Immobilized 4,5-dicyanoimidazole, its preparation and use

Abstract

The present invention provides immobilized 4,5-dicyanoimidazole, its preparation method, and use. The immobilized 4,5-dicyanoimidazole is obtained by radical copolymerization of a first comonomer, a second comonomer, and a third comonomer, where the first comonomer is divinylbenzene or ethylene glycol dimethacrylate, the second comonomer is styrene or methyl methacrylate, and the third comonomer is 2-vinyl-4,5-dicyanoimidazole. This solves the problem in the prior art that 4,5-dicyanoimidazole is difficult to separate from the reaction system after activation in the coupling reaction, and is therefore applicable to the field of 4,5-dicyanoimidazole immobilization.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application is based on and claims priority from a Chinese application with CN application number 202211203439.5 filed on September 29, 2022, the disclosure of which is hereby incorporated in its entirety into this application.

[0002] The present invention relates to the field of 4,5-dicyanoimidazole immobilization, and in particular to immobilized 4,5-dicyanoimidazole, its preparation method and use. [Background technology]

[0003] Small nucleic acids refer to oligonucleotide molecules, including small interfering nucleic acids (siRNA), antisense nucleic acids (ASO), microRNA (miRNA), and nucleic acid aptamers. Small nucleic acid drugs are composed of nucleotides and represent a new class of drugs that are distinct from small molecule drugs and antibody drugs. Compared with traditional chemical drug molecules, small nucleic acid drugs have advantages such as strong target specificity, high efficacy, long-lasting drug action, simple drug design, and a wide range of candidate targets. The main types of small nucleic acid drugs are siRNA drugs and antisense nucleic acid drugs. Both primarily act on cytoplasmic mRNA, regulating protein expression through base-complementary recognition and inhibition of target mRNA, thereby achieving the therapeutic goal of disease.

[0004] In recent years, people's demand for small nucleic acid drugs has increased. In 1998, the U.S. Food and Drug Administration (FDA) approved the market launch of the first small nucleic acid (oligonucleotide) drug, Vitravene, for the treatment of cytomegalovirus retinitis. With the daily success of clinical projects and the potential market demand for oligonucleotide-based drugs, it is extremely important to develop safe, environmentally clean, and cost-effective methods for synthesizing these molecules. In the synthesis of oligonucleotides, one key step is the coupling of ribose, deoxyribose moieties, and phosphoramidite compounds. In laboratories, researchers often use 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (shown in Formula A), which is more active and can react and couple with the hydroxyl group on the glycosyl furan ring without the need for additional activation reagents. In laboratories, this reaction can be performed directly using an automated solid-phase synthesizer.

[0005] [ka]

[0006] However, 2-cyanoethyldiisopropylchlorophosphoramidite is expensive, unstable at room temperature, and prone to explosion when heated. The use of such raw materials increases the cost of industrial production and poses potential risks. The inexpensive and safer 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (shown in Formula B) can be used as an alternative to the above raw materials, but this raw material has low reactivity and must be activated using an amphoteric activator, such as 1H-tetrazole or 4,5-dicyanoimidazole (4,5-DCI).

[0007] However, 1H-tetrazole is expensive, highly toxic, and potentially explosive, making its widespread use in industrial production difficult. While 4,5-DCI is a relatively safe activator and widely used in many phosphorylation reactions, its main drawback is its poor water solubility, which makes it difficult to remove after the reaction (in contrast, 1H-tetrazole is water-soluble and can be removed by washing with water), requiring passage through a silica gel column. This has a negative impact on system expansion. Given 4,5-DCI's safety, low cost, easy availability, and good reaction activity, it remains difficult to completely replace it at this stage.

[0008] [ka] Summary of the Invention [Problem to be solved by the invention]

[0009] The main object of the present invention is to provide immobilized 4,5-dicyanoimidazole, its preparation method and use, in order to solve the problem in the prior art that 4,5-dicyanoimidazole is difficult to separate from the reaction system after activation of the coupling reaction. [Means for solving the problem]

[0010] To achieve the above object, according to a first aspect of the present invention, there is provided an immobilized 4,5-dicyanoimidazole, which is obtained by radical copolymerization of a first comonomer, a second comonomer, and a third comonomer, wherein the first comonomer is divinylbenzene or ethylene glycol dimethacrylate, the second comonomer is styrene or methyl methacrylate, and the third comonomer is 2-vinyl-4,5-dicyanoimidazole.

[0011] Furthermore, in the immobilized 4,5-dicyanoimidazole, the mass ratio of the first comonomer, the second comonomer, and the third comonomer is (10-50):(10-50):(10-80), and the mass ratio of the total mass of the first comonomer and the second comonomer to the third comonomer is ≦9:1, preferably the mass ratio of the first comonomer, the second comonomer, and the third comonomer is (20-30):(20-30):(40-60), and more preferably the mass ratio of the first comonomer, the second comonomer, and the third comonomer is 1:1:2.

[0012] Furthermore, the solubility of the immobilized 4,5-dicyanoimidazole in N,N-dimethylformamide, ether, acetonitrile, alcohol, ester, and chloroalkane is less than 1 g / L. Preferably, the infrared spectrum of the immobilized 4,5-dicyanoimidazole has the following characteristic absorptions: multiple peaks at 3149-2488, 2242, 1911, 1728, 1644, 1574, 1510, and 1370 cm. -1 The method for preparing the infrared detection sample of immobilized 4,5-dicyanoimidazole is the potassium bromide tableting method.

