CN113388894A - Method for functionalizing aryl olefin of On-DNA in construction of DNA coding compound library - Google Patents
Method for functionalizing aryl olefin of On-DNA in construction of DNA coding compound library Download PDFInfo
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 49
- -1 aryl olefin Chemical class 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 29
- JRZJOMJEPLMPRA-UHFFFAOYSA-N olefin Natural products CCCCCCCC=C JRZJOMJEPLMPRA-UHFFFAOYSA-N 0.000 title claims abstract description 15
- 238000010276 construction Methods 0.000 title claims abstract description 12
- 238000006243 chemical reaction Methods 0.000 claims abstract description 36
- 150000008049 diazo compounds Chemical class 0.000 claims abstract description 15
- RMVRSNDYEFQCLF-UHFFFAOYSA-N thiophenol Chemical compound SC1=CC=CC=C1 RMVRSNDYEFQCLF-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000011941 photocatalyst Substances 0.000 claims abstract description 13
- 125000000524 functional group Chemical group 0.000 claims abstract description 9
- 150000002148 esters Chemical class 0.000 claims abstract description 8
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims abstract description 5
- 108020004414 DNA Proteins 0.000 claims description 35
- 239000000243 solution Substances 0.000 claims description 31
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 150000001336 alkenes Chemical class 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 125000003118 aryl group Chemical group 0.000 claims description 10
- 125000000217 alkyl group Chemical group 0.000 claims description 9
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 9
- 125000001424 substituent group Chemical group 0.000 claims description 9
- 125000004185 ester group Chemical group 0.000 claims description 8
- 229910052736 halogen Inorganic materials 0.000 claims description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 125000003545 alkoxy group Chemical group 0.000 claims description 6
- 125000000304 alkynyl group Chemical group 0.000 claims description 6
- 238000007306 functionalization reaction Methods 0.000 claims description 6
- 150000002367 halogens Chemical class 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- 125000000547 substituted alkyl group Chemical group 0.000 claims description 6
- 125000003107 substituted aryl group Chemical group 0.000 claims description 6
- 238000003786 synthesis reaction Methods 0.000 claims description 6
- 125000003172 aldehyde group Chemical group 0.000 claims description 5
- 230000015572 biosynthetic process Effects 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 5
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 4
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 4
- 125000000051 benzyloxy group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])O* 0.000 claims description 4
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 230000035484 reaction time Effects 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- VQGHOUODWALEFC-UHFFFAOYSA-N 2-phenylpyridine Chemical group C1=CC=CC=C1C1=CC=CC=N1 VQGHOUODWALEFC-UHFFFAOYSA-N 0.000 claims description 2
- TXNLQUKVUJITMX-UHFFFAOYSA-N 4-tert-butyl-2-(4-tert-butylpyridin-2-yl)pyridine Chemical compound CC(C)(C)C1=CC=NC(C=2N=CC=C(C=2)C(C)(C)C)=C1 TXNLQUKVUJITMX-UHFFFAOYSA-N 0.000 claims description 2
- GSNUFIFRDBKVIE-UHFFFAOYSA-N DMF Natural products CC1=CC=C(C)O1 GSNUFIFRDBKVIE-UHFFFAOYSA-N 0.000 claims description 2
- FXHOOIRPVKKKFG-UHFFFAOYSA-N N,N-Dimethylacetamide Chemical compound CN(C)C(C)=O FXHOOIRPVKKKFG-UHFFFAOYSA-N 0.000 claims description 2
- SPEUIVXLLWOEMJ-UHFFFAOYSA-N acetaldehyde dimethyl acetal Natural products COC(C)OC SPEUIVXLLWOEMJ-UHFFFAOYSA-N 0.000 claims description 2
- 125000003368 amide group Chemical group 0.000 claims description 2
- 125000002619 bicyclic group Chemical group 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 claims 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 claims description 2
- 229960002143 fluorescein Drugs 0.000 claims description 2
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 claims description 2
- 229910052739 hydrogen Inorganic materials 0.000 claims description 2
- 239000001257 hydrogen Substances 0.000 claims description 2
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims description 2
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 2
- 229910052751 metal Inorganic materials 0.000 claims description 2
- 239000002184 metal Substances 0.000 claims description 2
- CXKWCBBOMKCUKX-UHFFFAOYSA-M methylene blue Chemical compound [Cl-].C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 CXKWCBBOMKCUKX-UHFFFAOYSA-M 0.000 claims description 2
- 229960000907 methylthioninium chloride Drugs 0.000 claims description 2
- 125000002950 monocyclic group Chemical group 0.000 claims description 2
- 239000000178 monomer Substances 0.000 claims description 2
- 239000002773 nucleotide 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
