Oligonucleic acid-pyrazole compound and synthesis method thereof

Abstract

The present invention provides an oligonucleic acid-pyrazole compound shown in formula I and a synthesis method thereof. The synthesis method of the present invention uses an oligonucleic acid-diketone compound shown in formula III and a hydrazine reagent shown in formula II as raw materials, reacts in a solvent, and obtains an oligonucleic acid-pyrazole compound shown in formula I. The method has little damage to DNA, good universality, simple operation, mild conditions, and can produce oligonucleic acid-pyrazole compounds in high yield, providing an important basis for expanding and enriching the DELT compound library.

Description

Technical Field

[0001] The invention belongs to the field of gene encoding compound library construction, and specifically relates to an oligonucleic acid-pyrazole compound and a synthesis method thereof. Background Art

[0002] At present, the exploration of lead compounds in pharmaceutical research is constantly entering an unknown field. Therefore, it is increasingly difficult to find suitable chemicals to combine with existing compounds and screen them. Traditional high-throughput screening has the disadvantages of limited number of compound libraries, long time and high cost. This limits the timeliness of lead compound discovery (AA Shelat, RK Guy, Nat. Chem. Biol. 2007, 3, 442.). Brenner and Lerner proposed in 1992 a method for bioactivity screening using gene-encoded compound library technology (DELT), which combines molecular biology technology, adds a DNA tag to each compound at the molecular level, and can generate up to 100 million compound libraries in a short period of time through combinatorial chemical synthesis methods. The compound mixtures labeled one by one by this DNA are directly screened for bioactivity (S. Brenner; RA Lerner. Proc. Natl. Acad. Sci. USA, 1992, 89, 5381-5383.), using a very small amount of DNA-encoded compound library and target protein for biological screening, and then eluting the adsorbed compounds, replicating and amplifying them through PCR technology, and sequencing and decoding the amplified DNA sequences to find the corresponding specific compounds. This technology not only breaks through the high cost of traditional high-throughput biological screening and the bottleneck of compound chemical space, and realizes a leap in the chemical structure space and quantity of the screened compounds, but also can simultaneously screen lead compounds in a simple and efficient manner under multiple biological screening conditions, becoming an increasingly popular method for discovering lead compounds.

[0003] In order to successfully screen small molecule compounds with affinity for biological target proteins, building diversity in chemical structure is an important guarantee for DELT to successfully screen. However, due to the particularity of DNA chemical structure, it can only be stable under certain conditions (temperature, pH, ion concentration, solvent, etc.). At the same time, the chemical reactions used in the construction of gene-encoded compound libraries also require higher yields. Therefore, the types of chemical reactions that can be used in gene-encoded compound libraries are very limited (RJ Fair, RT Walsh, CD Hupp. Bioorg. Med. Chem. Lett., 2021, 51, 128-339.). Therefore, the more types of chemical reactions that can be successfully applied to gene-encoded compound libraries, the richer the conditions, the more choices there are when designing and synthesizing gene-encoded compound libraries, and the richer the diversity of the resulting compound library. This technology has now become a new pillar of drug discovery. At the same time, there are more and more reports on new DNA synthesis reactions. Therefore, developing more synthesis methods that can be applied to gene-encoded compound libraries is also one of the important tasks in the field of DELT (RM. Franzini, C. Randolph. J Med. Chem., 2016, 59, 6629-6644; P. Dickson, T. Kodadek. Org. Biomol. Chem., 2019, 17, 4676-4688; K. S. Chinese, A. Brunschweiger. Tet. Lett. 2020, 61, 151889; PRFitzgerald, BMPaegel. Chem. Rev. 2021, 121, 7155-7177; Y. Shi, YR Wu, WNZhang, et al. RSC Adv., 2021, 11, 2359-2376.).

