A method for synthesizing On-DNA aryl alkyne lead compounds

Abstract

The present invention provides a method for synthesizing an On-DNA aryl alkyne lead compound, and belongs to the field of gene-encoded compound library construction. The method uses an oligonucleic acid-terminal alkyne compound and an arylboronic acid as raw materials, and reacts in the presence of a copper catalyst and a base to obtain an On-DNA aryl alkyne lead compound. The method for synthesizing an On-DNA aryl alkyne lead compound of the present invention has low cost, little damage to DNA, good universality, simple operation, mild conditions, and can obtain On-DNA aryl alkyne compounds in high yield, and has broad application prospects in constructing a gene-encoded compound library.

Description

Technical Field

[0001] The invention belongs to the field of gene encoding compound library construction, and specifically relates to a method for synthesizing On-DNA aryl alkyne lead compounds. Background Art

[0002] In drug development, high-throughput screening for biological targets helps to quickly obtain lead compounds, but traditional high-throughput screening based on single molecules has the disadvantages of limited number of compound libraries, long time consumption, and high cost. This limits the discovery of lead compounds to a certain extent. In 1992, Brenner and Lerner creatively proposed a method for using gene-encoded compound library technology (DELT) for biological activity screening. It combines the characteristics of combinatorial chemistry and molecular biology technology. By adding a special technology of a DNA tag to each compound, the combination-split method (Split and pool) is used to obtain a compound library of up to 100 million in a short time. DELT not only breaks through the high cost bottleneck of traditional high-throughput screening, but also achieves a leap in the chemical structure, space and quantity of the screened compounds, and uses a new biological screening mode similar to phenotypic screening. Compared with traditional high-throughput screening, the gene-encoded compound library greatly increases the number of compounds and the diversity of chemical structure space.

[0003] One of the main tasks of gene-encoded compound library technology is the development of chemical reactions on DNA (referred to as On-DNA chemical reactions). At present, some On-DNA chemical reactions have been reported. For example, Shanghai WuXi AppTec New Drug Development Co., Ltd. (Chinese patent application with application number CN201910609569.0) discloses a method for obtaining On-DNA aromatic compounds through Suzuki coupling reaction: using On-DNA aromatic halides as substrates, in the presence of Pd catalysts, ligands and bases, reacting with organic potassium trifluoroborate reagents to obtain On-DNA aromatic compounds. This method increases the diversity of DNA-encoded compound libraries of On-DNA aromatic halides, has high reaction yields, wide substrate universality, mild conditions, and convenient operation, and is suitable for the synthesis of DNA-encoded compound libraries in multi-well plates. Chengdu Pioneer Pharmaceutical Development Co., Ltd. (Chinese patent application with application number CN201910590679.7) discloses a method for synthesizing On-DNA aryl benzyl substituted compounds, which uses On-DNA aldehyde compounds as raw materials, reacts with indole under alkaline conditions to generate On-DNA indole alcohol compounds, and then reduces the On-DNA indole alcohol compounds to indole alkylated compounds by 1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylic acid diethyl ester under acidic conditions. The more types of chemical reactions that can be achieved on the gene-encoded compound library, the richer the conditions, the more choices there are when designing and synthesizing the gene-encoded compound library, and the richer the diversity of the resulting compound library. However, the types of On-DNA chemical reactions currently reported are limited, and they cannot meet the broad demand for the development of lead compound discovery.

[0004] Aryl alkyne compounds refer to a class of compounds containing directly connected aromatic and alkyne structures. These compounds are important raw materials or intermediates in organic synthesis. At the same time, studies have found that aryl alkyne compounds have a variety of biological activities. For example, the compound TolaSure (BM-3103) is a wound healing drug for acute induced wounds caused by punch biopsy being developed by BioMendics in the United States (Related literature: Safety and Efficacy of Topical TolaSure Targeting Aggregated Mutant Keratin in Severe Epidermolysis Bullosa Simplex (NCT05062070) ClinicalTrials.gov Web Site 2021, October 04); Another example is compound X, which is a drug with antimalarial properties and anticancer activity discovered by the Central Research Institute of Medicinal Aromatic Plants in India (Related literature: Kumari, P.; Misra, K.; Sisodia, BS; et al. Planta Med 2009, 75 (1), 59).