- 229910052760 oxygen Inorganic materials 0.000 claims description 2
- AZJPTIGZZTZIDR-UHFFFAOYSA-L rose bengal Chemical compound [K+].[K+].[O-]C(=O)C1=C(Cl)C(Cl)=C(Cl)C(Cl)=C1C1=C2C=C(I)C(=O)C(I)=C2OC2=C(I)C([O-])=C(I)C=C21 AZJPTIGZZTZIDR-UHFFFAOYSA-L 0.000 claims description 2
- 229930187593 rose bengal Natural products 0.000 claims description 2
- 229940081623 rose bengal Drugs 0.000 claims description 2
- STRXNPAVPKGJQR-UHFFFAOYSA-N rose bengal A Natural products O1C(=O)C(C(=CC=C2Cl)Cl)=C2C21C1=CC(I)=C(O)C(I)=C1OC1=C(I)C(O)=C(I)C=C21 STRXNPAVPKGJQR-UHFFFAOYSA-N 0.000 claims description 2
- 125000000472 sulfonyl group Chemical group *S(*)(=O)=O 0.000 claims description 2
- 229910052717 sulfur Inorganic materials 0.000 claims description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 2
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 claims description 2
- 150000001408 amides Chemical class 0.000 claims 1
- 125000003636 chemical group Chemical group 0.000 claims 1
- 125000000753 cycloalkyl group Chemical group 0.000 claims 1
- 230000000379 polymerizing effect Effects 0.000 claims 1
- 239000000758 substrate Substances 0.000 abstract description 4
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- 238000006452 multicomponent reaction Methods 0.000 abstract 1
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- 238000012216 screening Methods 0.000 description 10
- 238000001946 ultra-performance liquid chromatography-mass spectrometry Methods 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000011161 development Methods 0.000 description 5
- 150000002611 lead compounds Chemical class 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- JBBKWIFIXPBQGZ-UHFFFAOYSA-N 2-diazonio-1-phenylmethoxyethenolate Chemical compound [N-]=[N+]=CC(=O)OCC1=CC=CC=C1 JBBKWIFIXPBQGZ-UHFFFAOYSA-N 0.000 description 3
- 230000009471 action Effects 0.000 description 3
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- 238000012163 sequencing technique Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- WLHCBQAPPJAULW-UHFFFAOYSA-N 4-methylbenzenethiol Chemical compound CC1=CC=C(S)C=C1 WLHCBQAPPJAULW-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical group N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
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- 230000004071 biological effect Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
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- FANCTJAFZSYTIS-IQUVVAJASA-N (1r,3s,5z)-5-[(2e)-2-[(1r,3as,7ar)-7a-methyl-1-[(2r)-4-(phenylsulfonimidoyl)butan-2-yl]-2,3,3a,5,6,7-hexahydro-1h-inden-4-ylidene]ethylidene]-4-methylidenecyclohexane-1,3-diol Chemical compound C([C@@H](C)[C@@H]1[C@]2(CCCC(/[C@@H]2CC1)=C\C=C\1C([C@@H](O)C[C@H](O)C/1)=C)C)CS(=N)(=O)C1=CC=CC=C1 FANCTJAFZSYTIS-IQUVVAJASA-N 0.000 description 1
- QBTROWHSMGZXCV-RQURQNPSSA-N (6r,7r)-1-[(4s,5r)-4-acetyloxy-5-methyl-3-methylidene-6-phenylhexyl]-4,7-dihydroxy-6-(11-phenoxyundecoxy)-2,8-dioxabicyclo[3.2.1]octane-3,4,5-tricarboxylic acid Chemical compound C([C@@H](C)[C@H](OC(C)=O)C(=C)CCC12[C@@H]([C@@H](OCCCCCCCCCCCOC=3C=CC=CC=3)C(O1)(C(O)=O)C(O)(C(O2)C(O)=O)C(O)=O)O)C1=CC=CC=C1 QBTROWHSMGZXCV-RQURQNPSSA-N 0.000 description 1
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 1
- 241001156002 Anthonomus pomorum Species 0.000 description 1
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- 230000010933 acylation Effects 0.000 description 1
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- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
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- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 125000001995 cyclobutyl group Chemical group [H]C1([H])C([H])([H])C([H])(*)C1([H])[H] 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
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- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
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Classifications
-
- 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
- C40B50/10—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 involving encoding steps
-
- 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
- C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
Abstract
The invention discloses a method for functionalizing aryl olefin of On-DNA in the construction of a DNA coding compound library, which realizes the multicomponent reaction of functionalizing aryl olefin of On-DNA through a visible light catalysis strategy, and can obtain the product of functionalizing aryl olefin of On-DNA after reacting aryl olefin, diazo compound, hans ester, p-toluene thiophenol and photocatalyst of On-DNA under the irradiation of visible light with certain wavelength at a certain temperature for a period of time. The method has the advantages of mild reaction conditions, high yield and good substrate functional group compatibility, and is suitable for synthesizing the DNA coding compound library by using a porous plate.