[0004] Pyrazole compounds are an important class of five-membered nitrogen heterocyclic compounds with important biological activities, such as antibacterial, antipyretic, analgesic, neuroprotective, anti-tumor and anti-inflammatory effects. Therefore, they have attracted the attention of the medical community and have been widely used in the synthesis and development of drug targets. At present, some pyrazole compounds have been used in clinical research or developed into marketed drugs. At the same time, pyrazole compounds play a pivotal role in pesticides and medicines due to their high efficiency, low toxicity and structural diversity.

[0005] Pyrazole compounds have a wide range of biological activities: anti-cancer and anti-tumor, treatment of Alzheimer's disease, antibacterial, anticonvulsant, etc. For example: celecoxib has anti-inflammatory, analgesic and antipyretic effects, and is used to treat rheumatic, rheumatoid arthritis and osteoarthritis. It can also be used for postoperative analgesia, toothache, dysmenorrhea, and the incidence of gastrointestinal adverse reactions, bleeding and ulcers is lower than that of other non-selective non-steroidal anti-inflammatory drugs. Ruxolitinib is the first oral drug approved by the US Food and Drug Administration (FDA) for the treatment of myelofibrosis; crizotinib is a targeted therapeutic drug for non-small cell lung cancer developed by Pfizer Pharmaceuticals in the United States and was approved for marketing in the United States in August 2011. In addition, a class of 2,3-dihydro-1H-pyrazole compounds also has major biological activities. For example, compound -642458 is an inhibitor of type IX carbonic anhydrase (F. Saczewski, A. Innocenti, J. Slawinski, et al. Bioorg. Med. Chem. 2008, 16, 3933).

[0006]

[0007] Under the premise that DELT is in urgent need of further enrichment, there is no report on oligonucleic acid-pyrazole compounds and their synthesis methods. Although there are reports on the preparation of pyrazole small molecule compounds by reacting diketone with hydrazine, the ethanol reflux conditions used therein, if directly applied to the synthesis of oligonucleic acid-pyrazole compounds, will lead to DNA denaturation (ARMalviya, VKZala, ZM Gadhawala. International Journal of Science and Research (IJSR) 2016, 5, 833-836.).

[0008] The main known methods for forming pyrazole compounds on DNA are: 1). Brunschweige's research group used the catalyst resistance of the self-made hexathymidine "hexT" adapter oligonucleotide to efficiently synthesize pyrazole compounds (MK H. Salamon, O. Bugain, K. Jung, A. Gohla, LJ Doetsch, D. Santos, A. Bhat, B. Wagner, A. Brunschweiger. Chem. Sci., 2017, 8, 3356-3361.), but their reaction conditions are relatively harsh, which can easily cause DNA denaturation and destroy DNA activity, and cannot be applied in the synthesis of commonly used gene encoding libraries. ; 2). Lu Xiaojie and his research team reported in 2020 that α, β unsaturated ketone and hydrazine reagent were first heated at 60 ° C for 2 h under pH = 9.5 or pH = 5.5 to form pyrazoline compounds, and then DDQ was added for oxidation to obtain pyrazole compounds (SX Liu, JJ Qi, WW Lu, X. Wang, XJ Lu. Org. Lett. 2021, 23, 908-913.). Although the reaction conditions of this method are relatively mild and the functional group compatibility is good, it requires heating and the introduction of oxidation operations, has many experimental steps, and is relatively cumbersome. 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 are prone to cause DNA denaturation and damage DNA activity, making the development of chemical reactions for the construction of gene-encoded compound libraries face many difficulties.

[0009] Therefore, developing a method for synthesizing oligonucleic acid-pyrazole compounds with little DNA damage and high yield is of great significance for filling the gap of oligonucleic acid-pyrazole compounds in DELT and enriching DELT. Summary of the invention

[0010] The purpose of the present invention is to provide a method for synthesizing an oligonucleic acid-pyrazole compound.