[0005]

[0006] The Chinese patent application with application number CN201910609568.6 discloses a method for preparing On-DNA aryl alkyne compounds in the construction of a DNA-encoded compound library, and the route is as follows. The method reacts an On-DNA aryl terminal alkyne compound with a small molecule aryl bromide in the presence of diisopropylamine and tetrakistriphenylphosphine palladium in a mixed solvent of ultrapure water and dimethylacetamide at 65°C for 3 hours to obtain an On-DNA aryl alkyne compound. However, on the one hand, the method uses an expensive and rare palladium catalyst, which increases the preparation cost; on the other hand, the method needs to be heated to 65°C. Studies have shown that the palladium catalyst itself also has a certain degree of damage to DNA, and this degree of damage will be further deepened under heating conditions (Malone, MLand Paegel, BMACS Comb. Sci. 2016, 18, 182-187.), so the method increases the risk of DNA chain damage. Once the DNA chain is damaged, the subsequent amplification work will not be completed, so that the On-DNA aryl alkyne compound loses its application value in the construction of a gene-encoded compound library. In fact, it is precisely because of the differences between DNA chemistry and ordinary chemical reactions that many ordinary small molecule chemical reactions have harsh conditions and can easily cause DNA denaturation and destroy DNA activity, which to a certain extent limits the construction and development of DNA-encoded compound libraries.

[0007]

[0008] The Chinese patent application with application number CN202210032980.8 also discloses a method for synthesizing aryl alkyne lead compounds, and the route is as follows. The method first uses iodinated alkynes as the starting material to prepare oligonucleic acid-iodinated alkynes, and then reacts the oligonucleic acid-iodinated alkynes with a boric acid reagent in the presence of a copper catalyst and a base at 25°C for 24 hours to obtain an On-DNA aryl alkyne lead compound. Although this method does not use expensive and rare palladium catalysts and reduces the reaction temperature, on the one hand, the raw material iodinated alkynes used in this method are not easy to buy and need to be prepared by themselves, which increases time and economic costs; on the other hand, the raw material iodinated alkynes used in this method have poor stability, are easy to lose iodine or hydrolyze, and are only suitable for reactions under mild conditions. The scope of application is limited, which limits the diversity of On-DNA aryl alkyne compounds that can be synthesized.

[0009]

[0010] Therefore, it is of great significance to develop a method for synthesizing On-DNA aryl alkyne compounds with low cost, wide application range, little DNA damage and high yield. Summary of the invention

[0011] The purpose of the present invention is to provide a new method for synthesizing On-DNA aryl alkyne lead compounds, and the use of the synthesis method in constructing a gene-encoded compound library.

[0012] The present invention provides a method for synthesizing an On-DNA aryl alkyne lead compound, the method comprising the following steps:

[0013]

[0014] Using the compound represented by formula I and the compound represented by formula II as raw materials, reacting in the presence of a base and a copper catalyst to obtain an On-DNA aryl alkyne lead compound represented by formula III;

[0015] Among them, DNA is a single-stranded or double-stranded nucleotide chain;

[0016] X is the connection unit;

[0017] L is no or connected unit;

[0018] Ring A is selected from a 5- to 6-membered heteroaromatic ring, a benzene ring, an aromatic condensed ring, and an aromatic heterocondensed ring;

[0019] m is an integer from 1 to 4;

[0020] R aEach independently selected from unsubstituted or substituted with one or more R d Substituted C 1-6 Alkyl, C 1-6 Alkoxy, OR c , halogen, cyano, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclic group, or two of them a Connect into a ring;

[0021] R c is selected from 3-6 membered saturated cycloalkyl and 3-6 membered saturated heterocyclic group;

[0022] R d Each is independently selected from cyano and halogen.

[0023] Furthermore, the equivalent ratio of the compound represented by formula I, the compound represented by formula II, the base and the copper catalyst is 1:(1-1000):(1-500):(0.1-100);

[0024] The base is any one of cesium carbonate, potassium carbonate, sodium carbonate, potassium bicarbonate, sodium bicarbonate, cesium hydroxide, potassium hydroxide, sodium hydroxide, lithium hydroxide, potassium phosphate, sodium phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, cesium fluoride, potassium tert-butoxide, sodium tert-butoxide, triethylamine, n-butylamine, isobutylamine, 4-dimethylaminopyridine, pyridine, N,N-diisopropylethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, N,N,N',N'-tetramethylethylenediamine, 1,1,3,3-tetramethylguanidine, N,N-dicyclohexylmethylamine, dicyclohexylamine, tetrahydropyrrole, inorganic salt buffer, and organic base buffer, or a mixture of any two or more thereof;

