Isocyan reversible blocking nucleotide and method for cutting off isocyan reversible group in isocyan reversible blocking nucleotide

Abstract

The invention provides an isocyanide reversible blocking nucleotide and a method for cutting off an isocyanide reversible group in the isocyanide reversible blocking nucleotide. The isocyanide reversible blocking nucleotide has a structure as shown in a formula I. According to the invention, by setting a blocking group R1, two deoxyribonucleotides with 3-site hydroxyl blocked by isocyano groups are provided. The blocking group can be cut by an orthogonal reagent tetrazine compound, so that the'restart-stop 'process of enzymatic synthesis is controlled, and the efficient and accurate synthesis of a specific nucleotide sequence is further realized.

Description

Technical Field

[0001] The invention belongs to the technical field of DNA synthesis, and particularly relates to a method for reversibly blocking isocyanate nucleotides and removing an isocyanate reversible group in the reversibly blocking isocyanate nucleotides. Background Art

[0002] DNA synthesis technology plays a vital role in many fields, including life sciences, biomedicine, materials science, and information storage. Although the first-generation phosphoramidite synthesis method can synthesize longer DNA sequences, as the chain length increases, synthesis errors gradually accumulate, resulting in a limited synthesis length, usually between 200-300 nucleotides. To overcome this limitation, the second-generation DNA synthesis technology uses high-throughput solid-phase chip strategies such as photochemical, electrochemical synthesis, and inkjet printing to achieve large-scale DNA synthesis. However, it still faces the problems of low single-sequence synthesis capacity and difficult separation.

[0003] The third generation of DNA synthesis technology is an enzyme-catalyzed reaction that does not rely on DNA templates. It has the characteristics of high efficiency, high accuracy, low substrate consumption and environmental friendliness, which provides new possibilities for breaking through the limitations of chemical synthesis. In particular, TdT enzyme, as an enzyme that can rapidly synthesize long-chain DNA without a template, is active in the presence of divalent metal ions Mg. 2+ 、Co 2+ When present, nucleotides can be randomly added to the 3'-hydroxyl termini of oligonucleotide chains, showing great application potential.

[0004] However, to precisely control TdT-catalyzed DNA sequence extension, a reversible blocking group must be added to the 3-position of the nucleotide monomer. While currently known blocking groups, such as methyl, 2'-nitrobenzyl, allyl, azidomethyl, and amino groups, offer some control, they present limitations in catalytic rate, precision, deprotection efficiency, and cost, limiting their widespread adoption in large-scale applications.

[0005] Therefore, how to provide a new nucleotide containing a blocking group has become a technical problem that needs to be solved urgently. Summary of the Invention

[0006] To address the shortcomings of the prior art, the present invention aims to provide a method for isocyanide-reversibly blocked nucleotides and for cleaving the isocyanide-reversible group in isocyanide-reversibly blocked nucleotides. By providing a blocking group R1, the present invention provides two deoxyribonucleotides in which the 3-hydroxyl group is blocked by an isocyanide group. This blocking group can be cleaved by an orthogonal tetrazine compound, thereby controlling the "restart-stop" process of enzymatic synthesis and achieving efficient and accurate synthesis of specific nucleotide sequences.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides an isocyanate reversible blocking nucleotide having a structure shown in the following formula I:

[0009]

[0010] Among them, R1 represents The wavy lines indicate the junction sites;

[0011] R2 is -H or -OH;

[0012] The base group represents a C3-C20 heteroaryl group.

[0013] By providing a blocking group R1, the present invention provides two deoxyribonucleotides with an isocyanate group blocking the 3-hydroxyl group. This blocking group can be cleaved by an orthogonal reagent, a tetrazine compound, thereby controlling the "restart-stop" process of enzymatic synthesis, thereby achieving efficient and accurate synthesis of specific nucleotide sequences. Furthermore, the isocyanate group is small in size, relatively stable in structure, easy to synthesize, low in cost, and has good affinity for TdT enzyme, facilitating TdT enzyme-catalyzed synthesis reactions. Furthermore, the use of tetrazine compounds for orthogonal cleavage is rapid.

[0014] The following are preferred technical solutions of the present invention, but are not intended to limit the technical solutions provided by the present invention. Through the following preferred technical solutions, the objectives and beneficial effects of the present invention can be better achieved and realized.

[0015] As a preferred technical solution of the present invention, the base group represents any one of the following groups:

[0016]

[0017] The dotted lines indicate the connection sites.

[0018] As a preferred technical solution of the present invention, the isocyanate reversibly blocking nucleotide includes the following compounds:

[0019]

[0020]

[0021] The present invention does not have any special restrictions on the preparation method of the isocyanate reversible blocking nucleotide described in the first aspect, and any commonly used preparation method in the art is applicable, including but not limited to: the preparation method of the isocyanate reversible blocking nucleotide comprises the following steps:

[0022] The R2 is -H, and the preparation method includes method A, which includes the following steps:

[0023]

[0024] The R2 is -OH, and the preparation method includes method B, which includes the following steps:

[0025]

[0026] The specific process is as follows:

[0027] (1) In the presence of an acid-binding agent and solvent A, compound A1 reacts with tert-butyldimethylsilyl chloride (TBSCl) to obtain compound A2; (2) in the presence of solvent B, compound A2 reacts with N,N-dimethylformamide dimethyl acetal to obtain compound A3;

[0028] (3) In the presence of solvent C, compound A3 and sodium hydride are activated, and then compound NC-OTs or isocyanuric acid is added to the reaction system to continue the reaction to obtain compound A4;

[0029] (4) Compound A4 reacts with tetrabutylammonium fluoride trihydrate (TBAF) in the presence of solvent C to obtain compound A5;

[0030] (5) Compound A5, trimethyl phosphate, and tri-n-butylamine are mixed, phosphorus oxychloride is added thereto for reaction, and then a mixture of acetonitrile, tri-n-butylamine, and ammonium pyrophosphate is added thereto, and the reaction is continued to obtain the isocyanide reversible blocking nucleotide (Compound A6);

[0031] Wherein, the acid binding agent in step (1) includes imidazole and / or pyridine.

[0032] The solvent A in step (1) comprises anhydrous dimethylformamide (DMF) and / or anhydrous pyridine.

[0033] The reaction in step (1) is carried out in a protective gas atmosphere, wherein the protective gas comprises nitrogen.

[0034] After the reaction in step (1), a post-processing step is also included, and the post-processing method includes filtration and pulping purification.

[0035] The solvent B in step (2) includes methanol.

[0036] The reaction in step (2) further includes a post-processing step, and the post-processing step includes drying.

[0037] The solvent C in step (3) includes anhydrous dimethylformamide (DMF).

[0038] The reaction in step (3) is carried out in a protective gas atmosphere, wherein the protective gas includes nitrogen.

[0039] The solvent D in step (4) includes tetrahydrofuran (THF).

[0040] The reaction in step (4) further includes a post-processing step, and the post-processing method includes drying and purification.

[0041] The reaction in step (5) is carried out in a protective gas atmosphere, wherein the protective gas comprises nitrogen.

[0042] The reaction in step (4) further includes a post-processing step, and the post-processing method includes purification and drying.

[0043] It should be noted that the present invention has no limitation on the process conditions of each step in the above preparation method, and the process conditions commonly used in the art (reaction temperature, reaction time, etc.) are applicable.

[0044] In a second aspect, the present invention provides a method for removing a reversible blocking group in the isocyanate reversibly blocking nucleotide as described in the first aspect, the method comprising the following steps:

[0045] After the isocyanide reversible blocking nucleotide reacts with the tetrazine compound, deprotection is performed to complete the removal of the reversible blocking group in the isocyanide reversible blocking nucleotide.

