CN113943979A - Method for preparing On-DNA thioether compound from On-DNA aryl halide - Google Patents

Method for preparing On-DNA thioether compound from On-DNA aryl halide Download PDF

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CN113943979A
CN113943979A CN202010673235.2A CN202010673235A CN113943979A CN 113943979 A CN113943979 A CN 113943979A CN 202010673235 A CN202010673235 A CN 202010673235A CN 113943979 A CN113943979 A CN 113943979A
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李进
纪跃
戴东良
罗华东
刘观赛
万金桥
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Hitgen Inc
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Abstract

The invention relates to a method for preparing an On-DNA thioether compound from an On-DNA aryl halide, which takes the On-DNA aryl halide as a substrate and performs coupling reaction with thiophenol or mercaptan under the action of a Cu catalyst, a ligand, alkali and an additive to construct a C-S bond. The reaction method provided by the invention provides a new method for the coupling reaction of the On-DNA aryl halide, enhances the diversity of the DNA coding compound library, has wide substrate universality, high yield, mild conditions and convenient operation, and is suitable for the synthesis of the DNA coding compound library by using a multi-hole plate.

Description

Method for preparing On-DNA thioether compound from On-DNA aryl halide
Technical Field
The invention belongs to the technical field of coding compound libraries, and particularly relates to a method for constructing a C-S bond by coupling reaction of On-DNA aryl halide and thiophenol or mercaptan in construction of a DNA coding compound library.
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.
At present, the construction of DNA coding compound library has various metal-promoted coupling reactions to construct C-C, C-N bonds. For example, reactions to build a C-C bond are with: suzuki coupling reaction, light-promoted decarboxylation coupling, C-H bond activation, RCM reaction and the like. The reactions for constructing the C-N bond are: buchwald coupling reaction, mu Llmann coupling reaction and the like.
However, there is no reported method suitable for C-S coupling in the synthesis of libraries of DNA encoding compounds. In order to address the gap in the C-S construction method, it is desirable to develop a method suitable for C-S coupling in the synthesis of libraries of DNA-encoding compounds, and for the synthesis of libraries of DNA-encoding compounds in multiwell plates.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for preparing an On-DNA thioether compound from an On-DNA aryl halide through a coupling reaction, which has the advantages of mild reaction conditions, high selectivity, high yield and simple post-treatment, is suitable for the production of a DNA coding compound library, and can obviously improve the diversity of molecules of the compound library.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a method for preparing On-DNA thioether compounds from On-DNA aryl halides is characterized in that the On-DNA aryl halides are used as substrates and are subjected to coupling reaction with thiophenol or mercaptan under the action of a Cu catalyst, a ligand, alkali and an additive to obtain the On-DNA thioether compounds, wherein the On-DNA aryl halides have a structural formula of DNA-Ar-X, and the thiophenol or mercaptan has a structural formula of RSH;
wherein the DNA in the structural formula comprises a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and/or unmodified nucleotide monomers, and the nucleotide chain is connected with Ar through one or more chemical bonds or groups; the length of the single-chain or double-chain nucleotide chain is 10-200 bp; -X of the On-DNA aryl halide is attached to the ring of Ar, X is chlorine, bromine or iodine;
wherein, the DNA in the structural formula is connected with Ar through one chemical bond or a plurality of chemical bonds or groups; when the chemical bond is one, the DNA in the structural formula is directly connected with Ar; multiple chemical bonds or groups are defined as multiple bonds spaced between the DNA and Ar in the structural formula, e.g., the DNA and Ar are linked by a methylene group (-CH)2-) are linked, i.e. linked by two chemical bonds; or the DNA and the Ar are connected through a carbonyl (-CO-) to the amino of the DNA, and are also connected through two chemical bonds; or DNA and Ar through a methyleneAlkoxycarbonyl (-CH)2CO-) is attached to the amino group of the DNA, i.e., by three consecutive chemical bonds.
Wherein Ar in the structural formula is selected from an optionally substituted aromatic ring or aromatic heterocycle with the molecular weight of less than 1000; r is selected from groups with molecular weight below 1000 and directly connected with sulfydryl.
Preferably; ar is selected from the following groups:
Figure BDA0002584805320000021
wherein, the Ar can have one OR more stituents which are hydrogen, halogen, carboxyl, cyano, hydroxyl, alkyl, stituted alkyl, alkoxy, stituted alkoxy, -C (O) OR1、-OC(O)R1、-C(O)R2Any one or more of the random combinations of R1Is selected from C1～C20Alkyl, said R2Selected from hydrogen or C1～C20An alkyl group; the alkyl group is C1-C20Linear or branched alkyl of (a); the alkoxy is C1-C20A straight or branched alkoxy group of (a);
the number of the substituent groups of the substituted alkyl is one or more, and the substituent groups of the substituted alkyl are one or more independently selected from halogen, cyano and carboxyl;
the number of the substituent of the substituted alkoxy is one or more, and the substituent of the substituted alkoxy is one or more independently selected from halogen, cyano and carboxyl.
