CN112778380A - Method for synthesizing On-DNA azide - Google Patents
Method for synthesizing On-DNA azide Download PDFInfo
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- CN112778380A CN112778380A CN202011200333.0A CN202011200333A CN112778380A CN 112778380 A CN112778380 A CN 112778380A CN 202011200333 A CN202011200333 A CN 202011200333A CN 112778380 A CN112778380 A CN 112778380A
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- 150000001540 azides Chemical class 0.000 title claims abstract description 68
- 238000000034 method Methods 0.000 title claims abstract description 29
- 230000002194 synthesizing effect Effects 0.000 title claims description 6
- 150000001875 compounds Chemical class 0.000 claims abstract description 62
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 44
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 44
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 31
- -1 amino compound Chemical class 0.000 claims abstract description 27
- 238000005580 one pot reaction Methods 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 335
- 238000006243 chemical reaction Methods 0.000 claims description 136
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 117
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 44
- 239000000872 buffer Substances 0.000 claims description 41
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 40
- 125000000217 alkyl group Chemical group 0.000 claims description 15
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 125000003118 aryl group Chemical group 0.000 claims description 12
- 229910052802 copper Inorganic materials 0.000 claims description 12
- 239000010949 copper Substances 0.000 claims description 12
- 239000000126 substance Substances 0.000 claims description 12
- 125000001424 substituent group Chemical group 0.000 claims description 12
- 125000006618 5- to 10-membered aromatic heterocyclic group Chemical group 0.000 claims description 9
- 229910052736 halogen Inorganic materials 0.000 claims description 8
- 150000002367 halogens Chemical class 0.000 claims description 8
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 8
- 125000003545 alkoxy group Chemical group 0.000 claims description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 125000000547 substituted alkyl group Chemical group 0.000 claims description 6
- 239000007810 chemical reaction solvent Substances 0.000 claims description 5
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 5
- 125000000623 heterocyclic group Chemical group 0.000 claims description 5
- 125000004122 cyclic group Chemical group 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 claims description 4
- QPLDLSVMHZLSFG-UHFFFAOYSA-N Copper oxide Chemical compound [Cu]=O QPLDLSVMHZLSFG-UHFFFAOYSA-N 0.000 claims description 3
- 229910021595 Copper(I) iodide Inorganic materials 0.000 claims description 3
- 125000005037 alkyl phenyl group Chemical group 0.000 claims description 3
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims description 3
- LSXDOTMGLUJQCM-UHFFFAOYSA-M copper(i) iodide Chemical compound I[Cu] LSXDOTMGLUJQCM-UHFFFAOYSA-M 0.000 claims description 3
- OPQARKPSCNTWTJ-UHFFFAOYSA-L copper(ii) acetate Chemical compound [Cu+2].CC([O-])=O.CC([O-])=O OPQARKPSCNTWTJ-UHFFFAOYSA-L 0.000 claims description 3
- 125000005842 heteroatom Chemical group 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- 230000035484 reaction time Effects 0.000 claims description 3
- 229910021589 Copper(I) bromide Inorganic materials 0.000 claims description 2
- 229910021591 Copper(I) chloride Inorganic materials 0.000 claims description 2
- 125000003342 alkenyl group Chemical group 0.000 claims description 2
- 125000004390 alkyl sulfonyl group Chemical group 0.000 claims description 2
- CREXVNNSNOKDHW-UHFFFAOYSA-N azaniumylideneazanide Chemical group N[N] CREXVNNSNOKDHW-UHFFFAOYSA-N 0.000 claims description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 claims description 2
- BERDEBHAJNAUOM-UHFFFAOYSA-N copper(I) oxide Inorganic materials [Cu]O[Cu] BERDEBHAJNAUOM-UHFFFAOYSA-N 0.000 claims description 2
- AQMRBJNRFUQADD-UHFFFAOYSA-N copper(I) sulfide Chemical compound [S-2].[Cu+].[Cu+] AQMRBJNRFUQADD-UHFFFAOYSA-N 0.000 claims description 2
- OMZSGWSJDCOLKM-UHFFFAOYSA-N copper(II) sulfide Chemical compound [S-2].[Cu+2] OMZSGWSJDCOLKM-UHFFFAOYSA-N 0.000 claims description 2
- NKNDPYCGAZPOFS-UHFFFAOYSA-M copper(i) bromide Chemical compound Br[Cu] NKNDPYCGAZPOFS-UHFFFAOYSA-M 0.000 claims description 2
- 229940045803 cuprous chloride Drugs 0.000 claims description 2
- KRFJLUBVMFXRPN-UHFFFAOYSA-N cuprous oxide Chemical compound [O-2].[Cu+].[Cu+] KRFJLUBVMFXRPN-UHFFFAOYSA-N 0.000 claims description 2
