Method for synthesizing On-DNA aryl sulfide structure compound through photochemical reaction

Abstract

The invention belongs to the field of lead compound library construction in medical research, and particularly relates to a construction method of an On-DNA aryl sulfide structure compound, which comprises the step of reacting On-DNA aryl mercaptan or On-DNA aryl thiophenol with a halide to generate the On-DNA aryl sulfide structure compound, the step is a photocatalytic reaction at room temperature, and the on-DNA aryl sulfide structure compound is obtained. The wavelength of the light source for reaction is 315-455 nanometers, the solvent for reaction comprises an organic solvent and water, and the organic solvent in the solvent is one or more of dimethyl sulfoxide, N, N-dimethylformamide and dimethylacetamide. The oligomeric nucleic acid-aryl sulfide structure compound with rich structures can be obtained at a time under the reaction conditions, the reaction conditions are wide in adaptability and obviously superior to the prior art, the oligomeric nucleic acid-aryl sulfide structure compound is suitable for being used as a reaction raw material of a first-stage or second-stage halide, and the conversion rate of a third-stage iodide can also reach 90% or above.

Description

Technical Field

[0001] The present application belongs to the field of lead compound library construction in medical research, and specifically relates to a method for constructing On-DNA aryl thioether structure compounds. Background Art

[0002] In the field of new drug development, high-throughput screening for biological targets is one of the main means to quickly obtain lead compounds. However, traditional high-throughput screening based on single molecules takes a long time, requires huge equipment investment, and has a limited number of library compounds (millions). In addition, the construction of a compound library requires decades of accumulation, which limits the efficiency and possibility of discovering lead compounds. The DNA-encoded compound library technology (DEL technology) that has emerged in recent years combines combinatorial chemistry and molecular biology techniques. At the molecular level, each compound is added with a DNA tag, which can synthesize a compound library of up to 100 million in a very short time. DEL technology has continued to develop in the past 10 years, and some recently reported DNA-encoded library screening based on active proteins and living cells has broadened the scope of this exciting technology. The development of new reactions compatible with DNA has enriched the construction space of various compounds with drug properties and promoted the construction of new DEL libraries.

[0003] Aromatic sulfides are commonly found in a wide range of bioactive natural products and drugs, such as Thymitaq, which is a non-competitive thymidylate synthase inhibitor that interacts with the folate cofactor binding site of the enzyme and can be used to treat liver cancer and nasopharyngeal carcinoma. Axitinib is a multi-target small molecule inhibitor, with the main targets being VEGFR, Kit, and PDGFR. Axitinib is an anti-tumor drug, and is clinically used mainly for adult patients with advanced renal cell carcinoma (RCC) who have previously failed a tyrosine kinase inhibitor or cytokine therapy. Nelfinavir mesylate is an effective, orally bioavailable HIV-1 protease inhibitor used for HIV infection and is a broad-spectrum anticancer agent.

[0004] However, after consulting many existing literatures, it was found that the conditions for synthesizing small molecule aromatic sulfides are not suitable for the reaction of tertiary halides with aromatic thiols (phenols) to generate aromatic sulfides, and even the conversion rate of secondary halides is relatively low. Therefore, it is necessary to develop a method for preparing On-DNA aromatic sulfide structure compounds with broad applicability to meet the needs of DEL technology for the construction and screening of lead compounds. Summary of the invention

[0005] In order to solve the above technical problems, the present application provides an On-DNA chemical reaction with wide substrate adaptability compared with the above prior art, that is, synthesizing On-DNA aryl sulfide structure compounds by photochemical reaction.

[0006] The specific method for preparing the On-DNA aryl thioether structure compound comprises: the step of reacting On-DNA aryl thiol or On-DNA aryl thiophenol with a halide to generate the On-DNA aryl thioether structure compound, the reaction equation is: Where R 1 is selected from benzyl, substituted or unsubstituted aryl or heteroaryl, and cycloaryl, wherein the substituted R 1 The substituent is halogen, alkyl or alkoxy; R 2 It is a substituted or unsubstituted primary alkyl, secondary alkyl, tertiary alkyl, benzyl and aryl, cycloaryl, heterocyclic aryl, or cycloheteroaryl; the substituent of the substituted primary alkyl is alkyl, alkenyl, alkynyl, cyano, hydroxyl, halogen, carboxyl, cycloalkyl, cycloalkyloxy; the halogen is monosubstituted or trisubstituted fluorine or chlorine; the cycloalkyl is a three- or four-membered ring; the cycloalkoxy is a cyclobutaneoxy; the secondary alkyl is a chain, cyclic, heterocyclic and spiroheterocyclic secondary alkyl; the cyclic is a three- to six-membered ring; the heterocyclic is a four- to six-membered heterocyclic ring, and its heteroatom is O, N, S; the spiroheterocyclic is an oxadiquadruple spiro ring; The substituents of the substituted secondary alkyl are ester, carboxyl, and alkyl; the tertiary alkyl is a chain or cyclic tertiary alkyl, and the cyclic is a four-membered bridge ring, a cubic ring, or a six-membered bridge ring; the substituents of the substituted tertiary alkyl are ester, cyano, halogen, and carboxyl; the halogen is disubstituted or trisubstituted fluorine; the substituent of the benzyl is iodine; the substituent of the aryl is alkyl, alkoxy, halogen, trifluoromethoxy, cyano, acetyl, aldehyde, alkenyl, methylamino, carboxyl, alkynyl, and aryl; the halogen is fluorine, chlorine, or bromine; the cyclic aromatic group is naphthyl; the heterocyclic aromatic group is a pyridine ring group; the cyclic heteroaromatic group is a benzopyridine ring group; The solvent comprises an organic solvent and water, wherein the organic solvent in the solvent is one or more of dimethyl sulfoxide, N,N-dimethylformamide and dimethylacetamide; The wavelength of the light source is 315 nanometers to 455 nanometers; The temperature is not higher than 40°C.

