On-DNA arylmethylthio compound and boric acid compound C-C coupling method

Abstract

The invention relates to a method for C-C coupling of an On-DNA (deoxyribonucleic acid) arylmethylthio compound and a boric acid compound in a DNA (deoxyribonucleic acid) coding compound library, which comprises the following steps: taking the On-DNA arylmethylthio compound as a raw material, and carrying out C-C coupling on the On-DNA arylmethylthio compound and the boric acid compound in the presence of alkali, a ligand, a reducing agent and a palladium catalyst. The method for the reaction of the On-DNA arylmethylthio compound and the boric acid compound can be carried out in a mixed phase of an organic solvent / water phase, post-treatment is simple, conditions are mild, diversified DNA coding compound libraries can be obtained in a short time at high yield, the types of C-C coupling substrates are expanded, and the method is suitable for industrial production. And the method is suitable for synthesis of a DNA coding compound by a porous plate.

Description

Technical Field

[0001] The present invention belongs to the technical field of coded compound libraries, and particularly relates to a method for CC coupling of an On-DNA arylmethylthio compound and a boronic acid compound in a DNA coded compound library. Background Art

[0002] In drug development, especially in new drug development, high-throughput screening for biological targets is one of the main means of quickly obtaining lead compounds. However, traditional high-throughput screening based on single molecules requires a long time, huge equipment investment, a limited number of library compounds (millions), and the construction of compound libraries requires decades of accumulation, which limits the efficiency and possibility of discovering lead compounds. In recent years, DNA-encoded compound library technology (WO2005058479, WO2018166532, CN103882532) has emerged. It combines combinatorial chemistry and molecular biology techniques, adds a DNA tag to each compound at the molecular level, and can synthesize up to 100 million compound libraries in a very short time. It has become the trend of next-generation compound library screening technology and has begun to be widely used in the pharmaceutical industry, producing many positive effects (Accounts of Chemical Research, 2014, 47, 1247-1255).

[0003] DNA-encoded compound libraries (DNA-encoded compound libraries) rapidly generate large compound libraries through combinatorial chemistry and can screen for lead compounds at high throughput, making lead compound screening unprecedentedly fast and efficient. One of the challenges in constructing DNA-encoded compound libraries is the need to synthesize chemically diverse small molecules on DNA with high yield. Since DNA requires certain conditions (solvent, pH, temperature, and ion concentration) to maintain stability, the on-DNA reactions used to construct DNA-encoded compound libraries also need to have high yields. Therefore, the reagent types, reaction types, and reaction conditions of the chemical reactions performed on DNA (referred to as on-DNA reactions) directly affect the richness and selectivity of DNA-encoded compound libraries. Therefore, developing chemical reactions compatible with DNA has become a long-term exploration and research direction of DNA-encoded compound library technology, directly affecting the application and commercial value of DNA-encoded compound libraries.

[0004] Substituted arylmethylthio compounds can be obtained by CC coupling reactions between arylmethylthio compounds and boronic acid compounds. This can enrich the topological structures of drug compounds and expand the substrate types for CC coupling. Introducing these arylmethylthio groups into DNA-encoded compound libraries can further expand the diversity of the compound library and improve the probability of screening effective compounds. However, there are currently no reported methods for synthesizing on-DNA substituted arylmethylthio compounds from on-DNA arylmethylthio compounds. Therefore, it is desirable to develop a novel synthesis method for on-DNA substituted arylmethylthio compounds that is suitable for large-scale multiwell plate processing, thereby increasing the diversity of DNA-encoded compound libraries and further enhancing the application value of DNA-encoded compound library technology. Summary of the Invention

[0005] The present invention provides a method for CC coupling of an On-DNA arylmethylthio compound and a boronic acid compound, characterized in that: the method uses the On-DNA arylmethylthio compound as a raw material, reacts with the boronic acid compound in the presence of a base, a ligand, a reducing agent, and a palladium catalyst to obtain an On-DNA product; wherein the structural formula of the On-DNA arylmethylthio compound is The structural formula of the boric acid compound is: The structural formula of the On-DNA product is

[0006] In the structural formula, the DNA comprises a single-stranded or double-stranded nucleotide chain obtained by polymerization of artificially modified and / or unmodified nucleotide monomers, and the nucleotide chain is connected to the remaining part of the compound through one or more chemical bonds or groups; the length of the DNA is 10 to 200 bp.