[0011] The present invention provides a method for synthesizing an oligonucleic acid-pyrazole compound, which uses an oligonucleic acid-diketone compound shown in formula III and a hydrazine reagent shown in formula II as raw materials, reacts in a solvent to obtain an oligonucleic acid-pyrazole compound shown in formula I; the reaction formula is as follows:

[0012]

[0013] Wherein, L is the connection unit;

[0014] R 1 C 1～10 alkyl;

[0015] R 2 For R a , R b A substituted or unsubstituted fused ring aromatic group, a fused heteroaromatic group or a 3- to 8-membered hydrocarbon group, wherein Ra , R b are independently selected from: hydrogen, C 1～5 Alkyl, C 1～5 Alkoxy, hydroxy, halogen, ester, alkynyl, or R a , R b Connect into a ring;

[0016] R 3 For H, R 3a None, or R 3 , R 3a C 1～5 Alkyl and R 3 , R 3a same;

[0017] or, R 2 and R 3 Connected into ring A, and R 2 and R 3a Connect into ring A.

[0018] Furthermore, the molar ratio of the oligonucleic acid-diketone compound represented by the above formula III to the hydrazine reagent represented by the formula II is: 1:(1-500), preferably 1:(5-500);

[0019] and / or the reaction temperature is 0 to 90°C; preferably 0 to 50°C;

[0020] And / or the reaction time is 1 to 24 hours; preferably 1 to 10 hours;

[0021] And / or the reaction is carried out at a pH of 3.5 to 9.5; preferably at a pH of 3.5 to 7.0;

[0022] and / or the solvent 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, benzyl alcohol, The invention relates to any one or a mixture of ether, dimethyl sulfide, diethyl sulfide, ethylene glycol dimethyl ether, ethylene glycol diethyl 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, and sodium dihydrogen phosphate buffer solution; preferably, the solvent of the reaction is a mixture of water, acetonitrile and sodium dihydrogen phosphate buffer solution.

[0023] Furthermore, the molar ratio of the oligonucleic acid-diketone compound represented by the above formula III to the hydrazine reagent represented by the formula II is 1:50;

[0024] and / or the reaction temperature is 25°C;

[0025] And / or the reaction time is 5 hours;

[0026] And / or the reaction is carried out at a pH of 3.5 to 6.0;

[0027] And / or the solvent is a mixture of water, acetonitrile and sodium dihydrogen phosphate buffer solution, and the total content of acetonitrile is not higher than 50%.

[0028] Further, the compound represented by formula II is: The compound shown in formula I is

[0029] Or, the compound shown in formula II is: The compound shown in formula I is Where R 3a C 1～5 alkyl;

[0030] Or, the compound shown in formula II is The compound shown in formula I is

[0031] Furthermore, the above L is wherein p is an integer of 0 to 3, q ​​is an integer of 1 to 3, and r is an integer of 0 to 3; preferably, L is

[0032] Furthermore, the compound of formula I is one of the following compounds:

[0033]

[0034] The present invention also provides an oligonucleic acid-pyrazole compound represented by Formula I:

[0035]

[0036] Wherein, L is the connection unit;

[0037] R 1 C 1～10 alkyl;

[0038] R 2 For R a , R b A substituted or unsubstituted fused ring aromatic group, a fused heteroaromatic group or a 3- to 8-membered hydrocarbon group, wherein R a , R b are independently selected from: hydrogen, C 1～5 Alkyl, C 1～5 Alkoxy, hydroxy, halogen, ester, alkynyl, or R a , R b Connect into a ring;

[0039] R 3a None or C 1～5 Alkyl; or, R 2 and R 3a Connect into ring A.

[0040] Furthermore, the above L is wherein p is an integer of 0 to 3, q ​​is an integer of 1 to 3, and r is an integer of 0 to 3; preferably, L is

[0041] Further, the compound shown in formula I is Where R 3a C 1～5 alkyl.

[0042] Furthermore, R 2 For R a , R b A substituted or unsubstituted fused ring aromatic group, a nitrogen-fused heteroaromatic group or a 5- to 6-membered ring hydrocarbon group, wherein R a , R b are independently selected from: hydrogen, C 1～3 Alkyl, methoxy, hydroxy, halogen, ester, alkynyl; or R a , R b Connected into a saturated ring;

[0043] and / or R3a is methyl;

[0044] and / or Ring A is a 3- to 6-membered saturated ring.