[0007] In some embodiments of the present application, the experimenters tried organic solvents DMSO, DMF, DMA, MeCN, PrOH and DME, and the experimental results showed that the mixed solvents of DMSO, DMF and DMA with water were significantly better than the mixed solvents of MeCN, PrOH and DME with water. The experimental results showed that the volume ratio of water to organic solvent had an effect on the conversion rate of 1:4>3:7>1:9, and 1:4 had the highest conversion rate.

[0008] In some embodiments of the present application, experimenters tried multiple light source wavelengths within the range of ultraviolet lamps. Experiments have shown that, within the wavelength range of ultraviolet to white light conversion, when the wavelength increases to 405 nanometers, the conversion rate decreases with the increase of wavelength. The applicant believes that the light source wavelength is preferably 315 to 455 nanometers, preferably 360 to 405 nanometers, and more preferably 360 to 370 nanometers.

[0009] In some embodiments of the present application, the reaction temperature has little effect on the conversion rate as long as the DNA chain is not broken. For ease of operation, 0°C to 40°C is a more convenient operating temperature according to different conditions such as different regions and climates.

[0010] In some embodiments of the present application, the conversion rate is reduced in the absence of an antioxidant, but it can still be achieved. Therefore, the applicant believes that a higher conversion rate can be obtained under the condition of an antioxidant. Surprisingly, sodium ascorbate is the best antioxidant in the experiment of the present application, and the concentration of the antioxidant is preferably 5-15 mmol / L, more preferably 5-10 mmol / L.

[0011] In some embodiments of the present application, the reaction can still proceed smoothly without adding DTT, and adding DTT at a final concentration of 0.1-1.0 mmol / L helps to obtain a higher conversion rate.

[0012] In summary, the reaction conditions of the present application can obtain oligonucleic acid-aryl thioether structure compounds with rich structures at one time, and the adaptability of the reaction conditions is significantly better than that of the prior art. It is not only suitable for primary or secondary halides as reaction raw materials, but the conversion rate of tertiary iodides can also reach more than 90%. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : S3g 1 H NMR spectrum (DMSO-d6, 400 MHz).

[0014] Figure 2 : S3i 1 H NMR spectrum (DMSO-d6, 400 MHz).

[0015] Figure 3 : S3n 1 H NMR spectrum (DMSO-d6, 400 MHz).

[0016] Figure 4-15 :Oligonucleic acid-thiol (phenol) compounds 1a-1d, 1g-1i, 1k-1o.

[0017] Figure 16-22 :Oligonucleic acid-thiol (phenol) compounds 1e, 1f, 1j, 1p-1s.

[0018] Fig.23 :Oligonucleic acid-thiol (phenol) compounds 1t.

[0019] Figure 24-128 : Oligonucleic acid-aryl thioether structure compounds 3a-3t and 3aa-3de.

[0020] Figure 129: 129a is the LCMS spectrum of raw material S4a; 129b is the LCMS spectrum of raw material 2cu; 129c is the LCMS spectrum of product S6a of comparative example 1.1; 129d is the LCMS spectrum of raw material 2bl; 129e is the LCMS spectrum of product S6b of comparative example 1.2.

[0021] Figure 130: 130a is the LCMS spectrum of the product 3cu of Comparative Example 2.1; 130b is the LCMS spectrum of the product 3bl of Comparative Example 2.2.

