe in the identity of a fluorescent group, and the photophysical property of the fluorescent dye is an important expression of the performance of the probe. Currently, fluorescent probes for labeling or derivatization mainly include rhodamine, fluorescein, phthalaldehyde and the like, wherein the fluorescein compound occupies an important position in the field of biological research. However, the preparation method of the fluorescent dye modified deoxynucleoside solid phase carrier is still immature.
Disclosure of Invention
The application mainly solves the problem that the preparation method of the fluorescent dye modified deoxynucleoside solid phase carrier is not available at present.
Based on the above problems, the present application proposes the following solutions:
A preparation method of a fluorescent dye modified deoxynucleoside solid phase carrier comprises the following steps:
S1, fully dissolving deoxynucleoside into a first organic solvent, adding 4,4' -dimethoxy triphenylchloromethane, and fully reacting to separate a compound 2;
s2, fully dissolving the compound 2 in the step S1 into a second organic solvent, adding a first condensing agent and a diamine trifluoroacetamide derivative, and fully reacting to separate a compound 3;
s3, fully dissolving the compound 3 in the step S2 into a methanol solution, adding an alkaline substance, and fully reacting to separate a compound 4;
S4, fully dissolving the compound 4 in the step S3 into a third organic solvent, adding fluorescent dye activated ester, and fully reacting to separate a compound 5;
s5, fully dissolving the compound 5 in the step S4 into a fourth organic solvent, sequentially adding anhydride and organic base, and fully reacting to separate a compound 6;
S6, fully dissolving the compound 6 in the step S5 into a fifth organic solvent, sequentially adding a second condensing agent and a solid phase carrier, and separating out the fluorescent dye modified deoxynucleoside solid phase carrier after full reaction.
As a further improvement of the present application, in step S1, the first organic solvent may be, but is not limited to, any one of pyridine, DMF, dichloromethane, ethyl acetate, and tetrahydrofuran.
As a further improvement of the present application, in step S2, the second organic solvent may be, but is not limited to, any one of DMF, dichloromethane, chloroform, ethyl acetate, diethyl ether, acetonitrile.
As a further improvement of the present application, in step S2, the first condensing agent may be, but is not limited to, any one of DCC, EDC, HOBt, HBTu, HATu, pyBop.
As a further improvement of the present application, in step S2, the diamine-based trifluoroacetamide derivative may be any one of ethylenediamine trifluoroacetamide, propylenediamine trifluoroacetamide, butylenediamine trifluoroacetamide, pentylene diamine trifluoroacetamide, hexamethylenediamine trifluoroacetamide, but not limited thereto.
As a further improvement of the present application, in step S3, the alkaline substance may be at least one of, but not limited to, sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, ammonia water, methylamine.
As a further improvement of the present application, in step S4, the third organic solvent may be, but is not limited to, any one of DMF, dichloromethane, chloroform, ethyl acetate, diethyl ether, acetonitrile.
As a further improvement of the present application, in step S4, the fluorescent dye activated ester may be any one of, but not limited to, FAM activated ester, HEX activated ester, TET activated ester, cyanine dye activated ester, rhodamine dye activated ester.
As a further improvement of the application, in step S4, the temperature of the reaction is 60-90 ℃.
As a further improvement of the present application, in step S5, the fourth organic solvent may be, but is not limited to, any one of DMF, dichloromethane, chloroform, ethyl acetate, diethyl ether, acetonitrile.
As a further improvement of the present application, in step S5, the organic base may be, but is not limited to, any one of triethylamine, pyridine, diisopropylamine, diisopropylethylamine.
As a further improvement of the present application, in step S5, the acid anhydride may be any one of, but not limited to, succinic anhydride, glutaric anhydride, and diethanol anhydride.
As a further improvement of the present application, in step S6, the fifth organic solvent may be, but is not limited to, any one of DMF, dichloromethane, chloroform, ethyl acetate, diethyl ether, acetonitrile.