[0046] As described, R1 represents R2 is -H, the tetrazine compound is 3,6-di(2-pyridyl)-1,2,4,5-tetrazine, and the process for removing the reversible blocking group in the isocyanate reversibly blocked nucleotide comprises the following steps:

[0047]

[0048] As mentioned above, R1 represents R2 is -H, the tetrazine compound is 3,6-di(2-pyridyl)-1,2,4,5-tetrazine, and the process for removing the reversible blocking group in the isocyanate reversibly blocked nucleotide comprises the following steps:

[0049]

[0050] As a preferred technical solution of the present invention, the tetracyano compound includes any one or a combination of at least two of 3,6-bis(2-pyridyl)-1,2,4,5-tetrazine, 3,6-dimethyl-1,2,4,5-tetrazine, 3,6-dimethoxy-1,2,4,5-tetrazine, bis(methylthio)-1,2,4,5-tetrazine, 3,6-di-p-tolyl-1,2,4,5-tetraazabenzene or 3,6-bis(3,5-dimethyl-1H-pyrazol-1-yl)-1,2,4,5-tetrazine.

[0051] Preferably, the molar ratio of the isocyanate reversible blocking nucleotide to the tetrazine compound is 1:(10-50), for example, 1:10, 1:13, 1:16, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45 or 1:50.

[0052] As a preferred technical solution of the present invention, the reaction temperature is 45-55°C, for example, it can be 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C or 55°C.

[0053] Preferably, the reaction time is 3 to 5 minutes, for example, 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes or 5 minutes.

[0054] Preferably, the reaction is carried out at a pH of 9.5 to 10.5, for example, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4 or 10.5.

[0055] In the present invention, the method for adjusting the pH of the reaction to 9.5 to 10.5 can be: adding sodium bicarbonate solution to the reaction system to adjust the pH of the reaction system to 9.5 to 10.5

[0056] Preferably, the reaction is carried out in the presence of a solvent, and the solvent includes any one or a combination of at least two of DMF, methanol, DMSO, THF, acetonitrile or water.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] By providing a blocking group R1, the present invention provides two deoxyribonucleotides with an isocyanate group blocking the 3-hydroxyl group. This blocking group can be cleaved by an orthogonal reagent, a tetrazine compound, thereby controlling the "restart-stop" process of enzymatic synthesis, thereby achieving efficient and accurate synthesis of specific nucleotide sequences. Furthermore, the isocyanate group is small in size, relatively stable in structure, easy to synthesize, low in cost, and has good affinity for TdT enzyme, facilitating TdT enzyme-catalyzed synthesis reactions. Furthermore, the use of tetrazine compounds for orthogonal cleavage is rapid. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The isocyanate provided by the present invention is used to reversibly block nucleotides in the DNA enzymatic synthesis process;

[0060] Figure 2 This is the result characterization of the TdT enzymatic DNA extension experiment provided in Application Example 1 of the present invention;

[0061] Figure 3This is the result of the deprotection experiment provided in Application Example 2 of the present invention;

[0062] Figure 4 This is the result characterization of the PUP enzymatic RNA extension experiment provided in Application Example 3 of the present invention;

[0063] Figure 5 This is the result characterization of the deprotection experiment provided in Application Example 4 of the present invention. DETAILED DESCRIPTION

[0064] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0065] For the convenience of understanding the present invention, the present invention is given below with examples. It should be understood by those skilled in the art that the examples are only for the purpose of helping to understand the present invention and should not be regarded as specific limitations of the present invention.

[0066] Preparation Example 1

[0067] This preparation example provides the compound NC-OTs and its synthesis method, and the synthesis method is as follows:

[0068]

[0069] (1) Synthesis of Compound NC-1

[0070] 10 g of ethyl formate was weighed, and 9.22 g of 3-amino-1-propanol was added under ice bath conditions, and stirred for 15 min. The reaction solution was removed from the ice bath and heated at 50° C. for 3 h. The oily product was obtained by spin drying.

[0071] (2) Synthesis of compound NC-OTs

[0072] Weigh 1 g of NC-1, add it to 20 mL of dry pyridine and stir at 0°C, weigh 7.4 g of TsCl, dissolve it in 20 mL of anhydrous dichloromethane, and slowly add it dropwise to the reaction solution. The reaction is continued for 4 hours. The reaction solution is quenched with ice water, extracted with ethyl acetate, and the organic phase is dried and purified by normal phase column chromatography using dichloromethane and n-hexane to obtain 1.5 g of compound NC-OTs.

[0073] Characterization data of compound NC-OTs: 1 H NMR (400MHz, CDCl3): δ (ppm) 7.79 (d, 2H), 7.37 (d, 2H), 4.16 (t, 2H), 3.50-3.48 (m, 2H), 2.45 (s, 3H), 2.07-1.95 (m, 2H).

[0074] Example 1

[0075] This example provides compound A6 and its synthesis method, which is as follows:

[0076]

[0077] (1) Synthesis of Compound A2

[0078] Weigh 25 g of compound A1 and 16.26 g of imidazole, add 250 mL of anhydrous DMF (dimethylformamide) to dissolve, replace with nitrogen, and precool at 10°C;

[0079] 18 g of TBSCl (tert-butyldimethylsilyl chloride) was dissolved in 50 mL of anhydrous DMF and slowly added dropwise to the mixture of Compound A1 and imidazole using a constant pressure addition funnel. After the addition, the reaction solution was moved to room temperature and reacted overnight. The reaction solution was added to ice water to quench the reaction, and filtered to obtain a white solid as the crude product of Compound A2. The white solid was purified by slurrying with n-hexane methyl tert-ether and filtered to obtain 32 g of Compound A2 as a white solid.

[0080] (2) Synthesis of Compound A3

[0081] 10 g of compound A2 was weighed and dissolved in 100 mL of methanol. 9.78 g of N,N-dimethylformamide dimethyl acetal was then added and stirred at room temperature overnight. After the reaction, the reaction solution was dried by rotary evaporation to obtain 11.5 g of compound A3.

[0082] (3) Synthesis of Compound A4

[0083] Weigh 1 g of compound A3, dissolve it in 5 mL of anhydrous DMF, replace the atmosphere with nitrogen, and stir at 5°C. Add 85 mg of NaH to activate for 15 min. Add 1.14 g of compound NC-OTs and continue the reaction for 1 h. Quench the reaction with water, extract the organic phase with ethyl acetate, and dry it. Purify it by normal phase silica gel column chromatography with dichloromethane and ethyl acetate to obtain 153 mg of compound A4.

[0084] (4) Synthesis of Compound A5

[0085] 100 mg of compound A4 was weighed and dissolved in 2 mL of THF (tetrahydrofuran). 107 mg of TBAF (tetrabutylammonium fluoride trihydrate) was added, and the mixture was stirred at 10°C overnight. The reaction was quenched by adding water, and the organic phase was extracted with ethyl acetate and dried. The organic phase was purified on normal phase silica gel using dichloromethane and methanol to obtain 50 mg of compound A5.

[0086] (5) Synthesis of Compound A6

[0087] 50 mg of compound A5 was weighed, dissolved in 1 mL of trimethyl phosphate, and then 57 mg of tri-n-butylamine was added. After nitrogen replacement, the mixture was stirred at 0°C. 19 μL of phosphorus oxychloride was added, and the reaction was allowed to proceed for 1 h. Then, a mixture of 115 mg of tri-n-butylamine and 183 mg of ammonium pyrophosphate dissolved in 5 mL of acetonitrile was added. The reaction was continued for 10 min, and then 10 mL of 0.1 M sodium bicarbonate solution was added to quench the reaction. 1 mL of concentrated ammonia was added and stirred at room temperature overnight. After removing the ammonia, the reaction solution was diluted to 200 mL and purified by DEAE weak anion exchange column chromatography using 1 M TEAB (triethylammonium carbonate) and pure water. The fraction was spin-dried and then subjected to reverse phase preparative purification and lyophilization to obtain 20 mg of compound A6.