[0022] Fig.131 :Liquid chromatography-mass spectrometry confirmed the test results of the link integrity between the oligonucleic acid-aryl thioether structure compound 3bw and Tag A. DETAILED DESCRIPTION

[0023] BHT: 2,6-di-tert-butyl-4-methylphenol; TBHQ: tert-butylhydroquinone; DMSO: dimethyl sulfoxide; DMF: N,N-dimethylformamide; DMA: dimethylacetamide; MeCN: acetonitrile; i PrOH: isopropanol; DME: dimethyl ether; DTT: dithiothreitol; Oligonucleic acid-thiol (phenol) compounds are direct raw materials for preparing On-DNA aryl thioether structure compounds. The preparation method of oligonucleic acid-thiol (phenol) compounds is as follows: (1) HP-Linker-NHFmoc is prepared from HP-NH2 (commercially available reagent), (2) HP-Linker-NH2 is then prepared from HP-Linker-NHFmoc, and (3) in the third step, HP-Linker-NH2 is converted into oligonucleic acid-thiol (phenol) compounds. Alternatively, HP-Linker-COOH is prepared from HP-NH2 (commercially available reagent), and then HP-Linker-COOH is converted into oligonucleic acid-thiol (phenol) compounds. This application uses the same method as CN118461019A to prepare the structures of HP-Linker-NH2 and HP-Linker-COOH, so the structural confirmation diagrams of HP-NH2, HP-Linker-NHFmoc, HP-Linker-NH2 and HP-Linker-COOH are omitted.

[0024] 1. Synthesis of oligonucleotide-Linker-NH2 raw material (HP-Linker-NH2) 1.1 Preparation of HP-Linker-NHFmoc from HP-NH2 (commercially available reagent) has the following reaction equation: Specifically, 100.0 nanomoles of HP-NH2 (commercially available product) was dissolved in deionized water to prepare a 1.0 mmol / L solution (100.0 microliters, 100.0 nanomoles, 1.0 equivalent). 40.0 equivalents of S1 (commercially available product) in DMSO solution (concentration: 200.0 mmol / L), 250.0 equivalents of sodium tetraborate (Na2B4O7) buffer solution with pH = 9.5 (concentration: 250.0 mmol / L), and 40.0 equivalents of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) aqueous solution (concentration: 200.0 mmol / L) were mixed, and the mixture was fully mixed with a vortex oscillator. The above mixture was then added to the HP-NH2 solution, mixed evenly, and reacted at 4°C for 1 hour. After the reaction was completed, 10% of the total volume of 5.0 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 4000.0rpm for half an hour and pour off the supernatant. The remaining precipitate is dissolved in deionized water to obtain a solution of HP-Linker-NHFmoc. HP-Linker-NHFmoc is detected by liquid chromatography-mass spectrometry, and its molecular weight is 5406.

[0025] 1.2 Preparation of HP-Linker-NH2 from HP-Linker-NHFmoc: 100.0 nanomoles of HP-Linker-NHFmoc were dissolved in deionized water to prepare 1.0 mmol / L (100.0 microliters, 100.0 nanomoles, 1.0 equivalents), and 36.0 microliters of 10% piperidine aqueous solution were added thereto. The two were mixed evenly and reacted at room temperature for 1 hour. After the reaction was completed, a 5.0 mol / L sodium chloride solution with a total volume of 10% was added to the reaction solution. Then, 3 times the total volume of anhydrous ethanol was added, and after oscillation, the reaction was placed in a refrigerator at -80°C and frozen for 2 hours. After that, centrifuged at a speed of 4000.0rpm for half an hour, and the supernatant was poured out. The remaining precipitate was dissolved in deionized water to obtain a solution of oligonucleotide-Linker-NH2 (HP-Linker-NH2). HP-Linker-NH2 was detected by liquid chromatography-mass spectrometry, and its molecular weight was 5184.

[0026] 2. Preparation of HP-Linker-COOH from HP-NH2: Specifically, 100.0 nanomoles of HP-NH2 are dissolved in deionized water to prepare a 1.0 mmol / L solution (100.0 microliters, 100.0 nanomoles, 1.0 equivalent). 40.0 equivalents of S2 (commercially available product) in DMSO solution (concentration: 200.0 mmol / L), 250.0 equivalents of sodium tetraborate (Na2B4O7) buffer solution with pH = 9.5 (concentration: 250.0 mmol / L), and 40.0 equivalents of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) aqueous solution (concentration: 200.0 mmol / L) are mixed, and the mixture is fully mixed with a vortex oscillator. Then the above mixture is added to the HP-NH2 solution, mixed evenly, and reacted at 4°C for 1 hour. After the reaction is completed, 10% of the total volume of 5.0 mol / L sodium chloride solution is 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 4000.0rpm for half an hour, and pour out the supernatant. The remaining precipitate is dissolved in deionized water to obtain a solution of HP-Linker-COOH. HP-Linker-COOH is detected by liquid chromatography-mass spectrometry, and its molecular weight is 5230.

[0027] 3. Sources or synthesis methods of raw materials used to prepare oligonucleic acid-thiol (phenol) compounds: The present application prepares oligonucleic acid-thiol (phenol) compounds by reacting the following molecules with HP-Linker-NH2 or with HP-Linker-COOH: Except for S3g, S3i, and S3n, which are homemade, the rest are commercially available. The reaction equations for preparing S3g, S3i, and S3n are: Where a is HCl, NaNO2, Na2S.9H2O, S8, NaOH, 0℃ to rt; where b is DMSO, 80℃.