- 229940112669 cuprous oxide Drugs 0.000 claims description 2
- 125000005059 halophenyl group Chemical group 0.000 claims description 2
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 2
- 239000012046 mixed solvent Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000000178 monomer Substances 0.000 claims description 2
- 150000007524 organic acids Chemical class 0.000 claims description 2
- 150000007530 organic bases Chemical class 0.000 claims description 2
- 238000006467 substitution reaction Methods 0.000 claims description 2
- NQPHMXWPDCSHTE-UHFFFAOYSA-N trifluoromethanesulfonyl azide Chemical compound FC(F)(F)S(=O)(=O)N=[N+]=[N-] NQPHMXWPDCSHTE-UHFFFAOYSA-N 0.000 claims description 2
- 101150054335 DNA-R gene Proteins 0.000 claims 2
- QTMDXZNDVAMKGV-UHFFFAOYSA-L copper(ii) bromide Chemical compound [Cu+2].[Br-].[Br-] QTMDXZNDVAMKGV-UHFFFAOYSA-L 0.000 claims 2
- 125000003837 (C1-C20) alkyl group Chemical group 0.000 claims 1
- 125000006552 (C3-C8) cycloalkyl group Chemical group 0.000 claims 1
- 229910021590 Copper(II) bromide Inorganic materials 0.000 claims 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 claims 1
- 229940076286 cupric acetate Drugs 0.000 claims 1
- 229960003280 cupric chloride Drugs 0.000 claims 1
- 229960004643 cupric oxide Drugs 0.000 claims 1
- WQPDQJCBHQPNCZ-UHFFFAOYSA-N cyclohexa-2,4-dien-1-one Chemical compound O=C1CC=CC=C1 WQPDQJCBHQPNCZ-UHFFFAOYSA-N 0.000 claims 1
- QEYREOIRYJXJGY-UHFFFAOYSA-N n-diazo-1h-imidazole-2-sulfonamide Chemical group [N-]=[N+]=NS(=O)(=O)C1=NC=CN1 QEYREOIRYJXJGY-UHFFFAOYSA-N 0.000 claims 1
- 238000006116 polymerization reaction Methods 0.000 claims 1
- 238000010276 construction Methods 0.000 abstract description 3
- 238000011065 in-situ storage Methods 0.000 abstract description 3
- 239000008367 deionised water Substances 0.000 description 114
- 229910021641 deionized water Inorganic materials 0.000 description 114
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 76
- 229910021538 borax Inorganic materials 0.000 description 75
- 235000010339 sodium tetraborate Nutrition 0.000 description 75
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 description 75
- 108020004414 DNA Proteins 0.000 description 66
- 229910000365 copper sulfate Inorganic materials 0.000 description 39
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 38
- 150000001412 amines Chemical class 0.000 description 38
- 235000011089 carbon dioxide Nutrition 0.000 description 38
- 238000012869 ethanol precipitation Methods 0.000 description 38
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 38
- FKVFDMXDAIOOGY-UHFFFAOYSA-N n-diazo-1h-imidazole-2-sulfonamide;hydrochloride Chemical compound Cl.[N-]=[N+]=NS(=O)(=O)C1=NC=CN1 FKVFDMXDAIOOGY-UHFFFAOYSA-N 0.000 description 38
- 239000011780 sodium chloride Substances 0.000 description 38
- 239000006228 supernatant Substances 0.000 description 38
- 230000008014 freezing Effects 0.000 description 37
- 238000007710 freezing Methods 0.000 description 37
- 239000002244 precipitate Substances 0.000 description 37
- 208000012839 conversion disease Diseases 0.000 description 35
- 125000004432 carbon atom Chemical group C* 0.000 description 5
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 150000002611 lead compounds Chemical class 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 125000003277 amino group Chemical group 0.000 description 3
- 229940125904 compound 1 Drugs 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 2
- 125000004429 atom Chemical group 0.000 description 2
- 230000001588 bifunctional effect Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 238000009509 drug development Methods 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 238000013537 high throughput screening Methods 0.000 description 2
- 125000001183 hydrocarbyl group Chemical group 0.000 description 2
- 238000012917 library technology Methods 0.000 description 2
- 125000005647 linker group Chemical group 0.000 description 2
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 150000003254 radicals Chemical class 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical group CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- UHBGYHOPZPPYTP-UHFFFAOYSA-N 1H-imidazole sulfuryl diazide Chemical group S(=O)(=O)(N=[N+]=[N-])N=[N+]=[N-].N1C=NC=C1 UHBGYHOPZPPYTP-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 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
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 239000005751 Copper oxide Substances 0.000 description 1