[0088] Characterization data of compound A6: 1 H NMR (400MHz, DMSO-d6): δ (ppm) 8.53 (s, 1H), 8.14 (s, 1H), 7.29 (s, 2H), 6.32 (dd, 1H), 4.36 ( d,1H),4.13(d,1H),3.95(ddd,2H),3.59(dt,4H),2.90(d,1H),2.43(dd,1H),1.89(t,2H).

[0089] Example 2

[0090] This example provides compound A9 and its synthesis method, which is as follows:

[0091]

[0092] (1) Synthesis of Compound A7

[0093] 1 g of compound A3 was weighed and dissolved in 5 mL of anhydrous DMF. 570 mg of CDI (carbonyldiimidazole) was added and the mixture was stirred at room temperature for 1 h. 500 mg of isocyanuric acid was added and the reaction was allowed to proceed overnight at room temperature. The reaction was quenched by adding water, and the organic phase was extracted with ethyl acetate and dried. The organic phase was purified by normal phase column chromatography using dichloromethane and ethyl acetate, and the fraction was dried to obtain 325 mg of compound A7.

[0094] (2) Synthesis of Compound A8

[0095] 100 mg of compound A7 was weighed, 2 mL of THF was added to dissolve it, and the mixture was stirred at 10°C. 200 mg of TBAF (tetrabutylammonium fluoride trihydrate) was weighed, 1 mL of THF was added to dissolve it, and the mixture was added to the reaction solution. The reaction was allowed to react overnight at 10°C. The reaction solution was quenched with water, and the organic phase was extracted with ethyl acetate and dried. The organic phase was purified by normal phase column chromatography using dichloromethane and methanol, and the fraction was dried to obtain 32 mg of compound A8.

[0096] (3) Synthesis of Compound A9

[0097] 50 mg of compound A8 was weighed and dissolved in 1 mL of trimethyl phosphate. 50 mg of tri-n-butylamine was added. After nitrogen replacement, the mixture was placed at 0°C with low temperature stirring. 16 μL of phosphorus oxychloride was added and the reaction was allowed to proceed for 1 h. A mixture of 105 mg of tri-n-butylamine and 163 mg of ammonium pyrophosphate dissolved in 5 mL of acetonitrile was added and the reaction was continued for 10 min. 10 mL of 0.1 M sodium bicarbonate solution was then added to quench the reaction. 1 mL of concentrated ammonia was added and stirred at room temperature overnight. After removing the ammonia, the reaction solution was diluted to 200 mL and purified by DEAE weak anion exchange column chromatography using 1 M TEAB and pure water. The fraction was dried and then subjected to reverse phase preparative purification and lyophilization to obtain 17 mg of compound A9.

[0098] Characterization data of compound A9: 1 H NMR (400MHz, DMSO-d6): δ (ppm) 8.57 (s, 1H), 8.12 (s, 1H), 7.30 (s, 2H), 6.32 (dd, 1H), 4.2 7–4.07(m,3H),3.90–3.70(m,2H),3.58(d,2H),3.05(dt,1H),2.59(dd,1H),1.96(s,2H).

[0099] Example 3

[0100] This example provides compound C6 and its synthesis method, which is as follows:

[0101]

[0102] (1) Synthesis of Compound C2

[0103] Weigh 25 g of compound C1, add 15 g of imidazole, dissolve in 300 mL of anhydrous DMF, and stir at 10 °C;

[0104] 19 g of TBSCl was weighed and dissolved in 50 mL of anhydrous DMF. The mixture was slowly added dropwise to the mixture solution of the above compound C1 and imidazole using a constant pressure addition funnel. After the addition, the reaction solution was moved to room temperature and stirred. The reaction was continued at room temperature overnight. The reaction solution was added to ice water to quench the reaction and filtered to obtain a white solid. The white solid was slurried with n-hexane and tert-methyl ether and filtered to obtain 33 g of white powdery solid C2.

[0105] (2) Synthesis of Compound C3

[0106] 10 g of compound C2 was weighed, dissolved in 100 mL of methanol, and 17 g of DMF-DMA (N,N-dimethylformamide dimethyl acetal) was added. The mixture was reacted at room temperature overnight, and the reaction solution was dried to obtain 11.6 g of compound C3.

[0107] (3) Synthesis of Compound C4

[0108] Weigh 1 g of compound C3, dissolve it in 10 mL of anhydrous DMF, replace the atmosphere with nitrogen, and stir at 0°C. Add 121 mg of NaH in three portions and react for 15 minutes. Weigh 1.5 g of compound NC-OTs, dissolve it in 3 mL of anhydrous DMF, and add it to the above reaction solution. Continue the reaction for 2 hours. Add water to quench the reaction solution, extract the organic phase with ethyl acetate, and dry it. Purify it by normal phase column chromatography with dichloromethane and ethyl acetate, and dry the fraction to obtain 165 mg of compound C4.

[0109] (4) Synthesis of Compound C5

[0110] 100 mg of compound C4 was weighed and dissolved in 2 mL of THF. The mixture was stirred at 10°C and 150 mg of TBAF was added to react overnight. The reaction was quenched by adding water. The organic phase was extracted with ethyl acetate and dried. The organic phase was purified by normal phase column chromatography using dichloromethane and ethyl acetate, and the fraction was dried to obtain 53 mg of compound C5.

[0111] (5) Synthesis of Compound C6

[0112] 50 mg of compound C5 was weighed, dissolved in 2 mL of trimethyl phosphate, 50 mg of tri-n-butylamine was added, and the mixture was stirred at -5 °C after nitrogen replacement. 15.6 μL of phosphorus oxychloride was added, and after reacting for 1 h, 122 mg of tri-n-butylamine and 195 mg of ammonium pyrophosphate were dissolved in 5 mL of acetonitrile, and the reaction was continued for 10 min. 10 mL of 0.1 M sodium bicarbonate solution was added to quench the reaction, 1 mL of concentrated ammonia was added, and the mixture was stirred at room temperature overnight. After removing the ammonia, the reaction solution was diluted to 200 mL and purified by DEAE weak anion exchange column chromatography using 1 M TEAB and pure water. The fraction was dried and then purified by reverse phase preparative purification and lyophilized to obtain 17 mg of compound C6.

[0113] Characterization data of compound C6: 1 H NMR (400MHz, DMSO-d6): δ (ppm) 7.64 (d, 1H), 7.15 (s, 2H), 6.09 (dd, 1H), 5.65 (d, 1H), 4.16–3 .99(m,3H),3.84–3.65(m,2H),3.52(tt,2H),2.34(ddd,1H),2.02(ddd,1H),1.89(dqd,2H).

[0114] Example 4

[0115] This example provides compound C9 and its synthesis method, and its synthesis method is as follows:

[0116]

[0117] (1) Synthesis of Compound C7

[0118] 1 g of compound C3 was weighed and dissolved in 10 mL of anhydrous DMF. 817 mg of CDI was added and reacted at room temperature for 1 h. 500 mg of isocyanuric acid was added and reacted overnight. The reaction was quenched by adding water. The organic phase was extracted with ethyl acetate and dried. The organic phase was purified by normal phase column chromatography with dichloromethane and ethyl acetate, and the fraction was dried to obtain 207 mg of compound C7.

