Immobilized 4,5-dicyanoimidazole, method for producing the same, 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) The present application claims priority to and is based on the Chinese application with Chinese application number 202211203439.5 filed on September 29, 2022, and the disclosure content of said Chinese application is hereby incorporated into the present application in its entirety.

[0002] The present invention relates to the field of immobilization of 4,5-dicyanoimidazole, and specifically to immobilized 4,5-dicyanoimidazole, a method for producing the same and use thereof. [Background Art]

[0003] Small nucleic acids refer to oligonucleotide molecules including small interfering nucleic acids (siRNA), antisense nucleic acids (ASO), microRNAs (miRNA) and nucleic acid aptamers. Small nucleic acid drugs are composed of nucleotides and are an entirely new category of drugs completely different from small molecule drugs and antibody drugs. Compared with traditional chemical drug molecules, small nucleic acid drugs have the advantages of strong target specificity, high efficiency, sustained drug action, convenient drug design, abundant candidate targets and the like. The main small nucleic acid drugs are siRNA drugs and antisense nucleic acid drugs, both of which mainly act on cytoplasmic mRNA, and achieve regulation of protein expression through base complementary recognition and inhibition of target mRNA, thereby achieving the purpose of treating diseases.

[0004] In recent years, there has been an increasing demand for small nucleic acid drugs. In 1998, the U.S. Food and Drug Administration (FDA) approved the market introduction of Vitravene, a first small nucleic acid (oligonucleotide) drug, for the treatment of cytomegalovirus retinitis. With the daily success of clinical projects and the potential market demand for oligonucleotide-based drugs, it is crucial to develop safe, environmentally clean, and cost-effective methods for synthesizing these molecules. One important step in oligonucleotide synthesis is the coupling of ribose, deoxyribose moieties, and phosphoramidite compounds. In the laboratory, a phosphorylation reagent commonly used by researchers is 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (shown in formula A), which is more active and can be coupled by reacting with the hydroxyl group on the glycosylfuran ring without the need for additional activating agents. In the laboratory, this reaction can be carried out directly using an automated solid-phase synthesizer.

[0005] [ka]

[0006] However, 2-cyanoethyldiisopropylchlorophosphoramidite is expensive, unstable at room temperature, and prone to explosion upon heating. The use of such raw materials increases the cost of industrial production and poses potential risks. A cheaper and safer alternative, 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphodiamidite (shown in formula B), can be used as a substitute for the above raw materials, but this raw material has low reactivity and requires activation with an amphoteric activator, such as 1H-tetrazole or 4,5-dicyanoimidazole (4,5-DCI).

[0007] Here, 1H-tetrazole is expensive, highly toxic, and potentially explosive, making it difficult to expand its application in industrial production. 4,5-DCI is widely used in many phosphorylation reactions as a relatively safe activator, but its main drawback is that it is poorly soluble in water and difficult to remove after the reaction (unlike 1H-tetrazole, which is water-soluble and can be removed by washing with water), requiring passage through a silica gel column. Therefore, it negatively impacts system expansion. From the standpoint of safety, low cost, availability, and good reaction activity, complete substitution of 4,5-DCI remains difficult at this stage.

[0008] [ka] [Overview of the project] [Problems that the invention aims to solve]

[0009] The main objective of the present invention is to provide immobilized 4,5-dicyanoimidazole, a method for producing the same, and a method for using the same, in order to solve the problem in the prior art of difficulty in separating 4,5-dicyanoimidazole from the reaction system after activation of the coupling reaction. [Means for solving the problem]

[0010] To achieve the above objective, according to a first aspect of the present invention, an immobilized 4,5-dicyanoimidazole is provided, which is 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.

[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), the mass ratio of the total mass of the first and second comonomers to the third comonomer is ≤9:1, preferably the mass ratio of the first, second and third comonomers is (20-30):(20-30):(40-60), and more preferably the mass ratio of the first, second and third comonomers is 1:1:2.

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

[0013] To achieve the above objective, a second aspect of the present invention provides a method for producing immobilized 4,5-dicyanoimidazole, the method comprising the step of carrying out a radical copolymerization reaction with 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 amount of the first comonomer added is 10 to 50 parts, and the amount of the second comonomer added is 10 to 50 parts, but the sum of the amounts of the first and second comonomers added does not exceed 90 parts, and the amount of 2-vinyl-4,5-dicyanoimidazole added is 10 to 80 parts, preferably 20 to 30 parts of the first comonomer, 20 to 30 parts of the second comonomer, and 40 to 60 parts of 2-vinyl-4,5-dicyanoimidazole, more preferably 25 parts of the first comonomer, 25 parts of the second comonomer, and 50 parts of 2-vinyl-4,5-dicyanoimidazole.