[0007] In the structural formula, DNA and L are linked via one or more chemical bonds. A single chemical bond refers to a direct link between DNA and L; multiple chemical bonds refer to a link between DNA and L separated by multiple chemical bonds. For example, DNA and L are linked via a methylene group (-CH2-), meaning they are linked via two chemical bonds; or DNA and L are linked via a carbonyl group (-CO-) to the amino group of DNA, also via two chemical bonds; or DNA and L are linked via a methylene carbonyl group (-CH2CO-) to the amino group of DNA, also via three consecutive chemical bonds.

[0008] The L is selected from -(L Y ) q -; q is an integer from 1 to 30; each L Yare independently selected from CH2, C(O), O, S, S(O), S(O)2, NH, 3-10 membered cycloalkyl, 3-10 membered heterocycloalkyl, 6-10 membered aromatic ring, 5-10 membered aromatic heterocycle; or L is absent; the absence of L means that DNA and R are directly linked by a covalent bond;

[0009] R is selected from 4-10 membered heterocycloalkyl, 6-10 membered aromatic ring, 5-10 membered aromatic heterocycle; the aromatic ring, aromatic heterocycle, heterocycloalkyl is optionally replaced by one, two or three independent R 1a replace;

[0010] Each R 1a are independently selected from hydrogen, halogen, cyano, nitro, hydroxyl, -C 1～6 Alkyl, halogen-substituted -C 1～6 Alkyl, -C 0～2 Alkylene-OH, -C 0～2 Alkylene-O(C 1～6 alkyl);

[0011] R 1 、R 2 are independently selected from hydrogen, -C 1～6 alkyl;

[0012] R 3 Selected from -C 2～6 alkenyl, 3-10 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aromatic ring, 5-10 membered aromatic heterocycle; the alkenyl, cycloalkyl, heterocycloalkyl, aromatic ring, aromatic heterocycle are optionally substituted by one, two or three independent R 1c replace;

[0013] Each R 1c are independently selected from hydrogen, halogen, cyano, nitro, hydroxyl, -C 1～6 Alkyl, halogen-substituted -C 1～6 Alkyl, -C 0～2 Alkylene-OH, -C 0～2 Alkylene-O(C 1～6 Alkyl), -OC 0～2 Alkylene-(5-10 membered aromatic ring), -C 0～2 Alkylene-(3-10 membered cycloalkyl), -C 0～2 Alkylene-(4- to 10-membered heterocycloalkyl);

[0014] Or, R 1 、R 2 The atoms directly connected to it together form a 5-10 membered heterocyclic ring; the heterocyclic ring is optionally replaced by one, two or three independent R 1b replace;

[0015] Or, R 1、R 3 The atoms directly connected to it together form a 5-10 membered heterocyclic ring; the heterocyclic ring is optionally replaced by one, two or three independent R 1b replace;

[0016] Each R 1b are independently selected from hydrogen, halogen, cyano, nitro, hydroxyl, -C 1～6 Alkyl, halogen-substituted -C 1～6 Alkyl, -C 0～2 Alkylene-OH, -C 0～2 Alkylene-O(C 1～6 alkyl).

[0017] As a preference: said L is selected from

[0018] Preferably, the R is selected from a 6-membered aromatic heterocycle, a 9-membered aromatic heterocycle, a 10-membered aromatic heterocycle, or a 9-membered heterocycloalkyl group; the aromatic heterocycle or heterocycloalkyl group is optionally replaced by one, two, or three independent R 1a replace;

[0019] Each R 1a Each is independently selected from hydrogen, halogen, methyl, -C1 alkylene-OH.

[0020] More specifically: said R is selected from

[0021] As a preference: the On-DNA arylmethylthio compound is specifically selected from:

[0022] As a preference: R 1 、R 2 are each independently selected from hydrogen;

[0023] R 3 is selected from vinyl, 6-membered cycloalkyl, 9-membered heterocycloalkyl, benzene ring, 10-membered aromatic ring, 5-membered aromatic heterocycle, 6-membered aromatic heterocycle, 9-membered aromatic heterocycle; the alkenyl, cycloalkyl, heterocycloalkyl, aromatic ring, aromatic heterocycle are optionally substituted by one, two or three independent R 1c replace;

[0024] Each R 1c Each of them is independently selected from hydrogen, methyl, ethyl, isopropyl, methoxy, trifluoromethyl, -O-C1 alkylene-(6-membered aromatic ring), 3-membered cycloalkyl, and 4-membered cycloalkyl.