[0025] The copper catalyst is any one of copper acetate, copper sulfate, copper chloride, copper nitrate, copper carbonate, cuprous iodide, copper-β-cyclodextrin complex, bis(2,4-pentanedionate)copper, copper acetylacetonate, tetra(acetonitrile)copper tetrafluoroborate, dichloro(1,10-phenanthroline)copper, bis(8-hydroxyquinoline)copper, copper trifluoromethanesulfonate, bis(trifluoro-2,4-pentanedionate)copper, copper perchlorate, tetra(acetonitrile)copper hexafluorophosphate, cuprous acetate, cupric bromide, cupric fluoride, cuprous bromide, cuprous chloride, cuprous chloride-bis(lithium chloride) complex, and cuprous bromide dimethyl sulfide complex, or a mixture of any two or more thereof;

[0026] The solvent of the reaction is water, methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, tert-butanol, amyl alcohol, cyclohexanol, 2-fluoroethanol, 2,2-difluoroethanol, 2,2,2-trifluoroethanol, hexafluoroisopropanol, benzyl alcohol, ethylene glycol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, glycerol, ethyl ether, propylene oxide, isopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, tetrahydropyran, 1,4-dioxane, anisole, dimethyl sulfide, diethyl sulfide, ethylene glycol dimethyl ether, ethylene glycol diethyl Any one of ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, acetone, cyclohexanone, dichloromethane, chloroform, chlorobenzene, 1,2-dichloroethane, ethyl acetate, n-hexane, cyclohexane, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 4-methoxypyridine, toluene, xylene, inorganic salt buffer, organic base buffer, or a mixture of any two or more thereof;

[0027] The reaction temperature is 0-40° C., and the reaction time is 1-48 hours.

[0028] Furthermore, the equivalent ratio of the compound represented by formula I, the compound represented by formula II, the base and the copper catalyst is 1:(1-200):(1-100):(0.1-50);

[0029] The base is one of potassium carbonate and sodium carbonate or a mixture of the two;

[0030] The copper catalyst is any one of cuprous iodide, tetra(acetonitrile)copper hexafluorophosphate, and cuprous chloride-bis(lithium chloride) complex, or a mixture of any two or more thereof;

[0031] The solvent of the reaction is a mixture of water, methanol and acetonitrile;

[0032] The reaction temperature is 25° C. and the reaction time is 24 hours.

[0033] Furthermore, the equivalent ratio of the compound represented by formula I, the compound represented by formula II, the base and the copper catalyst is 1:200:15:10;

[0034] The base is potassium carbonate;

[0035] The copper catalyst is cuprous iodide.

[0036] Furthermore, X is selected from NHCO, CONH, NH, and CO.

[0037] Further, L is selected from none, C 1-4 Alkylene, Not substituted or replaced by one or more R bSubstituted 5- to 6-membered heteroaromatic ring, unsubstituted or substituted with one or more R b Substituted benzene ring;

[0038] Among them, L1 is selected from C 1-2 Alkylene, L2 is selected from C 1-2 Alkylene, Y is selected from O or S;

[0039] R b are each independently selected from halogen, C 1-3 alkyl;

[0040] Ring A is selected from a 5- to 6-membered heteroaromatic ring, a benzene ring, an aromatic condensed ring, and an aromatic heterocondensed ring;

[0041] m is 1, 2 or 3;

[0042] R a Each independently selected from unsubstituted or substituted with one or more R d Substituted C 1-4 Alkyl, C 1-4 Alkoxy, OR c , halogen, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclic group, or two of them a Connected to form a ring, the ring is a 5-6 membered saturated ring;

[0043] R c is selected from 3-6 membered saturated cycloalkyl and 3-6 membered saturated heterocyclic group;

[0044] R d Each is independently selected from cyano and halogen.

[0045] Further, X is NHCO, and the synthesis method of the compound represented by Formula I comprises the following steps:

[0046]

[0047] The compound represented by formula Ia and the compound represented by formula Ib are used as raw materials for reaction to obtain the compound represented by formula I.

[0048] Furthermore, the reaction is carried out under the action of a base and a condensation reagent, and the equivalent ratio of the compound represented by Formula Ia, the compound represented by Formula Ib, the base and the condensation reagent is 1: (40-160): (100-400): (25-100);

[0049] The reaction temperature is 0-40°C and the reaction time is 0.5-10 hours;

[0050] The solvent of the reaction is water, methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, tert-butanol, amyl alcohol, cyclohexanol, 2-fluoroethanol, 2,2-difluoroethanol, 2,2,2-trifluoroethanol, hexafluoroisopropanol, benzyl alcohol, ethylene glycol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, glycerol, ethyl ether, propylene oxide, isopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, tetrahydropyran, 1,4-dioxane, anisole, dimethyl sulfide, diethyl sulfide, ethylene glycol dimethyl ether, ethylene glycol diethyl Any one of ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, acetone, cyclohexanone, dichloromethane, chloroform, chlorobenzene, 1,2-dichloroethane, ethyl acetate, n-hexane, cyclohexane, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 4-methoxypyridine, toluene, xylene, inorganic salt buffer, organic base buffer, or a mixture of any two or more thereof.