[0028] A more specific method for preparing S3g, S3i, and S3n is as follows: 0.3 g (1.3 eq.) of sodium nitrite (NaNO2) is dissolved in 1.0 ml of ice-cooled aqueous solution, and then dropwise added to a mixed solution of 1.0 ml of an aqueous solution of 3.3 mmol (1.0 eq.) of aminocarboxylic acid and 0.6 ml of concentrated hydrochloric acid (HCl) at 0°C, and the mixture is stirred at 5°C for 30 minutes. Then, 1.04 g (1.3 eq.) of sodium sulfide nonahydrate (Na2S.9H2O) in 1.0 ml of cooled aqueous solution, 138.0 mg (0.16 eq.) of sulfur (S8), and 0.3 ml of a 10.0 mol / L sodium hydroxide (NaOH) aqueous solution are added in sequence. The mixture is stirred at room temperature for 2 hours, and then acidified to pH 2 with hydrochloric acid. The resulting precipitate is collected by filtration, washed with water, dried under high vacuum, and can be entered into the next step without further purification.

[0029] The solid obtained in the previous step was dissolved in 5.0 ml of DMSO and stirred at 80°C overnight. Purification by reverse phase column chromatography gave the corresponding yellow solid disulfide. Figure 1-3 .

[0030] S3G Preparation of: Disulfide S3g (131.0 mg, 23%) was obtained from 3-amino-5-methylbenzoic acid (500.0 mg, 3.31 mmol) as a yellow solid. 1 H NMR(400MHz,DMSO-d6)δ13.11(s,2H),7.90–7.84(m,2H),7.69–7.66(m,2H),7.61–7.56(m,2H),2.35(s,6H).MS(ESI-MS)m / z:333[MH] + . S3i Preparation of: Disulfide (S3i, 197.0 mg, 35%) was obtained from 3-amino-5-fluorobenzoic acid (500.0 mg, 3.22 mmol) as a yellow solid. 1H NMR(400MHz, DMSO-d6)δ13.53(s,2H),7.91(t,J=1.6Hz,2H),7.72(dt,J=8.8,2.1Hz,2H),7.62–7.54(m,2H).MS(ESI-MS)m / z:341[MH] + . S3n Preparation of: Disulfide (S3n, 134.0 mg, 24%) was obtained from 4-amino-3-fluorobenzoic acid (500.0 mg, 3.22 mmol) as a yellow solid. 1 H NMR(400MHz,DMSO-d6)δ13.40(s,2H),7.91–7.64(m,6H).MS(ESI-MS)m / z:341[MH] + . 4. Preparation of oligonucleic acid-thiol (phenol) compounds 4.1 Preparation of oligonucleic acid-thiol (phenol) compounds from HP-Linker-NH2 4.1.1 Synthesis of oligonucleic acid-thiol (phenol) compound raw material (1a) prepared from HP-Linker-NH2 50.0 nanomoles of HP-Linker-NH2 were dissolved in deionized water to prepare 1.0 mmol / L (50.0 μL, 50.0 nanomoles, final concentration 0.2 mmol / L). Mix the DMSO solution of disulfide S3a (commercial product) (200.0 mmol / L, 50.0 μL, final concentration of 40.0 mmol / L), EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1,200.0 mmol / L, 50.0 μL, final concentration of 240.0 mmol / L), HOAt (1-hydroxy-7-azobenzotriazole, 240.0 mmol / L, 50.0 μL, final concentration of 48.0 mmol / L) and DIEA (N,N-diisopropylethylamine, 1,200.0 mmol / L, 50.0 μL, final concentration of 240.0 mmol / L), and mix the mixture thoroughly with a vortex oscillator. Then add the above mixture to the solution of HP-Linker-NH2, mix well and react at 25°C for 1 hour. After the reaction is completed, add 10% of the total volume of 5.0 mol / L sodium chloride solution 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 -80°C refrigerator for 2 hours. After that, centrifuge at 4000.0 rpm for half an hour, and pour out the supernatant. The remaining precipitate is treated with DTT to cleave the disulfide bond (patent: CN 114853822B), and then precipitated with sodium chloride / ethanol and dissolved in deionized water to obtain a solution of 1a, whose molecular weight is 5334.9 and the yield is 97%.

[0031] The invention team of this application adopted the method for preparing raw material 1a to synthesize other oligonucleic acid-thiol (phenol) compounds (1b-1d, 1g-1i, 1k-1o). The test results of 1a-1d, 1g-1i, 1k-1o are shown in Figure 4-15 .