[0025] Or, R 1 、R 2 The atoms directly connected to it together form a 5-membered heterocyclic ring; the heterocyclic ring is optionally replaced by one, two or three independent R 1breplace;

[0026] Or, R 1 、R 3 The atoms directly connected to it together form a 9-membered heterocyclic ring; the heterocyclic ring is optionally replaced by one, two or three independent R 1b replace;

[0027] Each R 1b are independently selected from hydrogen, methyl, ethyl, propyl, and isopropyl.

[0028] Specifically: the R 3 Selected from

[0029] As a preference: the boric acid compound is selected from:

[0030]

[0031] At the same time, the present invention provides a method for constructing a CC coupling between an On-DNA arylmethylthio compound and a boronic acid compound, comprising the following steps: adding 10-1000 times the molar equivalent of the boronic acid compound, 10-1000 times the molar equivalent of the base, 10-100 times the molar equivalent of the ligand, 10-100 times the molar equivalent of the reducing agent, and 1-10 times the molar equivalent of the palladium catalyst to an On-DNA arylmethylthio compound solution with a molar equivalent of 1 and a molar concentration of 0.5-5 mM, and reacting at 10°C to 100°C for 0.5-24 hours.

[0032] Furthermore, the base is selected from sodium borate, lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, sodium phosphate, potassium phosphate, sodium hydrogen phosphate, potassium hydrogen phosphate, N-methylmorpholine, triethylamine, diisopropylethylamine, DBU (1,8-diazabicycloundec-7-ene), 4-dimethylaminopyridine, 2,6-lutidine, N-methylimidazole; preferably, the base is cesium hydroxide.

[0033] Furthermore, the palladium catalyst of the reaction is Pd2(dba)3, tetrakistriphenylphosphine palladium, palladium acetate, palladium trifluoroacetate, elemental palladium (palladium black), sSPhosPd-G2; preferably, the catalyst is sSPhos-Pd-G2.

[0034] Furthermore, the ligand required for the palladium catalyst is selected from triphenylphosphine, triaryl-substituted phosphine, tricyclohexylphosphine, trialkyl-substituted phosphine, monoaryldialkylphosphine and other commonly used phosphine ligands for palladium catalysis.

[0035] Furthermore, the reducing agent is Na2SO3, FeSO4, sodium ascorbyl palmitate, tetrahydroxydiboron, sodium borohydride, or sodium cyanoborohydride; preferably, the reducing agent is sodium ascorbyl palmitate.

[0036] Furthermore, the sulfur ligand required for the methylthio group is selected from sulfur ligands such as copper thiophene-2-carboxylate, cuprous iodide, cuprous chloride, cuprous bromide, cuprous sulfate, and cuprous cyanide. Preferably, the sulfur ligand is copper thiophene-2-carboxylate.

[0037] Furthermore, the reaction is carried out in a solvent, which is any one or a mixed solvent of water, methanol, ethanol, acetonitrile, dimethyl sulfoxide, dimethylacetamide, inorganic salt buffer, organic acid buffer, and organic base buffer; preferably, the reaction solvent contains water and dimethylacetamide solution.

[0038] Furthermore, the reaction temperature of the reaction is 10°C to 100°C; preferably, the reaction temperature is 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, or 100°C.

[0039] Furthermore, the reaction time is 0.5 to 24 hours; preferably, the reaction time is 1 hour, 2 hours, 4 hours, 8 hours, 10 hours, 16 hours, 18 hours, or 20 hours.

[0040] Furthermore, in the method, the molar equivalent of the On-DNA arylmethylthio compound is 1, the molar equivalent of the boronic acid compound is 10-1000, and the molar equivalent of the base is 10-1000; preferably, the equivalent of the boronic acid compound is 50, 100, 150, 200, 300, 400, 500, 600, 800, 1000, and the equivalent of the base is 50, 100, 200, 300, 400, 500, 600, 800, 1000; the molar equivalent of the ligand is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100; the molar equivalent of the reducing agent is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100; and the molar equivalent of the palladium catalyst is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0041] Most preferably, the molar equivalent of the boronic acid compound is 150, the molar equivalent of the base is 100, the molar equivalent of the ligand is 20, the molar equivalent of the reducing agent is 20, and the molar equivalent of the palladium catalyst is 2.