[0119] (2) Synthesis of Compound C8

[0120] 100 mg of compound C7 was weighed and dissolved in 5 mL of THF. 160 mg of TBAF was added and reacted at room temperature overnight. The reaction solution was quenched with water, and the organic phase was extracted with ethyl acetate and dried. The organic phase was purified by normal phase column chromatography using dichloromethane and methanol, and the fraction was dried to obtain 42 mg of compound C8.

[0121] (3) Synthesis of Compound C9

[0122] 100 mg of compound C8 was weighed, dissolved in 2 mL of trimethyl phosphate, and 70 mg of tri-n-butylamine was added. After nitrogen replacement, the mixture was placed at -5°C with stirring, and 41 μL of phosphorus oxychloride was added. After reacting for 1 hour, 202 mg of tri-n-butylamine and 350 mg of ammonium pyrophosphate were dissolved in 5 mL of acetonitrile and the mixture was continued to react for 10 minutes. 10 mL of 0.1 M sodium bicarbonate solution was added to quench the reaction, and 1 mL of concentrated ammonia was added and stirred at room temperature overnight. After removing the ammonia, the reaction solution was diluted to 200 mL and purified by DEAE weak anion exchange column chromatography using 1 M TEAB and pure water. The fraction was dried and then purified by reverse phase preparative purification and lyophilized to obtain 95 mg of compound C9.

[0123] Characterization data of compound C9: 1 H NMR (400MHz, DMSO-d6): δ (ppm) 7.76 (d, 1H), 7.13 (s, 2H), 6.17 (dd, 1H), 5.63 (d, 1H), 4.26–4 .11(m,3H),3.74–3.69(m,2H),3.55(tt,2H),2.33(ddd,1H),2.06(ddd,1H),1.93(dqd,2H).

[0124] Example 5

[0125] This example provides compound G6 and its synthesis method, which is as follows:

[0126]

[0127] (1) Synthesis of Compound G2

[0128] Weigh 25 g of compound G1, dissolve it in 300 mL of anhydrous DMF, add 16 g of imidazole, replace the atmosphere with nitrogen, and stir at 10°C;

[0129] Weigh 16.2 g of TBSCl, dissolve it in 50 mL of anhydrous DMF, add the resulting solution to a constant pressure addition funnel, and slowly add it dropwise to the mixture of compound G1 and imidazole using the constant pressure addition funnel. After the addition, bring the reaction solution to room temperature and react overnight. Quench the reaction solution by adding ice water and filter to obtain a white solid. Slurry the solid with n-hexane and petroleum ether, purify the solid, and filter to obtain 30.13 g of compound G2.

[0130] (2) Synthesis of Compound G3

[0131] 10 g of compound G2 was weighed and dissolved in 100 mL of methanol. 10 g of DMF-DMA was added and stirred at room temperature overnight. The reaction solution was spin-dried to obtain 11.4 g of compound G3.

[0132] (3) Synthesis of Compound G4

[0133] Weigh 1 g of compound G3, dissolve it in 5 mL of anhydrous DMF, replace the atmosphere with nitrogen, and stir at 10 °C. Add 109 mg of NaH in three portions and react for 30 min. Weigh 1.65 g of NC-OTs compound, dissolve it in 2 mL of anhydrous DMF, and add it to the reaction solution. Stir at room temperature overnight. After adding water to quench the reaction, extract the organic phase with ethyl acetate and dry it. Purify it by normal phase column chromatography with dichloromethane and ethyl acetate, and dry the fraction to obtain 107 mg of compound G4.

[0134] (4) Synthesis of Compound G5

[0135] 100 mg of compound G4 was weighed and dissolved in 5 mL of THF. The mixture was stirred at 10°C, and 170 mg of TBAF was added and stirred overnight. The reaction was quenched by adding water, and the organic phase was extracted with ethyl acetate and dried. The organic phase was purified by normal phase column chromatography using dichloromethane and ethyl acetate, and the fraction was dried to obtain 42 mg of compound G5.

[0136] (5) Synthesis of Compound G6

[0137] 100 mg of compound G5 was weighed and dissolved in 5 mL of trimethyl phosphate. 147 mg of tri-n-butylamine was added, and the mixture was moved to -5°C with stirring after nitrogen replacement. 46.8 μL of phosphorus oxychloride was added, and after reacting for 2 h, 222 mg of tri-n-butylamine and 350 mg of ammonium pyrophosphate were dissolved in 5 mL of acetonitrile, and the reaction was continued for 10 min. 10 mL of 0.1 M sodium bicarbonate solution was added to quench the reaction, and 1 mL of concentrated ammonia was added and stirred at room temperature overnight. After removing the ammonia, the reaction solution was diluted to 200 mL and purified by DEAE weak anion exchange column chromatography using 1 M TEAB and pure water. The fraction was dried and then purified by reverse phase preparative purification and lyophilized to obtain 45 mg of compound G6.

[0138] Characterization data of compound G6: 1 H NMR (400MHz, DMSO-d6): δ (ppm) 8.05 (s, 1H), 6.25 (dd, 1H), 4.65 (dt, 1H), δ 4.23–4.0 2(m,3H),3.93–3.65(m,2H),3.57(d,2H),3.11(dt,1H),2.61(dd,1H),1.93(s,2H).

[0139] Example 6

[0140] This example provides compound G9 and its synthesis method, which is as follows:

[0141]

[0142] (1) Synthesis of Compound G7

[0143] 1 g of compound G3 was weighed and dissolved in 5 mL of anhydrous DMF. 550 mg of CDI was added and stirred at room temperature overnight. 580 mg of isocyanuric acid was added and stirring was continued at room temperature overnight. The reaction solution was quenched with water, and the organic phase was extracted with ethyl acetate and dried. The organic phase was purified by normal phase column chromatography using dichloromethane and ethyl acetate, and the fraction was dried to obtain 420 mg of compound G7.

[0144] (2) Synthesis of Compound G8

[0145] 100 mg of compound G7 was weighed and dissolved in 2 mL of tetrahydrofuran. The mixture was stirred at 10°C, and 135 mg of TBAF was added and stirred overnight. The reaction solution was quenched with water, and the organic phase was extracted with ethyl acetate and dried. The organic phase was purified by normal phase column chromatography with dichloromethane and methanol, and the fraction was dried to obtain 35 mg of compound G8.

[0146] (3) Synthesis of Compound G9

[0147] 50 mg of compound G8 was weighed and dissolved in 2 mL of trimethyl phosphate. 50 mg of tri-n-butylamine was added, and the mixture was stirred at -10°C after nitrogen replacement. 19 μL of phosphorus oxychloride was added, and the reaction was allowed to proceed for 1.5 h. 2 mL of acetonitrile was added to dissolve 105 mg of tri-n-butylamine and 158 mg of ammonium pyrophosphate, and the reaction was continued for 10 min. 10 mL of 0.1 M sodium bicarbonate solution was added to quench the reaction. 1 mL of concentrated ammonia was added and stirred at room temperature overnight. After removing the ammonia, the reaction solution was diluted to 200 mL and purified by DEAE weak anion exchange column chromatography using 1 M TEAB and pure water. The fraction was dried and then purified by reverse phase preparative purification and lyophilized to obtain 12 mg of compound G9.

[0148] Characterization data of compound G9: 1 H NMR (400MHz, DMSO-d6): δ (ppm) 8.03 (s, 1H), 6.22 (dd, 1H), 4.63 (dt, 1H), δ 4.19–3.9 9(m,3H),3.91–3.58(m,2H),3.52(d,2H),3.14(dt,1H),2.65(dd,1H),1.91(s,2H).