[0032] 4.1.2 Synthesis of oligonucleic acid-thiol (phenol) compound raw material (1e) prepared from HP-Linker-NH2 Dissolve 50.0 nanomoles of HP-Linker-NH2 in deionized water to make 1.0 mmol / L (50.0 microliters, 50.0 nanomoles, final concentration 0.2 mmol / L). Mix the DMSO solutions of disulfide S3e (commercial product) (200.0 mmol / L, 50.0 microliters, final concentration 40.0 mmol / L), EDCI (1,200.0 mmol / L, 50.0 microliters, final concentration 240.0 mmol / L), HOAt (240.0 mmol / L, 50.0 microliters, final concentration 48.0 mmol / L) and DIEA (1,200.0 mmol / L, 50.0 microliters, final concentration 240.0 mmol / L), and mix the mixture thoroughly with a vortex oscillator. Then add the above mixture to the HP-Linker-NH2 solution, mix well, and react at 25°C for 1 hour. After the reaction is completed, add 10% of the total volume of 5.0 mol / L sodium chloride solution 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 -80°C refrigerator for 2 hours. After that, centrifuge at 4000.0rpm for half an hour, and pour out the supernatant. The remaining precipitate is dissolved in deionized water to obtain a 1e solution, whose molecular weight is 5338.9 and the yield is >99%.

[0033] The invention team of this application adopted the method of preparing raw material 1e to synthesize other oligonucleic acid-thiol (phenol) compounds (1f, 1j, 1p-1s). The test results of 1e, 1f, 1j, 1p-1s are shown in Figure 16-22 .

[0034] 4.2 Synthesis of raw materials (1t) of oligonucleic acid-thiol (phenol) compounds prepared from HP-Linker-COOH Dissolve 50.0 nanomoles of HP-Linker-COOH in deionized water to make 1.0 mmol / L (50.0 microliters, 50.0 nanomoles, final concentration 0.1 mmol / L). Mix HATU (O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, 50.0 mmol / L, 50.0 microliters, final concentration 5.0 mmol / L), DIEA (50.0 mmol / L, 50.0 microliters, final concentration 5.0 mmol / L) and S3t (50. mmol / L, 50.0 microliters, final concentration 5.0 mmol / L) in DMSO, and mix the mixture thoroughly with a vortex oscillator. Add the above mixture to the HP-Linker-COOH solution, mix well, and react at 25°C for 2 hours. After the reaction is completed, add 10% of the total volume of 5.0 mol / L sodium chloride solution 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 -80°C refrigerator for 2 hours. After that, centrifuge at 4000.0rpm for half an hour and pour off the supernatant. The remaining precipitate is dissolved in deionized water to obtain a 1t solution with a molecular weight of 5393.8 and a yield of 86%. The 1t test results are shown in Fig.23 .

[0035] Examples 1-18 Take the following reaction as an example to optimize the reaction conditions The specific reaction conditions and reaction results are shown in Table 1.

[0036] Table 1 Notes: a Final concentration.

[0037] b The conversion rate was determined by LC-MS.

[0038] c No nitrogen.

[0039] d BHT: 2,6-di-tert-butyl-4-methylphenol.

[0040] e TBHQ: tert-butylhydroquinone.

[0041] fGeneral reaction conditions: 1k (10.0 nmol, 1.0 eq., 0.1 mmol / L final concentration), sodium ascorbate (10.0 mmol / L final concentration), DTT (dithiothreitol, 0.5 mmol / L final concentration) and 2df (10.0 mmol / L final concentration) in solvent water / DMSO (v:v=1:4) were irradiated with UV lamp (365 nm, 5 W) at 25°C for 10 min.

[0042] g No DTT.

[0043] As for the light source, the applicant must first explain that the light source described in this application is ultraviolet light. It is well known to those skilled in the art that the maximum wavelength of ultraviolet light is usually 400nm, but it is found in experimental studies that when the wavelength is 405nm, the conversion rate decreases significantly, which may be caused by the lack of energy of the long wave, so the wavelength between 365 and 455 is intercepted for detailed description. Comparing Examples 1-4, we can see that under the condition that other conditions remain unchanged, the conversion rate of On-DNA aryl sulfide structure compound 3k is higher than that of others under the irradiation of ultraviolet light source with a wavelength of 365 nanometers. Under the same conditions, as the wavelength increases, the conversion rate decreases, and according to the wavelength range of the light source actually used and the experimental results, the applicant believes that the wavelength of the light source is preferably 315 to 455 nanometers, preferably 360 to 405, and more preferably 360 to 370 nanometers.

[0044] Regarding the inert protection conditions, the nitrogen described in this embodiment is only a representative of the inert protection, not all. By comparing Examples 2 and 5, we can see that when other reaction conditions remain unchanged, without the protection of inert gas (nitrogen), the conversion rate of the On-DNA aryl sulfide structure compound 3k will be significantly reduced; therefore, the applicant believes that the preferred reaction condition is to be carried out under the protection of inert gas.

[0045] By comparing Examples 2, 6-7, we can see that when other reaction conditions remain unchanged, sodium ascorbate is used as an antioxidant, and the conversion rate of On-DNA aryl sulfide structure compound 3k is higher than that of others. The applicant believes that the preferred antioxidants are tert-butylhydroquinone (TBHQ) and sodium ascorbate, among which sodium ascorbate is the best antioxidant in this technical solution.