As a further improvement of the present application, in step S6, the second condensing agent may be, but is not limited to, any one of DCC, EDC, HOBt, HBTu, HATu, pyBop.
As a further improvement of the present application, in step S6, the solid support may be, but is not limited to, any one of CPG and aminomethyl polystyrene resin.
As a further improvement of the present application, the deoxynucleotide may be any one of, but not limited to, adenine deoxynucleotide, thymine deoxynucleotide, cytosine deoxynucleotide and guanine deoxynucleotide.
As a further improvement of the present application, the deoxynucleoside may be, but is not limited to, 5- (2-carboxyvinyl) -2' -deoxyuridine.
The application has the beneficial effects that the application provides the preparation method of the fluorescent dye modified deoxynucleoside solid phase carrier, which is simple and convenient, has high yield and realizes the purpose of high-efficiency fluorescent diagnosis.
Drawings
FIG. 1 shows the synthesis reaction process of fluorescent dye modified deoxynucleoside solid phase carrier.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the technical solutions of the present application will be clearly and completely described below with reference to specific embodiments of the present application and the accompanying drawings. It should be apparent that the described embodiments are only some embodiments of the present application and not all embodiments are not intended to limit the scope of the present application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
The application provides a preparation method of a fluorescent dye modified deoxynucleoside solid phase carrier, which comprises the following steps:
s1, fully dissolving deoxynucleoside into a first organic solvent, adding 4,4' -dimethoxy triphenylchloromethane, fully reacting, separating out a compound 2, and preferably, carrying out the reaction at room temperature, wherein the separating steps comprise the steps of carrying out first-step decompression and first-step extraction sequentially after the full reaction, collecting a first organic phase, removing the solvent, purifying residues through the first step to obtain the compound 2, and further, the extracting solution applied in the first-step extraction is a mixed solution of water and dichloromethane, wherein the solvent removing process in the step is reduced pressure evaporation, and the first-step purification process can be, but is not limited to, chromatography, column chromatography and the like.
S2, fully dissolving the compound 2 in the step S1 into a second organic solvent, adding a first condensing agent and a diamine trifluoroacetamide derivative, fully reacting, separating the compound 3, preferably, placing the reaction at room temperature, sequentially performing first-step quenching after the full reaction, performing second-step extraction, collecting a second organic phase, removing the solvent, purifying the residue through the second step to obtain the compound 3, further, using water as a solution for the first-step quenching, using ethyl acetate as an extraction liquid for the second-step extraction, and performing reduced pressure evaporation as a process for removing the solvent in the second-step purification, wherein the process for the second-step purification can be, but is not limited to, chromatography, column chromatography and the like.
S3, fully dissolving the compound 3 in the step S2 into a methanol solution, adding an alkaline substance, fully reacting, and separating the compound 4, wherein the reaction is preferably carried out at room temperature, and the separation step is that the concentration and the drying are carried out after the full reaction.
And S4, fully dissolving the compound 4 in the step S3 into a third organic solvent, adding fluorescent dye activated ester, fully reacting, and separating the compound 5, wherein preferably, the reaction is carried out under a heating condition, the solvent is removed after the full reaction, dichloromethane is added into residues, the residues are washed three times with water, the third organic phase is collected and concentrated, and finally, the compound 5 is obtained after the third purification, and further, the solvent removing process in the step is reduced pressure evaporation, and the third purification process can be, but is not limited to, chromatography, column chromatography and the like.
S5, fully dissolving the compound 5 in the step S4 into a fourth organic solvent, sequentially adding anhydride and organic base, fully reacting, and separating out the compound 6, wherein preferably, the reaction is carried out at room temperature, the separation step comprises the steps of quenching in the second step and extracting in the third step in sequence, collecting the fourth organic phase, and then removing the solvent to obtain the compound 6, and further, the liquid used for the second step quenching is water, and the process for removing the solvent is reduced pressure evaporation.