[0042] Furthermore, the order of adding materials in the reaction is to first add the On-DNA arylmethylthio compound, then add the boric acid compound, base, ligand and reducing agent, and finally add the palladium catalyst.

[0043] Furthermore, the method is used for batch multi-well plate operations.

[0044] Furthermore, the method is used for the synthesis of DNA-encoded compound libraries in multi-well plates.

[0045] The method of the present invention enables CC coupling reactions between On-DNA arylmethylthio compounds and boronic acid compounds in DNA-encoded compound libraries. It can be widely applied to various On-DNA arylmethylthio backbone compounds, increasing the diversity of pyrimidine- and pyridine-based backbones and enabling the large-scale introduction of various substituted boronic acid compounds as modules. The method offers high yield, produces a single product, can be performed in an organic solvent / aqueous mixed aqueous phase, is simple to operate, is environmentally friendly, and is suitable for the synthesis of DNA-encoded compound libraries using multiwell plates.

[0046] Definitions of terms used in the present invention: Unless otherwise stated, the initial definitions provided for groups or terms in this document apply to the groups or terms throughout the specification; for terms that are not specifically defined herein, they should be given the meaning that a person skilled in the art would give them based on the disclosure and context.

[0047] "Substitution" means that the hydrogen atoms in a molecule are replaced by other different atoms or groups; or the lone pair of electrons in the atoms in the molecule are replaced by other atoms or groups. For example, the lone pair of electrons on the S atom can be replaced by an O atom to form

[0048] "Optionally substituted" means that "substitution" may but need not occur, and the description includes instances where it occurs and instances where it does not occur.

[0049] The minimum and maximum carbon atom content in the hydrocarbon group is indicated by a prefix, for example, the prefix (Ca~C b )alkyl refers to any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, C1-C 20 The alkyl group refers to a straight-chain or branched alkyl group containing 1 to 20 carbon atoms.

[0050] "Alkyl" refers to a saturated hydrocarbon chain having a specified number of member atoms. Alkyl groups can be straight or branched. Representative branched alkyl groups have one, two, or three branches. Alkyl groups may optionally be substituted with one or more substituents as defined herein. Alkyl groups include methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, and tert-butyl), pentyl (n-pentyl, isopentyl, and neopentyl), and hexyl. Alkyl groups may also be part of other groups, such as -O(C 1～6 alkyl).

[0051] "Alkylene" refers to a divalent saturated aliphatic hydrocarbon radical having the specified number of member atoms.a ~ b Alkylene refers to an alkylene group having a to b carbon atoms. Alkylene groups include branched and straight chain hydrocarbon groups. For example, the term "propylene" can be exemplified by the following structure: Likewise, the term "dimethylbutylene" can be exemplified, for example, by any of the following structures:

[0052] The -C0~4 alkylene group of the present invention can be a C0 alkylene group, a C1 alkylene group (e.g., -CH2-), a C2 alkylene group (e.g., -CH2CH2-, etc.), a C3 alkylene group or a C4 alkylene group; the C0 alkylene group means that the group here does not exist and is connected in the form of a chemical bond, such as A-C0 alkylene-B means AB, that is, the A group and the B group are directly connected by a chemical bond.

[0053] The unsaturated group mentioned in the present invention refers to a group or molecule containing a carbon-carbon double bond, a carbon-carbon triple bond, a carbon-oxygen double bond, a carbon-sulfur double bond, a carbon-nitrogen triple bond, etc.

[0054] "Alkenyl" refers to a straight or branched chain hydrocarbon group having at least one site of vinyl unsaturation (>C=C<). For example, C a-b Alkenyl refers to an alkenyl group having a to b carbon atoms and is intended to include, for example, ethenyl, propenyl, isopropenyl, 1,3-butadienyl, and the like.

[0055] "Alkynyl" refers to a straight chain monovalent hydrocarbon radical or a branched monovalent hydrocarbon radical containing at least one triple bond. The term "alkynyl" is also intended to include those hydrocarbon radicals having one triple bond and one double bond. For example, C 2-6 Alkynyl is meant to include ethynyl, propynyl, and the like.