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 125000003282 alkyl amino group Chemical group 0.000 description 1
- 238000013459 approach Methods 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
- ODWXUNBKCRECNW-UHFFFAOYSA-M bromocopper(1+) Chemical compound Br[Cu+] ODWXUNBKCRECNW-UHFFFAOYSA-M 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 238000006757 chemical reactions by type Methods 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229940125782 compound 2 Drugs 0.000 description 1
- 229940126214 compound 3 Drugs 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 229910000431 copper oxide Inorganic materials 0.000 description 1
- 125000000753 cycloalkyl group Chemical group 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 125000006357 methylene carbonyl group Chemical group [H]C([H])([*:1])C([*:2])=O 0.000 description 1
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- BAVYZALUXZFZLV-UHFFFAOYSA-N mono-methylamine Natural products NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 description 1
- 125000002950 monocyclic group Chemical group 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- 125000003367 polycyclic group Chemical group 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000006268 reductive amination reaction Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
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- 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
- 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
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/08—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
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- Chemical Kinetics & Catalysis (AREA)
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Abstract
The invention relates to a method for converting an On-DNA amino compound into an On-DNA azide in situ through one-step reaction in the construction of a DNA coding compound library. The method has the advantages of few types of added reagents, high yield, single product and simple post-treatment, can introduce azide as a synthesis module in the synthesis of the DNA coding compound library on a large scale, and is suitable for the synthesis operation of the DNA coding compound library by a multi-hole plate.
Description
Technical Field
The invention belongs to the technical field of coding compound libraries, and particularly relates to a method for obtaining an On-DNA azide from an On-DNA amino compound in the presence of a copper reagent and an azide reagent.
Background
In drug development, especially new drug development, high-throughput screening for biological targets is one of the main means for rapidly obtaining lead compounds. However, traditional high throughput screening based on single molecules requires long time, large equipment investment, limited number of library compounds (millions), and the building of compound libraries requires decades of accumulation, limiting the efficiency and possibility of discovery of lead compounds. The recent DNA-encoded compound library technologies (WO2005058479, WO2018166532, CN103882532) combine the technologies of combinatorial chemistry and molecular biology, add a DNA tag to each compound on the molecular level, and synthesize up to hundred million levels of compound libraries in a very short time, which is a trend of the next generation compound library screening technology, and begin to be widely applied in the pharmaceutical industry, resulting in many positive effects (Accounts of Chemical Research,2014,47, 1247-.
The DNA coding compound library can rapidly generate a giant compound library through combinatorial chemistry, and can screen out a lead compound with high flux, so that the screening of the lead compound becomes unprecedented rapidness and high efficiency. One of the challenges in constructing libraries of DNA-encoding compounds is the need to synthesize chemically diverse small molecules on DNA in high yields. Because DNA can be kept stable under certain conditions (solvent, pH, temperature and ion concentration), the On-DNA reaction applied to the construction of the DNA coding compound library also needs higher yield. Therefore, the reagent type, reaction type and reaction condition of the chemical reaction (On-DNA reaction for short) carried out On DNA directly influence the richness and selectivity of the DNA coding compound library. Therefore, the development of chemical reactions compatible with DNA is also a long-term research and research direction of the current DNA coding compound library technology, and the application and commercial value of the DNA coding compound library are directly influenced.