S6, fully dissolving the compound 6 in the step S5 into a fifth organic solvent, sequentially adding a second condensing agent and a solid phase carrier, fully reacting, and separating out a fluorescent dye modified deoxynucleoside solid phase carrier, wherein the reaction is preferably carried out at room temperature for 10 hours, and the separation step is filtering and collecting solids.
The purpose of quenching the reaction in the above steps is that in the organic chemical reaction, a certain reaction substrate is excessive, and when the chemical reaction proceeds to a certain extent, the target product is already obtained, and the excessive reaction substrate continues to exist, and further reaction is generated to a by-product or the post-treatment operation is affected, so that it is required to quench it. The quenching is carried out by using another compound which is easier to react with the excessive substance, and removing the excessive substance from the system by reducing the reaction rate or destroying the reaction system so as to terminate the reaction, thereby achieving the purposes of reducing the generation of byproducts and ensuring the safety and convenience of post-treatment of experiments.
In the present application, since 5- (2-carboxyvinyl) -2 '-deoxyuridine is one of deoxynucleosides, 5- (2-carboxyvinyl) -2' -deoxyuridine is exemplified as follows:
a preparation method of a fluorescent dye modified deoxynucleoside solid phase carrier, the whole process of the synthesis reaction is shown in figure 1, comprises the following steps:
S1, placing 40g of a compound 1 in a dry flask, wherein the compound 1 is 5- (2-carboxyvinyl) -2 '-deoxyuridine, adding 300ml of pyridine into the flask for dissolution, then adding 45g of 4,4' -dimethoxy triphenylchloromethane, reacting at room temperature until the pyridine is completed, decompressing and distilling off the pyridine, extracting with water and 300ml of dichloromethane, collecting an organic phase, decompressing and distilling off the solvent, purifying a residue by column chromatography to obtain a compound 2, wherein the compound 2 is 52 g of pure white solid, the compound 1 is obtained by a reaction a, and the reaction a is
Nuclear magnetic detection HNMR of Compound 2 is ：1H NMR(DMSO)δ2.16-2.25(m,1H),2.30-2.36(m,1H),3.16(dd,1H),3.24(dd,1H),3.72(dd,6H),3.88-3.92(m,1H),4.21-4.26(m,1H),5.29(d,1H),6.17(dd,1H),6.78-6.87(m,5H),7.17-7.38(m,10H),8.03(s,1H),11.67(s,1H),12.1(w,1H).
S2, placing 52 g of the compound 2 in the step S1 into a dry flask, adding 300ml of DMF for dissolution, sequentially adding a condensing agent 27 g of DCC, 18g of HOBt, 24ml of triethylamine and 22 g of 1, 6-hexamethylenediamine trifluoroacetamide for reaction to completion at room temperature, adding 800 ml of water for quenching, extracting and layering 800 ml of ethyl acetate, collecting an organic phase, decompressing and distilling the solvent, purifying the residue by column chromatography to obtain 50 g of white solid which is the compound 3, and obtaining the compound 3 by the reaction b, wherein the reaction b is
Nuclear magnetic detection HNMR of Compound 3 is ：1H NMR(DMSO)δ1.23-.128(m,4H),1.38-1.48(m,4H),2.14-2.20(m,1H),2.30-2.36(m,1H),2.99-3.03(m,1H),3.10-3.19(m,5H),3.72(dd,6H),3.88-3.92(m,1H),4.21-4.26(m,1H),5.29(d,1H),6.17(t,1H),6.85-6.91(m,4H),7.05-7-4(m,11H),7.94(s,1H),8.01(t,1H),9.39(t,1H),11.60(s,1H).