[0149] Example 7

[0150] This example provides compound T6 and its synthesis method, which is as follows:

[0151]

[0152] (1) Synthesis of Compound T2

[0153] Weigh 25 g of compound T1, dissolve it in 180 mL of anhydrous pyridine, replace the atmosphere with nitrogen, and stir at 10 °C. Slowly add 33 mL of TMSCl dropwise and react for 1 h. Take 18 mL of benzoyl chloride, dilute it with 20 mL of anhydrous dichloromethane, and add it dropwise to the reaction solution. Continue to react for 2 h after the addition. Add water to quench the reaction. Add dilute hydrochloric acid to adjust the pH to weak acidity and continue stirring for 2 h. Extract the organic phase with ethyl acetate and dry it. Purify it by column chromatography with dichloromethane and methanol, and dry the fraction to obtain 28.4 g of compound T2.

[0154] (3) Synthesis of Compound T3

[0155] Weigh 10g of compound T2 and dissolve it in 150mL of anhydrous DMF. Add 4.7g of imidazole and stir at 10°C. Weigh 5.2g of TBSCl and dissolve it in 20mL of anhydrous DMF. Slowly add it dropwise to the reaction mixture. After the addition is complete, move the reaction mixture to room temperature and stir overnight. The reaction mixture is quenched by adding ice water. The organic phase is extracted with ethyl acetate and dried. Purify the organic phase by normal phase column chromatography using dichloromethane and methanol, and the fraction is dried to obtain 10.2g of compound T3.

[0156] (3) Synthesis of Compound T4

[0157] 1 g of compound T3 was weighed and dissolved in 10 mL of anhydrous DMF. After nitrogen replacement, the mixture was stirred at 0°C. 90 mg of NaH was added three times, and the reaction was allowed to proceed for 20 min. 900 mg of NC-OTs was weighed and dissolved in 2 mL of anhydrous DMF, and then added to the reaction solution. The mixture was moved to room temperature and reacted for 2 h. The reaction solution was quenched with water, and the organic phase was extracted with ethyl acetate and dried. The organic phase was purified by normal phase column chromatography using dichloromethane and ethyl acetate, and the fraction was dried to obtain 325 mg of compound T4.

[0158] (4) Synthesis of Compound T5

[0159] 100 mg of compound T4 was weighed and dissolved in 5 mL of THF. 135 mg of TBAF was weighed and added to the reaction solution, and the mixture was stirred at room temperature overnight. The reaction was quenched by adding water, and the organic phase was extracted with ethyl acetate and dried. The organic phase was purified by normal phase column chromatography using dichloromethane and methanol, and the fraction was dried to obtain 47 mg of compound T5.

[0160] (5) Synthesis of Compound T6

[0161] 100 mg of compound T5 was weighed and dissolved in 2 mL of trimethyl phosphate. 86 mg of tri-n-butylamine was added, and the mixture was stirred at -5°C after nitrogen replacement. 45 μL of phosphorus oxychloride was added and the reaction was allowed to proceed for 1.5 h. 5 mL of acetonitrile was then added to dissolve 209 mg of tri-n-butylamine and 330 mg of ammonium pyrophosphate, and the reaction was continued for 10 min. 10 mL of 0.1 M sodium bicarbonate solution was added to quench the reaction. 1 mL of concentrated ammonia was added and stirred at room temperature overnight. After degassing the ammonia, the reaction solution was diluted to 200 mL and purified by DEAE weak anion exchange column chromatography using 1 M TEAB and pure water. The fraction was dried and then purified by reverse phase preparative purification and lyophilized to obtain 67 mg of compound T6.

[0162] Characterization data of compound T6: 1 H NMR (400MHz, DMSO-d6): δ (ppm) 7.56 (d, 1H), 6.12 (dd, 1H), 4.13 (h, 1H), 3.94–3.56 (m, 5H), 3.51–3.35 (m, 2H), 2.14–1.93 (m, 2H), 1.77–1.72 (m, 3H).

[0163] Example 8

[0164] This example provides compound T9 and its synthesis method, which is as follows:

[0165]

[0166] (1) Synthesis of Compound T7

[0167] 1 g of compound T3 was weighed and dissolved in 5 mL of anhydrous DMF. 528 mg of CDI was added and reacted at room temperature for 1 h. 400 mg of isocyanuric acid was added and stirred at room temperature overnight. The reaction solution was quenched with water, and the organic phase was extracted with ethyl acetate and dried. The organic phase was purified by normal phase column chromatography using dichloromethane and ethyl acetate, and the fraction was dried to obtain 230 mg of compound T7.

[0168] (2) Synthesis of Compound T8

[0169] 100 mg of compound T7 was weighed and dissolved in 5 mL of THF. 150 mg of TBAF was added and stirred overnight. The reaction solution was quenched with water, and the organic phase was extracted with ethyl acetate and dried. The organic phase was purified by normal phase column chromatography using dichloromethane and methanol, and the fraction was dried to obtain 52 mg of compound T8.

[0170] (3) Synthesis of Compound T9

[0171] 100 mg of compound T8 was weighed and dissolved in 2 mL of trimethyl phosphate. 70 mg of tri-n-butylamine was added, and the mixture was stirred at -5°C after nitrogen replacement. 40 μL of phosphorus oxychloride was added and the reaction was allowed to proceed for 1 h. 5 mL of acetonitrile was then added to dissolve 250 mg of tri-n-butylamine and 300 mg of ammonium pyrophosphate, and the reaction was continued for 10 min. 10 mL of 0.1 M sodium bicarbonate solution was added to quench the reaction. 1 mL of concentrated ammonia was added and stirred at room temperature overnight. After degassing the ammonia, the reaction solution was diluted to 200 mL and purified by DEAE weak anion exchange column chromatography using 1 M TEAB and pure water. The fraction was dried and then purified by reverse phase preparative purification and lyophilized to obtain 39 mg of compound T9.

[0172] Characterization data of compound T9: 1 H NMR (400MHz, DMSO-d6): δ (ppm) 7.76 (d, 1H), 6.22 (dd, 1H), 4.13 (h, 1H), 3.91–3.52 (m, 5H), 3.54–3.358 (m, 2H), 2.14–1.93 (m, 2H), 1.77–1.72 (m, 3H).

[0173] Example 9

[0174] This example provides compound AA6 and its synthesis method, which is as follows:

[0175]

[0176] (1) Synthesis of Compound AA2

[0177] Weigh 25 g of compound AA1, dissolve it in 200 mL of anhydrous pyridine, and place it in ice water to cool and stir;

[0178] Another 42 g of TBSCl was dissolved in 50 mL of anhydrous DMF and slowly added dropwise to the anhydrous pyridine solution of the above compound AA1. After the addition, the mixture was moved to room temperature and stirred overnight. The reaction solution was quenched by adding ice water and filtered. The filter residue was dissolved in ethyl acetate and mixed with silica gel. The sample was purified by column chromatography with dichloromethane and methanol, and the fraction was spin-dried to obtain 20 g of compound AA2.

[0179] (2) Synthesis of Compound AA3

[0180] 10 g of compound AA2 was weighed and dissolved in 100 mL of methanol. 12 g of DMF-DMA was added and stirred at room temperature overnight. The reaction solution was then dried to obtain 11 g of compound AA3.

[0181] (3) Synthesis of Compound AA4

[0182] Weigh 5 g of compound AA3, add 20 mL of anhydrous DMF to dissolve, after nitrogen replacement, move to 0 ° C low temperature with stirring, add 435 mg of NaH in 3 times, and react for 30 minutes after the addition. Weigh 5.24 g of NC-OTs and dissolve it in 5 mL of anhydrous DMF and add it to the reaction solution. Continue to react for 1 hour. After the reaction, water is added to quench the reaction. The organic phase is extracted with ethyl acetate and dried, and purified by normal phase column chromatography with petroleum ether and ethyl acetate. The fraction is dried to obtain 3 g of compound AA4.