[0013] To achieve the above object, according to a second aspect of the present invention, there is provided a method for producing immobilized 4,5-dicyanoimidazole, the method comprising the step of performing a radical copolymerization reaction of 2-vinyl-4,5-dicyanoimidazole using a first comonomer and a second comonomer, wherein the first comonomer is divinylbenzene or ethylene glycol dimethacrylate, and the second comonomer is styrene or methyl methacrylate.

[0014] Furthermore, in the radical copolymerization reaction, the input mass of the first comonomer is 10 to 50 parts, the input mass of the second comonomer is 10 to 50 parts, but the sum of the input masses of the first comonomer and the second comonomer does not exceed 90 parts, and the input mass of 2-vinyl-4,5-dicyanoimidazole is 10 to 80 parts, preferably the input mass of the first comonomer is 20 to 30 parts, the input mass of the second comonomer is 20 to 30 parts, and the input mass of 2-vinyl-4,5-dicyanoimidazole is 40 to 60 parts, and more preferably the input mass of the first comonomer is 25 parts, the input mass of the second comonomer is 25 parts, and the input mass of 2-vinyl-4,5-dicyanoimidazole is 50 parts.

[0015] Furthermore, the first comonomer, the second comonomer, and 2-vinyl-4,5-dicyanoimidazole are placed in a reaction solvent to carry out a radical copolymerization reaction, where the reaction solvent includes one or more of methyl t-butyl ether, acetonitrile, and N,N-dimethylformamide, and the radical copolymerization reaction is preferably carried out in a nitrogen gas or rare gas atmosphere.

[0016] Furthermore, the radical initiator for the radical copolymerization reaction includes one or more of azobisisobutyronitrile, azobisisoheptanonitrile, or benzoyl peroxide, and the reaction temperature for the radical copolymerization reaction is preferably 65°C to 90°C, and the reaction time for the radical copolymerization reaction is preferably 10 hours to 20 hours.

[0017] To achieve the above object, according to a third aspect of the present invention, there is provided a coupling method, which comprises the step of activating and coupling a phosphoramide with a hydroxyl group using the above immobilized 4,5-dicyanoimidazole or immobilized 4,5-dicyanoimidazole produced by the above method for producing immobilized 4,5-dicyanoimidazole.

[0018] Furthermore, immobilized 4,5-dicyanoimidazole is used to activate the phosphoramide and hydroxyl groups for coupling reaction, and the conversion rate of the coupling reaction is 90% to 94%. Preferably, the compound having a hydroxyl group includes a dimethoxytrityl-protected nucleoside or a dimethoxytrityl-protected deoxynucleoside. Preferably, the compound having a phosphoramide includes a phosphoramidite compound. Preferably, the phosphorylating reagent includes 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite.

[0019] Additionally, the coupling method includes the step of performing a sequential coupling reaction using immobilized 4,5-dicyanoimidazole.

[0020] To achieve the above object, according to a fourth aspect of the present invention, there is provided use of the above immobilized 4,5-dicyanoimidazole, or the preparation method, or the coupling method in a coupling reaction between a phosphoramide and a hydroxyl group and / or in oligonucleotide synthesis. [Effects of the Invention]

[0021] Using the technical solution of the present invention, 4,5-dicyanoimidazole is prepared into immobilized 4,5-dicyanoimidazole, which has highly cross-linked properties, is insoluble in common organic solvents, and can be removed from the reaction system by simple filtration after activation of the coupling reaction. DETAILED DESCRIPTION OF THE INVENTION

[0022] The examples and features of the examples in the present application can be combined with each other as long as they are not contradictory. The present invention will be described in detail below with reference to examples.

[0023] As mentioned in the Background Art section, 4,5-DCI is a relatively safe activator and is widely used in many phosphorylation reactions. However, it has the major drawback of being easily soluble in organic solvents and poorly soluble in water, making it difficult to remove after the reaction, making it difficult to use in reactions that expand the system.

[0024] Therefore, the present inventors attempted to prepare immobilized 4,5-dicyanoimidazole by radical copolymerization of 2-vinyl-4,5-dicyanoimidazole to immobilized 4,5-dicyanoimidazole, which is advantageous for use in phosphorylation reactions. Therefore, a series of protection methods are proposed in this application.

[0025] In a first exemplary embodiment of the present application, there is provided an immobilized 4,5-dicyanoimidazole, the immobilized 4,5-dicyanoimidazole being obtained by a radical copolymerization reaction from a first comonomer, a second comonomer, and a third comonomer, wherein the first comonomer is divinylbenzene or ethylene glycol dimethacrylate, the second comonomer is styrene or methyl methacrylate, and the third comonomer is 2-vinyl-4,5-dicyanoimidazole.

[0026] In a preferred embodiment, in the immobilized 4,5-dicyanoimidazole, the mass ratio of the first comonomer, the second comonomer, and the third comonomer is (10-50):(10-50):(10-80), and the mass ratio of the total mass of the first comonomer and the second comonomer to the third comonomer is ≦9:1, preferably the mass ratio of the first comonomer, the second comonomer, and the third comonomer is (20-30):(20-30):(40-60), and more preferably the mass ratio of the first comonomer, the second comonomer, and the third comonomer is 1:1:2.