On-DNA azide is an important intermediate, and at present, two main approaches for synthesizing the On-DNA azide are as follows: firstly, the On-DNA azide is prepared by chemical reactions such as condensation, reductive amination and the like of a DNA compound and a bifunctional reagent containing azide, but the bifunctional reagent is less; secondly, starting from a DNA aryl halogenated compound, reacting with an azide reagent to generate an On-DNA aryl azide, wherein the range of a reaction substrate is limited to aryl; both reactions greatly limit the types of On-DNA azides that can be obtained.
It is therefore desirable to develop a new method for the in situ conversion of On-DNA amino compounds to On-DNA azides and to adapt to large-scale multi-well plate procedures for the construction of DNA coding compound libraries.
Disclosure of Invention
The invention discloses a method for synthesizing an On-DNA azide, which comprises the steps of taking an On-DNA amino compound as a raw material, and carrying out one-step reaction in a reaction solvent in the presence of a copper reagent and an azide reagent to obtain the On-DNA azide; the structural formula of the On-DNA amino compound is DNA-R1-NH2The structural formula of the On-DNA azide is DNA-R1-N3；
The DNA in the structural formula comprises a single-stranded or double-stranded nucleotide chain obtained by polymerizing an artificially modified and/or unmodified nucleotide monomer, the nucleotide chain is connected with the rest part of the compound through one or more chemical bonds or groups, and the length of the DNA is 10-200 bp.
R1Selected from groups having a molecular weight below 1000 that are directly attached to the DNA and amino nitrogen atoms or are absent.
Wherein, the DNA and R in the structural formula1Is connected by a chemical bond or a plurality of chemical bonds, and when a chemical bond is connected, the DNA and the R in the structural formula1Directly connecting; when multiple chemical bonds are present, they refer to DNA and R in the structural formula1Are connected with a plurality of chemical bonds at intervals, for example, DNA and R1Through a methylene group (-CH)2-) are connected, i.e. byTwo chemical bonds are connected; or DNA and R1The amino group of the DNA is connected with the amino group of the DNA through a carbonyl (-CO-) and is also connected through two chemical bonds; or DNA and R1Through a methylene carbonyl group (-CH)2CO-) is attached to the amino group of the DNA, again by three consecutive chemical bonds.
Said R1Selected from alkyl, substituted alkyl, 5-10 membered aryl, substituted 5-10 membered aryl, 5-10 membered aromatic heterocyclic group, substituted 5-10 membered aromatic heterocyclic group, 5-10 membered heterocyclic group; wherein:
the alkyl group is C1～C20Alkyl or C3～C8A cycloalkyl group; the number of substituents of the substituted alkyl group is one or more; the substituent of the substituted alkyl is one or more of halogen, carboxyl, nitro, amino, guanidyl, (methylamine acyl) amino, alkoxy, halogenated phenyl, phenyl substituted by cyano, alkyl phenyl, phenyl substituted by hydroxyl, 5-10-membered heterocyclic group, 5-10-membered aromatic heterocyclic group, alkenyl and alkylsulfonyl which are mutually independent;
the number of the substituent for substituting the 5-to 10-membered aryl is one or more, and the substituents for substituting the 5-to 10-membered aryl are independently selected from halogen, cyano, nitro, carboxyl, alkoxy and C1～C20One or more of alkyl, trifluoromethyl and phenyl;
the number of the substituents for substituting the 5-to 10-membered aromatic heterocyclic group is one or more; the substituents for substituting the 5-to 10-membered aromatic heterocyclic group are independently selected from halogen, cyano, nitro, carboxyl, alkoxy and C1～C20One or more of alkyl and trifluoromethyl.
Preferably, the method comprises the following steps: On-DNA amino compounds are specifically selected from: DNA-NH2、
A method for synthesizing an On-DNA azide, comprising the reaction steps of: adding 1-100 times molar equivalent of copper reagent and 1-100 times molar equivalent of azide reagent into an On-DNA amino compound solution with molar equivalent of 1 and molar concentration of 0.1-5mM, and reacting for 0.5-24 hours at 20-100 ℃.