[0183] (4) Synthesis of Compound AA5

[0184] Weigh 1 g of compound AA4, add 10 mL of THF, stir and dissolve, place at 10 ° C and stir at low temperature, weigh 1.25 g of TBAF, add 3 mL of THF to dissolve, and then add to the reaction solution, stir at low temperature overnight, add water to quench the reaction, spin off THF, extract the organic phase with ethyl acetate, and dry it. Purify it by normal phase column chromatography with dichloromethane and methanol, and dry the fraction to obtain 453 mg of compound AA5.

[0185] (5) Synthesis of Compound AA6

[0186] Weigh 100 mg of compound AA5, dissolve it in 4 mL of trimethyl phosphate, add 0.15 mL of tri-n-propylamine, replace the atmosphere with nitrogen, move to 0 ° C and stir at low temperature, add 47 μL of phosphorus oxychloride, react for 1 hour, add a mixed solution of 280 mg of ammonium pyrophosphate and 0.3 mL of tri-n-propylamine dissolved in 2 mL of anhydrous acetonitrile, continue the reaction for 20 minutes, quench the reaction with 10 mL of 0.1 M sodium bicarbonate solution, add 1 mL of concentrated ammonia solution and stir at room temperature overnight. After removing the ammonia, the reaction solution was diluted to 200 mL and purified by DEAE weak anion exchange column chromatography with 1 M TEAB and pure water. After the fraction was dried, it was purified by reverse phase preparative purification and lyophilized to obtain 53 mg of compound AA6.

[0187] Characterization data of compound AA6: 1 H NMR (400MHz, DMSO-d6): δ (ppm) 8.49 (s, 1H), 8.17 (s, 1H), 7.33 (s, 2H), 6.42 (dd, 1H), 4.66 (s, 1H) ,4.26(d,1H),4.17(d,1H),3.88(ddd,2H),3.62(dt,3H),3.11(d,1H),2.49(dd,1H),1.97(t,2H).

[0188] Example 10

[0189] This example provides compound AA9 and its synthesis method, which is as follows:

[0190]

[0191] (1) Synthesis of Compound AA7

[0192] 5 g of compound AA3 was weighed and dissolved in 20 mL of anhydrous DMF. 2.56 g of CDI was added and reacted for 1 h. 2.4 g of isocyanuric acid was added and the reaction was continued at room temperature overnight. The reaction solution was quenched with water, and the organic phase was extracted with ethyl acetate and dried. The organic phase was purified by normal phase column chromatography with petroleum ether and ethyl acetate, and the fraction was dried to obtain 3.1 g of compound AA7.

[0193] (2) Synthesis of Compound AA8

[0194] Weigh 1 g of compound AA7, dissolve it in 10 mL of THF, stir at 10°C, add 1.2 g of TBAF dissolved in 5 mL of THF, stir and react overnight, quench the reaction solution with water, spin-dry the THF, extract the organic phase with ethyl acetate and spin-dry it, purify it by normal phase column chromatography with dichloromethane and methanol, and spin-dry the fraction to obtain 438 mg of compound AA8.

[0195] (3) Synthesis of Compound AA9

[0196] Weigh 100 mg of compound AA8, dissolve it in 3 mL of trimethyl phosphate, add 0.13 mL of tri-n-propylamine, replace the atmosphere with nitrogen, move to -10 ° C and stir at low temperature, add 42 μL of phosphorus oxychloride, react for 1.5 h, add a mixed solution of 315 mg of ammonium pyrophosphate and 0.27 mL of tri-n-propylamine dissolved in 2 mL of anhydrous acetonitrile, continue the reaction for 20 min, quench the reaction with 10 mL of 0.1 M sodium bicarbonate solution, add 1 mL of concentrated ammonia solution and stir at room temperature overnight, spin off the ammonia, and dilute the reaction solution to 200 mL. Purify by DEAE weak anion exchange column chromatography with 1 M TEAB and pure water, spin-dry the fraction, perform reverse phase preparative purification, and lyophilize to obtain 35 mg of compound AA9.

[0197] Characterization data of compound AA9: 1 H NMR (400MHz, DMSO-d6): δ (ppm) 8.67 (s, 1H), 8.21 (s, 1H), 7.35 (s, 2H), 6.33 (dd, 1H), 4.73 (s, 1H) ),4.25–4.17(m,2H),3.95–3.72(m,2H),3.61(d,2H),3.11(dt,1H),2.62(dd,1H),1.99(s,2H).

[0198] Example 11

[0199] This example provides compound CC6 and its synthesis method, which is as follows:

[0200]

[0201] (1) Synthesis of compound CC2

[0202] Weigh 25 g of compound CC1, dissolve it in 200 mL of anhydrous pyridine, and stir at 0°C;

[0203] Weigh 47 g of TBSCl and dissolve it in 100 mL of anhydrous DMF. Slowly add this dropwise to the anhydrous pyridine solution of compound CC1. After addition, bring the reaction mixture to room temperature and allow to react overnight. Quench the reaction by adding ice water, filter the resulting residue, dissolve it in ethyl acetate, and purify it by normal-phase column chromatography using dichloromethane and methanol. The fraction is then spin-dried to dryness to yield 15.2 g of compound CC2.

[0204] (2) Synthesis of compound CC3

[0205] 5 g of compound CC3 was weighed and dissolved in 50 mL of methanol. 3.79 g of DMF-DMA was added and stirred at room temperature overnight. The reaction solution was spin-dried to obtain 5.6 g of compound CC3.

[0206] (3) Synthesis of compound CC4

[0207] Weigh 5 g of compound CC3, dissolve it in 20 mL of anhydrous DMF, place it at 0°C with stirring, add 455 mg of NaH in batches, and react for 30 minutes. Weigh 6.81 g of NC-OTs, dissolve it in 10 mL of anhydrous DMF, and add it to the reaction solution. After reacting for 1 hour, water is added to quench the reaction. The organic phase is extracted with ethyl acetate and dried, and purified by normal phase column chromatography with petroleum ether and ethyl acetate. The fraction is dried to obtain 2.37 g of compound CC4.

[0208] (4) Synthesis of compound CC5

[0209] Weigh 1 g of compound CC4, dissolve it in 10 mL of THF, and stir at 10°C. Weigh 1.5 g of TBAF, dissolve it in 5 mL of THF, and add it to the reaction solution. Stir overnight. Add water to quench the reaction solution. After removing THF, extract the organic phase with ethyl acetate and dry it. Purify it by normal phase column chromatography with dichloromethane and methanol, and dry the fraction to obtain 514 mg of compound CC5.

[0210] (5) Synthesis of compound CC6

[0211] 100 mg of compound CC5 was weighed, dissolved in 4 mL of trimethyl phosphate, and 0.1 mL of tri-n-propylamine was added. After nitrogen replacement, the mixture was moved to 0°C and stirred at low temperature. 50 μL of phosphorus oxychloride was added and reacted for 1 h. A mixed solution of 300 mg of ammonium pyrophosphate and 0.33 mL of tri-n-propylamine dissolved in 2 mL of anhydrous acetonitrile was added and the reaction was continued for 20 min. The reaction was quenched with 10 mL of 0.1 M sodium bicarbonate solution, and 1 mL of concentrated ammonia was added and stirred at room temperature overnight. After removing the ammonia, the reaction solution was diluted to 200 mL and purified by DEAE weak anion exchange column chromatography using 1 M TEAB and pure water. The fraction was dried and then purified by reverse phase preparative purification and lyophilized to obtain 57 mg of compound CC6.