Description
Technical Field
The invention belongs to the technical field of DNA coding compound libraries, and particularly relates to a method for functionalizing aryl olefin of On-DNA in the construction of a DNA coding compound library.
Drug screening has always been one of the most critical steps in the early stages of development of new drugs. However, the traditional screening scheme finds that the hit rate of the lead compound is low, the time is long, and the lead compound is one of the factors causing the long development period of new drugs, in 1992, Brenner and Lerner combine combinatorial chemistry and molecular biology to put forward the concept of a DNA coding compound library (reference: PNAS.1992,89,5381), and along with the rapid development of the second-generation sequencing technology, the screening of the lead compound is realized under the promotion that the sequencing flux is greatly improved and the sequencing cost is greatly reduced, so that the screening of the lead compound becomes unprecedented fast and efficient. Each small molecule in the coding library has a predetermined DNA sequence as a unique code, which can be subsequently identified by high throughput sequencing methods.
Compared with conventional compound libraries, DNA-encoded compound libraries have the following advantages: 1) the vast number of compounds, usually libraries of DNA-encoding compounds, can contain hundreds of millions of compounds, hundreds or even thousands of compounds compared to traditional libraries; 2) the cost of a single compound is greatly reduced, and the cost of the compound in each DNA coding compound library is only one thousandth to one ten thousandth of the traditional compound library; 3) the library building period is greatly shortened, at present, the period for building a new DNA coding compound library can be completed within several weeks, and the traditional compound library is usually accumulated for decades; 4) the compound structure is denser, and the structure of the DNA coding compound library is more aggregated compared with the traditional compound library because the bonding mode of the same batch of DNA coding compound library is uniform. 5) The cost of screening the biological activity is greatly reduced, and the screening mode of the library of the DNA coding compound library is compared with the single screening mode of the traditional compound library, so that the screening quantity of the biological activity can be greatly reduced, and the screening cost is greatly reduced.
Since the concept of DEL was introduced in 1992, it has attracted a great deal of attention from pharmaceutical companies. Many effective library construction methods have been developed and the part of lead compounds successfully screened against different targets has entered the clinical research stage. The concept of enzyme-catalyzed ligation of oligonucleotide tags is described in the DNA record library construction methods as originally proposed by Kinoshita and Nishigaki (ref: Nucleic Acids Symp. Ser. No. 1995,34,201); DNA template synthesis technology developed by professor David Liu at the university of Harvard (reference: Science 2004,305,1601); professor Dario Neri, Federal rational engineering, Zurich, Switzerland developed coded self-assembly chemistry (ESAC) technology (reference: Angew. chem. int. Ed.2007,46,4671); professor Lilaugh, hong Kong university, proposed a library of DNA dynamic compounds (DEDLs) (ref: J.Am.chem.Soc.2018,140, 15859); in addition, the method has a plurality of breakthrough progresses in the aspect of target screening, only fixed target protein can be screened initially, then free target protein in solution is screened, and in recent years, protein screening on the surface of cells and in the cells is also realized, so that the practicability of the technology is greatly improved.