S3, placing 50 g of the compound 3 in the step S2 into a dry flask, adding 100ml of methanol for dissolution, then adding 100ml of concentrated ammonia water, reacting at room temperature until the reaction is complete, concentrating until the reaction is dry to obtain a compound 4, directly using the product in the next reaction, and obtaining the compound 4 through a reaction c of the compound 3, wherein the reaction c is
S4, placing 44 g of the compound 4 in the step S3 into a flask, adding 400 ml of DMF for dissolution, then adding 40 g of FAM-NHS, heating the reaction to 80 ℃ overnight, evaporating solvent DMF under reduced pressure after the reaction is completed, adding 500 ml of dichloromethane for dissolution, washing the residue with water for 3 times, collecting an organic phase for concentration, purifying the residue by a column chromatography column to obtain 40 g of compound 5, and obtaining compound 5 from the compound 4 through a reaction d, wherein the reaction d is
Nuclear magnetic detection HNMR of Compound 5 is ：1H NMR(DMSO)δ1.23-.128(m,4H),1.32(s,18H),1.38-1.48(m,4H),2.14-2.20(m,1H),2.30-2.36(m,1H),2.99-3.03(m,1H),3.10-3.19(m,5H),3.72(dd,6H),3.88-3.92(m,1H),4.21-4.26(m,1H),5.29(d,1H),6.17(t,1H),6.8(dd,4H),6.85-6.91(m,4H),7.05-7.4(m,14H),7.94(s,1H),8.01(t,1H),8.40(d,1H),8.73(s,1H),9.39(t,1H),11.60(s,1H).
S5, placing 40 g of the compound 5 in the step S4 into a dry flask, adding 400 ml of dichloromethane for dissolution, sequentially adding 4.9 g of succinic anhydride and 9 ml of triethylamine, reacting at room temperature to completion, adding 200 ml of water for quenching, extracting and layering, collecting an organic phase, evaporating the solvent under reduced pressure to obtain a compound 6, directly using the product in the next reaction, and obtaining the compound 6 from the compound 5 through a reaction e, wherein the reaction e is
S6, placing 10 g of the compound 6 in the step S5 into a dry flask, adding dichloromethane for dissolution, then sequentially adding 8 g of DCC,5 g of HOBt and 10 ml of triethylamine, stirring and reacting at room temperature for 10 hours, filtering, collecting solids to obtain the compound 7, namely FAM-dT-CPG, and reacting f to obtain the compound 6:
In summary, the application provides a preparation method of a fluorescent dye modified deoxynucleoside solid phase carrier, which is simple and convenient, has high yield and achieves the purpose of high-efficiency fluorescent diagnosis. And FAM is a common fluorescein dye, and has excitation wavelength of 498nm and emission wavelength of 525nm, which are commonly used in fluorescent probes. FAM may be modified on deoxynucleoside dT to form FAM-dT phosphoramidite monomer, which may be labeled at any site of the probe during synthesis of DNA probe, or may be supported on a solid support to form FAM-dT-solid support, such as FAM-dT-CPG (PS), for 3' modification of DNA probe. The application particularly describes the synthesis method of the solid phase carrier of FAM-dT-CPG (PS).
Although the present disclosure describes embodiments, not every embodiment is described in terms of a single embodiment, and such description is for clarity only, and one skilled in the art will recognize that the embodiments may be combined in any suitable manner to form other embodiments that will be apparent to those skilled in the art.
The above list of detailed descriptions is only specific to practical embodiments of the present invention, and they are not intended to limit the scope of the present invention, and all equivalent embodiments or modifications that do not depart from the spirit of the present invention should be included in the scope of the present invention.