[0212] Characterization data of compound CC6: 1 H NMR (400MHz, DMSO-d6): δ (ppm) 7.71 (d, 1H), 7.13 (s, 2H), 6.13 (dd, 1H), 5.63 (d, 1H), 4.72 (s, 1H), 4.22–3.98(m,2H),3.86–3.67(m,2H),3.51(tt,2H),2.32(ddd,1H),2.05(ddd,1H),1.91(dqd,2H).

[0213] Example 12

[0214] This example provides compound CC9 and its synthesis method, the synthesis method is as follows:

[0215]

[0216] (1) Synthesis of compound CC7

[0217] 5 g of compound CC7 was weighed and dissolved in 10 mL of anhydrous DMF. 2.68 g of CDI was added and reacted at room temperature for 30 min. 2.5 g of isocyanuric acid propanol was added and reacted at room temperature overnight. Water was added to quench the reaction. The organic phase was extracted with ethyl acetate and dried. The organic phase was purified by normal phase column chromatography with n-hexane and ethyl acetate, and the fraction was dried to obtain 3.2 g of compound CC7.

[0218] (2) Synthesis of compound CC8

[0219] Weigh 1 g of compound CC7, dissolve it in 10 mL of THF, and stir at 10°C. Weigh 1.2 g of TBAF, dissolve it in 5 mL of THF, add it to the reaction solution, and stir overnight at low temperature. After the reaction solution is quenched with water, the THF is removed by rotation. The organic phase is extracted with ethyl acetate and dried. The organic phase is purified by normal phase column chromatography with dichloromethane and methanol, and the fraction is dried to obtain 426 mg of Huawei CC8.

[0220] (3) Synthesis of compound CC9

[0221] 100 mg of compound CC8 was weighed, dissolved in 3 mL of trimethyl phosphate, and 0.1 mL of tri-n-propylamine was added. After nitrogen replacement, the mixture was moved to 0°C and stirred at low temperature. 45 μL of phosphorus oxychloride was added, and the reaction was allowed to react for 1 h. A mixed solution of 400 mg of ammonium pyrophosphate and 0.28 mL of tri-n-propylamine dissolved in 2 mL of anhydrous acetonitrile was added, and the reaction was continued for 20 min. The reaction was quenched with 10 mL of 0.1 M sodium bicarbonate solution, and 1 mL of concentrated ammonia was added and stirred at room temperature overnight. After removing the ammonia, the reaction solution was diluted to 200 mL and purified by DEAE weak anion exchange column chromatography using 1 M TEAB and pure water. The fraction was dried and then purified by reverse phase preparative purification and lyophilized to obtain 37 mg of compound CC9.

[0222] Characterization data of compound CC9: 1 H NMR (400MHz, DMSO-d6): δ (ppm) 7.74 (d, 1H), 7.19 (s, 2H), 6.15 (dd, 1H), 5.59 (d, 1H), 4.67 (s, 1H), 4.23–4.12(m,2H),3.77–3.67(m,2H),3.49(tt,2H),2.35(ddd,1H),2.11(ddd,1H),1.95(dqd,2H).

[0223] Example 13

[0224] This example provides compound GG6 and its synthesis method, which is as follows:

[0225]

[0226] (1) Synthesis of compound GG2

[0227] Weigh 25 g of compound GG1, dissolve it in 300 mL of anhydrous pyridine, place it in ice water and stir at low temperature, weigh 40 g of TBSCl, dissolve it in 100 mL of anhydrous DMF, and slowly add it dropwise to the nucleoside solution. After the addition, move the reaction solution to room temperature and react overnight. The reaction solution is quenched by adding ice water and filtered. The filter residue is dissolved in ethyl acetate and mixed. It is purified by normal phase column chromatography with dichloromethane and ethyl acetate, and the fraction is spin-dried to obtain 17 g of compound GG2.

[0228] (2) Synthesis of compound GG3

[0229] 10 g of compound GG2 was weighed and dissolved in 100 mL of methanol. 7 g of DMF-DMA was added and stirred at room temperature overnight. The reaction solution was spin-dried to obtain 11 g of compound GG3.

[0230] (3) Synthesis of compound GG4

[0231] 5 g of compound GG3 was weighed, dissolved in 25 mL of anhydrous DMF, and stirred at 0°C after nitrogen replacement. 317 mg of sodium hydride was added in batches and the reaction was continued for 1 h. 6.3 g of NC-OTs was weighed, dissolved in 10 mL of anhydrous DMF, and added to the reaction solution. The reaction was continued for 1 h. The reaction solution was quenched with water, and the organic phase was extracted with ethyl acetate and dried. The organic phase was purified by normal phase column chromatography with n-hexane and ethyl acetate, and the fraction was dried to obtain 2.7 g of compound GG4.

[0232] (4) Synthesis of compound GG5

[0233] 1 g of compound GG4 was weighed and dissolved in 10 mL of THF. 1.4 g of TBAF was weighed and dissolved in 5 mL of THF and added to the reaction solution. The mixture was stirred at 10°C overnight. The reaction solution was quenched by adding water. After removing THF, the organic phase was extracted with ethyl acetate and dried. The organic phase was purified by normal phase column chromatography with dichloromethane and methanol, and the fraction was dried to obtain 518 mg of compound GG5.

[0234] (5) Synthesis of compound GG6

[0235] 100 mg of compound GG5 was weighed, dissolved in 3 mL of trimethyl phosphate, and 0.14 mL of tri-n-propylamine was added. After nitrogen replacement, the mixture was moved to 0°C and stirred at low temperature. 45 μL of phosphorus oxychloride was added, and the reaction was continued for 1.5 h. A mixed solution of 270 mg of ammonium pyrophosphate and 0.28 mL of tri-n-propylamine dissolved in 2 mL of anhydrous acetonitrile was added. The reaction was continued for 20 min. The reaction was quenched with 10 mL of 0.1 M sodium bicarbonate solution, and 1 mL of concentrated ammonia was added and stirred at room temperature overnight. After removing the ammonia, the reaction solution was diluted to 200 mL and purified by DEAE weak anion exchange column chromatography using 1 M TEAB and pure water. The fraction was dried and then purified by reverse phase preparative purification and lyophilized to obtain 47 mg of compound GG6.

[0236] Characterization data of compound GG6: 1H NMR (400MHz, DMSO-d6): δ (ppm) 8.02 (s, 1H), 6.22 (dd, 1H), 4.69 (s, 1H), 4.63 (dt, 1H), δ 4. 19–4.04(m,2H),3.92–3.61(m,2H),3.55(d,2H),3.13(dt,1H),2.63(dd,1H),1.89(s,2H).

[0237] Example 14

[0238] This example provides compound GG9 and its synthesis method, which is as follows:

[0239]

[0240] (1) Synthesis of compound GG7

[0241] 5 g of compound GG6 was weighed and dissolved in 20 mL of anhydrous DMF. 2.4 g of CDI was added and the reaction was continued for 1 h. 2.3 g of isocyanuric acid was added and the reaction was continued for 1 h. The reaction solution was quenched with water, and the organic phase was extracted with ethyl acetate and dried. The organic phase was purified by normal phase column chromatography with n-hexane and ethyl acetate, and the fraction was dried to obtain 3.4 g of compound GG7.

[0242] (2) Synthesis of compound GG8

[0243] 1 g of compound GG7 was weighed and dissolved in 10 mL of THF. 1.4 g of TBAF dissolved in 5 mL of THF was added, and the mixture was stirred at 10°C overnight. The reaction was quenched by adding water, and the organic phase was extracted with ethyl acetate and dried. The organic phase was purified by normal phase column chromatography with dichloromethane and methanol, and the fraction was dried to obtain 422 mg of compound GG8.