[0051] Further, the base is N,N-diisopropylethylamine, and the condensation reagent is 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate;

[0052] The equivalent ratio of the compound represented by formula Ia, the compound represented by formula Ib, the base and the condensation reagent is 1:80:200:50;

[0053] The reaction temperature is room temperature and the reaction time is 1 hour;

[0054] The solvent of the reaction is a mixture of water and dimethyl sulfoxide.

[0055] Furthermore, the On-DNA aryl alkyne lead compound is one of the following compounds:

[0056]

[0057] 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 in this document, the meaning that a person skilled in the art can give them should be given based on the disclosure and context.

[0058] The minimum and maximum carbon atom content in a hydrocarbon group is indicated by a prefix, for example, the prefix C a-b Alkyl refers to any alkyl group containing from "a" to "b" carbon atoms. 1-6 The alkyl group refers to a straight or branched chain alkyl group containing 1 to 6 carbon atoms.

[0059] "Heteroaromatic ring" or "heteroaryl" refers to a heteroaromatic group containing one or more heteroatoms. The heteroatoms referred to here include oxygen, sulfur and nitrogen. For example, furanyl, thienyl, pyridyl, pyrazolyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc.

[0060] "Fused cycloalkyl" refers to a polycyclic cycloalkyl group, wherein two rings of the polycyclic cycloalkyl group share two adjacent carbon atoms. "Aromatic fused ring" or "fused ring aryl" refers to a polycyclic fused cycloalkyl group wherein at least one ring is an aryl group.

[0061] "Heterofused ring" refers to a polycyclic heterocyclic group, wherein two rings in the polycyclic heterocyclic group share two adjacent carbon atoms or heteroatoms. "Aromatic heterofused ring" or "fused ring heteroaryl" refers to a polycyclic heterofused ring group in which at least one ring is a heteroaryl.

[0062] Halogen refers to fluorine, chlorine, bromine or iodine.

[0063] Room temperature refers to 25±5℃.

[0064] The present invention discloses a novel method for synthesizing an On-DNA aryl alkyne lead compound, wherein the method uses an oligonucleic acid-terminal alkyne compound and an aryl boronic acid as raw materials, and reacts in the presence of a copper catalyst and a base to obtain an On-DNA aryl alkyne lead compound. It is well known to those skilled in the art that DNA must remain stable under certain conditions, and the reaction applied to the construction of a DNA-encoded compound library needs to have a higher yield. Under the reaction conditions of the present invention, the product On-DNA aryl alkyne compound not only has a high yield, but also has good DNA integrity in the product. The integrity of the On-DNA aryl alkyne compound obtained in the present invention can not only be confirmed from liquid chromatography mass spectrometry, but also can be verified by performing a next step of DNA enzyme-catalyzed coupling reaction.

[0065] Compared with the existing methods for preparing On-DNA aryl alkyne lead compounds, the method of the present invention has the following advantages:

[0066] (1) Compared with iodine-substituted alkynes, the raw material terminal alkynes used in the present invention are all commercial products, which can be obtained by purchasing commercial products and are cheap, thus reducing time and economic costs;

[0067] (2) The present invention does not use expensive and rare palladium catalysts, but uses cheaper and more readily available copper catalysts, further reducing production costs;

[0068] (3) Compared with iodoalkynes, the terminal alkynes used as raw materials in the present invention are more stable and can be stably present even under acidic and alkaline conditions, and have a wider range of applications, thus broadening the types of On-DNA aryl alkyne lead compounds that can be synthesized;

[0069] (4) The method of the present invention has mild conditions and causes little damage to DNA.

[0070] In summary, the method for synthesizing On-DNA aryl alkyne lead compounds of the present invention has low cost, little damage to DNA, good universality, simple operation, mild conditions, and can prepare On-DNA aryl alkyne compounds in high yield.

[0071] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0072] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 : Liquid chromatography mass spectrum of DNA-NHFmoc.

[0074] Figure 2 : Liquid chromatography mass spectrum of DNA-NH2.

[0075] Figure 3 : Liquid chromatography-mass spectrum of S1-1.

[0076] Figure 4 : Liquid chromatography-mass spectrum of S1-2.

[0077] Figure 5 : Liquid chromatography-mass spectrum of S1-3.