[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, wherein the reaction solvent includes one or more of methyl t-butyl ether, acetonitrile, or N,N-dimethylformamide, and preferably the radical copolymerization reaction is carried out in a nitrogen gas or noble gas atmosphere.

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

[0017] To achieve the above objective, a third aspect of the present invention provides a coupling method which includes the step of activating and coupling a phosphoramide and a hydroxyl group using the immobilized 4,5-dicyanoimidazole or immobilized 4,5-dicyanoimidazole produced by the above method for producing immobilized 4,5-dicyanoimidazole.

[0018] Further, activated phosphoramide and a hydroxyl group are subjected to a coupling reaction using immobilized 4,5-dicyanoimidazole, and the conversion rate of the coupling reaction is 90% to 94%. Preferably, the compound having a hydroxyl group comprises a nucleoside protected with dimethoxytrityl or a deoxynucleoside protected with dimethoxytrityl, preferably the compound having phosphoramide comprises a phosphoramidite compound, and preferably the phosphorylating reagent comprises 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite.

[0019] Further, the coupling method comprises the step of carrying out a continuous coupling reaction using the 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-mentioned immobilized 4,5-dicyanoimidazole, the above-mentioned production method, or the above-mentioned coupling method in a coupling reaction between a phosphoramide and a hydroxyl group and / or oligonucleotide synthesis.

Effects of the Invention

[0021] When the technical solution of the present invention is used, 4,5-dicyanoimidazole is produced into immobilized 4,5-dicyanoimidazole. The immobilized 4,5-dicyanoimidazole has highly cross-linked properties, is insoluble in common organic solvents, and can be removed from the reaction system by a simple filtration means after activation of the coupling reaction.

Mode for Carrying Out the Invention

[0022] It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict. Hereinafter, the present invention will be described in detail with reference to embodiments.

[0023] As mentioned in the Background Art, 4,5-DCI is widely used as a relatively safe activator in many phosphorylation reactions, but it has a major drawback that it is easily soluble in organic solvents, poorly soluble in water, and cannot be easily removed after the reaction, so it is difficult to be used for scale-up reactions of the system.

[0024] Therefore, the inventors of the present application attempted to produce immobilized 4,5-dicyanoimidazole, and prepared 2-vinyl-4,5-dicyanoimidazole into immobilized 4,5-dicyanoimidazole by radical copolymerization reaction, which is advantageous for use in phosphorylation reactions. Accordingly, a series of protection methods of the present application have been proposed.

[0025] In a first exemplary embodiment of the present application, an immobilized 4,5-dicyanoimidazole is provided, which is obtained from a first comonomer, a second comonomer and a third comonomer by a radical copolymerization reaction, 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), 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), more preferably, the mass ratio of the first comonomer, the second comonomer and the third comonomer is 1:1:2.

[0027] In preferred examples, the solubility of 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 immobilized 4,5-dicyanoimidazole has the following characteristic absorptions: 3149-2488 (multiple peaks), 2242, 1911, 1728, 1644, 1574, 1510, and 1370 cm⁻¹. -1 The method for producing an infrared detection sample of immobilized 4,5-dicyanoimidazole is the potassium bromide tablet method, and the potassium bromide tablet method is a common method for producing infrared samples in the prior art.

[0028] The immobilized 4,5-dicyanoimidazole described above is a random copolymer obtained by a radical copolymerization reaction. Such immobilized 4,5-dicyanoimidazole has a solubility of less than 1 g / L in the above organic solvent and other general-purpose organic solvents, either individually or in mixtures, and further less than 0.1 g / L and 0.01 g / L. Therefore, after activation of the subsequent reaction with such immobilized 4,5-dicyanoimidazole, it can be removed by simple filtration and recycled back into the reaction. The mass of the third comonomer in the immobilized 4,5-dicyanoimidazole can be characterized using conventional methods for measuring the nitrogen element.

[0029] A second typical embodiment of the present application provides a method for producing immobilized 4,5-dicyanoimidazole, the method comprising the step of carrying out 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.