[0027] In a preferred embodiment, the solubility of the immobilized 4,5-dicyanoimidazole in N,N-dimethylformamide, ether, acetonitrile, alcohol, ester, and chloroalkane is less than 1 g / L, and preferably, the infrared spectrum of the immobilized 4,5-dicyanoimidazole has the following characteristic absorptions: 3149-2488 (multiple peaks), 2242, 1911, 1728, 1644, 1574, 1510, and 1370 cm -1 The method for preparing the infrared detection sample of immobilized 4,5-dicyanoimidazole is the potassium bromide tableting method, which is a common method for preparing infrared samples in the prior art.

[0028] The immobilized 4,5-dicyanoimidazole is a random copolymer obtained by radical copolymerization. The solubility of the immobilized 4,5-dicyanoimidazole in the above organic solvents and other commonly used organic solvents, either alone or in combination, is less than 1 g / L, even less than 0.1 g / L or 0.01 g / L. Therefore, after activation of the subsequent reaction with the immobilized 4,5-dicyanoimidazole, the immobilized 4,5-dicyanoimidazole can be removed by simple filtration and recycled for subsequent reactions. The mass of the third comonomer in the immobilized 4,5-dicyanoimidazole can be characterized using a conventional method for measuring nitrogen element.

[0029] In a second exemplary embodiment of the present application, there is provided a method for preparing immobilized 4,5-dicyanoimidazole, the method comprising: performing a radical copolymerization reaction on 2-vinyl-4,5-dicyanoimidazole using a first comonomer and a second comonomer, wherein the first comonomer is divinylbenzene or ethylene glycol dimethacrylate, and the second comonomer is styrene or methyl methacrylate.

[0030] After screening various crosslinkers commonly used in conventional technologies, the inventors found that 2-vinyl-4,5-dicyanoimidazole was subjected to radical copolymerization using divinylbenzene or ethylene glycol dimethacrylate and styrene or methyl methacrylate as crosslinkers to obtain high-performance immobilized 4,5-dicyanoimidazole. Such immobilized 4,5-dicyanoimidazole exhibits poor solubility in organic solvents and is insoluble in all common organic solvents and organic solvent mixtures. Therefore, after activation of the immobilized 4,5-dicyanoimidazole for subsequent reactions, the immobilized 4,5-dicyanoimidazole can be removed by simple filtration and recycled for subsequent reactions.

[0031] In a preferred embodiment, in the radical copolymerization reaction, the input mass of the first comonomer is 10 to 50 parts, the input mass of the second comonomer is 10 to 50 parts, but the sum of the input masses of the first comonomer and the second comonomer does not exceed 90 parts, and the input mass of 2-vinyl-4,5-dicyanoimidazole is 10 to 80 parts, preferably the input mass of the first comonomer is 20 to 30 parts, the input mass of the second comonomer is 20 to 30 parts, and the input mass of 2-vinyl-4,5-dicyanoimidazole is 40 to 60 parts, and more preferably the input mass of the first comonomer is 25 parts, the input mass of the second comonomer is 25 parts, and the input mass of 2-vinyl-4,5-dicyanoimidazole is 50 parts.

[0032] In this radical copolymerization reaction, by adjusting the amounts of the first and second comonomers added, it is possible to obtain immobilized 4,5-dicyanoimidazole, a product with excellent physical properties. The amount of the first comonomer used can affect the degree of crosslinking of the polymer product, and the above dosage contributes to improving the degree of crosslinking. More importantly, it can reduce the solubility of the resulting polymer product in organic solvents, facilitating subsequent removal by simple filtration and enabling recycling.

[0033] If the amount of the first comonomer added is too small, the degree of crosslinking of the product may be reduced, and the solubility of the resulting polymer product in organic solvents may increase, making it difficult to achieve the above-mentioned effects of simple removal and recycling. If the amount of the first comonomer added is too large, the proportion of the active component 4,5-dicyanoimidazole in the final product may decrease, potentially affecting catalytic activity. Furthermore, if the amount of the first comonomer added is too large, the polymer product may not swell easily in organic solvents during subsequent use, which may affect catalytic activity.

[0034] In the radical copolymerization reaction, the amount of the second comonomer contributes to adjusting the processability of the polymer product; if the amount of the second comonomer is too small, the polymer will be difficult to process. The amount of 2-vinyl-4,5-dicyanoimidazole added also affects the polymer performance; if the amount is too small, the catalytic activity of the product will decrease; if the amount is too large, the degree of crosslinking of the product will decrease and the solubility in organic solvents will increase, making it difficult to meet recycling requirements.

[0035] In a preferred embodiment, the first comonomer, the second comonomer, and 2-vinyl-4,5-dicyanoimidazole are placed in a reaction solvent to carry out a radical copolymerization reaction, where the reaction solvent includes, but is not limited to, one or more of methyl t-butyl ether, acetonitrile, or N,N-dimethylformamide, and the radical copolymerization reaction is preferably carried out in a nitrogen gas or noble gas atmosphere.