[0056] "Cycloalkyl" and "cycloalkane" refer to saturated or partially saturated cyclic groups having carbon atoms and no ring heteroatoms and having a single ring or multiple rings (including fused and combined). For polycyclic ring systems with aromatic and non-aromatic rings without ring heteroatoms, the term "cycloalkyl" is applicable when the point of attachment is at a non-aromatic carbon atom (e.g., 5,6,7,8,-tetrahydronaphthalene-5-yl). The term "cycloalkyl" includes cycloalkenyl groups such as cyclohexenyl. Examples of cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl, cyclooctyl, cyclopentenyl, and cyclohexenyl. Examples of cycloalkyl groups including polybicycloalkyl ring systems are bicyclohexyl, bicyclopentyl, bicyclooctyl, etc. For example

[0057] "Heterocycle", "heterocycloalkyl" and "heterocycloalkane" refer to a saturated ring or non-aromatic unsaturated cyclic group having a single ring or multiple rings (including fused and combined) containing at least one heteroatom; wherein the heteroatom refers to a nitrogen atom, an oxygen atom, or a sulfur atom;

[0058] "Aryl" and "aromatic ring" are used interchangeably to refer to an all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group with a conjugated π electron system, such as "C 6-10 The term "aryl" refers to a monocyclic or bicyclic aromatic group having 6 to 10 carbon atoms. Non-limiting examples of aryl include phenyl, naphthyl, and the like.

[0059] The "aromatic heterocycle" described in the present invention refers to an aromatic unsaturated ring containing at least one heteroatom; wherein the heteroatom refers to a nitrogen atom, an oxygen atom, a sulfur atom, etc. It is usually an aromatic monocyclic or bicyclic hydrocarbon containing multiple ring atoms, wherein one or more ring atoms are selected from O, N, and S heteroatoms. Preferably, there are one to three heteroatoms. Heterocyclic aromatic groups are represented by pyridyl, indolyl, quinoxalinyl, quinolyl, isoquinolyl, benzothiophenyl, benzofuranyl, benzothiophenyl, benzopyranyl, benzothiapyranyl, furyl, pyrrolyl, thiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, oxadiazolyl, benzimidazolyl, benzothiazolyl, benzoxazolyl

[0060] The "aromatic heterocycle" described in the invention also includes aromatic heterocycle and saturated heterocycle or saturated ring, for example:

[0061] The halogen is fluorine, chlorine, bromine or iodine.

[0062] The "halogen-substituted alkyl" mentioned in the present invention refers to an alkyl group in which one or more hydrogen atoms are replaced by halogen; for example, a halogen-substituted C 1～4 The alkyl group refers to an alkyl group containing 1 to 4 carbon atoms in which hydrogen atoms are substituted by one or more halogen atoms; examples include monofluoromethyl, difluoromethyl, and trifluoromethyl.

[0063] Alkoxy: refers to an alkyl group connected to an oxygen atom to form a substituent, for example, methoxy is -OCH3.

[0064] The "-OR", "-NRR" and the like described in the present invention refer to that the R group is connected to the oxygen atom or nitrogen atom via a single bond.

[0065] The oxygen atom in "-C(O)R", "-S(O)2R" and the like described in the present invention is connected to the carbon atom or the sulfur atom via a double bond.

[0066] In the present invention, the oxygen atom in "-C(O)R", "-S(O)2R", etc. is connected to the carbon atom or sulfur atom by a double bond, and the R group is connected to the oxygen atom or sulfur atom by a single bond. For example, "-S(O)(NH)R" means that the oxygen atom and the nitrogen atom are connected to the sulfur atom by a double bond, and the R group is connected to the sulfur atom by a single bond.

[0067] The present invention It means that the oxygen atom and sulfur atom are connected to the substitution position through a double bond.

[0068] The “---” in the description of the group of the present invention, It is used to describe the position of the substitution group. For example It means that the tetrahydropyrrole ring is fused to other rings in the structure through the "" position.

[0069] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0070] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 : The corresponding conversion rate distribution diagram of 16 On-DNA compounds obtained in Example 2 of the present invention.

[0072] Figure 2 : The corresponding conversion rate distribution diagram of 10 On-DNA compounds obtained in Example 3 of the present invention. DETAILED DESCRIPTION

[0073] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0074] In the present invention, DNA-NH2 is a DNA structure with an -NH2 linker formed by single-stranded or double-stranded DNA and a linker group, such as the DNA-NH2 structure of "compound 1" in WO2005058479. For example, the following DNA structure:

[0075]

[0076] Among them, A is adenine, T is thymine, C is cytosine, and G is guanine.