Preferably, the On-DNA aryl halide is specifically selected from the following structures:
Figure BDA0002584805320000031
the structural formula of the thiophenol or the mercaptan is RSH, wherein R is selected from C1～C20Alkyl or 0 to 5R3A substituted 5-to 12-membered aromatic or heteroaromatic ring; said R3Is any one or more of hydrogen, halogen, carboxyl, cyano, hydroxyl, alkyl, substituted alkyl and alkoxy; the alkyl group is C1-C20Linear or branched alkyl of (a); the alkoxy is C1-C20A straight or branched alkoxy group of (a);
the number of the substituents of the substituted alkyl group is one or more; the substituents of the substituted alkyl are independently selected from halogen and carboxyl.
Preferably, the alkoxy group is selected from C1-C6Alkoxy is selected from methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy and hexoxy.
Preferably, R is selected from 0-5R3A substituted 5-12 membered aromatic ring or aromatic heterocycle, wherein the 5-12 membered aromatic ring or aromatic heterocycle is selected from the following groups:
Figure BDA0002584805320000032
preferably, the thiophenol is specifically selected from the following structures:
Figure BDA0002584805320000041
a method for preparing On-DNA thioether compounds from On-DNA aryl halides comprises the following steps: adding mercaptan or thiophenol, alkali, additive, copper catalyst and ligand into aryl halide solution with molar equivalent of 1 and molar concentration of 0.5-5mM, and reacting at 20-80 deg.C for 2-48 hr until the reaction is finished.
The chemical reaction equation reacts as follows:
Figure BDA0002584805320000042
preferably, the copper catalyst is cuprous iodide, cupric acetylacetonate, cuprous oxide, ketone bromide, cuprous bromide, copper trifluoromethanesulfonyl, copper trifluoroacetate, copper sulfate, copper acetate or copper chloride, and further, the copper catalyst is cuprous iodide or copper trifluoroacetate.
Preferably, the ligand is 1, 10-phenanthroline (1,10-Phen) or bipyridine.
Preferably, the base is t-Bu4NOH，K3PO4，NaOH，KOH，CsOH，Na2CO3，K2CO3Or Cs2CO3Further, the alkali is NaOH.
Preferably, the additive is tetra-n-butylammonium iodide or tetra-n-butylammonium bromide, and further, the additive is tetra-n-butylammonium iodide.
Preferably, the reaction is carried out in a solvent, and the solvent is one or a mixture of water, DMSO, acetonitrile, DMA, acetone, methanol, NMP and THF; further, the solvent is H2And a mixed solvent of O and DMA.
Preferably, the reaction temperature is 40 ℃,50 ℃, 60 ℃, 70 ℃ or 80 ℃.
Preferably, the reaction time is 1 hour, 2 hours, 4 hours or 16 hours.
Preferably, in the method, the molar equivalent of the On-DNA aryl halide is 1, the molar equivalent of the copper catalyst is 5-50, the molar equivalent of the ligand is 10-100, the molar equivalent of the base is 100-1000, the molar equivalent of the thiophenol or the thiol is 50-1000, and the molar equivalent of the additive is 10-100; further, the molar equivalent of the copper catalyst is 50, the molar equivalent of the ligand is 100, the molar equivalent of the base is 500, the molar equivalent of the thiophenol or thiol is 100, and the molar equivalent of the additive is 50.
Further, the feeding sequence of the reaction is that the copper catalyst and the ligand are mixed in advance, mercaptan or thiophenol, alkali and an additive are added into the solution of the On-DNA aryl halide, and finally the mixed solution of the copper catalyst and the ligand is added.
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 the coupling reaction of the On-DNA aryl halogenated compound and the thiophenol or the mercaptan under the catalysis of the copper to construct the C-S bond in the construction of the DNA coding compound library, has high yield and single product, can be carried out in the mixed aqueous phase of an organic solvent/aqueous phase, has simple operation, and is suitable for the synthesis of the DNA coding compound library by using a porous plate.
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 is a straight or branched chain hydrocarbon radical derived from an alkane molecule by the removal of one hydrogen atom, e.g. methyl-CH3ethyl-CH2CH3(ii) a The alkyl group may also be part of another group, such as a C1-C6 alkoxy group.
Halogen is fluorine, chlorine, bromine or iodine.
Alkoxy groups: means that the alkyl radical is bound to an oxygen atom to form a substituent, e.g. methoxy is-OCH3。
5-12-membered aromatic ring: refers to an aromatic single ring or multiple rings composed of C atoms and containing no hetero atoms.