The DNA coding compound library technology has obvious advantages of speed, scale and cost, and simultaneously has some defects, such as relatively large molecular weight, insufficient chemical reaction types compatible with DNA, insufficient structural diversity and insufficient diversity of library building reagents, wherein the insufficient chemical reaction types become key factors for restricting the further development of the DNA coding compound library technology. The mature organic synthesis reactions currently applicable to the DNA coding compound library technology are quite limited and currently applied to the above common reactions including acylation, SNAr substitution, Suzuki-Miyaura coupling, reductive amination, Fmoc removal, and the like.
Products formed by these conventional chemical reactions, which are publicly reported, are often single-framework-deficient and C-richsp3-Csp3The development of such key frameworks has enabled the construction of C-richsp3-Csp3The DNA compatible chemistry of the core skeleton is significant to the field, only two cases of the existing method realize the aryl alkene functional group reaction of On-DNA by a visible light catalytic strategy, one case is that the aryl alkene of the On-DNA and small molecule conjugated alkene form two new C through a free radical pathsp3-Csp3A key, thereby successfully constructing a cyclobutane scaffold (the reference documents are Org.Lett.2020,22, 2908-2913); another example of the photo-redox catalyzed construction of C from an arylalkene of On-DNA with an N-aryl tertiary aminesp3-Csp3Keys (ref: org. Lett.2021,23, 3486-. However, the two methods have poor universality and strict requirements On substrate structures, and the method aims to find On-DNA with better universalityEfficient construction of aryl alkene functional group with Csp3-Csp3Skeletal products, therefore we developed the process of the present invention.
Disclosure of Invention
The invention aims to provide a method for aryl olefin functionalization of On-DNA in construction of a DNA coding compound library.
The purpose of the invention is realized by the following technical scheme: a method for aryl olefin functionalization of On-DNA in construction of a DNA coding compound library comprises the steps of mixing a solution of the On-DNA aryl olefin compound with the molar concentration of 0.1-2.0 mM, a diazo compound solution with the molar concentration of 10-500 molar equivalents, a Hans ester solution with the molar concentration of 10-500 molar equivalents, a p-toluene thiophenol solution with the molar concentration of 10-100 molar equivalents and a metal photocatalyst solution with the molar concentration of 0.1-10 molar equivalents, and reacting for 10-600 minutes at 20-80 ℃ under the irradiation of visible light with the wavelength of 400 and 550nm to ensure that olefin, diazo compound and Hans ester of the On-DNA aryl olefin compound react to obtain an aryl olefin functionalization product of the On-DNA;
wherein, the general structural formula of the On-DNA aryl alkene compound can be used
The formula of the prepared aryl alkene functionalized product of the On-DNA is shown as follows:
wherein, the On-DNA aryl alkene compound is an On-DNA compound obtained by connecting a chemical functional group containing alkene or alkene containing substituent groups with a DNA sequence through covalent bonds at the molecular level;
wherein the DNA is a single-stranded or double-stranded nucleotide chain obtained by polymerization of an artificially modified and/or unmodified nucleotide monomer;
wherein Ar is a monocyclic or bicyclic aromatic ring, DNA and Ar in the structural formula are connected through one or more chemical bonds, and alkene in the structure of the On-DNA aryl alkene compound is directly connected to the Ar ring;
wherein the diazo compound is a diazo ester compound, a diazo amide compound, a diazo ketone compound, a sulfonic ester diazo, a phosphoric ester diazo and the like.
Wherein the product
Y in the structural formula is ester group, amido group, carbonyl group, sulfonyl group or phosphoryl group, R in the structural formula3And R4Respectively selected from alkyl, substituted alkyl, aryl or substituted aryl, wherein the alkyl is C1～C10An alkyl group; the number of the substituent groups of the substituted alkyl is one or more, and the substituent groups of the substituted alkyl are independently selected from one or more of halogen, alkoxy, benzyloxy, vinyl, alkynyl, aldehyde group, ester group and cyano; the number of the substituent groups of the substituted aryl groups is one or more, and the substituent groups of the substituted aryl groups are mutually independent and are selected from one or more of halogen, alkoxy, benzyloxy, trifluoromethyl, amino, alkynyl, aldehyde group, ester group and cyano.