[0078] Figure 6 : Liquid chromatography-mass spectrum of S1-4.

[0079] Figure 7 : Liquid chromatography-mass spectrum of S1-5.

[0080] Figure 8 : Liquid chromatography-mass spectrum of S1-6.

[0081] Fig. 9 : Liquid chromatography-mass spectrum of S1-7.

[0082] Fig.10 : Liquid chromatography-mass spectrum of S1-8.

[0083] Fig.11 : Liquid chromatography-mass spectrum of S1-9.

[0084] Fig.12: Liquid chromatography-mass spectrum of S1-10.

[0085] Fig.13 : Liquid chromatography-mass spectrum of P1.

[0086] Fig.14 : Liquid chromatography-mass spectrum of P2.

[0087] Fig.15 : Liquid chromatography-mass spectrum of P3.

[0088] Fig.16 : Liquid chromatography-mass spectrum of P4.

[0089] Fig.17 : Liquid chromatography-mass spectrum of P5.

[0090] Fig.18 : Liquid chromatography-mass spectrum of P6.

[0091] Fig.19 : Liquid chromatography-mass spectrum of P7.

[0092] Fig. 20 : Liquid chromatography-mass spectrum of P8.

[0093] Fig.21 : Liquid chromatography-mass spectrum of P9.

[0094] Fig. 22 : Liquid chromatography-mass spectrum of P10.

[0095] Fig.23 : Liquid chromatography-mass spectrum of P11.

[0096] Fig.24 : Liquid chromatography-mass spectrum of P12.

[0097] Fig.25 : Liquid chromatography-mass spectrum of P13.

[0098] Fig.26 : Liquid chromatography-mass spectrum of P14.

[0099] Fig. 27 : Liquid chromatography-mass spectrum of P15.

[0100] Fig.28 : Liquid chromatography-mass spectrum of P16.

[0101] Fig.29 : Liquid chromatography-mass spectrum of P17.

[0102] Fig.30 : Liquid chromatography-mass spectrum of P18.

[0103] Fig.31 : Liquid chromatography-mass spectrum of P19.

[0104] Fig.32 : Liquid chromatography-mass spectrum of P20.

[0105] Fig.33 : Liquid chromatography-mass spectrum of P21.

[0106] Fig.34 : Liquid chromatography-mass spectrum of P22.

[0107] Fig.35 : Liquid chromatography-mass spectrum of P23.

[0108] Fig.36 : Liquid chromatography-mass spectrum of P24.

[0109] Fig.37 : Liquid chromatography-mass spectrum of P25.

[0110] Fig.38 : Liquid chromatography-mass spectrum of P26.

[0111] Fig.39 : Liquid chromatography-mass spectrum of P27.

[0112] Fig.40 : Liquid chromatography-mass spectrum of P2-1.

[0113] Fig.41 : The structure of HP.

[0114] Fig.42 : Structures, yields and molecular weights of oligonucleic acid-aryl alkyne compounds P1 to P27, with the yields indicated in brackets. DETAILED DESCRIPTION

[0115] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0116] The aryl boronic acid used in the embodiment of the present invention is one of the following S2-1 to S2-18, all of which are known compounds and can be obtained by purchasing commercial products:

[0117]

[0118] The oligonucleic acid-terminal alkyne compound used in the embodiment of the present invention is one of the following S1-1 to S1-10:

[0119]

[0120] The following is the route for synthesizing oligonucleic acid-terminal alkyne compounds S1-1 to S1-10:

[0121]

[0122] 100 nanomoles of HP (HP has the structure Fig.41 40 equivalents of the starting head fragment compound SM1 (commercial product, 200 mmol / L DMSO solution), 250 equivalents of sodium tetraborate (Na2B4O7) buffer solution with pH = 9.47 (250 mmol / L aqueous solution), and 40 equivalents of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) (200 mmol / L aqueous solution, 4000 nanomoles) were mixed. The mixture was fully mixed and added to the HP solution, mixed again and reacted at 4°C for 1 hour. After the reaction was completed, 10% of the total volume of 5 mol / L sodium chloride solution was added to the reaction solution, and then 3 times the total volume of anhydrous ethanol was added. After shaking evenly, the reaction solution was placed in a refrigerator at -80°C for 2 hours. After that, the mixture was centrifuged at 4000 rpm for half an hour, and the supernatant was discarded. The remaining precipitate was dried to obtain DNA-NHFmoc. The spectrum of DNA-NHFmoc was detected by liquid chromatography-mass spectrometry. Figure 1 As shown, its molecular weight is 5406.