[0030] The inventors screened various crosslinking agents commonly used in the prior art and found that by using divinylbenzene or ethylene glycol dimethacrylate, and styrene or methyl methacrylate as crosslinking agents and performing a radical copolymerization reaction with 2-vinyl-4,5-dicyanoimidazole, they could obtain a highly effective immobilized 4,5-dicyanoimidazole. Such immobilized 4,5-dicyanoimidazoles all have poor solubility in organic solvents and are insoluble in any common organic solvent or mixture of organic solvents. Therefore, after activating the subsequent reaction with the immobilized 4,5-dicyanoimidazole, it can be removed by simple filtration and recycled back into the reaction.

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

[0032] In this radical copolymerization reaction, by adjusting the amounts of the first and second comonomers added, immobilized 4,5-dicyanoimidazole, a product with excellent physical properties, can be obtained. The amount of the first comonomer used can affect the degree of crosslinking of the polymer product. The above dosage contributes to improving the degree of crosslinking, and more importantly, it can reduce the solubility of the obtained polymer product in organic solvents, making it easier to remove later 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 decrease, increasing the solubility of the resulting polymer product in organic solvents, making it difficult to achieve the aforementioned simple removal and recycling effects. If the amount of the first comonomer added is too large, it may lead to a decrease in the proportion of the active component 4,5-dicyanoimidazole in the final product, 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 can easily affect catalytic activity.

[0034] In radical copolymerization reactions, the amount of secondary comonomer added contributes to regulating the moldability of the polymer product; if too little secondary comonomer is added, the polymer becomes difficult to mold. The amount of 2-vinyl-4,5-dicyanoimidazole added also affects the performance of the polymer; if too little is added, the catalytic activity of the product decreases, while if too much is added, the degree of crosslinking of the product decreases, the solubility in organic solvents increases, and it becomes difficult to meet recycling requirements.

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

[0036] The above radical copolymerization reaction can be carried out in the above organic solvent or other general-purpose organic solvents. The first comonomer, the second comonomer, and 2-vinyl-4,5-dicyanoimidazole are all soluble in organic solvents. However, the immobilized 4,5-dicyanoimidazole produced after the radical copolymerization reaction has low solubility in the above reaction solvent. This reaction produces an insoluble polymer, which promotes the forward progress of the reaction while simultaneously facilitating the separation and purification of the product.

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

[0038] Using the above-mentioned radical initiator, the reaction can be initiated by generating radicals through decomposition after being heated. In the above radical copolymerization reaction, the reaction temperature is 65-90°C and the reaction time is 10-20 hours. The decomposition initiation temperature of a radical initiator, such as azobisisobutyronitrile, is around 64°C. Above 85°C, the decomposition rate is too fast, making it prone to explosive polymerization. Therefore, by performing 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 less likely to be generated.

[0039] The above copolymerization reaction is a precipitation copolymerization because no aqueous phase or dispersant is added. The product obtained by copolymerization is a fine powder with a particle size on the order of tens of microns. This reaction takes place over a fixed period of time, and there is no need to filter the generated product to determine whether the reaction has finished or not once the reaction time is up.

[0040] A third typical embodiment of the present application provides a coupling method comprising the step of activating and coupling a phosphoramide and 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 preferred embodiments, immobilized 4,5-dicyanoimidazole is used to activate the coupling reaction between the phosphoramide and the hydroxyl group, with a conversion rate of 90% to 94%. Preferably, the compound having the hydroxyl group includes a nucleoside protected with dimethoxytrityl (DMTr group) or a deoxynucleoside protected with a DMTr group. Preferably, the compound having the phosphoramide includes a phosphoramidite compound. Preferably, the phosphorylation reagent includes 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphodiamidite.

[0042] Using the immobilized 4,5-dicyanoimidazole described above, the coupling reaction between phosphoramide and a hydroxyl group can be activated, completing the crucial synthesis step for commercially valuable products such as oligonucleotides. This immobilized 4,5-dicyanoimidazole can catalyze the coupling reaction of inexpensive reaction starting materials, achieving a coupling reaction rate exceeding 90%. The immobilized 4,5-dicyanoimidazole is insoluble in common organic solvents, reducing post-treatment costs for the coupling reaction. Furthermore, it can be separated from the reaction system by physical means such as filtration, reducing separation costs. Simultaneously, the immobilized 4,5-dicyanoimidazole can be recycled, further reducing production costs.