Claims (3)
1. The preparation method of the fluorescent dye modified deoxynucleoside solid phase carrier is characterized by comprising the following steps:
S1, fully dissolving deoxynucleoside into a first organic solvent, adding 4,4 '-dimethoxy triphenylchloromethane, and fully reacting to separate a compound 2, wherein the deoxynucleoside is 5- (2-carboxyvinyl) -2' -deoxyuridine, and the first organic solvent is any one of pyridine, DMF, dichloromethane, ethyl acetate and tetrahydrofuran;
S2, fully dissolving the compound 2 in the step S1 into a second organic solvent, adding a first condensing agent and a diamine trifluoroacetamide derivative, and fully reacting to separate a compound 3, wherein the second organic solvent is any one of DMF, dichloromethane, chloroform, ethyl acetate, diethyl ether and acetonitrile, and the first condensing agent is any one of DCC, EDC, HOBt, HBTu, HATu, pyBop;
s3, fully dissolving the compound 3 in the step S2 into a methanol solution, adding an alkaline substance, and fully reacting to separate a compound 4;
S4, fully dissolving the compound 4 in the step S3 into a third organic solvent, adding fluorescent dye activated ester, and fully reacting at the temperature of 60-90 ℃ to separate out a compound 5, wherein the fluorescent dye activated ester is FAM activated ester, and the third organic solvent is any one of DMF, dichloromethane, chloroform, ethyl acetate, diethyl ether and acetonitrile;
S5, fully dissolving the compound 5 in the step S4 into a fourth organic solvent, sequentially adding anhydride and organic base, and fully reacting to separate a compound 6, wherein the anhydride is any one of succinic anhydride, glutaric anhydride and diethanol anhydride, the organic base is any one of triethylamine, pyridine, diisopropyl and diisopropylethylamine, and the fourth organic solvent is any one of DMF, dichloromethane, chloroform, ethyl acetate, diethyl ether and acetonitrile;
S6, fully dissolving the compound 6 in the step S5 into a fifth organic solvent, sequentially adding a second condensing agent and a solid phase carrier, and separating out a fluorescent dye modified deoxynucleoside solid phase carrier after full reaction, wherein the second condensing agent is any one of DCC, EDC, HOBt, HBTu, HATu, pyBop, the solid phase carrier is any one of controllable glass bead powder CPG and aminomethyl polystyrene resin, and the fifth organic solvent is any one of DMF, dichloromethane, chloroform, ethyl acetate, diethyl ether and acetonitrile.
2. The method for preparing a fluorescent dye-modified deoxynucleoside solid carrier as claimed in claim 1, wherein in the step S2, the diamine trifluoroacetamide derivative is any one of ethylenediamine trifluoroacetamide, propylenediamine trifluoroacetamide, butylenediamine trifluoroacetamide, pentylene diamine trifluoroacetamide and hexylenediamine trifluoroacetamide.
3. The method for preparing a fluorescent dye-modified deoxynucleoside solid carrier as claimed in claim 1, wherein in the step S3, the alkaline substance is at least one of sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, ammonia water and methylamine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110479298.9A CN115260261B (en) | 2021-04-30 | 2021-04-30 | A method for preparing a fluorescent dye-modified deoxynucleoside solid phase carrier |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110479298.9A CN115260261B (en) | 2021-04-30 | 2021-04-30 | A method for preparing a fluorescent dye-modified deoxynucleoside solid phase carrier |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115260261A CN115260261A (en) | 2022-11-01 |
| CN115260261B true CN115260261B (en) | 2024-12-13 |
Family
ID=83745888
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110479298.9A Active CN115260261B (en) | 2021-04-30 | 2021-04-30 | A method for preparing a fluorescent dye-modified deoxynucleoside solid phase carrier |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115260261B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2025074178A1 (en) * | 2023-10-03 | 2025-04-10 | Sony Group Corporation | Methods for preparing polymeric dyes with drug conjugates |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102219819A (en) * | 2011-04-19 | 2011-10-19 | 李辉 | Nucleotide monomer for improving Tm value of oligonucleotide chain and its preparation method and application |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6864059B2 (en) * | 1996-01-23 | 2005-03-08 | Affymetrix, Inc. | Biotin containing C-glycoside nucleic acid labeling compounds |