The 5-to 12-membered aromatic heterocyclic ring is a single ring or a plurality of rings having aromaticity and comprising 5 to 12 atoms of C, O, S, N etc.
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.
Drawings
FIG. 1: LC-Ms spectrum and Ms spectrum of compound 3 in example 1.
FIG. 2: LC-Ms spectrum and Ms spectrum of compound 4 in example 2.
FIG. 3: LC-Ms spectrum and Ms spectrum of compound 5 in example 3.
FIG. 4: LC-Ms and Ms of Compound 6 in example 4.
FIG. 5: LC-Ms spectrum and Ms spectrum of compound 7 in example 5.
FIG. 6: LC-Ms spectrum and Ms spectrum of compound 8 in example 6.
FIG. 7: LC-Ms spectrum and Ms spectrum of compound 9 in example 7.
FIG. 8: LC-Ms spectrum and Ms spectrum of compound 10b in example 8.
FIG. 9: LC-Ms and Ms of compound 11b in example 9.
FIG. 10: LC-Ms spectrum and Ms spectrum of compound 12b in example 10.
FIG. 11: LC-Ms spectrum and Ms spectrum of compound 13b in example 11.
FIG. 12: LC-Ms spectrum and Ms spectrum of compound 14b in example 12.
FIG. 13: LC-Ms spectrum and Ms spectrum of compound 15b in example 13.
Detailed Description
The technical solution of the present invention is fully and clearly described below with reference to specific examples. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.
DNA-NH in the present invention2with-NH formed by single-or double-stranded DNA and linker groups2DNA constructs for linkers, e.g. DNA-NH of "compound 1" in WO20050584792And (5) structure. Also for example the following DNA structure:
Figure BDA0002584805320000061
wherein A is adenine, T is thymine, C is cytosine, and G is guanine.
In the invention, the term "room temperature" means 20-25 ℃.
DMA: dimethylacetamide; HATU: 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate. DIPEA: n, N-diisopropylethylamine; CAS: 7087-68-5. BBS buffer: a borate buffered solution.
DMSO, DMSO: dimethyl sulfoxide; THF: tetrahydrofuran; NMP: n-methyl pyrrolidone; DDCC: diethyl dithiocarbamate (salt).
Example 1 Synthesis of On-DNA thioether Compound 3
Figure BDA0002584805320000062
Compound 1(20 μ L) was dissolved in borate buffered solution (pH 9.4,250mM) to a starting concentration of 1 mM; p-iodobenzoic acid (10. mu.L, 100 equivalents, 200mM DMA solution), HATU (5. mu.L, 100 equivalents, 400mM DMA solution) and DIPEA (5. mu.L, 100 equivalents, 400mM DMA solution) which were previously cooled in a refrigerator at-20 ℃ for 5 minutes were mixed, and then stored in a refrigerator at 4 ℃ for 5 minutes, the mixture was added to the compound1 solution, the reaction solution was thoroughly mixed by vortex shaking, and the reaction solution was left at room temperature for 12 hours.
After the reaction is finished: and (2) ethanol precipitation, namely 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, uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, centrifuging for half an hour at the rotating speed of 12000rpm, pouring out a supernatant, and dissolving the rest precipitate with deionized water to obtain the On-DNA aryl iodide compound 2.
On-DNA aryl iodide 2 (20. mu.L) was dissolved in deionized water to prepare a 1mM aqueous solution, and then thiophenol (10. mu.L, 400 equivalents, 200mM DMA solution), sodium hydroxide (10. mu.L, 500 equivalents, 1M aqueous solution), tetrabutylammonium iodide (10. mu.L, 50 equivalents, 100mM DMA solution) and a solution of premixed CuI (5. mu.L, 50 equivalents, 200mM DMA solution), 1,10-Phen (5. mu.L, 100 equivalents, 400mM DMA solution) were added to the solution in this order, mixed well, and reacted at 80 ℃ for 16 hours.
After the reaction is finished: (1) copper removal treatment: DDTC (20. mu.L, 100 equivalents, 100mM DMA solution) was added, mixed well and left for half an hour; (2) ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding anhydrous ethanol with the volume of 3 times of the total volume, 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 the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound; (3) alcohol washing: and adding 75% ethanol (80 mu L) into the sample treated in the previous step, uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, centrifuging at 12000rpm for half an hour, pouring out the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound with the conversion rate of 74%.
Example 2 Synthesis of On-DNA thioether Compound 4
Figure BDA0002584805320000071
On-DNA aryl iodide 2 (20. mu.L) was dissolved in deionized water to prepare a 1mM aqueous solution, and then thiophenol (10. mu.L, 400 equivalents, 200mM DMA solution), sodium hydroxide (10. mu.L, 500 equivalents, 1M aqueous solution), tetrabutylammonium iodide (10. mu.L, 50 equivalents, 100mM DMA solution) and a solution of premixed CuI (5. mu.L, 50 equivalents, 200mM DMA solution), 1,10-Phen (5. mu.L, 100 equivalents, 400mM DMA solution) were added to the solution in this order, mixed well, and reacted at 80 ℃ for 16 hours.