[0077] DIPEA: N,N-diisopropylethylamine; DIC: N,N'-diisopropylcarbodiimide; HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; DMA: dimethylacetamide; DMSO: dimethyl sulfoxide; DDTC: sodium diethyldithiocarbamate. Pd(dppf)Cl2: 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride. sSPhosPd G2: sodium chloro(2-dicyclohexylphosphino-2',6'-dimethoxy-1,1'-biphenyl-3'-sulfonate)[2-(2'-amino-1,1'-biphenyl)]palladium(II).

[0078] Example 1: Synthesis of On-DNA Arylmethylthio Compounds

[0079] Step 1. Synthesis of On-DNA Arylmethylthio Compounds

[0080]

[0081] Dissolve 1 in 250 mM borate buffer (pH 9.4) to a 1 mM concentration. Next, mix 50 equivalents of HATU (0.4 M in DMA), 50 equivalents of carboxymethylthio reagent (0.4 M in DMA), and 50 equivalents of DIPEA (0.4 M in DMA) at 0°C. Vortex the mixture thoroughly and store at 0°C for 5 minutes. Add the above mixture to the HP 1 solution, mix thoroughly, and react at room temperature for 0.5-1 hour.

[0082] After the reaction, ethanol precipitation was performed: 10% of the total volume of 5 M sodium chloride solution was added to the reaction solution, followed by the addition of 3 times the total volume of anhydrous ethanol. After vortexing, the reaction mixture was placed on dry ice for 0.5 hours, and then centrifuged at 12000 rpm for half an hour at low temperature (-4°C). The supernatant was discarded and the remaining precipitate was dissolved in deionized water to obtain a solution of On-DNA arylmethylthio compound 2. After quantification by OD on a microplate reader, LCMS was used to confirm that the conversion rate of compound 2 was 85%.

[0083] Step 2: On-DNA aryl-substituted methylthio compounds

[0084]

[0085] On-DNA arylmethylthio compound 2 was prepared into a 1 mM concentration solution (20 μL, 20 nmol) with deionized water, and phenylboric acid (3000 nmol, 150 eq, 200 mM DMA solution), cesium hydroxide (2000 nmol, 100 eq, 500 mM dd-H2O solution), copper thiophene-2-carboxylate (40 nmol, 20 eq, 10 mM DMA solution), sodium ascorbyl palmitate (400 nmol, 20 eq, 100 mM dd-H2O solution), and sSPhos-Pd-G2 (40 nmol, 2 eq, 10 mM DMA solution) were added to the solution in sequence, mixed well, and reacted at 80°C for 1 hour.

[0086] After the reaction, DDTC (800 nmol, 40 eq, 200 mM dd-H2O solution) was added to the reaction system, mixed evenly, and reacted at 80°C for 10 minutes. After the reaction, the mixture was centrifuged at 12000 rpm for 20 minutes at low temperature (-4°C), the supernatant was removed, 10% of the total volume of 5M sodium chloride solution was added to the supernatant, and then 3 times the total volume of anhydrous ethanol was added. After shaking evenly, the reaction was placed in dry ice for 0.5 hours, and then centrifuged at 12000 rpm for half an hour at low temperature (-4°C). The supernatant was discarded and the remaining precipitate was dissolved in deionized water to obtain a solution of On-DNA aryl-substituted methylthio compound 3. After quantification by OD on a microplate reader, LCMS was sent to confirm that the conversion rate of compound 3 was 80%.

[0087] Example 2: Synthesis of On-DNA Arylmethylthio Compounds

[0088] React the On-DNA arylmethylthio compound 2 with 16 boronic acid compounds respectively:

[0089]

[0090] On-DNA arylmethylthio compound 2 was prepared into a 1 mM concentration solution (20 μL, 20 nmol) with deionized water, and boric acid compound (3000 nmol, 150 eq, 200 mM DMA solution), cesium hydroxide (2000 nmol, 100 eq, 500 mM dd-H2O solution), copper thiophene-2-carboxylate (40 nmol, 20 eq, 10 mM DMA solution), sodium ascorbyl palmitate (400 nmol, 20 eq, 100 mM dd-H2O solution), and sSPhos-Pd-G2 (40 nmol, 2 eq, 10 mM DMA solution) were added to the solution in sequence, mixed well, and reacted at 80°C for 1 hour.