[0036] The radical copolymerization reaction can be carried out in the organic solvents listed above or other commonly used organic solvents. The first comonomer, the second comonomer, and 2-vinyl-4,5-dicyanoimidazole are all soluble in organic solvents, but the immobilized 4,5-dicyanoimidazole produced after the radical copolymerization reaction has low solubility in the reaction solvents listed above. Therefore, an insoluble polymer is produced by the reaction, which promotes the forward progress of the reaction and also makes it easier to separate and purify the product.

[0037] In a preferred embodiment, the radical initiator of the radical copolymerization reaction includes one or more of azobisisobutyronitrile, azobisisoheptanonitrile, or benzoyl peroxide, and the reaction temperature of the radical copolymerization reaction is preferably 65°C to 90°C, and the reaction time of the radical copolymerization reaction is preferably 10 hours to 20 hours.

[0038] The radical initiator can be used to generate radicals by decomposition after exposure to heat, thereby initiating a radical copolymerization reaction. In the radical copolymerization reaction, the reaction temperature is 65 to 90°C, and the reaction time is 10 to 20 hours. The decomposition temperature of a radical initiator, such as azobisisobutyronitrile, is about 64°C. If the temperature exceeds 85°C, the decomposition rate is too fast and explosive polymerization occurs. Therefore, by carrying out the radical copolymerization reaction within the above temperature range, the conversion rate of the radical copolymerization reaction is high, the reaction is fast, and by-products are not easily generated.

[0039] The copolymerization reaction is a precipitation copolymerization because no components such as an aqueous phase or a dispersant are added, and the copolymerization product is a fine powder with a particle size of the order of several tens of microns. The reaction is carried out for a certain period of time, and there is no need to filter the product or determine whether the reaction is complete or not when the reaction time has elapsed.

[0040] In a third exemplary embodiment of the present application, there is provided a coupling method, the coupling method comprising the step of activating and coupling a phosphoramide with a hydroxyl group using immobilized 4,5-dicyanoimidazole produced by the above-described method for producing immobilized 4,5-dicyanoimidazole or the above-described immobilized 4,5-dicyanoimidazole.

[0041] In a preferred embodiment, immobilized 4,5-dicyanoimidazole is used to activate the coupling reaction between phosphoramide and hydroxyl group, and the conversion rate of the coupling reaction is 90% to 94%. Preferably, the compound having a hydroxyl group includes a nucleoside protected with a dimethoxytrityl (DMTr group) or a deoxynucleoside protected with a DMTr group. Preferably, the compound having a phosphoramide includes a phosphoramidite compound. Preferably, the phosphorylating reagent includes 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite.

[0042] The immobilized 4,5-dicyanoimidazole can be used to activate the coupling reaction between phosphoramide and hydroxyl groups, completing a key step in the synthesis of commercially valuable products such as oligonucleotides. The immobilized 4,5-dicyanoimidazole can be used to catalyze the coupling reaction of inexpensive reactants, achieving a coupling reaction rate of over 90%. The immobilized 4,5-dicyanoimidazole is insoluble in common organic solvents, reducing the post-treatment costs of the coupling reaction. The immobilized 4,5-dicyanoimidazole can be separated from the reaction system by physical means such as filtration, reducing separation costs and allowing the immobilized 4,5-dicyanoimidazole to be recycled, further reducing production costs.

[0043] In a preferred embodiment, the coupling method comprises carrying out a cascaded coupling reaction using immobilized 4,5-dicyanoimidazole.

[0044] Taking advantage of the property that immobilized 4,5-dicyanoimidazole is insoluble in organic solvents, it can be immobilized in a continuous reactor. The reaction substrate is dissolved in the reaction solvent and brought into contact with the immobilized 4,5-dicyanoimidazole while flowing through the continuous reactor, thereby completing the reaction. This enables continuous industrial large-batch production and eliminates the need for separation such as filtration, further reducing the cost of product purification.

[0045] In a fourth exemplary embodiment of the present application, there is provided a use of the immobilized 4,5-dicyanoimidazole, or the method of preparation, or the coupling method, in a coupling reaction between a phosphoramide and a hydroxyl group and / or in oligonucleotide synthesis.

[0046] The beneficial effects of the present invention will be described in more detail below with reference to specific examples.

[0047] Example 1 Preparation of immobilized 4,5-dicyanoimidazole.

[0048] Under a nitrogen gas atmosphere, 0.5 g of divinylbenzene, 0.5 g of styrene, and 1 g of 2-vinyl-4,5-dicyanoimidazole (2-vinyl-4,5-DCI) were placed in a 25 mL two-neck flask, 10 mL of DMF was added as a solvent, and finally 25 mg of AIBN was added. The mixture was heated to 70 °C and reacted for 16 h. After overnight, heating was stopped. The product was filtered, washed with ethanol, and dried to obtain 1.47 g of product, designated C-1.

[0049] The product has a peak at 3149-2488 cm in the infrared spectrum. -1 Multiple peaks: 2242, 1911, 1728, 1644, 1574, 1510 and 1370 cm -1 The product was crosslinked and insoluble in common solvents, including but not limited to methanol, ethanol, acetonitrile, toluene, dichloromethane, ethyl ether, DMF, DMSO, and ethyl acetate. 0.1 g of the product was allowed to stand in 1 mL of each of the above solvents for 10 minutes, and both were washed, dried, and weighed before and after standing. No visible changes were observed, and the mass difference before and after standing accounted for less than 1% of the mass (i.e., the weight loss rate).