[0045] Furthermore, the above compound is one of the following compounds:

[0046]

[0047] The present invention also provides the use of the above-mentioned synthesis method in constructing a gene-encoded compound library.

[0048] Beneficial effects of the invention: The invention provides a method for synthesizing oligonucleic acid-pyrazole lead compounds on DNA, and the oligonucleic acid-pyrazole lead compounds are successfully prepared under specific pH conditions and temperature for a specific reaction time. The method has little damage to DNA, good universality, simple operation, mild conditions, and can prepare oligonucleic acid-pyrazole compounds with high yield, providing an important basis for expanding and enriching the DELT compound library.

[0049] The "DNA" in the structural formula of the present invention is a single-stranded or double-stranded oligonucleotide chain.

[0050] The minimum and maximum values ​​of the carbon atom content in the hydrocarbon groups of the present invention are indicated by prefixes, 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 chain or branched chain alkyl group containing 1 to 6 carbon atoms.

[0051] The "cyclic hydrocarbon group" of the present invention refers to a saturated or unsaturated pure carbon ring (the atoms constituting the ring skeleton are all C) or a heterocycle (a heterocycle is a pure carbon ring in which some carbon atoms are replaced by heteroatoms such as O, N, S to form a stable ring structure. For example, a methylene group -CH2- on a ring is replaced by O by C to form a stable structure of -O-, or replaced by N to form a stable structure of -NH-; or replaced by S to form a stable structure of -SO2-, etc.).

[0052] In the present invention, "the substituent is an ester group" means that two hydrogen atoms of -CH2- are replaced by =O to form a -(C=O)- structure, or the hydrogen atoms of -CH= are replaced by -(C=O)- structure.

[0053] The "condensed ring aromatic group" of the present invention refers to a polycyclic aromatic group in which two rings share two adjacent carbon atoms, such as naphthyl (i.e., a 6-membered aromatic ring and a 6-membered aromatic ring, or a benzene ring and a benzene ring), anthracenyl, and phenanthryl.

[0054] The "fused heteroaryl" of the present invention refers to a polycyclic aromatic ring / aromatic group containing at least one heteroatom (O, N or S), and two of the rings share two adjacent carbon atoms or heteroatoms. For example, a 5-membered aromatic ring or heteroaromatic ring (such as furan, thiophene, pyrrole, pyridine ring) and a 6-membered aromatic ring or heteroaromatic ring (such as benzene, pyridine, pyridazine, pyrimidine, pyrazine ring), such as indole, or a 6-membered aromatic ring or heteroaromatic ring and a 6-membered aromatic ring or heteroaromatic ring, such as quinoline; or more than 2 rings, such as acridine. "Nitrogen-fused heteroaryl" means that the heteroatom in the above-mentioned "fused heteroaryl" is N, and the number of heteroatoms is one or more.

[0055] The “organic solvent” of the present invention includes, but is not limited to, methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, tert-butanol, pentanol, 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 ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, N-methylpyrrolidone, N,N-dimethylformamide, N,N- dimethylacetamide, dimethyl sulfoxide (DMSO), 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.

[0056] 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.

[0057] 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

[0058] Figure 1 It is the liquid chromatography mass spectrum of the oligonucleic acid-COOH raw material (S2).

[0059] Figure 2 It is the liquid chromatography mass spectrum of oligonucleic acid-NHFmoc raw material (S3).

[0060] Figure 3 It is the liquid chromatography mass spectrum of oligonucleic acid-NH2 raw material (S4).

[0061] Figure 4It is the liquid chromatography mass spectrum of the oligonucleic acid-diketone compound raw material (S5-1).

[0062] Figure 5 It is the liquid chromatography mass spectrum of the oligonucleic acid-diketone compound raw material (S5-2).

[0063] Figure 6 It is a structural diagram of each hydrazine raw material (S6-1~S6-32).