[0091] After the reaction, DDTC (800 nmol, 40 eq, 200 mM dd-H2O solution) was added to the reaction system, mixed evenly, and reacted at 80°C for 10 minutes. After the reaction, the mixture was centrifuged at 12000 rpm for 20 minutes at low temperature (-4°C), the supernatant was removed, 10% of the total volume of 5M sodium chloride solution was added to the supernatant, and then 3 times the total volume of anhydrous ethanol was added. After shaking evenly, the reaction was placed in dry ice and frozen for 0.5 hours, and then centrifuged at 12000 rpm for half an hour at low temperature (-4°C), the supernatant was discarded, and the remaining precipitate was dissolved in deionized water to obtain a solution of 16 On-DNA product compounds. After quantification by OD on an enzyme-linked microplate reader, the conversion rate of the compound was confirmed by LCMS. The conversion rate of the 16 On-DNA product structures can be found in [1]. Figure 1 .

[0092] Example 3: Synthesis of On-DNA Arylmethylthio Compounds

[0093]

[0094] Ten On-DNA aromatic methylthio compounds were prepared into 1 mM concentration solutions (20 μL, 20 nmol) with deionized water, and phenylboronic acid (3000 nmol, 150 eq, 200 mM DMA solution), cesium hydroxide (2000 nmol, 100 eq, 500 mM dd-H2O solution), copper thiophene-2-carboxylate (40 nmol, 20 eq, 10 mM DMA solution), sodium ascorbyl palmitate (400 nmol, 20 eq, 100 mM dd-H2O solution), and sSPhos-Pd-G2 (40 nmol, 2 eq, 10 mM DMA solution) were added to the solutions in sequence, mixed well, and reacted at 80°C for 1 hour.

[0095] After the reaction, DDTC (800 nmol, 40 eq, 200 mM dd-H2O solution) was added to the reaction system, mixed evenly, and reacted at 80°C for 10 minutes. After the reaction, the mixture was centrifuged at 12000 rpm for 20 minutes at low temperature (-4°C), the supernatant was removed, 10% of the total volume of 5M sodium chloride solution was added to the supernatant, and then 3 times the total volume of anhydrous ethanol was added. After shaking evenly, the reaction was placed in dry ice and frozen for 0.5 hours, and then centrifuged at 12000 rpm for half an hour at low temperature (-4°C), the supernatant was discarded, and the remaining precipitate was dissolved in deionized water to obtain a solution of 10 On-DNA product compounds. After quantification by OD on an enzyme reader, the conversion rate of the compound was confirmed by LCMS. The conversion rate of the 10 On-DNA products can be found in Figure 2 .

[0096] This method showed good conversion rates in the reaction of one arylmethylthio compound and 16 boronic acids or ten arylmethylthio compounds and one boronic acid.

[0097] In summary, the present invention enables CC coupling of On-DNA arylmethylthio compounds with boronic acid compounds in the presence of a base by controlling reaction conditions such as solvent, temperature, and pH to produce On-DNA substituted arylmethylthio compounds. This method has a wide range of substrate applicability, can be performed in mixed organic solvent / aqueous aqueous phases, is simple to operate, is environmentally friendly, and is suitable for the synthesis of DNA-encoded compound libraries using multiwell plates.