[0050] The coupling reaction activated by immobilized 4,5-dicyanoimidazole is as follows:

[0051] 1. Coupling reaction of 5-methylcytidine derivatives activated by immobilized 4,5-dicyanoimidazole (immobilized 4,5-DCI, C-1) with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite:

[0052] [ka]

[0053] 100 mg of the immobilized 4,5-DCI resin was added to 2 mL of acetonitrile, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (36 μL, 0.12 mmol) was added dropwise. 72 mg of 5-methylcytidine derivative (0.10 mmol) was dissolved in 100 μL of dimethylformamide (DMF) and added dropwise to the system, which was then allowed to react overnight at room temperature. After completion of the reaction, HPLC analysis revealed that the conversion rate of the phosphorylated product of the 5-methylcytosine nucleoside derivative was 94%.

[0054] 2. Coupling reaction of 5-methyluridine derivatives activated by immobilized 4,5-DCI (C-1) with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite:

[0055] [ka]

[0056] 100 mg of the immobilized 4,5-DCI resin was added to 2 mL of acetonitrile, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (36 μL, 0.12 mmol) was added dropwise. 62 mg of 5-methyluridine derivative (0.10 mmol) was dissolved in 100 μL of DMF and added dropwise to the system, which was then allowed to react overnight at room temperature. After the reaction was completed, HPLC analysis revealed that the conversion rate of the phosphorylated product of the 5-methyluridine derivative was 91%.

[0057] 3. Coupling reaction of adenosine derivatives activated by immobilized 4,5-DCI (C-1) with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite:

[0058] [ka]

[0059] 100 mg of the immobilized 4,5-DCI resin was added to 2 mL of acetonitrile, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (36 μL, 0.12 mmol) was added dropwise. 73 mg of adenosine derivative (0.10 mmol) was dissolved in 100 μL of DMF and added dropwise to the system, which was then allowed to react overnight at room temperature. After completion of the reaction, HPLC analysis revealed that the conversion rate of the phosphorylated product of the adenosine derivative was 90%.

[0060] 4. Coupling reaction of guanosine derivatives activated by immobilized 4,5-DCI (C-1) with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite:

[0061] [ka]

[0062] 100 mg of the immobilized 4,5-DCI resin was added to 2 mL of acetonitrile, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (36 μL, 0.12 mmol) was added dropwise. 70 mg of guanosine derivative (0.10 mmol) was dissolved in 100 μL of DMF and added dropwise to the system, which was then allowed to react overnight at room temperature. After completion of the reaction, HPLC analysis revealed that the conversion rate of the phosphorylated product of the guanosine derivative was 93%.

[0063] Example 2 Under a nitrogen gas atmosphere, 0.4 g of divinylbenzene, 0.4 g of styrene, and 1.2 g of 2-vinyl-4,5-DCI were placed in a 25 mL two-neck flask, 10 mL of DMF was added as a solvent, and finally 25 mg of AIBN was added. The mixture was heated to 70 °C and reacted for 16 h. After overnight, heating was stopped. The product was filtered, washed with ethanol, and dried to obtain 1.71 g of product, designated C-2.

[0064] Coupling reaction of 5-methylcytidine derivatives activated by immobilized 4,5-DCI (C-2) with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite:

[0065] [ka]

[0066] 100 mg of the immobilized 4,5-DCI resin (C-2) was added to 2 mL of acetonitrile, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (36 μL, 0.12 mmol) was added dropwise. 72 mg of 5-methylcytidine derivative (0.10 mmol) was dissolved in 100 μL of dimethylformamide (DMF) and added dropwise to the system, which was then allowed to react overnight at room temperature. After completion of the reaction, HPLC analysis revealed that the conversion rate of the phosphorylated product of the 5-methylcytosine nucleoside derivative was 86%.

[0067] Example 3 Under a nitrogen gas atmosphere, 0.6 g of divinylbenzene, 0.6 g of styrene, and 0.8 g of 2-vinyl-4,5-DCI were placed in a 25 mL two-neck flask, 10 mL of DMF was added as a solvent, and finally 25 mg of AIBN was added. The mixture was heated to 70 °C and reacted for 16 h. After overnight, heating was stopped. The product was filtered, washed with ethanol, and dried to obtain 1.71 g of product, designated C-3.

[0068] Coupling reaction of 5-methylcytidine derivatives activated by immobilized 4,5-DCI (C-3) with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite:

[0069] [ka]

[0070] 100 mg of the immobilized 4,5-DCI resin (C-3) was added to 2 mL of acetonitrile, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (36 μL, 0.12 mmol) was added dropwise. 72 mg of 5-methylcytidine derivative (0.10 mmol) was dissolved in 100 μL of dimethylformamide (DMF) and added dropwise to the system, which was then allowed to react overnight at room temperature. After completion of the reaction, HPLC analysis revealed that the conversion rate of the phosphorylated product of the 5-methylcytosine nucleoside derivative was 85%.