After the reaction is finished: (1) copper removal treatment: DDTC (20 μ L,100 equiv., 100mM DMA solution) was added, mixed well and left for half an hour; (2) ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding anhydrous ethanol with the volume of 3 times of the total volume, 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 the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound; (3) alcohol washing: and adding 75% ethanol (80 mu L) into the sample treated in the previous step, uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, centrifuging at 12000rpm for half an hour, pouring out the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound with the conversion rate of 62%.
Example 3 Synthesis of On-DNA thioether Compound 5
Figure BDA0002584805320000081
On-DNA aryl iodide 2 (20. mu.L) was dissolved in deionized water to prepare a 1mM aqueous solution, and thiophenol (10. mu.L, 400 equivalents, 200mM DMA solution), sodium hydroxide (10. mu.L, 500 equivalents, 1M aqueous solution), tetrabutylammonium iodide (10. mu.L, 50 equivalents, 100mM DMA solution) and a solution of premixed CuI (5. mu.L, 50 equivalents, 200mM DMA solution), 1,10-Phen (5. mu.L, 100 equivalents, 400mM DMA solution) were added to the solution in this order. Mixing evenly, and reacting for 16 hours at 80 ℃.
After the reaction is finished: (1) copper removal treatment: DDTC (20 μ L,100 equiv., 100mM DMA solution) was added, mixed well and left for half an hour; (2) ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding anhydrous ethanol with the volume of 3 times of the total volume, 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 the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound; (3) alcohol washing: and adding 75% ethanol (80 mu L) into the sample treated in the previous step, uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, centrifuging at 12000rpm for half an hour, pouring out the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound with the conversion rate of 62%.
Example 4 Synthesis of On-DNA thioether Compound 6
Figure BDA0002584805320000082
On-DNA aryl iodide 2 (20. mu.L) was dissolved in deionized water to prepare a 1mM aqueous solution, and then thiophenol (10. mu.L, 400 equivalents, 200mM DMA solution), sodium hydroxide (10. mu.L, 500 equivalents, 1M aqueous solution), tetrabutylammonium iodide (10. mu.L, 50 equivalents, 100mM DMA solution) and a solution of premixed CuI (5. mu.L, 50 equivalents, 200mM DMA solution), 1,10-Phen (5. mu.L, 100 equivalents, 400mM DMA solution) were added to the solution in this order, mixed well, and reacted at 80 ℃ for 16 hours.
After the reaction is finished: (1) copper removal treatment: DDTC (20. mu.L, 100 equivalents, 100mM DMA solution) was added, mixed well and left for half an hour; (2) ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding anhydrous ethanol with the volume of 3 times of the total volume, 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 the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound; (3) alcohol washing: and adding 75% ethanol (80 mu L) into the sample treated in the previous step, uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, centrifuging at the rotating speed of 12000rpm for half an hour, pouring out the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound with the conversion rate of 63%.
Example 5 Synthesis of On-DNA thioether Compound 7
Figure BDA0002584805320000091
On-DNA aryl iodide 2 (20. mu.L) was dissolved in deionized water to prepare a 1mM aqueous solution, and then thiophenol (10. mu.L, 400 equivalents, 200mM DMA solution), sodium hydroxide (10. mu.L, 500 equivalents, 1M aqueous solution), tetrabutylammonium iodide (10. mu.L, 50 equivalents, 100mM DMA solution) and a solution of premixed CuI (5. mu.L, 50 equivalents, 200mM DMA solution), 1,10-Phen (5. mu.L, 100 equivalents, 400mM DMA solution) were added to the solution in this order, mixed well, and reacted at 80 ℃ for 16 hours.
After the reaction is finished: (1) copper removal treatment: DDTC (20 μ L,100 equiv., 100mM DMA solution) was added, mixed well and left for half an hour; (2) ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding anhydrous ethanol with the volume of 3 times of the total volume, 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 the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound; (3) alcohol washing: and adding 75% ethanol (80 mu L) into the sample treated in the previous step, uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, centrifuging at the rotating speed of 12000rpm for half an hour, pouring out the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound with the conversion rate of 75%.