[0123] (1.2) Synthesis of DNA-NH2

[0124]

[0125] Dissolve 100 nanomoles of DNA-NHFmoc in deionized water to prepare a 1 mmol / L solution (100 microliters, 100 nanomoles, 1 equivalent), add 36 microliters of 10% piperidine aqueous solution, mix the two evenly and react at room temperature for 1 hour. After the reaction is completed, add 10% of the total volume of 5 mol / L sodium chloride solution to the reaction solution. Then continue to add 3 times the total volume of anhydrous ethanol, shake evenly, and freeze the reaction solution in a -80°C refrigerator for 2 hours. Afterwards, centrifuge at 4000 rpm for half an hour and pour off the supernatant. The remaining precipitate is dried to obtain oligonucleic acid-NH2 (abbreviated as DNA-NH2). Detection using liquid chromatography-mass spectrometry, the spectrum of DNA-NH2 is as shown Figure 2 As shown, its molecular weight is 5184.

[0126] (1.3) Synthesis of oligonucleic acid-terminal alkyne compound S1

[0127]

[0128] Dissolve 100 nanomoles of DNA-NH2 in deionized water to prepare a 1 mmol / L DNA-NH2 solution (100 microliters, 100 nanomoles, 1 equivalent). Add 200 equivalents of N,N-diisopropylethylamine (200 mmol / L DMSO solution) and 50 equivalents of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (200 mmol / L DMSO solution) to 80 equivalents of acid-terminal alkyne compound 1 (200 mmol / L DMSO solution), mix the mixture thoroughly and add it to the DNA-NH2 solution, mix it again and react at room temperature for 1 hour. After the reaction is completed, add 10% of the total volume of 5 mol / L sodium chloride solution to the reaction solution. Then continue to add 3 times the total volume of anhydrous ethanol, shake it evenly, and place the reaction solution in a -80°C refrigerator for 2 hours. Afterwards, the mixture was centrifuged at 4000 rpm for half an hour, the supernatant was discarded, and the remaining precipitate was dried to obtain the oligonucleic acid-terminal alkyne compound S1. Compound S1 corresponds to compounds S1-1 to S1-10 according to the different L in the structure.

[0129] The spectra of S1-1 to S1-10 were detected by liquid chromatography-mass spectrometry. Figure 3-12 .

[0130] Example 1: Synthesis of oligonucleic acid-aryl alkyne compound P1

[0131]

[0132] Dissolve 1 nanomole of oligonucleic acid-terminal alkyne compound S1-1 (1 equivalent) in deionized water to prepare a 1 mmol / L solution, and add 200 equivalents of aryl boronic acid S2-1 (200 mmol / L methanol solution), 15 equivalents of potassium carbonate (20 mmol / L aqueous solution) and 10 equivalents of cuprous iodide (10 mmol / L acetonitrile solution). Mix the mixture evenly and react at 25°C for 24 hours. After the reaction is completed, add 2 microliters of 1 mol / L sodium diethyldithiocarbamate aqueous solution to the reaction solution and react at 70°C for 10 to 30 minutes. Then add 10% of the total volume of 5 mol / L sodium chloride aqueous solution and 3 times the total volume of anhydrous ethanol to the above reaction solution, shake and mix evenly, and place the reaction solution in a refrigerator at -80°C for 2 hours. After that, centrifuge at 4000rpm for half an hour, pour off the supernatant, and dry the remaining precipitate to obtain the oligonucleic acid-aryl alkyne compound P1. The molecular weight of the corresponding product was 5388, indicating that the oligonucleic acid-aryl alkyne compound P1 was successfully synthesized. The reaction yield was 88% as determined by liquid chromatography-mass spectrometry. Fig.13 .

[0133] Example 2-18: Synthesis of oligonucleic acid-aryl alkyne compounds P2 to P18

[0134] The synthetic method of Example 1 is referred to, except that the raw material aryl boronic acid S2-1 is replaced by S2-2 to S2-18, respectively, to obtain oligonucleic acid-aryl alkyne compounds P2 to P18, respectively.

[0135] Examples 19-27: Synthesis of oligonucleic acid-aryl alkyne compounds P19 to P27

[0136] The synthetic method of Example 1 is referred to, except that the raw material oligonucleic acid-terminal alkyne compound S1-1 is replaced by S1-2 to S1-10, respectively, to obtain oligonucleic acid-aryl alkyne compounds P19 to P27, respectively.

[0137] The molecular weight and reaction yield of the above product were detected by liquid chromatography-mass spectrometry. The corresponding spectra are shown in Figure 14-39 The structures and yields of oligonucleic acid-aryl alkyne compounds P1 to P27 are summarized in Fig.42 .