[0071] Example 4 Under a nitrogen gas atmosphere, 1.0 g of divinylbenzene, 0.5 g of styrene, and 0.5 g of 2-vinyl-4,5-DCI were placed in a 25 mL two-neck flask, 10 mL of DMF was added as a solvent, and finally 25 mg of AIBN was added. The mixture was heated to 70 °C and reacted for 16 h. After overnight, heating was stopped. The product was filtered, washed with ethanol, and dried to obtain 1.63 g of product, designated C-4.

[0072] Coupling reaction of 5-methylcytidine derivatives activated by immobilized 4,5-DCI (C-4) with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite:

[0073] [ka]

[0074] 100 mg of the immobilized 4,5-DCI resin (C-4) was added to 2 mL of acetonitrile, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (36 μL, 0.12 mmol) was added dropwise. 72 mg of 5-methylcytidine derivative (0.10 mmol) was dissolved in 100 μL of dimethylformamide (DMF) and added dropwise to the system, which was then allowed to react overnight at room temperature. After completion of the reaction, HPLC analysis revealed that the conversion rate of the phosphorylated product of the 5-methylcytosine nucleoside derivative was 42%.

[0075] Example 5 Under a nitrogen gas atmosphere, 0.5 g of divinylbenzene, 1.0 g of styrene, and 0.5 g of 2-vinyl-4,5-DCI were placed in a 25 mL two-neck flask, 10 mL of DMF was added as a solvent, and finally 25 mg of AIBN was added. The mixture was heated to 70 °C and reacted for 16 h. After overnight, heating was stopped. The product was filtered, washed with ethanol, and dried to obtain 1.59 g of product, designated C-5.

[0076] Coupling reaction of 5-methylcytidine derivatives activated by immobilized 4,5-DCI (C-5) with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite:

[0077] [ka]

[0078] 100 mg of the immobilized 4,5-DCI resin (C-5) was added to 2 mL of acetonitrile, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (36 μL, 0.12 mmol) was added dropwise. 72 mg of 5-methylcytidine derivative (0.10 mmol) was dissolved in 100 μL of dimethylformamide (DMF) and added dropwise to the system, which was then allowed to react overnight at room temperature. After completion of the reaction, HPLC analysis revealed that the conversion rate of the phosphorylated product of the 5-methylcytosine nucleoside derivative was 35%.

[0079] Example 6 Under a nitrogen gas atmosphere, 0.75 g of divinylbenzene, 0.75 g of styrene, and 0.5 g of 2-vinyl-4,5-DCI were placed in a 25 mL two-neck flask, 10 mL of DMF was added as a solvent, and finally 25 mg of AIBN was added. The mixture was heated to 70 °C and reacted for 16 h. After overnight, heating was stopped. The product was filtered, washed with ethanol, and dried to obtain 1.53 g of product, designated C-6.

[0080] Coupling reaction of 5-methylcytidine derivatives activated by immobilized 4,5-DCI (C-6) with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite:

[0081] [ka]

[0082] 100 mg of the immobilized 4,5-DCI resin (C-6) was added to 2 mL of acetonitrile, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (36 μL, 0.12 mmol) was added dropwise. 72 mg of 5-methylcytidine derivative (0.10 mmol) was dissolved in 100 μL of dimethylformamide (DMF) and added dropwise to the system, which was then allowed to react overnight at room temperature. After completion of the reaction, HPLC analysis revealed that the conversion rate of the phosphorylated product of the 5-methylcytosine nucleoside derivative was 38%.

[0083] (Comparative Example 1) Under a nitrogen gas atmosphere, 1.0 g of divinylbenzene and 1.0 g of 2-vinyl-4,5-DCI were placed in a 25 mL two-neck flask, 10 mL of DMF was added as a solvent, and finally 25 mg of azobisisobutyronitrile (AIBN) was added. The mixture was heated to 70 °C and reacted for 16 hours. After overnight, heating was stopped. In this comparative example, because styrene was not added, the polymerized product was difficult to mold.

[0084] Example 7 Under a nitrogen gas atmosphere, 0.5 g of divinylbenzene, 0.5 g of methyl methacrylate, and 1 g of 2-vinyl-4,5-DCI were placed in a 25 mL two-neck flask, 10 mL of DMF was added as a solvent, and finally 25 mg of AIBN was added. The mixture was heated to 70 °C and reacted for 16 h. After overnight, heating was stopped. The product was filtered, washed with ethanol, and dried to obtain 1.32 g of product, designated C-7.

[0085] Coupling reaction of 5-methylcytidine derivatives activated by immobilized 4,5-DCI (C-7) with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite:

[0086] [ka]

[0087] 100 mg of the immobilized 4,5-DCI resin (C-7) was added to 2 mL of acetonitrile, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (36 μL, 0.12 mmol) was added dropwise. 72 mg of 5-methylcytidine derivative (0.10 mmol) was dissolved in 100 μL of dimethylformamide (DMF) and added dropwise to the system, which was then allowed to react overnight at room temperature. After completion of the reaction, HPLC analysis revealed that the conversion rate of the phosphorylated product of the 5-methylcytosine nucleoside derivative was 76%.

[0088] Example 8 Under a nitrogen gas atmosphere, 0.5 g of ethylene glycol dimethacrylate, 0.5 g of styrene, and 1 g of 2-vinyl-4,5-dicyanoimidazole (2-vinyl-4,5-DCI) were placed in a 25 mL two-neck flask, 10 mL of DMF was added as a solvent, and finally 25 mg of AIBN was added. The mixture was heated to 70 °C and reacted for 16 h. After overnight, heating was stopped. The product was filtered, washed with ethanol, and dried to obtain 1.35 g of product, designated C-8.