Specifically, the On-DNA aryl alkenes include, but are not limited to, the following groups:
wherein R is1The functional group for connecting the DNA part can be specifically a group which can complementarily react with the functional group on the DNA, and can be any one of carboxyl, amino, aldehyde and aromatic halogen; r1Can be directly connected with the aromatic ring or connected with a plurality of chemical bonds at intervals; r2Selected from hydrogen, halogen, amino, cyano, hydroxyl, alkoxy, alkynyl, benzyloxy, ester group and C1～C10Alkyl radical, C1～C10Alkylene radical, C3～C7Any one of cycloalkyl radicals, R2The number on the ring is one or more; x is O, S, NH or alkyl substituted amino.
Specifically, the molar concentration of the On-DNA aryl alkene compound after being dissolved in water is 0.1-2.0 mM; preferably, the molar concentration of the On-DNA arylalkene compound after dissolution in water is 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1.0mM, 1.1mM, 1.2mM, 1.3mM, 1.4mM, 1.5mM, 1.6mM, 1.7mM, 1.8mM, 1.9mM, 2.0 mM; more preferably, the molarity after dissolution in water is 1.0 mM.
Specifically, the solvent used in the system refers to a common chemical solvent, and includes one or a mixture of several of water, acetonitrile, methanol, ethanol, isopropanol, DMF, DMSO, DMA, tetrahydrofuran, an inorganic salt buffer, an organic acid buffer, and an organic base buffer.
Specifically, the molar equivalent of the diazo compound is 10-500, preferably, the molar equivalent of the diazo compound is 10 equivalents, 20 equivalents, 40 equivalents, 60 equivalents, 80 equivalents, 100 equivalents, 150 equivalents, 200 equivalents, 300 equivalents, 400 equivalents, 500 equivalents; more preferably, the molar equivalents of the diazo compound are 100 equivalents.
Specifically, the molar equivalent of the hans-ester is 10-500, preferably, the molar equivalent of the hans-ester is 10 equivalents, 20 equivalents, 40 equivalents, 60 equivalents, 80 equivalents, 100 equivalents, 150 equivalents, 200 equivalents, 300 equivalents, 400 equivalents, 500 equivalents; more preferably, the molar equivalents of hans-esters are 100 equivalents.
Specifically, the photocatalyst is Ir (ppy)3、Ir(dtbbpy)(ppy)2PF6、Ru(bpy)3Cl2·6H2O、Ru(bpz)3(PF6)2、Ru(bpy)3(PF6)2Eosin Y, fluorescein, methylene blue, rose bengal; preferably, the photocatalyst is Ru (bpy)3Cl2·6H2O。
Specifically, the molar equivalent of the photocatalyst is 0.1-10 equivalents; preferably, the molar equivalent of the photocatalyst is 0.1 equivalent, 0.5 equivalent, 1.0 equivalent, 2.0 equivalents, 3.0 equivalents, 4.0 equivalents, 5.0 equivalents, 6.0 equivalents, 7.0 equivalents, 8.0 equivalents, 9.0 equivalents, 10.0 equivalents; more preferably, the molar equivalent of the photocatalyst is 1.0 equivalent.
Specifically, the molar equivalent of the p-toluene thiophenol is 10-100 equivalents; preferably, the molar equivalents of the p-toluenesulfonol are 10 equivalents, 20 equivalents, 30 equivalents, 40 equivalents, 50 equivalents, 60 equivalents, 70 equivalents, 80 equivalents, 90 equivalents, 100 equivalents; more preferably, the molar equivalent of the p-toluenesulphonol is 50 equivalents.
Specifically, the reaction temperature is 20-80 ℃; preferably, the reaction temperature is 20 ℃, 25 ℃,30 ℃, 35 ℃, 40 ℃, 45 ℃,50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃; more preferably, the reaction temperature is 25 ℃.
Specifically, the reaction time is 10-600 minutes; preferably, the reaction time is 10 minutes, 20 minutes, 40 minutes, 60 minutes, 80 minutes, 100 minutes, 120 minutes, 150 minutes, 180 minutes, 240 minutes, 300 minutes, 420 minutes, 480 minutes, 540 minutes, 600 minutes; more preferably, the reaction time is 180 minutes.