[0138] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0139] Experimental Example 1: Conditional screening experiment for synthesizing oligonucleic acid-aryl alkyne compounds

[0140]

[0141] Taking the synthesis of oligonucleic acid-aryl alkyne compound P2 as an example, the conditions such as the type of copper catalyst, the type of base, the ratio of raw materials, and the reaction time were screened. Specifically, referring to the method of synthesizing method P2 in Example 2, the only difference is that the type and equivalent of the copper catalyst, the type and equivalent of the base, the equivalent of the raw material S2-2, and the reaction time are controlled according to Table 1, and the yields of the compound P2 obtained under different conditions are compared.

[0142] Table 1. Results of the condition screening experiment for the synthesis of compound P2

[0143]

[0144] In Table 1, "b" of No. 14 indicates that the reaction time is 10 hours, and the reaction time of the remaining Nos. 1-13 and 15 is the same as that in Example 2, which is 24 hours.

[0145] According to the experimental results, it can be seen that under the reaction conditions of No. 12 (ie, the reaction conditions of Example 2), the yield of the obtained product P2 is the highest.

[0146] In fact, the reaction conditions of No. 15 are the same as the preferred reaction conditions for preparing the aryl alkyne lead compound P2 by reacting oligonucleic acid-iodinated alkyne with a boric acid reagent in the presence of a copper catalyst and a base in the Chinese patent application with application number CN202210032980.8. However, by comparing the reaction yields of No. 15 and No. 12, it can be seen that if P2 is prepared by the method of the present invention under the preferred reaction conditions for preparing P2 in CN202210032980.8, the yield of the obtained product is only 60%, which is significantly lower than the product yield of No. 15.

[0147] That is to say, the method of the present invention can not only overcome the disadvantages of using iodine alkynes as starting materials under reaction conditions No. 12 (i.e., the reaction conditions of Example 2), but also obtain On-DNA aryl alkyne lead compounds with high yield.

[0148] Experimental Example 2: Verification of the integrity of the oligonucleotide in the oligonucleotide-aryl alkyne compound of the present invention

[0149] The following is a linking experiment using the oligonucleotide-aryl alkyne compound P2 and Tag A (a short-chain oligonucleotide with molecular weights of 4064 and 5884, respectively) to verify the integrity of the oligonucleotide:

[0150]

[0151] Steps: Dissolve 1 nanomole of P2 in deionized water to prepare a 1 mmol / L solution (1 microliter, 1 nanomole, 1 equivalent), add 1.2 equivalents of TagA (1 mmol / L aqueous solution, 1.2 equivalents), 1 microliter of 10×T4 DNA ligation buffer solution and 0.5 microliter of T4 DNA ligase. Mix the above solutions evenly and react at room temperature for 1 hour. After the reaction is completed, add 10% of the total volume of 5 mol / L sodium chloride solution to the reaction solution, and then continue to add 3 times the total volume of anhydrous ethanol. After shaking evenly, place the reaction solution in a -80°C refrigerator and freeze it for 2 hours. Then centrifuge at 4000 rpm for half an hour and pour off the supernatant. The remaining precipitate is dissolved in deionized water and detected by liquid chromatography-mass spectrometry to confirm the product molecular weight. The liquid chromatography-mass spectrometry detection results of the reaction raw material P2 and the product P2-1 are shown respectively. Fig.14 and Fig.40 .

[0152] The results showed that the oligonucleic acid-aryl alkyne compound P2 and Tag A could successfully undergo DNA enzyme-catalyzed coupling reaction, indicating that the method for synthesizing compound P2 of the present invention did not damage the structure of the oligonucleic acid part; at the same time, liquid chromatography-mass spectrometry analysis showed that the reaction product had high purity, and mass spectrometry detection could accurately obtain experimental values ​​consistent with the calculated values, further verifying the good integrity of DNA.

[0153] In summary, the present invention provides a method for synthesizing On-DNA aryl alkyne lead compounds, which belongs to the field of gene-encoded compound library construction. The present invention discloses a novel method for synthesizing On-DNA aryl alkyne lead compounds, which uses oligonucleic acid-terminal alkyne compounds and arylboronic acid as raw materials, and reacts in the presence of a copper catalyst and a base to obtain On-DNA aryl alkyne lead compounds. The method for synthesizing On-DNA aryl alkyne lead compounds of the present invention has low cost, little damage to DNA, good universality, simple operation, mild conditions, and can produce On-DNA aryl alkyne compounds in high yield, and has broad application prospects in constructing gene-encoded compound libraries.