[0089] Coupling reaction of 5-methylcytidine derivatives activated by immobilized 4,5-DCI (C-8) with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite:

[0090] [ka]

[0091] 100 mg of the immobilized 4,5-DCI resin (C-8) was added to 2 mL of acetonitrile, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (36 μL, 0.12 mmol) was added dropwise. 72 mg of 5-methylcytidine derivative (0.10 mmol) was dissolved in 100 μL of dimethylformamide (DMF) and added dropwise to the system, which was then allowed to react overnight at room temperature. After completion of the reaction, HPLC analysis revealed that the conversion rate of the phosphorylated product of the 5-methylcytosine nucleoside derivative was 81%.

[0092] Example 9 Under a nitrogen gas atmosphere, 0.5 g of ethylene glycol dimethacrylate, 0.5 g of methyl methacrylate, and 1 g of 2-vinyl-4,5-DCI were placed in a 25 mL two-neck flask, 10 mL of DMF was added as a solvent, and finally 25 mg of AIBN was added. The mixture was heated to 70 °C and reacted for 16 h. After overnight, heating was stopped. The product was filtered, washed with ethanol, and dried to obtain 1.29 g of product, designated C-9.

[0093] Coupling reaction of 5-methylcytidine derivatives activated by immobilized 4,5-DCI(C-9) with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite:

[0094] [ka]

[0095] 100 mg of the immobilized 4,5-DCI resin (C-9) was added to 2 mL of acetonitrile, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (36 μL, 0.12 mmol) was added dropwise. 72 mg of 5-methylcytidine derivative (0.10 mmol) was dissolved in 100 μL of dimethylformamide (DMF) and added dropwise to the system, which was then allowed to react overnight at room temperature. After completion of the reaction, HPLC analysis revealed that the conversion rate of the phosphorylated product of the 5-methylcytosine nucleoside derivative was 75%.

[0096] (Comparative Example 2) Coupling reaction activated by free 4,5-DCI 1. Coupling reaction of 5-methylcytidine derivatives activated by free 4,5-DCI with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite:

[0097] [ka]

[0098] 13 mg (0.11 mmol) of 4,5-DCI was dissolved in 2 mL of acetonitrile, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (36 μL, 0.12 mmol) was added dropwise. 72 mg of 5-methylcytidine derivative (0.10 mmol) was dissolved in 100 μL of DMF and added dropwise to the above system, followed by overnight reaction at room temperature. After completion of the reaction, HPLC analysis revealed that the conversion rate of the phosphorylated product of the 5-methylcytidine derivative was 97%.

[0099] 2. Coupling reaction of 5-methyluridine derivatives activated by free 4,5-DCI with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite:

[0100] [ka]

[0101] 13 mg (0.11 mmol) of 4,5-DCI was dissolved in 2 mL of acetonitrile, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (36 μL, 0.12 mmol) was added dropwise. 62 mg of 5-methyluridine derivative (0.10 mmol) was dissolved in 100 μL of DMF and added dropwise to the above system, followed by overnight reaction at room temperature. After completion of the reaction, HPLC analysis revealed that the conversion rate of the phosphorylated product of the 5-methyluridine derivative was 95%.

[0102] 3. Coupling reaction of adenosine derivatives activated by free 4,5-DCI with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite:

[0103] [ka]

[0104] 13 mg (0.11 mmol) of 4,5-DCI was dissolved in 2 mL of acetonitrile, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (36 μL, 0.12 mmol) was added dropwise. 73 mg of adenosine derivative (0.10 mmol) was dissolved in 100 μL of DMF and added dropwise to the above system, followed by overnight reaction at room temperature. After completion of the reaction, HPLC analysis revealed that the conversion rate of the adenosine derivative to the phosphorylated product was 95%.

[0105] 4. Coupling reaction of guanosine derivatives activated by free 4,5-DCI with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite:

[0106] [ka]

[0107] 13 mg (0.11 mmol) of 4,5-DCI was dissolved in 2 mL of acetonitrile, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (36 μL, 0.12 mmol) was added dropwise. 70 mg of guanosine derivative (0.10 mmol) was dissolved in 100 μL of DMF and added dropwise to the above system, followed by overnight reaction at room temperature. After completion of the reaction, HPLC analysis revealed that the conversion of the phosphorylated product of the guanosine derivative was 96%.

[0108] 4,5-DCI, the substrate, and the phosphorus reagent are all easily soluble in acetonitrile and DMF, but poorly soluble in water. Therefore, it is difficult to remove them by washing with water or extraction after the reaction. Therefore, it is necessary to remove impurities by filtration using a nanofiltration membrane, which is troublesome.

[0109] When immobilized 4,5-DCI resin is used, the substrate conversion rate is substantially the same as that of small molecule 4,5-DCI, and the resin only needs to be separated by centrifugation or filtration, greatly simplifying the impurity removal process.

[0110] As is apparent from the above description, the above-described embodiments of the present invention achieve the following technical effects. The first and second comonomers were used to carry out radical copolymerization of 2-vinyl-4,5-dicyanoimidazole to produce immobilized 4,5-dicyanoimidazole, which was capable of catalyzing the coupling reaction of phosphoramide with hydroxyl groups, with no significant decrease in substrate conversion compared to activation with 4,5-DCI.