Specifically, the wavelength of the visible light is 400-550 nm; preferably, the visible light wavelength is 420nm, 450nm, 460nm, 480nm, 500nm, 550 nm; more preferably, the visible light wavelength is 450 nm.
In particular, the method is used for batch multi-well plate operations.
In particular, the method is used for the synthesis of libraries of DNA-encoding compounds for multi-well plates.
The invention provides a method for constructing an On-DNA aryl olefin functional group in a new DNA coding compound library by utilizing a visible light catalysis strategy, expands the DNA compatible chemical reaction types, has mild reaction conditions and high yield, and constructs a C-containing compoundsp3-Csp3The skeleton product and the reaction substrate have high universality, can be compatible with most of functional groups, and is suitable for synthesis of a DNA coding compound library by a multi-well plate.
Drawings
FIG. 1 shows the synthetic reaction scheme and the corresponding conversion rates for 8 On-DNA arylalkene compounds according to the present invention, wherein the conversion rates are based On the area of the TIC peak On UPLC-MS.
FIG. 2 shows the UPLC-MS total ion chromatogram of 3g of On-DNA arylalkene compound (the exact molecular weight of 3 g: 5078.9401, formula for calculating charge-to-mass ratio with four charges: [ M-4H ]/4, calculated to obtain molecular weight: 1268.73, observed molecular weight: 1268.73) in the example of the present invention
FIG. 3 shows the chemical reaction formula and the corresponding conversion rate of On-DNA aryl alkene functionalized products obtained by the reaction of 8 On-DNA aryl alkene compounds with benzyl diazoacetate and Hans ester under the action of p-toluenesulphonol and a photocatalyst in the embodiment of the present invention, based On the TIC peak area On UPLC-MS.
FIG. 4 is a UPLC-MS total ion chromatogram of On-DNA arylolefin functional group product 6g prepared by reacting 3g of On-DNA arylolefin compound with benzyl diazoacetate and Hans ester under the action of p-toluenesulphon and photocatalyst in the specific embodiment of the present invention (compound 6g exact molecular weight: 5229.0081, calculation formula with four charge-to-mass ratios: [ M-4H ]/4, calculated molecular weight: 1306.24, observed molecular weight: 1306.24)
FIG. 5 shows the specific structure and conversion rate of On-DNA aryl alkene functionalized product obtained by reacting DNA-NHCO-Ph-4-ethylene with different diazo compounds in the specific embodiment of the present invention, wherein the conversion rate is based On the TIC peak area On UPLC-MS.
FIG. 6 is a UPLC-MS total ion chromatogram of On-DNA aryl alkene functionalized product 8bw obtained by reaction of DNA-NHCO-Ph-4-ethylene with diazo compound 7bw in the embodiment of the present invention (exact molecular weight of compound 8 bw: 5318.9299, calculation formula with four charge-to-mass ratios: [ M-4H ]/4, calculated molecular weight: 1328.72, observed molecular weight: 1328.72)
The specific implementation mode is as follows:
the present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The described embodiments are only a part of the embodiments of the present invention, not all embodiments, and the processes, conditions, experimental methods, etc. for carrying out the present invention are general knowledge and common general knowledge in the art, and the present invention is not particularly limited.
EXAMPLE 1, Synthesis of 8 On-DNA arylalkene Compounds
Reacting DNA-NH2(Headpiece) was dissolved in 250mM boric acid buffer pH 9.4 to make a concentration of 1.0mM, TEA was used as a base to react with activated carboxylic acid 2 to obtain the corresponding On-DNA aryl alkene compound 3, which was then precipitated with ethanol, concentrated and dried and used in the next reaction without purification.
The ethanol precipitation method comprises the following steps: transferring the reaction solution into a 2.0mLEP tube, adding a 5M sodium chloride solution with the total reaction volume of 10%, adding cold ethanol with the volume 3 times of the total volume, pre-storing the mixture at-20 ℃, uniformly oscillating the mixture, freezing the mixture in a refrigerator at-80 ℃ for 2 hours, centrifuging the mixture at 4 ℃ at 13000RPM for 5 minutes, removing supernatant, dissolving precipitates in deionized water, and preparing a 1mM solution to obtain an On-DNA aryl alkene compound 3 solution for later use. Meanwhile, conversion rate of each molecule is detected by using UPLC-MS, 8 compounds 3 are synthesized in total, and specific structures and yield are shown in figure 1.