Further, the copper reagent is one or a mixture of more of copper powder, copper oxide, cuprous oxide, copper sulfide, cuprous sulfide, copper sulfate, copper acetate, copper chloride, copper bromide, cuprous chloride, cuprous bromide and cuprous iodide; preferably, the copper reagent is copper sulfate.
Further, the azide reagent is an imidazole sulfonyl azide or a salt thereof, a trifluoromethanesulfonyl azide or a salt thereof.
Further, the molar equivalent of the On-DNA amino compound is 1, the molar equivalent of the azide reagent is 1-100, and the molar equivalent of the copper reagent is 1-100; preferably, the azide reagent is present in a molar equivalent of 5 equivalents, 10 equivalents, 20 equivalents, 24 equivalents, 30 equivalents, 40 equivalents, or 50 equivalents; preferably, the copper reagent molar equivalent is 1 equivalent, 2 equivalents, 3 equivalents, 4 equivalents, 5 equivalents, 6 equivalents, 10 equivalents, 20 equivalents, or 40 equivalents.
Further, the reaction solvent is a water-containing mixed solvent of any one or more of water, methanol, ethanol, acetonitrile, an inorganic salt buffer solution, an organic acid buffer solution and an organic base buffer solution; preferably, the reaction solvent contains a sodium bicarbonate buffer or a sodium borate buffer.
Further, the reaction temperature of the reaction is 20-100 ℃; preferably, the reaction temperature is 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 80 ℃ or 100 ℃.
Further, the reaction time of the reaction is 0.5-24 hours; preferably, the reaction time is 0.5 hours, 1 hour, 2 hours, 4 hours or 20 hours.
Furthermore, the feeding sequence of the reaction is that the On-DNA amino compound is added firstly, and then the copper reagent and the azide reagent are added in sequence.
Further, the above method is used for batch multi-well plate operations.
Further, the above method is used for the synthesis of libraries of DNA-encoding compounds for multi-well plates.
The method can realize one-step reaction to convert the On-DNA amino compound into the On-DNA azide in situ. The method has the advantages of few types of added reagents, high yield, single product and simple post-treatment, can introduce azide as a synthesis module in a large scale, is suitable for On-DNA aliphatic amino compounds and aromatic amino compounds, and is suitable for large-scale multi-plate operation.
Definitions of terms used in connection with the present invention: the initial definitions provided herein for a group or term apply to that group or term throughout the specification unless otherwise indicated; for terms not specifically defined herein, the meanings that would be given to them by a person skilled in the art are to be given in light of the disclosure and the context.
"substituted" means that a hydrogen atom in a molecule is replaced by a different atom or molecule.
The minimum and maximum values of the carbon atom content in the hydrocarbon group are indicated by a prefix, e.g. prefix (Ca-C)b) Alkyl means any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, C1～C20The alkyl group is a straight-chain or branched alkyl group having 1 to 20 carbon atoms.
Alkyl means a straight or branched hydrocarbon radical in an alkane molecule, e.g. methyl-CH3ethyl-CH2CH3methylene-CH2-; the alkyl group may also be part of another group, such as C1～C6Alkoxy radical, C1～C6An alkylamino group.
Cycloalkyl groups: refers to saturated or partially saturated cyclic groups having multiple carbon atoms and no ring heteroatoms and having a single ring or multiple rings (including fused, bridged, and spiro ring systems).
The halogen is fluorine, chlorine, bromine or iodine.
Alkoxy radicalBy radical is meant an alkyl radical linked to the oxygen atom to form a substituent, e.g. methoxy being-OCH3。
The halophenyl group means a group in which H on a phenyl group is substituted with halogen.
Alkylphenyl refers to a group formed by substituting H on a phenyl group with an alkyl group.
Aryl means an aromatic monocyclic or multicyclic group consisting of C atoms, free of heteroatoms.
The term "arylheterocyclyl" refers to a single cyclic group or a plurality of cyclic groups having aromaticity composed of atoms such as C, O, S, N.