Example 6 Synthesis of On-DNA thioether Compound 8
Figure BDA0002584805320000101
On-DNA aryl iodide 2 (20. mu.L) was dissolved in deionized water to prepare a 1mM aqueous solution, and then thiophenol (10. mu.L, 400 equivalents, 200mM DMA solution), sodium hydroxide (10. mu.L, 500 equivalents, 1M aqueous solution), tetrabutylammonium iodide (10. mu.L, 50 equivalents, 100mM DMA solution) and a solution of premixed CuI (5. mu.L, 50 equivalents, 200mM DMA solution), 1,10-Phen (5. mu.L, 100 equivalents, 400mM DMA solution) were added to the solution in this order, mixed well, and reacted at 80 ℃ for 16 hours.
After the reaction is finished: (1) copper removal treatment: DDTC (20 μ L,100 equiv., 100mM DMA solution) was added, mixed well and left for half an hour; (2) ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding anhydrous ethanol with the volume of 3 times of the total volume, 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 the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound; (3) alcohol washing: and adding 75% ethanol (80 mu L) into the sample treated in the previous step, uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, centrifuging at 12000rpm for half an hour, pouring out the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound with the conversion rate of 51%.
Example 7 Synthesis of On-DNA thioether Compound 9
Figure BDA0002584805320000102
On-DNA aryl iodide 2 (20. mu.L) was dissolved in deionized water to prepare a 1mM aqueous solution, and thiophenol (10. mu.L, 400 equivalents, 200mM DMA solution), sodium hydroxide (10. mu.L, 500 equivalents, 1M aqueous solution), tetrabutylammonium iodide (10. mu.L, 50 equivalents, 100mM DMA solution) and a solution of premixed CuI (5. mu.L, 50 equivalents, 200mM DMA solution), 1,10-Phen (5. mu.L, 100 equivalents, 400mM DMA solution) were added to the solution in this order. Mixing evenly, and reacting for 16 hours at 80 ℃.
After the reaction is finished: (1) copper removal treatment: DDTC (20. mu.L, 100 equivalents, 100mM DMA solution) was added, mixed well and left for half an hour; (2) ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding anhydrous ethanol with the volume of 3 times of the total volume, 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 the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound; (3) alcohol washing: and adding 75% ethanol (80 mu L) into the sample treated in the previous step, uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, centrifuging at 12000rpm for half an hour, pouring out the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound with the conversion rate of 79%.
Example 8 Synthesis of On-DNA thioether Compound 10b
Figure BDA0002584805320000111
Compound 1(20 μ L) was dissolved in borate buffered solution (pH 9.4,250mM) to a starting concentration of 1 mM; ortho-iodobenzoic acid (10. mu.L, 100 equivalents, 200mM DMA solution), HATU (5. mu.L, 100 equivalents, 400mM DMA solution) and DIPEA (5. mu.L, 100 equivalents, 400mM DMA solution) which had been previously cooled for 5 minutes in a refrigerator at-20 ℃ were mixed, and then stored in a refrigerator at 4 ℃ for 5 minutes, the above mixture was added to the compound1 solution, the reaction solution was thoroughly mixed by vortex shaking, and the reaction solution was left at room temperature for 12 hours.
After the reaction is finished: and (5) ethanol precipitation. Adding a 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 the supernatant, and dissolving the rest precipitate with deionized water to obtain the On-DNA aryl iodide compound.
An On-DNA aryl iodide compound 10a (20. mu.L) was dissolved in deionized water to prepare a 1mM aqueous solution, and then thiophenol (10. mu.L, 400 equivalents, 200mM DMA solution), sodium hydroxide (10. mu.L, 500 equivalents, 1M aqueous solution), tetrabutylammonium iodide (10. mu.L, 50 equivalents, 100mM DMA solution) and a solution of premixed CuI (5. mu.L, 50 equivalents, 200mM DMA solution), 1,10-Phen (5. mu.L, 100 equivalents, 400mM DMA solution) were added to the solution in this order, mixed well, and reacted at 80 ℃ for 16 hours.
After the reaction is finished: (1) copper removal treatment: DDTC (20. mu.L, 100 equivalents, 100mM DMA solution) was added, mixed well and left for half an hour; (2) ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding anhydrous ethanol with the volume of 3 times of the total volume, 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 the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound; (3) alcohol washing: and adding 75% ethanol (80 mu L) into the sample treated in the previous step, uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, centrifuging at 12000rpm for half an hour, pouring out the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound with the conversion rate of 64%.
Example 9 Synthesis of On-DNA thioether Compound 11b
Figure BDA0002584805320000121
Compound 1(20 μ L) was dissolved in borate buffered solution (pH 9.4,250mM) to a starting concentration of 1 mM; carboxylic acid (10. mu.L, 100 equivalents, 200mM DMA solution), HATU (5. mu.L, 100 equivalents, 400mM DMA solution) and DIPEA (5. mu.L, 100 equivalents, 400mM DMA solution) which had been previously cooled in a refrigerator at-20 ℃ for 5 minutes were mixed, and then stored in a refrigerator at 4 ℃ for 5 minutes, the mixture was added to the compound1 solution, and the reaction solution was thoroughly mixed by vortexing and left at room temperature for 12 hours.