EXAMPLE 2, functionalization of aryl-olefin of 8 On-DNA with benzyl diazoacetate and hans ester under the action of p-toluenesulfophenol and photocatalyst
To a 0.5mL Ep tube were added diazo compounds 4 (10. mu.L, 1. mu. mol,100mM dimethyl sulfoxide solution, 100 equivalents), hanster 5 (10. mu.L, 1. mu. mol,100mM dimethyl sulfoxide solution, 100 equivalents), p-tolylthiophenol (5. mu.L, 0.5. mu. mol,100mM dimethyl sulfoxide solution, 50 equivalents), Ru (bpy)3Cl2·6H2O (2. mu.L, 10nmol,5mM dimethyl sulfoxide solution, 1 equivalent), water (10. mu.L) and the above On-DNA arylalkene compound 3 solution (10. mu.L, 10nmol,1mM aqueous solution, 1 equivalent) were mixed well, and then the EP tube was placed in a glass test tube, replaced with nitrogen gas for 10min, and reacted at 25 ℃ under blue light for 3 hours.
After the reaction is finished, an ethanol precipitation method is adopted:
transferring the reaction liquid into a 1.5mL EP tube, adding a 5M sodium chloride solution with the total reaction volume of 10%, adding cold ethanol with the volume 3 times of the total volume and pre-stored at-20 ℃, uniformly oscillating, freezing in a refrigerator at-80 ℃ for 2 hours, centrifuging at 13000RPM for 5min at 4 ℃, removing the supernatant, drying in vacuum to remove residual solvent, adding 100 mu L of deionized water to prepare a 0.1mM aqueous solution for UPLC-MS detection, and determining the conversion rate of each reaction, wherein the specific structure and the conversion rate of the product are shown in figure 3.
Example 3 On- DNA Arylolefin 3a and 80 diazo Compounds 7 to achieve On-DNA Arylolefin functionalization
80 diazo compounds 7 (10. mu.L, 1. mu. mol,100mM dimethyl sulfoxide solution, 100 equivalents) were added to corresponding 80 wells in a 96-well plate, followed by hanster 5 (10. mu.L, 1. mu. mol,100mM dimethyl sulfoxide solution, 100 equivalents), p-toluthiol (5. mu.L, 0.5. mu. mol,100mM dimethyl sulfoxide solution, 50 equivalents), Ru (bpy)3Cl2·6H2O (2. mu.L, 10nmol,5mM dimethyl sulfoxide solution, 1 equivalent), water (10. mu.L) and the above On-DNA arylalkene compound 3a solution (10. mu.L, 10nmol,1mM aqueous solution, 1 equivalent) were mixed well, replaced with nitrogen for 10min, and reacted in a shaker at 25 ℃ for 3 hours under blue light.
After the reaction is finished, an ethanol precipitation method is adopted:
adding a 5M sodium chloride solution accounting for 10% of the total volume of the reaction solution into each hole of a 96-hole plate, sealing a membrane, uniformly shaking, adding cold ethanol with the volume 3 times of the total volume and pre-stored at-20 ℃, uniformly shaking, freezing in a refrigerator at-80 ℃ for 2 hours, centrifuging at 4 ℃ for 30 minutes under the centrifugal force of 4000G, removing supernatant, dissolving precipitates with deionized water, obtaining a product by vacuum drying, finally adding 100 mu L deionized water to prepare a 0.1mM aqueous solution for UPLC-MS detection, and determining the conversion rate of each reaction, wherein the specific structure and the conversion rate of the product are shown in figure 5.
In conclusion, the invention successfully develops a new DNA compatible chemical reaction, utilizes a visible light catalysis strategy to realize the method for constructing the aryl alkene functional group of the On-DNA in the DNA coding compound library, has mild reaction conditions, high yield and high substrate universality, can be compatible with most of functional groups, and is suitable for the synthesis of the DNA coding compound library by using a multi-hole plate. The embodiments and figures listed are only some representative embodiments of the invention, the invention is not limited to the above embodiments, variations and advantages that can be realized by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention, and the scope of protection is realized by the appended claims.