[0098] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

Claims

1. A method for CC coupling of an On-DNA arylmethylthio compound and a boronic acid compound, characterized in that: The method uses an On-DNA arylmethylthio compound as a raw material, and reacts it with a boric acid compound in the presence of a base, a ligand, a reducing agent, and a palladium catalyst to obtain an On-DNA product; wherein the On-DNA arylmethylthio compound has the structural formula: The structural formula of the boric acid compound is: The structural formula of the On-DNA product is Wherein, the DNA in the structural formula comprises a single-stranded or double-stranded nucleotide chain obtained by polymerization of artificially modified and / or unmodified nucleotide monomers, and the nucleotide chain is connected to the rest of the compound through one or more chemical bonds or groups; The L is selected from -(L Y ) q -; q is an integer from 1 to 30; each L Y are independently selected from CH2, C(O), O, S, S(O), S(O)2, NH, 3-10 membered cycloalkyl, 3-10 membered heterocycloalkyl, 6-10 membered aromatic ring, 5-10 membered aromatic heterocycle; or L is absent; R is selected from 4-10 membered heterocycloalkyl, 6-10 membered aromatic ring, 5-10 membered aromatic heterocycle; the heterocycloalkyl, aromatic ring, aromatic heterocycle are optionally replaced by one, two or three independent R 1a replace; Each R 1a are independently selected from hydrogen, halogen, cyano, nitro, hydroxyl, -C 1～6 Alkyl, halogen-substituted -C 1～6 Alkyl, -C 0～2 Alkylene-OH, -C 0～2 Alkylene-O(C 1～6 alkyl); R 1 、R 2 are independently selected from hydrogen, -C 1～6 alkyl; R 3 Selected from -C 2～6 alkenyl, 3-10 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aromatic ring, 5-10 membered aromatic heterocycle; the alkenyl, cycloalkyl, heterocycloalkyl, aromatic ring, aromatic heterocycle are optionally substituted by one, two or three independent R 1c replace; Each R 1c are independently selected from hydrogen, halogen, cyano, nitro, hydroxyl, -C 1～6 Alkyl, halogen-substituted -C 1～6 Alkyl, -C 0～2 Alkylene-OH, -C 0～2 Alkylene-O(C 1～6 Alkyl), -OC 0～2 Alkylene-(5-10 membered aromatic ring), -C 0～2 Alkylene-(3-10 membered cycloalkyl), -C 0～2 Alkylene-(4- to 10-membered heterocycloalkyl); Or, R 1 、R 2 The atoms directly connected to it together form a 5-10 membered heterocyclic ring; the heterocyclic ring is optionally replaced by one, two or three independent R 1b replace; Or, R 1 、R 3 The atoms directly connected to it together form a 5-10 membered heterocyclic ring; the heterocyclic ring is optionally replaced by one, two or three independent R 1b replace; Each R 1b are independently selected from hydrogen, halogen, cyano, nitro, hydroxyl, -C 1～6 Alkyl, halogen-substituted -C 1～6 Alkyl, -C 0～2 Alkylene-OH, -C 0～2 Alkylene-O(C 1～6 alkyl).

2. The method according to claim 1, wherein: The reaction method comprises the following steps: adding 10-1000 times the molar equivalent of a boric acid compound, 10-1000 times the molar equivalent of a base, a ligand, a reducing agent, and a palladium catalyst to an On-DNA arylmethylthio compound solution with a molar equivalent of 1 and a molar concentration of 0.5-5 mM, and reacting at 10° C. to 100° C. for 0.5-24 hours.

3. The method according to claim 2, wherein: The base is selected from sodium borate, lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, sodium phosphate, potassium phosphate, sodium hydrogen phosphate, potassium hydrogen phosphate, N-methylmorpholine, triethylamine, diisopropylethylamine, DBU (1,8-diazabicycloundec-7-ene), 4-dimethylaminopyridine, 2,6-lutidine or N-methylimidazole.

4. The method according to claim 2, wherein: The palladium catalyst is selected from Pd2(dba)3, tetrakistriphenylphosphine palladium, palladium acetate, palladium trifluoroacetate, elemental palladium or sSPhos-Pd-G2.

5. The method according to claim 2, wherein: The reducing agent is selected from Na2SO3, FeSO4, sodium ascorbyl palmitate, tetrahydroxydiboron, sodium borohydride or sodium cyanoborohydride.

6. The method according to claim 2, wherein: The ligand is selected from copper thiophene-2-carboxylate, cuprous iodide, cuprous chloride, cuprous bromide, cuprous sulfate or cuprous cyanide.

7. The method according to claim 2, wherein: The reaction is carried out in a solvent, which is any one or a mixed solvent of water, methanol, ethanol, acetonitrile, dimethyl sulfoxide, dimethylacetamide, inorganic salt buffer, organic acid buffer, and organic base buffer.

8. The method according to claim 2, wherein: The reaction temperature of the reaction is 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, and 100°C.

9. The method according to claim 2, wherein: The reaction time of the reaction is 1 hour, 2 hours, 4 hours, 8 hours, 10 hours, 16 hours, 18 hours and 20 hours.

10. The method according to claim 2, wherein: In the method, the molar equivalent of the On-DNA arylmethylthio compound is 1, the molar equivalent of the boronic acid compound is 50, 100, 150, 200, 300, 400, 500, 600, 800, 1000, the molar equivalent of the base is 50, 100, 200, 300, 400, 500, 600, 800, 1000; the molar equivalent of the ligand is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100; the molar equivalent of the reducing agent is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100; and the molar equivalent of the palladium catalyst is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

11. The method according to any one of claims 1 to 10, characterized in that The method is used for batch multiwell plate operations.

12. The method according to any one of claims 1 to 10, characterized in that The method is used for the synthesis of DNA-encoded compound libraries in multi-well plates.

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