[0111] The above description is merely a preferred embodiment of the present invention, and is not intended to limit the present invention. Various modifications and variations of the present invention are possible by those skilled in the art. Any modifications, equivalent replacements, improvements, etc. made within the scope of the present invention should be included in the protection scope of the present invention.

Claims

1. an immobilized 4,5-dicyanoimidazole, the immobilized 4,5-dicyanoimidazole being obtained by a radical copolymerization reaction from a first comonomer, a second comonomer, and a third comonomer; 1. An immobilized 4,5-dicyanoimidazole, wherein the first comonomer is divinylbenzene or ethylene glycol dimethacrylate, the second comonomer is styrene or methyl methacrylate, and the third comonomer is 2-vinyl-4,5-dicyanoimidazole.

2. In the immobilized 4,5-dicyanoimidazole, the mass ratio of the first comonomer, the second comonomer, and the third comonomer is (10 to 50):(10 to 50):(10 to 80), and the mass ratio of the total mass of the first comonomer and the second comonomer to the third comonomer is ≦9:1; Preferably, the mass ratio of the first comonomer to the second comonomer to the third comonomer is (20-30):(20-30):(40-60); More preferably, the mass ratio of the first comonomer to the second comonomer and the third comonomer is 1:1:

2.

3. The solubility of the immobilized 4,5-dicyanoimidazole in N,N-dimethylformamide, ether, acetonitrile, alcohol, ester, and chloroalkane is all less than 1 g / L; Preferably, the infrared spectrum of the immobilized 4,5-dicyanoimidazole has the following characteristic absorption: 3149 cm -1 -2488cm -1 Multiple peaks, 2242 cm -1 , 1911 cm -1 , 1728cm -1 , 1644cm -1 , 1574cm -1 , 1510cm -1 and 1370 cm -1 The immobilized 4,5-dicyanoimidazole according to claim 1 or 2, characterized in that the method for producing the infrared detection sample of the immobilized 4,5-dicyanoimidazole is a potassium bromide tableting method.

4. A method for producing immobilized 4,5-dicyanoimidazole, comprising the steps of: conducting a radical copolymerization reaction of 2-vinyl-4,5-dicyanoimidazole with a first comonomer and a second comonomer; A method for producing immobilized 4,5-dicyanoimidazole, characterized in that the first comonomer is divinylbenzene or ethylene glycol dimethacrylate, and the second comonomer is styrene or methyl methacrylate.

5. In the radical copolymerization reaction, the input mass of the first comonomer is 10 parts to 50 parts, the input mass of the second comonomer is 10 parts to 50 parts, but the sum of the input masses of the first comonomer and the second comonomer does not exceed 90 parts, and the input mass of the 2-vinyl-4,5-dicyanoimidazole is 10 parts to 80 parts; Preferably, the weight amount of the first comonomer is 20 to 30 parts, the weight amount of the second comonomer is 20 to 30 parts, and the weight amount of the 2-vinyl-4,5-dicyanoimidazole is 40 to 60 parts; More preferably, the input mass of the first comonomer is 25 parts, the input mass of the second comonomer is 25 parts, and the input mass of the 2-vinyl-4,5-dicyanoimidazole is 50 parts.

6. carrying out the radical copolymerization reaction by adding the first comonomer, the second comonomer, and the 2-vinyl-4,5-dicyanoimidazole to a reaction solvent, wherein the reaction solvent comprises one or more of methyl t-butyl ether, acetonitrile, or N,N-dimethylformamide; 5. The method according to claim 4, wherein the radical copolymerization reaction is preferably carried out in an atmosphere of nitrogen gas or a rare gas.

7. the radical initiator of the radical copolymerization reaction comprises one or more of azobisisobutyronitrile, azobisisoheptanonitrile, or benzoyl peroxide; Preferably, the reaction temperature of the radical copolymerization reaction is 65°C to 90°C, The method according to claim 4, wherein the reaction time of the radical copolymerization reaction is preferably 10 to 20 hours.

8. A coupling method, comprising the step of activating and coupling a phosphoramide with a hydroxyl group using the immobilized 4,5-dicyanoimidazole according to any one of claims 1 to 3 or the immobilized 4,5-dicyanoimidazole produced by the method for producing immobilized 4,5-dicyanoimidazole according to any one of claims 4 to 7.

9. The compound having a hydroxyl group includes a dimethoxytrityl-protected nucleoside or a dimethoxytrityl-protected deoxynucleoside, Preferably, the compound having a phosphoramide includes a phosphoramidite compound, 9. The coupling method according to claim 8, wherein the phosphorylating reagent preferably comprises 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite.

10. The coupling method according to claim 8 or 9, characterized in that the coupling method comprises a step of carrying out a continuous coupling reaction using the immobilized 4,5-dicyanoimidazole.

11. Use of the immobilized 4,5-dicyanoimidazole according to any one of claims 1 to 3, or the preparation method according to any one of claims 4 to 7, or the coupling method according to any one of claims 8 to 10 in coupling reactions of phosphoramides with hydroxyl groups and / or in oligonucleotide synthesis.

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