[0064] Figure 7 It is a structural diagram of the oligonucleic acid-pyrazole compounds (P1-P34) of the present invention.

[0065] Figure 8 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P1 of the present invention.

[0066] Fig. 9 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P2 of the present invention.

[0067] Fig.10 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P3 of the present invention.

[0068] Fig.11 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P4 of the present invention.

[0069] Fig.12 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P5 of the present invention.

[0070] Fig.13 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P6 of the present invention.

[0071] Fig.14 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P7 of the present invention.

[0072] Fig.15 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P8 of the present invention.

[0073] Fig.16 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P9 of the present invention.

[0074] Fig.17 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P10 of the present invention.

[0075] Fig.18 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P11 of the present invention.

[0076] Fig.19It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P12 of the present invention.

[0077] Fig. 20 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P13 of the present invention.

[0078] Fig.21 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P14 of the present invention.

[0079] Fig. 22 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P15 of the present invention.

[0080] Fig.23 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P16 of the present invention.

[0081] Fig.24 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P17 of the present invention.

[0082] Fig.25 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P18 of the present invention.

[0083] Fig.26 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P19 of the present invention.

[0084] Fig. 27 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P20 of the present invention.

[0085] Fig.28 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P21 of the present invention.

[0086] Fig.29 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P22 of the present invention.

[0087] Fig.30 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P23 of the present invention.

[0088] Fig.31 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P24 of the present invention.

[0089] Fig.32 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P25 of the present invention.

[0090] Fig.33 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P26 of the present invention.

[0091] Fig.34It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P27 of the present invention.

[0092] Fig.35 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P28 of the present invention.

[0093] Fig.36 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P29 of the present invention.

[0094] Fig.37 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P30 of the present invention.

[0095] Fig.38 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P31 of the present invention.

[0096] Fig.39 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P32 of the present invention.

[0097] Fig.40 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P33 of the present invention.

[0098] Fig.41 It is the liquid chromatography mass spectrum of the oligonucleic acid-pyrazole compound P34 of the present invention.

[0099] Fig.42 It is a liquid chromatography mass spectrum of the product A-P3 after the oligonucleic acid-pyrazole compound P3 of the present invention is connected with TagA. DETAILED DESCRIPTION

[0100] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products. All "equivalents" in the present specification refer to molar equivalents.

[0101] 1. Synthesis of oligonucleic acid-COOH raw material (S2)

[0102] 1.1 Synthesize oligonucleic acid-COOH raw material (S2) according to the following reaction formula:

[0103]

[0104] 100 nanomoles of HP (S1, HP has the structure Figure 140 equivalents of the starting head fragment compound (commercial product) in DMSO solution (concentration: 200 mmol / L), 250 equivalents of sodium tetraborate (Na2B4O7) buffer solution (concentration: 250 mmol / L) at pH = 9.5, and 40 equivalents of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) aqueous solution (concentration: 200 mmol / L) were mixed, and the mixture was fully mixed with a vortex oscillator. The above mixture was then added to the HP solution, mixed evenly, 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. Then, 3 times the total volume of anhydrous ethanol was added, and after oscillation, the reaction solution was placed in a refrigerator at -80°C for 2 hours. Afterwards, the mixture was centrifuged at 4000 rpm for half an hour, and the supernatant was discarded. The remaining precipitate was dissolved in deionized water to obtain a DNA-COOH solution. The spectrum of DNA-COOH (S2) was detected by liquid chromatography-mass spectrometry. Figure 1 As shown, its molecular weight is 5241.