After the reaction is finished: ethanol precipitation: adding a 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 the supernatant, and dissolving the rest precipitate with deionized water to obtain the On-DNA aryl iodide compound.
On-DNA aryl iodide compound 11a (20. mu.L) was dissolved in deionized water to prepare a 1mM aqueous solution, and thiophenol (10. mu.L, 400 equivalents, 200mM DMA solution), sodium hydroxide (10. mu.L, 500 equivalents, 1M aqueous solution), tetrabutylammonium iodide (10. mu.L, 50 equivalents, 100mM DMA solution) and a solution of premixed CuI (5. mu.L, 50 equivalents, 200mM DMA solution), 1,10-Phen (5. mu.L, 100 equivalents, 400mM DMA solution) were added to the solution in this order. Mixing evenly, and reacting for 16 hours at 80 ℃.
After the reaction is finished: (1) copper removal treatment: DDTC (20. mu.L, 100 equivalents, 100mM DMA solution) was added, mixed well and left for half an hour; (2) ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding anhydrous ethanol with the volume of 3 times of the total volume, 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 the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound; (3) alcohol washing: and adding 75% ethanol (80 mu L) into the sample treated in the previous step, uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, centrifuging at 12000rpm for half an hour, pouring out the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound with the conversion rate of 53%.
Example 10 Synthesis of On-DNA thioether Compound 12b
Figure BDA0002584805320000122
Compound 1(20 μ L) was dissolved in borate buffered solution (pH 9.4,250mM) to a starting concentration of 1 mM; carboxylic acid (10. mu.L, 100 equivalents, 200mM DMA solution), HATU (5. mu.L, 100 equivalents, 400mM DMA solution) and DIPEA (5. mu.L, 100 equivalents, 400mM DMA solution) were mixed each previously cooled in a refrigerator at-20 ℃ for 5 minutes, and then stored in a refrigerator at 4 ℃ for 5 minutes, the mixture was added to the solution of Compound1, and the reaction solution was thoroughly mixed by vortexing. The reaction solution was left at room temperature for 12 hours.
After the reaction is finished: and (5) ethanol precipitation. Adding a 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 the supernatant, and dissolving the rest precipitate with deionized water to obtain the On-DNA aryl iodide compound.
On-DNA aryl iodide compound 12a (20. mu.L) was dissolved in deionized water to prepare a 1mM aqueous solution, and thiophenol (10. mu.L, 400 equivalents, 200mM DMA solution), sodium hydroxide (10. mu.L, 500 equivalents, 1M aqueous solution), tetrabutylammonium iodide (10. mu.L, 50 equivalents, 100mM DMA solution) and a solution of premixed CuI (5. mu.L, 50 equivalents, 200mM DMA solution), 1,10-Phen (5. mu.L, 100 equivalents, 400mM DMA solution) were added to the solution in this order. Mixing evenly, and reacting for 16 hours at 80 ℃.
After the reaction is finished: (1) copper removal treatment: DDTC (20 μ L,100 equiv., 100mM DMA solution) was added, mixed well and left for half an hour; (2) ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding anhydrous ethanol with the volume of 3 times of the total volume, 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 the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound; (3) alcohol washing: and adding 75% ethanol (80 mu L) into the sample treated in the previous step, uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, centrifuging at 12000rpm for half an hour, pouring out the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound with the conversion rate of 57%.
Example 11 Synthesis of On-DNA thioether Compound 13b
Figure BDA0002584805320000131
Compound 1(20 μ L) was dissolved in borate buffered solution (pH 9.4,250mM) to a starting concentration of 1 mM; carboxylic acid (10. mu.L, 100 equivalents, 200mM DMA solution), HATU (5. mu.L, 100 equivalents, 400mM DMA solution) and DIPEA (5. mu.L, 100 equivalents, 400mM DMA solution) were mixed each previously cooled in a refrigerator at-20 ℃ for 5 minutes, and then stored in a refrigerator at 4 ℃ for 5 minutes, the mixture was added to the solution of Compound1, and the reaction solution was thoroughly mixed by vortexing. The reaction solution was left at room temperature for 12 hours.
After the reaction is finished: and (5) ethanol precipitation. Adding a 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 the supernatant, and dissolving the rest precipitate with deionized water to obtain the On-DNA aryl iodide compound.
On-DNA aryl iodide compound 13a (20. mu.L) was dissolved in deionized water to prepare a 1mM aqueous solution, and thiophenol (10. mu.L, 400 equivalents, 200mM DMA solution), sodium hydroxide (10. mu.L, 500 equivalents, 1M aqueous solution), tetrabutylammonium iodide (10. mu.L, 50 equivalents, 100mM DMA solution) and a solution of premixed CuI (5. mu.L, 50 equivalents, 200mM DMA solution), 1,10-Phen (5. mu.L, 100 equivalents, 400mM DMA solution) were added to the solution in this order. Mixing evenly, and reacting for 16 hours at 80 ℃.