Claims (8)
1. A method for aryl olefin functionalization of On-DNA in construction of a DNA coding compound library is characterized in that a solution of the On-DNA aryl olefin compound with a molar concentration of 0.1-2.0 mM is mixed with a diazo compound solution with 10-500 molar equivalents, a hans ester solution with 10-500 molar equivalents, a p-toluene thiophenol solution with 10-100 molar equivalents and a metal photocatalyst solution with 0.1-10 molar equivalents, and the mixture is subjected to a reaction for 10-600 minutes at 20-80 ℃ under the irradiation of visible light with 400-;
wherein, the On-DNA aryl alkene compound is an On-DNA compound obtained by connecting an alkene-containing chemical group with a DNA sequence through a covalent bond at the molecular level; the DNA is a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and/or unmodified nucleotide monomers; the diazo compound is a diazo ester compound, a diazo amide compound, a diazo ketone compound, a sulfonic ester diazo, a phosphoric ester diazo and the like.
2. The method of claim 1, wherein the On-DNA arylalkene compound has the general structural formula:
the general structural formula of the prepared On-DNA alkene functional group product is as follows:
wherein Ar is a monocyclic or bicyclic aromatic ring, and DNA in the structural formula is connected with Ar through any one or more of alkyl, amide, amino, aldehyde group, aromatic halide and carboxyl;
wherein Y in the structural formula is ester group, amido group, carbonyl group, sulfonyl group or phosphoryl group, and R in the structural formula3And R4Respectively selected from alkyl, substituted alkyl, aryl or substituted aryl, wherein the alkyl is C1～C10An alkyl group; the number of the substituent groups of the substituted alkyl groups is one or more, and the substituent groups of the substituted alkyl groups are independently selected from one or more of halogen, alkoxy, benzyloxy, vinyl, alkynyl, aldehyde group, ester group and cyano; the number of the substituent groups of the substituted aryl groups is one or more, and the substituent groups of the substituted aryl groups are mutually independent and are selected from one or more of halogen, alkoxy, benzyloxy, trifluoromethyl, amino, alkynyl, aldehyde group, ester group and cyano.
3. The method of claim 1 or 2, wherein the On-DNA aryl alkene compound is selected from the following structures:
wherein R is1The functional group for connecting the DNA part can be specifically a group which can complementarily react with the functional group on the DNA, and can be any one of carboxyl, amino, aldehyde and aromatic halogen; r1Can be directly connected with the aromatic ring or connected with a plurality of chemical bonds at intervals; r2Selected from hydrogen, halogen, amino, cyano, hydroxyl, alkoxy, alkynyl, benzyloxy, ester group and C1～C10Alkyl radical, C1～C10Alkylene radical, C3～C7In cycloalkyl radicalsAny one of R2The number on the ring is one or more; x is O, S, NH or alkyl substituted amino.
4. The method of claim 1, wherein the On-DNA arylalkene compound is present in a molar concentration of 1.0 mM; the molar equivalent of the diazo compound solution is 100 equivalents; the molar equivalent of the hans ester solution is 100 equivalents; the molar equivalent of the p-toluene thiophenol solution is 50 equivalents; the molar equivalent of the photocatalyst is 1.0 equivalent; the reaction temperature is 25 ℃; the reaction time is 180 minutes; the visible light wavelength is 450 nm.
5. The method according to claim 1, wherein the solvent used in the system refers to a common chemical solvent, and includes one or more of water, acetonitrile, methanol, ethanol, isopropanol, DMF, DMSO, DMA, tetrahydrofuran, inorganic salt buffer, organic acid buffer, and organic base buffer.
6. The method of claim 1, wherein the photocatalyst is Ir (ppy)3、Ir(dtbbpy)(ppy)2PF6、Ru(bpy)3Cl2·6H2O、Ru(bpz)3(PF6)2、Ru(bpy)3(PF6)2Eosin Y, fluorescein, methylene blue, rose bengal.
7. The method of any one of claims 1 to 6 used in a batch multiwell plate operation.
8. The method of any one of claims 1 to 6 for the synthesis of a library of DNA encoding compounds for multi-well plates.
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