[0105] 2. Synthesis of oligonucleic acid-NH2 raw material (S4)

[0106] 2.1 Synthesize oligonucleic acid-NHFmoc raw material (S3) according to the following reaction formula:

[0107]

[0108] 100 nanomoles of HP (S1, HP has the structure Figure 140 equivalents of the starting head fragment compound (commercial product) in DMSO solution (concentration: 200 mmol / L), 250 equivalents of sodium tetraborate (Na2B4O7) buffer solution (concentration: 250 mmol / L) at pH = 9.5, and 40 equivalents of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) aqueous solution (concentration: 200 mmol / L) were mixed, and the mixture was fully mixed with a vortex oscillator. The above mixture was then added to the HP solution, mixed evenly, 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. Then, 3 times the total volume of anhydrous ethanol was added, and after oscillation, the reaction solution was placed in a refrigerator at -80°C for 2 hours. Afterwards, the mixture was centrifuged at 4000 rpm for half an hour, and the supernatant was discarded. The remaining precipitate was dissolved in deionized water to obtain a DNA-NHFmoc solution. The spectrum of DNA-NHFmoc (S3) was detected by liquid chromatography-mass spectrometry. Figure 2 As shown, its molecular weight is 5406.

[0109] 2.2 Synthesize oligonucleic acid-NH2 raw material (S4) according to the following reaction formula:

[0110]

[0111] Dissolve 100 nanomoles of DNA-NHFmoc (S2) in deionized water to make 1 mmol / L (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 in a refrigerator at -80°C for 2 hours. After that, centrifuge at 4000 rpm for half an hour and pour off the supernatant. The remaining precipitate is dissolved in deionized water to obtain a solution of oligonucleic acid-NH2 (S4, abbreviated as DNA-NH2). The spectrum of DNA-NH2 (S4) detected by liquid chromatography-mass spectrometry is as shown Figure 3 As shown, its molecular weight is 5184.

[0112] The representative structure of S1 is:

[0113]

[0114] However, it should be specially noted that the method of the present invention is not limited to the DNA chain of the specific S1 structure mentioned above, but can be a single-stranded or double-stranded deoxyribonucleotide chain obtained by polymerization of other artificially modified or unmodified deoxyribonucleotide monomers, and the length of the chain is not limited.

[0115] 3. Synthesis of oligonucleic acid-diketone compound raw material (S5-1)

[0116]

[0117] 10 nanomoles of DNA-COOH (S2) were dissolved in deionized water to prepare 1 mmol / L (10 μL, 1 equivalent), and 160 μL of DMSO, 100 equivalents of 1-(4-piperidinyl-1,3-butanedione) in DMSO (concentration: 200 mmol / L, 10 μL), 80 equivalents of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, concentration: 250 mmol / L, 10 μL) and 200 equivalents of N,N-diisopropylethylamine (DIEA, concentration: 250 mmol / L, 10 μL) were added, and the mixture was thoroughly mixed with a vortex oscillator and reacted at 25° C. for 2 hours. After the reaction was completed, 10% of the total volume of 5 mol / L sodium chloride solution was added to the reaction solution. Then, continue to add anhydrous ethanol 3 times the total volume, shake evenly, and place the reaction solution in a -80°C refrigerator for 2 hours. After that, centrifuge at 4000rpm for half an hour, and pour out the supernatant. The remaining precipitate is dissolved in deionized water to obtain a solution of oligonucleic acid-diketone compound raw material (S5-1). The spectrum of the oligonucleic acid-diketone compound raw material (S5-1) detected by liquid chromatography-mass spectrometry is as follows Figure 4 As shown, its molecular weight is 5394 and the yield is 83%.

[0118] 4. Synthesis of oligonucleic acid-diketone compound raw material (S5-2)

[0119]

[0120] Dissolve 10 nanomoles of DNA-NH2(S4) in deionized water to make 1 mmol / L (10 microliters, 1 equivalent), and add 200 equivalents of DMSO solution of 4,6-dioxoheptanoic acid (commercial product) (concentration: 200 mmol / L), 250 equivalents of sodium tetraborate (Na2B4O7) buffer solution with pH = 9.5 (concentration: 250 mmol / L), and 150 equivalents of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) aqueous solution (concentration: 200 mmol / L), mix well and react at 25°C for 12 hours. After the reaction is completed, add 5 mol / L sodium chloride solution with a total volume of 10% to the reaction solution. Then, continue to add 3 times the total volume of anhydrous ethanol, shake evenly, and place the reaction solution in a refrigerator at -80°C for 2 hours. After that, centrifuge at 4000rpm for half an hour and pour off the supernatant. The remaining precipitate is dissolved in deionized water to obtain a solution of the oligonucleic acid-diketone compound raw material (S5-2). The spectrum of the oligonucleic acid-diketone compound raw material (S5-2) is detected by liquid chromatography-mass spectrometry. Figure 5 As shown, its molecular weight is 5323 and the yield is 91%.