[0046] By comparing Examples 2 and 8-10, we can see that when other reaction conditions remain unchanged, when the final concentration of sodium ascorbate is 0-10 mmol / L, the conversion rate of On-DNA aryl sulfide structure compound 3k increases with the increase of the final concentration of sodium ascorbate. When the final concentration of sodium ascorbate continues to increase to 15 mmol / L, the conversion rate of On-DNA aryl sulfide structure compound 3k decreases slightly. According to the above results, the technical solution of the present application can still be achieved in the absence of sodium ascorbate, preferably 5-15 mmol / L, more preferably 5-10 mmol / L.

[0047] By comparing Examples 2 and 11-15, we can see that the effect of solvent on the conversion rate of On-DNA aryl sulfide structure compound 3k is that the effect of mixed solvents of DMSO, DMF and DMA with water is significantly better than that of mixed solvents of MeCN, PrOH, and DME with water. The applicant believes that the organic solvent for the reaction is selected from any one of DMSO, DMF or DMA.

[0048] By comparing Examples 2, 16-17, we can see that the effect of the volume ratio of water to organic solvent on the conversion rate of On-DNA aryl sulfide structure compound 3k is 1:4>3:7>1:9. Based on this experimental result, the applicant believes that the ratio of water to organic solvent used in the reaction is 1:9-3:7.

[0049] By comparing Examples 16 and 18, we can see that when other reaction conditions remain unchanged and DTT is not added, the conversion rate of On-DNA aryl thioether structure compound 3k will decrease, and the types and amounts of impurities will increase. Therefore, it is preferred to add DTT at a final concentration of 0.1-1.0 mmol / L.

[0050] Example 19 Synthesis of oligonucleic acid-aryl thioether structure compound (3a) using oligonucleic acid-thiol (phenol) compound raw material (1a) Oligonucleic acid-thiol (phenol) compound (1a, 10.0 nanomoles, 1.0 equivalents, 1.0 mmol / L aqueous solution, 10.0 μL, 0.1 mmol / L final concentration), sodium ascorbate (100.0 mmol / L aqueous solution, 10.0 μL, 10.0 mmol / L final concentration), DTT (dithiothreitol, 10.0 mmol / L DMSO solution, 5.0 μL, 0.5 mmol / L final concentration), 2df (100.0 mmol / L DMSO solution, 10.0 μL, 10.0 mmol / L final concentration) and 65.0 μL DMSO were added to a 0.6 ml centrifuge tube. The mixture was thoroughly mixed by vortexing, and then shaken and irradiated (365 nm, 50 W UV lamp) at 25° C. for 10 minutes. After the reaction, 10% of the total volume of 5.0 mol / L 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, 12000 rpm, 15 minutes), the supernatant was poured off, and the remaining precipitate was the product oligonucleotide-aryl sulfide structure compound (3a, Fig.24 ), detected by liquid chromatography-mass spectrometry, the molecular weight was 5445.5, and the yield was 81%.

[0051] Example 20-123 The difference from Example 19 is that oligonucleic acid-thiol (phenol) compounds (1b-1t) are used as raw materials to react with different halides (2aa-2df) to synthesize other representative oligonucleic acid-aryl thioether structure compounds (3b-3t, 3aa-3de). The specific structures are shown in Table 2. The detection results of 3a-3t and 3aa-3de are shown in Figure 24-128 .

[0052] Table 2 Comparative Example 1 Synthesis of small molecule aryl sulfide Operation: Purge nitrogen gas into a 2.0 ml centrifuge tube three times. Then add cesium carbonate (Cs2CO3, 4.0 mg, 12.2 μmol, 1.5 equivalents), iodide (2.0 mg, 8.1 μmol, 1.0 equivalents), p-toluene thiophenol (S4a, 1.5 mg, 12.2 μmol, 1.5 equivalents, Fig.129a) and 62 μl DMSO. The reaction mixture was purged with nitrogen three times. The mixture was thoroughly mixed by vortexing, and then shaken and irradiated (26 W white light) at 25°C for 1 hour. Reference 1 is: Liu, B., Lim, CH, & Miyake, GM (2017). JACS, 139(39), 13616–13619. Comparative Example 1.1 The iodide is p-iodoacetophenone (2cu) When the iodide is p-iodoacetophenone (2cu, Fig.129b ), the reaction formula is: It can be seen from LCMS that when the iodide is an aromatic iodide, the target product can be obtained according to the conditions in the literature. Fig.129c .

[0053] Comparative Example 1.2 The iodide is a tertiary alkyl iodide 1-(difluoromethyl)-3-iodobicyclo[1.1.1]pentane (2bl) The difference from Comparative Example 1.1 is that the iodide is a tertiary alkyl iodide 1-(difluoromethyl)-3-iodobicyclo[1.1.1]pentane (2bl, Fig.129d ), the reaction formula is LCMS analysis spectrum see Fig.129e , from the LCMS analysis, it was concluded that the unknown peak was a newly generated peak, but there was no MS signal, so it was impossible to indicate whether the target product was generated.