[0043] In preferred embodiments, the coupling method includes the step of performing a sequential coupling reaction using immobilized 4,5-dicyanoimidazole.

[0044] By utilizing the property that immobilized 4,5-dicyanoimidazole is insoluble in organic solvents, it can be immobilized in a continuous reaction apparatus. The reaction substrate dissolves in the reaction solvent and is brought into contact with the immobilized 4,5-dicyanoimidazole while the reaction is running through the continuous reaction apparatus to complete the reaction. This enables large-batch industrial production using continuous processes, eliminates the need for separation such as filtration, and further reduces the cost of product purification.

[0045] A fourth typical embodiment of the present application provides the use of the immobilized 4,5-dicyanoimidazole, or a method for producing it, or a coupling method in a coupling reaction between a phosphoramide and a hydroxyl group and / or in the synthesis of oligonucleotides.

[0046] The beneficial effects of this invention will be explained in more detail below with specific examples.

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

[0048] Under a nitrogen 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-necked flask. 10 mL of DMF was added as the solvent, and finally 25 mg of AIBN was added. The mixture was heated to 70 °C and the system was reacted for 16 hours. After one night, heating was stopped. The product was filtered, washed with ethanol, and dried to obtain 1.47 g of product, which was named C-1.

[0049] The product exhibits an infrared spectrum of 3149–2488 cm⁻¹. -1 Multiple peaks: 2242, 1911, 1728, 1644, 1574, 1510 and 1370 cm -1 The product exhibited characteristic absorption. 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 left in 1 mL of each of the above solvents for 10 minutes, and both samples were washed, dried, and weighed before and after standing. Visual observation showed no change, and the percentage of the mass difference before and after standing (i.e., weight loss rate) was less than 1% in all cases.

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

[0051] 1. Coupling reaction between a 5-methylcytidine derivative activated by immobilized 4,5-dicyanoimidazole (immobilized 4,5-DCI,C-1) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphodiamidite:

[0052] [ka]

[0053] 100 mg of the immobilized 4,5-DCI resin described above 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), added dropwise to the above system, and reacted overnight at room temperature. After the reaction was complete, analysis by HPLC showed that the conversion rate of the phosphorylation product of the 5-methylcytosine nucleoside derivative was 94%.

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

[0055] [ka]

[0056] 100 mg of the immobilized 4,5-DCI resin described above 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 LDF and added dropwise to the above system, and the reaction was allowed to proceed overnight at room temperature. After the reaction was complete, analysis by HPLC showed that the conversion rate of the phosphorylation product of the 5-methyluridine derivative was 91%.

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

[0058] [ka]

[0059] 100 mg of the immobilized 4,5-DCI resin described above 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 LDMF and added dropwise to the above system, and the reaction was allowed to proceed overnight at room temperature. After the reaction was complete, analysis by HPLC showed that the conversion rate of the phosphorylated product of the adenosine derivative was 90%.

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

[0061] [ka]

[0062] 100 mg of the immobilized 4,5-DCI resin described above 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 LDF and added dropwise to the above system, and the reaction was allowed to proceed overnight at room temperature. After the reaction was complete, analysis by HPLC showed that the conversion rate of the phosphorylated product of the guanosine derivative was 93%.

[0063] (Example 2) Under a nitrogen 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-necked flask. 10 mL of DMF was added as the solvent, and finally 25 mg of AIBN was added. The mixture was heated to 70 °C and the system was reacted for 16 hours. After one night, heating was stopped. The product was filtered, washed with ethanol, and dried to obtain 1.71 g of product, which was named C-2.

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

[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), added dropwise to the above system, and reacted overnight at room temperature. After the reaction was complete, analysis by HPLC showed that the conversion rate of the phosphorylation product of the 5-methylcytosine nucleoside derivative was 86%.

[0067] (Example 3) Under a nitrogen 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-necked flask. 10 mL of DMF was added as the solvent, and finally 25 mg of AIBN was added. The mixture was heated to 70 °C and the system was reacted for 16 hours. After one night, heating was stopped. The product was filtered, washed with ethanol, and dried to obtain 1.71 g of product, which was named C-3.

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

[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), added dropwise to the system, and reacted overnight at room temperature. After the reaction was complete, analysis by HPLC showed that the conversion rate of the phosphorylation product of the 5-methylcytosine nucleoside derivative was 85%.