[0121] Example 1: Synthesis of oligonucleic acid-pyrazole compound (P1) using oligonucleic acid-diketone compound raw material (S5-1)

[0122]

[0123] To a 0.5 nanomole aqueous solution of an oligonucleic acid-dione compound (S5-1, 1 mmol / L aqueous solution, 1 equivalent, 0.5 nanomole, 0.5 microliter), sodium dihydrogen phosphate buffer (250 mmol / L aqueous solution, 250 equivalents, 0.5 microliter) at pH = 3.5 and a 3-hydrazinoquinoline hydrochloride solution (50 mol / L acetonitrile solution, 50 equivalents, 0.5 microliter) were added, and the mixture was thoroughly mixed by vortexing and reacted at 25° C. for 5 hours. After the reaction was completed, a 5 mol / L sodium chloride solution of 10% of the total volume and 3 times of anhydrous ethanol were added to the reaction solution, and after being shaken evenly, it was placed in a refrigerator at -80° C. for 2 hours, and then high-speed refrigerated centrifugation (4° C., 12,000 rpm, 15 minutes) was performed, and the supernatant was discarded. The remaining precipitate was the product oligonucleic acid-pyrazole compound (P1), which was detected by liquid chromatography-mass spectrometry. The detection results are shown in FIG. Figure 8 , molecular weight is 5516, and the yield is 86%.

[0124] The S6-1 compound is replaced by Figure 6 The raw materials (S6-2 to S6-32) shown in the figure are used to synthesize other representative oligonucleic acid-pyrazole compounds ( Figure 7 , P2~P32), the test results are shown in Figure 9-Figure 39 .

[0125] Example 2: Synthesis of oligonucleic acid-pyrazole compound (P33) using oligonucleic acid-diketone compound raw material (S5-2)

[0126]

[0127] To an aqueous solution of 0.5 nanomoles of oligonucleic acid-dione compound (1 millimole / liter aqueous solution, 1 equivalent, 0.5 nanomoles, 0.5 microliters), sodium dihydrogen phosphate buffer (250 millimole / liter aqueous solution, 250 equivalents, 0.5 microliters) at pH = 3.5 and 3-hydrazinoquinoline hydrochloride solution (50 mol / liter acetonitrile solution, 50 equivalents, 0.5 microliters) were added, and the mixture was thoroughly mixed by vortexing and reacted at 25° C. for 5 hours. After the reaction was completed, 10% of the total volume of 5 mol / liter sodium chloride solution and 3 times of anhydrous ethanol were added to the reaction solution, and after being shaken evenly, it was placed in a refrigerator at -80° C. for 2 hours, and then high-speed refrigerated centrifugation (4° C., 12,000 rpm, 15 minutes) was performed, and the supernatant was discarded. The remaining precipitate was the product oligonucleic acid-pyrazole compound (P1), which was detected by liquid chromatography-mass spectrometry. The detection results are shown in Fig.40 , molecular weight is 5454.2, and the yield is 95%.

[0128] In addition, the oligonucleic acid-diketone compound raw material (S5-2) and the hydrazine reagent S6-24 (structure see Figure 6 ) Synthesized oligonucleic acid-pyrazole compounds (P34, see Figure 7 ), the test results are shown in Fig.41 .

[0129] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0130] Experimental Example 1: Screening of Synthesis Conditions of Oligonucleic Acid-Pyrazole Compounds of the Present Invention

[0131] The method for synthesizing the oligonucleic acid-pyrazole compound P1 in Example 1 was referred to, except that the following parameters were controlled according to Table 1: different pH values, reaction temperatures (° C.).