[0054] Comparative Examples 1.1-2 show that the experimental conditions of Reference 1 cannot satisfy the reaction of tertiary iodide.

[0055] Comparative Example 2 Synthesis of On-DNA Aryl Sulfide under Reference Conditions Operation: Purge nitrogen gas three times into a 0.6 ml centrifuge tube. Then add cesium carbonate (C s2 CO3, 100.0 mmol / L H2O solution, 50.0 equivalents, 0.5 μL), iodide (100.0 mmol / L DMSO solution, 100.0 equivalents, 1.0 μL), oligonucleotide-thiol (phenol) compound (1k, 1.0 mmol / L H2O solution, 1.0 nanomolar, 1.0 μL) and 7.5 μL DMSO. The reaction mixture was purged with nitrogen three times. The mixture was thoroughly mixed by vortexing, and then shaken and irradiated (26W white light) at 25°C for 1 hour. The reaction conditions of reference 1 were still used: Liu, B., Lim, CH, & Miyake, GM (2017). JACS, 139(39), 13616–13619. Comparative Example 2.1 The iodide is p-iodoacetophenone (2cu) When the iodide is p-iodoacetophenone (2cu, Fig.129b), the reaction equation is It can be seen from LCMS that when the iodide is an aromatic iodide, the target product can be obtained according to the conditions in the literature. Fig.130a , the conversion rate is 31%. The conversion rate of Example 76 of the present application is 91%, which is significantly lower than the conversion rate of Example 76 of the present application.

[0056] Comparative Example 2.2 The iodide is a tertiary alkyl iodide 1-(difluoromethyl)-3-iodobicyclo[1.1.1]pentane (2bl) When the iodide is a tertiary alkyl iodide 1-(difluoromethyl)-3-iodobicyclo[1.1.1]pentane (2bl), the reaction formula is It can be seen from LCMS that when the iodide is a tertiary alkyl iodide 1-(difluoromethyl)-3-iodobicyclo[1.1.1]pentane (2bl), the reaction is carried out under the conditions of the reference literature. The reaction results are compared with the results of Example 113. Fig.130b ,pass Fig.130b We can see that under this reaction condition, product 3bl was not obtained because the elution time was different from that of product 3bl. Therefore, the applicant concluded that the target product was not obtained, and the conversion rate of Example 113 of the present application was 94%.

[0057] In summary, by comparing Examples 76 and 113 of the present application with Comparative Examples 1-2, the reaction conditions of the technical solution of the present application are mild, and the range of the wavelength of light is different. According to the literature (Liu, B., Lim, CH, & Miyake, GM (2017). JACS, 139 (39), 13616-13619), the wavelength of light is white light, and it is well known to those skilled in the art that the wavelength range of white light is 400-760 nanometers, and the synthesis of small molecule aromatic sulfides needs to be carried out under alkaline conditions. In addition to being different from the conditions for synthesizing small molecule aromatic sulfides, the technical solution of the present application has also achieved unexpected results, that is, under the mild reaction conditions of the present application, the adaptability is also much greater than that of synthesizing small molecule aromatic sulfides. Combined with the reaction raw materials (Table 2) of the present application, 2bj to 2br are tertiary halides, and by comparing with this comparative example, under the reaction conditions for synthesizing small molecule aromatic sulfides, tertiary halides cannot be achieved.

[0058] Verification Example Integrity Verification Experiment of Oligonucleic Acid in Oligonucleic Acid-Aryl Sulfide Structure Compound of the Present Invention Oligonucleotide-aryl thioether structure compound 3bw and Tag A (short-chain oligonucleotide, molecular weights of the two chains are 4064 and 5884, respectively) are linked: 1.0 nanomole 3bw is dissolved in deionized water to prepare a 1.0 mmol / L solution (1.0 μL, 1.0 nanomole, 1.0 equivalent), and 1.2 equivalents of Tag A (1.0 mmol / L aqueous solution, 1.2 μL), 1.0 μL 10×T4 DNA linking buffer solution and 0.5 μL 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 5.0 mol / L sodium chloride solution is added to the reaction solution, and then 3 times the total volume of anhydrous ethanol is added. After oscillation, the reaction is placed in a refrigerator at -80°C for 2 hours. After centrifugation at 4000 rpm for half an hour, the supernatant is poured out. 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 15307.7. The mass spectrometry results are shown in Fig.131 .

[0059] The results of LCMS analysis show that the oligonucleic acid-aryl thioether structure compound 3bw can be successfully coupled with Tag A. This shows that the DNA chain integrity of the oligonucleic acid-aryl thioether structure compound obtained by the synthesis method described in this application is good. The molecular weight can be accurately displayed by LCMS mass spectrometry, further confirming that the nucleotides are not damaged. Therefore, the reaction method of the present invention will not damage the basic structure and activity of DNA.