After the reaction is finished: (1) copper removal treatment: DDTC (20 μ L,100 equiv., 100mM DMA solution) was added, mixed well and left for half an hour; (2) ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding anhydrous ethanol with the volume of 3 times of the total volume, 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 the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound; (3) alcohol washing: and adding 75% ethanol (80 mu L) into the sample treated in the previous step, uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, centrifuging at 12000rpm for half an hour, pouring out the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound with the conversion rate of 64%.
Example 12 Synthesis of On-DNA thioether Compound 14b
Figure BDA0002584805320000141
Compound 1(20 μ L) was dissolved in borate buffer (pH 9.4,250mM) to give an initial concentration of 1mM, carboxylic acid (10 μ L,100 equivalents, 200mM DMA solution), HATU (5 μ L,100 equivalents, 400mM DMA solution) and DIPEA (5 μ L,100 equivalents, 400mM DMA solution) were mixed and placed in a refrigerator at-20 ℃ for 5 minutes, and then placed in a refrigerator at 4 ℃ for 5 minutes, the mixture was added to the solution of compound1, the reaction mixture was mixed well by vortexing, and the reaction mixture was left at room temperature for 12 hours.
After the reaction is finished: and (5) ethanol precipitation. Adding a 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 the supernatant, and dissolving the rest precipitate with deionized water to obtain the On-DNA aryl iodide compound.
On-DNA aryl iodide 14a (20. mu.L) was dissolved in deionized water to prepare a 1mM aqueous solution, and then thiophenol (10. mu.L, 400 equivalents, 200mM DMA solution), sodium hydroxide (10. mu.L, 500 equivalents, 1M aqueous solution), tetrabutylammonium iodide (10. mu.L, 50 equivalents, 100mM DMA solution) and a solution of premixed CuI (5. mu.L, 50 equivalents, 200mM DMA solution), 1,10-Phen (5. mu.L, 100 equivalents, 400mM DMA solution) were added to the solution in this order, mixed well, and reacted at 80 ℃ for 16 hours.
After the reaction is finished: (1) copper removal treatment: DDTC (20 μ L,100 equiv., 100mM DMA solution) was added, mixed well and left for half an hour; (2) ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding anhydrous ethanol with the volume of 3 times of the total volume, 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 the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound; (3) alcohol washing: and adding 75% ethanol (80 mu L) into the sample treated in the previous step, uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, centrifuging at 12000rpm for half an hour, pouring out the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound with the conversion rate of 62%.
Example 13 Synthesis of On-DNA thioether Compound 15b
Figure BDA0002584805320000151
Compound 1(20 μ L) was dissolved in borate buffer (pH 9.4,250mM) to give an initial concentration of 1mM, carboxylic acid (10 μ L,100 equivalents, 200mM DMA solution), HATU (5 μ L,100 equivalents, 400mM DMA solution) and DIPEA (5 μ L,100 equivalents, 400mM DMA solution) were mixed and placed in a refrigerator at-20 ℃ for 5 minutes, and then stored in a refrigerator at 4 ℃ for 5 minutes, the mixture was added to the solution of compound1, and the reaction mixture was vortexed and mixed thoroughly. The reaction solution was left at room temperature for 12 hours.
After the reaction is finished: ethanol precipitation: adding a 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 the supernatant, and dissolving the rest precipitate with deionized water to obtain the On-DNA aryl iodide compound.
The On-DNA aryl iodide compound 12a (20. mu.L) was dissolved in deionized water to prepare a 1mM aqueous solution, and then thiophenol (10. mu.L, 400 equivalents, 200mM DMA solution), sodium hydroxide (10. mu.L, 500 equivalents, 1M aqueous solution), tetrabutylammonium iodide (10. mu.L, 50 equivalents, 100mM DMA solution) and a solution of premixed CuI (5. mu.L, 50 equivalents, 200mM DMA solution), 1,10-Phen (5. mu.L, 100 equivalents, 400mM DMA solution) were added to the solution in this order, mixed well, and reacted at 80 ℃ for 16 hours.
After the reaction is finished: (1) copper removal treatment: DDTC (20 μ L,100 equiv., 100mM DMA solution) was added, mixed well and left for half an hour; (2) ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution, then continuously adding anhydrous ethanol with the volume of 3 times of the total volume, 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 the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound; (3) alcohol washing: and adding 75% ethanol (80 mu L) into the sample treated in the previous step, uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, centrifuging at 12000rpm for half an hour, pouring out the supernatant, and dissolving the rest precipitate with deionized water to obtain an On-DNA thioether compound with the conversion rate of 52%.