The heterocyclic group is a monocyclic or polycyclic hydrocarbon group having at least one saturated or unsaturated nonaromatic property selected from O, S, N.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Description of the drawings:
FIG. 1 is the LC-Ms and Ms spectra of Compound1 of example 1.
Detailed Description
The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.
DNA-NH in the present invention2Is formed by single-stranded or double-stranded DNA and a linker group and has-NH2DNA constructs for linkers, e.g. DNA-NH of "compound 1" in WO20050584792And (5) structure. Also for example the following DNA structure:
wherein A is adenine, T is thymine, C is cytosine, and G is guanine.
Example 1 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in a methanol buffer solution (sodium bicarbonate, 50mM) of sodium bicarbonate buffer solution to prepare a 0.25mM concentration solution (40. mu.L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution) and imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM methanol: deionized water solution at a volume ratio of 1: 1) were sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding anhydrous ethanol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out a supernatant, dissolving the rest precipitate with deionized water to obtain a compound1, and determining the reaction conversion rate to be 99% by LCMS. The theoretical molecular weight is 5210.0, and the actual molecular weight is 5209.6.
Example 2 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding anhydrous ethanol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out a supernatant, dissolving the rest precipitate with deionized water to obtain a compound 2, and determining the reaction conversion rate to be 99% by LCMS. The theoretical molecular weight is 5210.0, and the actual molecular weight is 5209.6.
Example 3 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 20 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding anhydrous ethanol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out a supernatant, dissolving the rest precipitate with deionized water to obtain a compound 3, and determining the reaction conversion rate to be 99% by LCMS. Theoretical molecular weight: 5210.0, molecular weight observed: 52109.6.
example 4 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 40 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 99%. The theoretical molecular weight is 5210.0, and the actual molecular weight is 5209.6.
Example 5 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 80 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 99%. The theoretical molecular weight is 5210.0, and the actual molecular weight is 5209.6.
Example 6 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) were sequentially added to the solution, mixed well, and then the reaction solution was left to react at 100 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 99%. The theoretical molecular weight is 5210.0, and the actual molecular weight is 5209.6.
Example 7 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 1 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 99%. The theoretical molecular weight is 5210.0, and the actual molecular weight is 5209.6.
Example 8 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 2 hours.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 99%. The theoretical molecular weight is 5210.0, and the actual molecular weight is 5209.6.
Example 9 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 4 hours.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 99%. The theoretical molecular weight is 5210.0, and the actual molecular weight is 5209.6.
Example 10 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 20 hours.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 99%. The theoretical molecular weight is 5210.0, and the actual molecular weight is 5209.6.
Example 11 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), cuprous iodide (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) were sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 90%. Theoretical molecular weight is 5210.0, actualThe molecular weight is 5209.6.
Example 12 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (50nmol, 5 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 99%. The theoretical molecular weight is 5210.0, and the actual molecular weight is 5209.6.
Example 13 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (100nmol, 10 equivalents, 120mM deionized water solution) were sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 10% sodium chloride solution of 5M into the solution, adding anhydrous ethanol 3 times of the total volume, shaking, freezing in dry ice for 2 hr, and rotating at 12000rpmCentrifuging for half an hour, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 99%. The theoretical molecular weight is 5210.0, and the actual molecular weight is 5209.6.
Example 14 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (400nmol, 40 equivalents, 120mM deionized water solution) were sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 99%. The theoretical molecular weight is 5210.0, and the actual molecular weight is 5209.6.
Example 15 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding to the solutionAdding 10% of 5M sodium chloride solution, adding 3 times of anhydrous ethanol, uniformly oscillating, freezing the reaction in dry ice for 2 hours, centrifuging at 12000rpm for half an hour, pouring out the supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 99%. The theoretical molecular weight is 5210.0, and the actual molecular weight is 5209.6.
Example 16 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 75%. The theoretical molecular weight is 5399.0, and the actual molecular weight is 5397.1.
Example 17 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed 84% conversion. The theoretical molecular weight is 5375.0, and the actual molecular weight is 5374.0.
Example 18 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 71%. The theoretical molecular weight is 5416.0, and the actual molecular weight is 5415.0.