| JP2013039060A (en) * | 2011-08-12 | 2013-02-28 | Saitama Univ | Linker for evolving protein with enzyme-like activity, and method for screening such protein using the linker |
| US20130066063A1 (en) * | 2011-09-09 | 2013-03-14 | John Cooke Hodges | Bicyclo[6.1.0]non-4-yne regents for chemical modification of oligonucleotides |
| JP5598784B2 (en) * | 2012-08-30 | 2014-10-01 | 株式会社ダナフォーム | Target nucleic acid analysis method, kit and analytical instrument |
| CN105348343B (en) * | 2015-11-25 | 2019-01-04 | 北京大学 | The deoxynucleoside phosphoramidite monomeric compound and its preparation method and application of chromophore modification |
| WO2018052063A1 (en) * | 2016-09-15 | 2018-03-22 | Ｎｅｃソリューションイノベータ株式会社 | Nucleoside derivative or salt thereof, reagent for synthesizing polynucleotide, method for producing polynucleotide, polynucleotide, and method for producing binding nucleic acid molecule |
| EP3514237B1 (en) * | 2016-09-15 | 2021-07-14 | NEC Solution Innovators, Ltd. | Secretory immunoglobulin a (siga)-binding nucleic acid molecule, sensor for siga analysis, and analysis method for siga |
| CN111018930A (en) * | 2019-12-06 | 2020-04-17 | 深圳大学 | TPE-2'-deoxynucleoside, fluorescent gel and preparation method and application thereof |
-
2021
- 2021-04-30 CN CN202110479298.9A patent/CN115260261B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102219819A (en) * | 2011-04-19 | 2011-10-19 | 李辉 | Nucleotide monomer for improving Tm value of oligonucleotide chain and its preparation method and application |
Non-Patent Citations (1)
| Title |
|---|
| Development of Photochemical DNA/RNA Manipulation Toward Its Application for Nanotechnology;Kenzo Fujimoto，等;《ransactions of the Materials R esearch Society of Japan》;20101231;第35卷(第1期);第85-89页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115260261A (en) | 2022-11-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114634497B (en) | Cysteine/homocysteine response AIE fluorescent probe and preparation method and application thereof | |
| CN115260261B (en) | A method for preparing a fluorescent dye-modified deoxynucleoside solid phase carrier | |
| CN115504897A (en) | Synthetic method of methotrexate intermediate | |
| CN108251516A (en) | DNA single-molecule sequencings method and sequencing system | |
| CN104003902B (en) | Triazenes linkage unit synthetic and the purposes in DNA sequencing thereof | |
| CN108191880B (en) | A kind of preparation method and application of fluorescent dye with carbazole-rhodamine hybrid structure | |
| CN117567286B (en) | A fluorescent probe that specifically recognizes chiral threonine, its preparation and application | |
| CN115260235A (en) | Preparation method of fluorescent dye phosphoramidite monomer | |
| CN109721621B (en) | A kind of preparation method of sialic acid recognition conductive polymer precursor | |
| CN116239493B (en) | Synthesis method of Linker compound | |
| CN105295896A (en) | Specifically marked capsaicin fluorescent probe and synthetic method and application thereof | |
| CN112390839B (en) | Triazene four-color fluorescence reversible termination nucleotide sequencing reagent, DNA single-molecule sequencing system and sequencing method | |
| WO2004029578A2 (en) | Energy transfer dyes, terminators and use thereof | |
| CN115197237B (en) | Method for purifying fluorescein isomer | |
| CN109929003A (en) | The tetraphenyl ethylene compound of the glycosyl units containing sialic acid, preparation method and application | |
| CN100460416C (en) | Method for preparing adenosine | |
| CN118812543B (en) | Fluorescent probe for detecting bisulfide ions and preparation method and application thereof | |
| WO2020102980A1 (en) | Preparation method for nucleotide for sequencing | |
| CN1940563A (en) | Fluorescent marking reagent | |
| CN116606336B (en) | 2′-O-substituted nucleosides with high stability and affinity and their applications | |
| CN120987964A (en) | A method for preparing a photocrosslinking agent compound | |
| CN111205210A (en) | Synthetic method of 2-methyl pyrroline | |
| CN115925656B (en) | A method for industrial synthesis of 5-HMF and its derivatives | |
| CN118754902B (en) | A fluorescent probe based on silicon-substituted rhodamine derivatives and its preparation method and application | |
| CN117417268A (en) | Preparation method of 4-cyano-2-methoxybenzaldehyde |
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 |