[0132] The product yield of the oligonucleic acid-pyrazole compound P1 obtained under different parameters was calculated. The results are shown in Table 1.

[0133] Table 1 Product yields of oligonucleic acid-pyrazole compound P1 synthesized under different conditions

[0134] serial number Different pH S6 equivalent Reaction temperature (℃) Reaction time (h) Product yield 1 9.5 50 25 5 50％ 2 9.5 50 40 5 45％ 3 9.5 50 50 5 50％ 4 8.0 50 25 5 53％ 5 7.0 50 25 5 67％ 6 6.0 50 25 5 68％ 7 3.5 50 25 5 95％

[0135] It can be seen from Table 1 that under the specific reaction conditions of the embodiments of the present invention, the yield of the oligonucleic acid-pyrazole compound P1 obtained is significantly improved, which can be as high as 95%.

[0136] Experimental Example 2: Integrity verification experiment of oligonucleic acid in oligonucleic acid-pyrazole compounds of the present invention

[0137]

[0138] Oligonucleic acid-pyrazole compound P3 and TagA (short-chain oligonucleotide, molecular weight of two chains are 4064 and 5884 respectively) link: 1 nanomole of P3 is dissolved in deionized water to prepare a solution with a concentration of 1 mmol / L (1 μl, 1 nanomole, 1 equivalent), and 1.2 equivalents of TagA (1 mmol / L aqueous solution, 1.2 μl, 1.2 equivalents), 1 μl of 10x T4 DNA linking buffer solution and 0.5 μl of T4 DNA linking enzyme are added thereto. Then the above solutions are mixed evenly and reacted at room temperature for 1 hour. After the reaction is completed, 10% of the total volume of 5 mol / L sodium chloride solution is added to the reaction solution, and then 3 times the total volume of anhydrous ethanol is added. After shaking evenly, the reaction is placed in a refrigerator at -80°C and frozen for 2 hours. After that, centrifuge at 4000rpm for half an hour and pour off the supernatant. The remaining precipitate was dissolved in deionized water and the molecular weight of the product was confirmed by liquid chromatography-mass spectrometry. The molecular weight was 15393. The mass spectrometry results are shown in Fig.42 .

[0139] The LCMS analysis results show that the oligonucleic acid-pyrazole compound P3 can be successfully coupled with TagA. This shows that the DNA chain integrity of the oligonucleic acid-pyrazole compound obtained by the synthesis method described in the present invention is good. The LCMS mass spectrum can accurately display its molecular weight, further confirming that its nucleotides are not damaged. Therefore, the reaction method in the present invention will not damage the basic structure and activity of DNA.

[0140] In summary, the present invention provides a method for synthesizing oligonucleic acid-pyrazole lead compounds on DNA, reacting under specific pH conditions and temperature for a specific time to successfully produce oligonucleic acid-pyrazole lead compounds. The method has little damage to DNA, good universality, simple operation, mild conditions, and can produce oligonucleic acid-pyrazole compounds in high yield, providing an important basis for expanding and enriching the DELT compound library.

Claims

1. A method for synthesizing an oligonucleic acid-pyrazole compound, characterized in that: It uses the oligonucleic acid-diketone compound shown in formula III and the hydrazine reagent shown in formula II as raw materials, reacts in a solvent to obtain the oligonucleic acid-pyrazole compound shown in formula I; the reaction formula is as follows: Wherein, the compound of formula I is one of the following compounds: The molar ratio of the oligonucleic acid-diketone compound represented by formula III to the hydrazine reagent represented by formula II is 1:50; The reaction temperature is 25°C; The reaction time is 5 hours; The reaction is carried out at pH 3.5; The solvent is a mixture of water, acetonitrile and sodium dihydrogen phosphate buffer solution, and the total content of acetonitrile is not higher than 50%.

2. Use of the synthesis method according to claim 1 in constructing a library of gene-encoded compounds.

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