Claims

1. A method for synthesizing On-DNA aryl alkyne lead compounds, characterized in that: The method comprises the following steps: The compound represented by formula I and the compound represented by formula II are used as raw materials, and reacted in the presence of a base and a copper catalyst to obtain an On-DNA aryl alkyne lead compound represented by formula III; the equivalent ratio of the compound represented by formula I, the compound represented by formula II, the base and the copper catalyst is 1:200:15:10; the base is one or a mixture of two of potassium carbonate and sodium carbonate; the copper catalyst is cuprous iodide; the reaction temperature is 0-40° C.; the reaction solvent is a mixture of water, methanol and acetonitrile; Among them, DNA is a single-stranded or double-stranded nucleotide chain; X is the connection unit; L is no or connected unit; Ring A is selected from a 5- to 6-membered heteroaromatic ring, a benzene ring, an aromatic condensed ring, and an aromatic heterocondensed ring; m is an integer from 1 to 4; R a Each independently selected from unsubstituted or substituted with one or more R d Substituted C 1-6 Alkyl, C 1-6 Alkoxy, OR c , halogen, cyano, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclic group, or two of them a Connect into a ring; R c is selected from 3-6 membered saturated cycloalkyl and 3-6 membered saturated heterocyclic group; R d Each is independently selected from cyano and halogen.

2. The method according to claim 1, characterized in that: The reaction temperature is 0-40° C., and the reaction time is 1-48 hours.

3. The method according to claim 2, characterized in that: The reaction time is 24 hours.

4. The method according to claim 3, characterized in that: The base is potassium carbonate.

5. The method according to any one of claims 1 to 4, characterized in that: X is selected from NHCO, CONH, NH, CO.

6. The method according to any one of claims 1 to 4, characterized in that: L is selected from none, C 1-4 Alkylene, Not substituted or replaced by one or more R b Substituted 5- to 6-membered heteroaromatic ring, unsubstituted or substituted with one or more R b Substituted benzene ring; Among them, L1 is selected from C 1-2 Alkylene, L2 is selected from C 1-2 Alkylene, Y is selected from O or S; R b are each independently selected from halogen, C 1-3 alkyl; Ring A is selected from a 5- to 6-membered heteroaromatic ring, a benzene ring, an aromatic condensed ring, and an aromatic heterocondensed ring; m is 1, 2 or 3; R a Each independently selected from unsubstituted or substituted with one or more R d Substituted C 1-4 Alkyl, C 1-4 Alkoxy, OR c , halogen, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclic group, or two of them a Connected to form a ring, the ring is a 5-6 membered saturated ring; R c is selected from 3-6 membered saturated cycloalkyl and 3-6 membered saturated heterocyclic group; R d Each is independently selected from cyano and halogen.

7. The method according to any one of claims 1 to 4, characterized in that: X is NHCO, and the synthesis method of the compound shown in Formula I comprises the following steps: The compound represented by formula Ia and the compound represented by formula Ib are used as raw materials for reaction to obtain the compound represented by formula I.

8. The method according to claim 7, characterized in that: The reaction is carried out under the action of a base and a condensation agent, and the equivalent ratio of the compound represented by Formula Ia, the compound represented by Formula Ib, the base and the condensation agent is 1: (40-160): (100-400): (25-100); The reaction temperature is 0-40°C and the reaction time is 0.5-10 hours; The solvent of the reaction is water, methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, tert-butanol, amyl alcohol, cyclohexanol, 2-fluoroethanol, 2,2-difluoroethanol, 2,2,2-trifluoroethanol, hexafluoroisopropanol, benzyl alcohol, ethylene glycol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, glycerol, ethyl ether, propylene oxide, isopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, tetrahydropyran, 1,4-dioxane, anisole, dimethyl sulfide, diethyl sulfide, ethylene glycol dimethyl ether, ethylene glycol diethyl Any one of ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, acetone, cyclohexanone, dichloromethane, chloroform, chlorobenzene, 1,2-dichloroethane, ethyl acetate, n-hexane, cyclohexane, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 4-methoxypyridine, toluene, xylene, inorganic salt buffer, organic base buffer, or a mixture of any two or more thereof.

9. The method according to claim 8, characterized in that: The base is N,N-diisopropylethylamine, and the condensation reagent is 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; The equivalent ratio of the compound represented by formula Ia, the compound represented by formula Ib, the base and the condensation reagent is 1:80:200:50; The reaction temperature is room temperature and the reaction time is 1 hour; The solvent of the reaction is a mixture of water and dimethyl sulfoxide.

10. The method according to any one of claims 1 to 4, characterized in that: The On-DNA aryl alkyne lead compound is one of the following compounds:

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