[0071] (Example 4) Under a nitrogen 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-necked flask. 10 mL of DMF was added as the solvent, and finally 25 mg of AIBN was added. The mixture was heated to 70 °C and the system was reacted for 16 hours. After one night, heating was stopped. The product was filtered, washed with ethanol, and dried to obtain 1.63 g of product, which was named C-4.

[0072] Coupling reaction between a 5-methylcytidine derivative activated by immobilized 4,5-DCI(C-4) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphodiamidite:

[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), added dropwise to the above system, and reacted overnight at room temperature. After the reaction was complete, analysis by HPLC showed that the conversion rate of the phosphorylation product of the 5-methylcytosine nucleoside derivative was 42%.

[0075] (Example 5) Under a nitrogen 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-necked flask. 10 mL of DMF was added as the solvent, and finally 25 mg of AIBN was added. The mixture was heated to 70 °C and the system was reacted for 16 hours. After one night, heating was stopped. The product was filtered, washed with ethanol, and dried to obtain 1.59 g of product, which was named C-5.

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

[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), added dropwise to the above system, and reacted overnight at room temperature. After the reaction was complete, analysis by HPLC showed that the conversion rate of the phosphorylation product of the 5-methylcytosine nucleoside derivative was 35%.

[0079] (Example 6) Under a nitrogen 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-necked flask. 10 mL of DMF was added as the solvent, and finally 25 mg of AIBN was added. The mixture was heated to 70 °C and the system was reacted for 16 hours. After one night, heating was stopped. The product was filtered, washed with ethanol, and dried to obtain 1.53 g of product, which was named C-6.

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

[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), added dropwise to the above system, and reacted overnight at room temperature. After the reaction was complete, analysis by HPLC showed that the conversion rate of the phosphorylation 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-necked 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 the system was reacted for 16 hours. After one night, heating was stopped. In this comparative example, since styrene was not added, the polymerized product was difficult to mold.

[0084] (Example 7) Under a nitrogen 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-necked flask. 10 mL of DMF was added as the solvent, and finally 25 mg of AIBN was added. The mixture was heated to 70 °C and the system was reacted for 16 hours. After one night, heating was stopped. The product was filtered, washed with ethanol, and dried to obtain 1.32 g of product, which was named C-7.

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

[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), added dropwise to the system, and reacted overnight at room temperature. After the reaction was complete, analysis by HPLC showed that the conversion rate of the phosphorylation product of the 5-methylcytosine nucleoside derivative was 76%.

[0088] (Example 8) Under a nitrogen 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-necked flask. 10 mL of DMF was added as the solvent, and finally 25 mg of AIBN was added. The mixture was heated to 70 °C and the system was reacted for 16 hours. After one night, heating was stopped. The product was filtered, washed with ethanol, and dried to obtain 1.35 g of product, which was named C-8.

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

[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), added dropwise to the system, and reacted overnight at room temperature. After the reaction was complete, analysis by HPLC showed that the conversion rate of the phosphorylation product of the 5-methylcytosine nucleoside derivative was 81%.

[0092] (Example 9) Under a nitrogen 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-necked flask. 10 mL of DMF was added as the solvent, and finally 25 mg of AIBN was added. The mixture was heated to 70 °C and the system was reacted for 16 hours. After one night, heating was stopped. The product was filtered, washed with ethanol, and dried to obtain 1.29 g of product, which was named C-9.

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

[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), added dropwise to the above system, and reacted overnight at room temperature. After the reaction was complete, analysis by HPLC showed that the conversion rate of the phosphorylation 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 a 5-methylcytidine derivative activated by free 4,5-DCI with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphodiamidite:

[0097] [ka]

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

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

[0100] [ka]

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

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

[0103] [ka]

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

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

[0106] [ka]

[0107] 4,5-DCI 13 mg (0.11 mmol) 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 LDF, added dropwise to the above system, and reacted overnight at room temperature. After the reaction was complete, analysis by HPLC showed that the conversion rate of the phosphorylated product of the guanosine derivative was 96%.

[0108] 4,5-DCI, the substrate, and the phosphorus reagent are all readily soluble in acetonitrile and DMF, but poorly soluble in water. Therefore, they are difficult to remove by washing or extraction after the reaction, requiring filtration through a nanofiltration membrane to remove impurities, which is cumbersome.