Claims (12)

1. A method for preparing an On-DNA thioether compound from an On-DNA aryl halide is characterized by comprising the following steps: taking an On-DNA aryl halide as a substrate, and carrying out a coupling reaction with thiophenol or mercaptan under the action of a Cu catalyst, a ligand, alkali and an additive to obtain an On-DNA thioether compound, wherein the On-DNA aryl halide has a structural formula of DNA-Ar-X, and the thiophenol or mercaptan has a structural formula of RSH;
wherein the DNA in the structural formula comprises a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and/or unmodified nucleotide monomers, and the nucleotide chain is connected with Ar through one or more chemical bonds or groups; the length of the single-stranded or double-stranded nucleotide chain is 5-200 bp; -X of the On-DNA aryl halide is attached to the ring of Ar, X is chlorine, bromine or iodine;
wherein Ar in the structural formula is selected from an optionally substituted aromatic ring or aromatic heterocycle; r is selected from groups with molecular weight below 1000 and directly connected with sulfydryl.
2. The method of claim 1, wherein Ar is selected from the group consisting of:
Figure FDA0002584805310000011
wherein, the Ar can have one OR more stituents which are hydrogen, halogen, carboxyl, cyano, hydroxyl, alkyl, stituted alkyl, alkoxy, stituted alkoxy, -C (O) OR1、-OC(O)R1、-C(O)R2Any one or more of the random combinations of R1Is selected from C1～C20Alkyl, said R2Selected from hydrogen or C1～C20An alkyl group; the alkyl group is C1-C20Linear or branched alkyl of (a); the alkoxy is C1-C20A straight or branched alkoxy group of (a);
the number of the substituent groups of the substituted alkyl is one or more, and the substituent groups of the substituted alkyl are one or more independently selected from halogen, cyano and carboxyl;
the number of the substituent of the substituted alkoxy is one or more, and the substituent of the substituted alkoxy is one or more independently selected from halogen, cyano and carboxyl.
3. The method of claim 1, wherein R is selected from C1～C20Alkyl or 0 to 5R3A substituted 5-to 12-membered aromatic or heteroaromatic ring; said R3Is any one or more of hydrogen, halogen, carboxyl, cyano, hydroxyl, alkyl, substituted alkyl and alkoxy; the alkyl group is C1-C20Linear or branched alkyl of (a); the alkoxy is C1-C20A straight or branched alkoxy group of (a); the number of the substituents of the substituted alkyl group is one or more; the substituents of the substituted alkyl are independently selected from halogen and carboxyl.
4. The method of claim 1, wherein: the reaction steps are as follows: adding mercaptan or thiophenol, alkali, additive, copper catalyst and ligand into aryl halide solution with molar equivalent of 1 and molar concentration of 0.5-5mM, and reacting at 20-80 deg.C for 1-24 hr until the reaction is finished.
5. The method of claim 4, wherein: the copper catalyst is selected from cuprous iodide, copper acetylacetonate, cuprous oxide, ketone bromide, cuprous bromide, copper trifluoromethanesulfonyl, copper trifluoroacetate, copper sulfate, copper acetate or copper chloride; the ligand is 1, 10-phenanthroline or bipyridine; the alkali is t-Bu4NOH，K3PO4，NaOH，KOH，CsOH，Na2CO3，K2CO3Or Cs2CO3(ii) a The additive is tetra-n-butylammonium iodide or tetra-n-butylammonium bromide.
6. The method of claim 4, wherein: the reaction solution is a mixed solvent containing one or more of water, DMSO, acetonitrile, DMA, acetone, methanol, NMP and THF.
7. The method of claim 4, wherein: the reaction temperature is 40 ℃,50 ℃, 60 ℃, 70 ℃ or 80 ℃.
8. The method of claim 4, wherein: the reaction time is 2 hours, 4 hours, 6 hours, 8 hours, or 16 hours.
9. The method of claim 4, wherein: the molar equivalent of the On-DNA aryl halide is 1, the molar equivalent of the copper catalyst is 5-50, the molar equivalent of the ligand is 10-100, the molar equivalent of the base is 100-1000, the molar equivalent of the thiophenol or the thiol is 50-1000, and the molar equivalent of the additive is 10-100.
10. The method of claim 4, wherein: the feeding sequence of the reaction is that the copper catalyst and the ligand are mixed in advance, mercaptan or thiophenol, alkali and an additive are added into the solution of the On-DNA aryl halide, and finally the mixed solution of the copper catalyst and the ligand is added.
11. The method according to any one of claims 1-10, wherein: the method is used for batch multi-well plate operations.
12. The method according to any one of claims 1-10, wherein: the method is used for the synthesis of libraries of DNA-encoding compounds for multi-well plates.
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