[0060] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for preparing an On-DNA aryl thioether structure compound, characterized in that: The method for preparing an On-DNA aryl thioether structure compound comprises the steps of reacting an On-DNA aryl thiol or an On-DNA aryl thiophenol with a halide to generate an On-DNA aryl thioether structure compound, and the reaction equation is: Where R 1 is selected from benzyl, substituted or unsubstituted aryl or heteroaryl, and cycloaryl, wherein the substituted R 1 The substituent is halogen, alkyl or alkoxy; R 2 It is a substituted or unsubstituted primary alkyl, secondary alkyl, tertiary alkyl, benzyl and aryl, cycloaryl, heterocyclic aryl, or cycloheteroaryl; the substituent of the substituted primary alkyl is alkyl, alkenyl, alkynyl, cyano, hydroxyl, halogen, carboxyl, cycloalkyl, cycloalkyloxy; the halogen is monosubstituted or trisubstituted fluorine or chlorine; the cycloalkyl is a three- or four-membered ring; the cycloalkoxy is a cyclobutaneoxy; the secondary alkyl is a chain, cyclic, heterocyclic and spiroheterocyclic secondary alkyl; the cyclic is a three- to six-membered ring; the heterocyclic is a four- to six-membered heterocyclic ring, and its heteroatom is O, N, S; the spiroheterocyclic is an oxadiquadruple spiro ring; The substituents of the substituted secondary alkyl are ester, carboxyl, and alkyl; the tertiary alkyl is a chain or cyclic tertiary alkyl, and the cyclic is a four-membered bridge ring, a cubic ring, or a six-membered bridge ring; the substituents of the substituted tertiary alkyl are ester, cyano, halogen, and carboxyl; the halogen is disubstituted or trisubstituted fluorine; the substituent of the benzyl is iodine; the substituent of the aryl is alkyl, alkoxy, halogen, trifluoromethoxy, cyano, acetyl, aldehyde, alkenyl, methylamino, carboxyl, alkynyl, and aryl; the halogen is fluorine, chlorine, or bromine; the cyclic aromatic group is naphthyl; the heterocyclic aromatic group is a pyridine ring group; the cyclic heteroaromatic group is a benzopyridine ring group; The solvent comprises an organic solvent and water, wherein the organic solvent in the solvent is one or more of dimethyl sulfoxide, N,N-dimethylformamide and dimethylacetamide; The wavelength of the light source is 315 nanometers to 455 nanometers; The temperature is not higher than 40°C.

2. The method for preparing an On-DNA aryl sulfide structure compound according to claim 1, characterized in that: The R 1 is a substituted or unsubstituted aryl group, a six-membered aryl group; said R 1 The heteroatom in the substituted or unsubstituted heterocyclic aromatic group is N, and the number of the heteroatom is 1; the R 1 The substituted or unsubstituted cycloaryl is a benzo five-membered heterocyclic aryl, wherein the heteroatom in the benzo five-membered heterocyclic aryl is N or S, and the number of the heteroatoms is 1 or 2; wherein R 1 The substituent is fluorine, C1 alkyl or alkoxy.

3. The method for preparing an On-DNA aryl thioether structure compound according to claim 1, characterized in that: The volume ratio of water to organic solvent is 1:9 to 3:

7.

4. The method for preparing an On-DNA aryl sulfide structure compound according to claim 1, characterized in that: The wavelength of the light source is 360-405 nanometers.

5. The method for preparing an On-DNA aryl sulfide structure compound according to claim 1, characterized in that: The temperature is from 0°C to room temperature.

6. The method for preparing an On-DNA aryl sulfide structure compound according to claim 1, characterized in that: The reaction conditions for the reaction of On-DNA arylthiol or On-DNA arylthiophenol with a halide also include an atmosphere protected by an inert gas.

7. The method for preparing an On-DNA aryl sulfide structure compound according to claim 1, characterized in that: The reaction conditions of the reaction of On-DNA aryl thiol or On-DNA aryl thiophenol with the halide also include adding an antioxidant into the reaction system, wherein the antioxidant is one of sodium ascorbate, 2,6-di-tert-butyl-4-methylphenol, or tert-butylhydroquinone.

8. The method for preparing an On-DNA aryl sulfide structure compound according to claim 6, characterized in that: The concentration of the antioxidant in the reaction system is 5 mmol / L to 15 mmol / L.

9. The method for preparing an On-DNA aryl sulfide structure compound according to claim 1, characterized in that: The reaction conditions for the reaction of On-DNA arylthiol or On-DNA arylthiophenol with a halide further include adding dithiothreitol into the reaction system.

10. An On-DNA aryl thioether structure compound prepared by the method for preparing an On-DNA aryl thioether structure compound according to any one of claims 1 to 9.

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