[0109] When using immobilized 4,5-DCI resin, the substrate conversion rate is substantially equivalent to that of small molecule 4,5-DCI, and the impurity removal process is greatly simplified by simply separating the resin by centrifugation or filtration.

[0110] As is clear from the above description, the above embodiment of the present invention achieves the following technical effects. A radical copolymerization reaction was carried out with 2-vinyl-4,5-dicyanoimidazole using the first and second comonomers to produce immobilized 4,5-dicyanoimidazole. This immobilized 4,5-dicyanoimidazole could catalyze the coupling reaction between a phosphoramide and a hydroxyl group, and the substrate conversion rate was not significantly reduced compared to activation with 4,5-DCI.

[0111] The above description is merely a preferred embodiment of the present invention and does not limit it, and various modifications and variations of the present invention are possible for those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made without departing from the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The immobilized 4,5-dicyanoimidazole is obtained from a first comonomer, a second comonomer, and a third comonomer by a radical copolymerization reaction. An immobilized 4,5-dicyanoimidazole characterized in that 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. The immobilized 4,5-dicyanoimidazole according to claim 1, wherein 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.

3. The immobilized 4,5-dicyanoimidazole according to claim 2, characterized in that the mass ratio of the first comonomer, the second comonomer, and the third comonomer is (20-30):(20-30):(40-60).

4. The immobilized 4,5-dicyanoimidazole according to claim 3, characterized in that the mass ratio of the first comonomer, the second comonomer, and the third comonomer is 1:1:

2.

5. The infrared spectrum of said immobilized 4,5-dicyanoimidazole has the following characteristic absorptions: 3149 cm -1 -2488 cm -1 multiple peaks, 2242 cm -1 , 1911 cm -1 , 1728 cm -1 , 1644 cm -1 , 1574 cm -1 , 1510 cm -1 and 1370 cm -1 , the method for preparing an infrared detection sample of said immobilized 4,5-dicyanoimidazole is potassium bromide tableting, characterized in that the immobilized 4,5-dicyanoimidazole according to claim 1.

6. A method for producing immobilized 4,5-dicyanoimidazole, The step includes carrying out a radical copolymerization reaction with 2-vinyl-4,5-dicyanoimidazole using the first comonomer and the 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.

7. The manufacturing method according to claim 6, characterized in that, in the radical copolymerization reaction, the amount of the first comonomer added is 10 to 50 parts, the amount of the second comonomer added is 10 to 50 parts, the sum of the amounts of the first and second comonomers added does not exceed 90 parts, and the amount of the 2-vinyl-4,5-dicyanoimidazole added is 10 to 80 parts.

8. The manufacturing method according to claim 7, characterized in that the amount of the first comonomer added is 20 to 30 parts, the amount of the second comonomer added is 20 to 30 parts, and the amount of the 2-vinyl-4,5-dicyanoimidazole added is 40 to 60 parts.

9. The manufacturing method according to claim 8, characterized in that the amount of the first comonomer added is 25 parts, the amount of the second comonomer added is 25 parts, and the amount of the 2-vinyl-4,5-dicyanoimidazole added is 50 parts.

10. The production method according to claim 6, wherein the first comonomer, the second comonomer, and the 2-vinyl-4,5-dicyanoimidazole are placed in a reaction solvent and the radical copolymerization reaction is carried out, wherein the reaction solvent includes one or more of methyl t-butyl ether, acetonitrile, or N,N-dimethylformamide.

11. The radical initiator for the radical copolymerization reaction comprises one or more of azobisisobutyronitrile, azobisisoheptanonitrile, or benzoyl peroxide. The reaction temperature for the radical copolymerization reaction is 65°C to 90°C. The manufacturing method according to claim 6, characterized in that the reaction time for the radical copolymerization reaction is 10 to 20 hours.

12. A coupling method characterized by comprising the step of activating and coupling a phosphoramide and a hydroxyl group using immobilized 4,5-dicyanoimidazole according to any one of claims 1 to 5, or the immobilized 4,5-dicyanoimidazole produced by a method for producing immobilized 4,5-dicyanoimidazole according to any one of claims 6 to 11.

13. The coupling method according to claim 12, characterized in that the compound having a hydroxyl group includes a nucleoside protected with dimethoxytrityl or a deoxynucleoside protected with dimethoxytrityl.

14. The coupling method according to claim 12, characterized in that it includes the step of performing a continuous coupling reaction using the immobilized 4,5-dicyanoimidazole.

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