Example 19 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 67%. The theoretical molecular weight is 5452.0, and the actual molecular weight is 5450.0.
Example 20 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 74%. The theoretical molecular weight is 5437.0, and the actual molecular weight is 5435.5.
Example 21 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 99%. The theoretical molecular weight is 5335.0, and the actual molecular weight is 5334.5.
Example 22 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 81%. The theoretical molecular weight is 5402.0, and the actual molecular weight is 5401.3.
Example 23 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 73%. The theoretical molecular weight is 5397.0, and the actual molecular weight is 5397.0.
Example 24 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 10% sodium chloride solution 5M, adding anhydrous ethanol 3 times of the total volume, shaking, freezing in dry ice for 2 hr, centrifuging at 12000rpm for half an hour, removing supernatant, and precipitatingDissolving the starch with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 68%. The theoretical molecular weight is 5412.0, and the actual molecular weight is 5411.2.
Example 25 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 51%. The theoretical molecular weight is 5460.0, and the actual molecular weight is 5458.6.
Example 26 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 10% of the total volume of 5M sodium chloride solution to the solution, and continuingAdding absolute ethyl alcohol with the volume 3 times of the total volume, after uniform oscillation, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out the supernatant, and dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 33%. The theoretical molecular weight is 5421.0, and the actual molecular weight is 5420.1.
Example 27 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 94%. The theoretical molecular weight is 5421.0, and the actual molecular weight is 5421.4.
Example 28 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed 84% conversion. The theoretical molecular weight is 5362.0, and the actual molecular weight is 5361.4.
Example 29 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 94%. The theoretical molecular weight is 5428.0, and the actual molecular weight is 5427.1.
Example 30 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10 nmol). To the solution, copper sulfate (40nmol,4 equivalents, 100mM deionized water solution) and imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) were added in this order, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 75%. The theoretical molecular weight is 5428.0, and the actual molecular weight is 5427.1.
Example 31 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 69%. The theoretical molecular weight is 5492.0, and the actual molecular weight is 5491.7.
Example 32 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 80%. The theoretical molecular weight is 5488.0, and the actual molecular weight is 5488.1.
Example 33 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 76%. The theoretical molecular weight is 5428.0, and the actual molecular weight is 5426.
Example 34 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 86%. The theoretical molecular weight is 5453.0, and the actual molecular weight is 5452.1.
Example 35 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed that the reaction conversion was 75%. The theoretical molecular weight is 5503.0, and the actual molecular weight is 5502.0.
Example 36 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 80%. The theoretical molecular weight is 5503.0, and the actual molecular weight is 5502.0.
Example 37 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10 nmol). To the solution, copper sulfate (40nmol,4 equivalents, 100mM deionized water solution) and imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) were added in this order, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 10% sodium chloride solution of 5M into the solution, adding anhydrous ethanol 3 times of the total volume, oscillating, and mixingThe reaction is frozen in dry ice for 2 hours, then centrifuged for half an hour at 12000rpm, the supernatant is poured off, the rest precipitate is dissolved by deionized water to obtain DNA-N3Solution, LCMS confirmed 84% conversion. The theoretical molecular weight is 5453.0, and the actual molecular weight is 5452.1.
Example 38 Synthesis of On-DNA Azide
The DNA amine-based compound was dissolved in sodium borate buffer (sodium borate 250mM, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), copper sulfate (40nmol,4 equivalents, 100mM deionized water solution), imidazole sulfonyl azide hydrochloride (240nmol, 24 equivalents, 120mM deionized water solution) was sequentially added to the solution, mixed well, and then the reaction solution was left to react at 60 ℃ for 0.5 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain DNA-N3Solution, LCMS confirmed reaction conversion to 78%. The theoretical molecular weight is 5466.0, and the actual molecular weight is 5465.5.
In conclusion, the method controls conditions such as reagent solvent, temperature, time and the like of the reaction, and obtains the On-DNA azide with high yield from the On-DNA amino compound in the presence of the copper reagent and the azide reagent. The method has the advantages of few types of added reagents, single product, simple post-treatment and suitability for operation by using a multi-hole plate.
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