[0244] (3) Synthesis of compound GG9

[0245] 100 mg of compound GG8 was weighed, dissolved in 4 mL of trimethyl phosphate, and 0.13 mL of tri-n-propylamine was added. After nitrogen replacement, the mixture was stirred at 0°C. 40 μL of phosphorus oxychloride was added, and the reaction was continued for 1.5 h. A mixed solution of 250 mg of ammonium pyrophosphate and 0.21 mL of tri-n-propylamine dissolved in 2 mL of anhydrous acetonitrile was added. The reaction was continued for 20 min. The reaction was quenched with 10 mL of 0.1 M sodium bicarbonate solution, and 1 mL of concentrated ammonia was added and stirred at room temperature overnight. After removing the ammonia, the reaction solution was diluted to 200 mL and purified by DEAE weak anion exchange column chromatography using 1 M TEAB and pure water. The fraction was dried and then purified by reverse phase preparative purification and lyophilized to obtain 44 mg of compound GG9.

[0246] Characterization data of compound GG9: 1H NMR (400MHz, DMSO-d6): δ (ppm) 7.99 (s, 1H), 6.25 (dd, 1H), 4.74 (s, 1H), 4.61 (dt, 1H), δ 4. 17–3.97(m,2H),3.92–3.60(m,2H),3.49(d,2H),3.11(dt,1H),2.62(dd,1H),1.89(s,2H).

[0247] Example 15

[0248] This example provides compound U6 and its synthesis method, which is as follows:

[0249]

[0250]

[0251] (1) Synthesis of Compound U2

[0252] Weigh 25 g of compound U1, add 250 mL of pyridine to dissolve, stir at 0 ° C, slowly add 65 mL of TMSCl, move to room temperature and stir for 4 hours, then move the reaction solution back to 0 ° C and stir, weigh 21.6 g of BzCl and add 50 mL of anhydrous DMF to dissolve, slowly add dropwise to the reaction solution, move to room temperature, stir overnight, add water to quench the reaction, extract with ethyl acetate and dry the organic phase, dissolve the organic phase in methanol, add 0.1 M hydrochloric acid solution, adjust the pH to 6.5, stir and react for 4 hours, dry, dissolve in ethyl acetate and mix, purify by normal phase column chromatography with dichloromethane and methanol, and dry the fraction to obtain 30 g of compound U2.

[0253] (2) Synthesis of Compound U3

[0254] Weigh 10 g of compound U2, dissolve it in 200 mL of anhydrous pyridine, and stir at 0°C. Weigh 13 g of TBSCl, dissolve it in 25 mL of anhydrous DMF, and slowly add it dropwise to the reaction solution. After the addition, move to room temperature and stir overnight. The reaction solution is quenched with ice water and filtered to obtain a residue. The residue is dissolved in ethyl acetate and mixed with the sample, and then purified by normal phase column chromatography with dichloromethane and ethyl acetate. The fraction is spin-dried to obtain 5.1 g of compound U3.

[0255] (3) Synthesis of Compound U4

[0256] 5g of compound U3 was dissolved in 25mL of anhydrous DMF. After nitrogen replacement, the mixture was stirred at 0°C. 400mg of NaH was added in three portions. The reaction was allowed to react for 30 minutes. 6.33g of NC-OTs was dissolved in 10mL of anhydrous DMF and added to the reaction mixture. The reaction was continued for 1 hour. The reaction mixture was quenched with water, extracted with ethyl acetate, and the organic phase was dried. The organic phase was purified by normal-phase column chromatography using dichloromethane and ethyl acetate, and the fraction was dried to afford 3.12g of compound U4.

[0257] (4) Synthesis of Compound U5

[0258] 1 g of compound U4 was weighed and dissolved in 10 mL of THF. 1.4 g of TBAF was weighed and dissolved in 5 mL of THF and added to the reaction solution. The mixture was stirred at 10°C overnight. The reaction solution was quenched by adding water. The THF was removed by vortexing and the organic phase was extracted with ethyl acetate and dried. The organic phase was purified by normal phase column chromatography with dichloromethane and methanol, and the fraction was dried to obtain 322 mg of compound U5.

[0259] (5) Synthesis of Compound U6

[0260] 100 mg of compound U5 was weighed and dissolved in 3 mL of trimethyl phosphate. 0.18 mL of tripropylamine was added, and the mixture was moved to -5°C with stirring after nitrogen replacement. 44 μL of phosphorus oxychloride was added, and after reacting for 1 h, a mixed solution of 263 mg of ammonium pyrophosphate and 0.27 mL of tri-n-propylamine dissolved in 2 mL of anhydrous acetonitrile was added. The reaction was continued for 20 min. The reaction was quenched with 10 mL of 0.1 M sodium bicarbonate solution, and 1 mL of concentrated ammonia was added and stirred at room temperature overnight. After removing the ammonia, the reaction solution was diluted to 200 mL and purified by DEAE weak anion exchange column chromatography using 1 M TEAB and pure water. The fraction was dried and then purified by reverse phase preparative purification and lyophilized to obtain 36 mg of compound U6.

[0261] Characterization data of compound U6: 1 H NMR (400MHz, DMSO-d6): δ (ppm) 2.07-2.19 (m, 2H), 3.65-3.77 (m, 2H), 4.13 (m, 1H), 4 .21-4.37(m,6H),4.74(s,1H),5.53(d,1H),5.87(d,1H),7.67(d,1H),10.03(s,1H).

[0262] Example 16

[0263] This example provides compound U9 and its synthesis method, which is as follows:

[0264]

[0265] (1) Synthesis of Compound U7

[0266] 5 g of compound U3 was weighed and dissolved in 25 mL of anhydrous DMF. 2.44 g of CDI was added and reacted for 1 h, followed by 2.21 g of isocyanuric acid propanol. The reaction was continued for 2 h. The reaction solution was quenched with water, extracted with ethyl acetate, and the organic phase was dried by spin-drying. The organic phase was purified by normal phase column chromatography using dichloromethane and ethyl acetate, and the fraction was dried to obtain 3.1 g of compound U7.

[0267] (2) Synthesis of Compound U8

[0268] 1 g of compound U7 was weighed and dissolved in 10 mL of THF. 1.4 g of TBAF was weighed and dissolved in 2 mL of THF and added to the reaction solution. The mixture was stirred at 10°C overnight. Water was added to the reaction solution to quench the reaction. The THF was removed by vortexing. The organic phase was extracted with ethyl acetate and dried. The organic phase was purified by normal phase column chromatography with dichloromethane and methanol, and the fraction was dried to obtain 415 mg of compound U8.

[0269] (3) Synthesis of Compound U9

[0270] 100 mg of compound U8 was weighed, dissolved in 2 mL of trimethyl phosphate, and 0.17 mL of tripropylamine was added. After atmospheric replacement, the mixture was moved to -5°C and stirred at low temperature. 40 μL of phosphorus oxychloride was added and the reaction was allowed to react for 1.5 h. A mixed solution of 300 mg of ammonium pyrophosphate and 0.25 mL of tri-n-propylamine dissolved in 2 mL of anhydrous acetonitrile was added and the reaction was continued for 20 min. The reaction was quenched by adding 10 mL of 0.1 M sodium bicarbonate solution, and 1 mL of concentrated ammonia was added and stirred at room temperature overnight. After degassing the ammonia, the reaction solution was diluted to 200 mL and purified by DEAE weak anion exchange column chromatography using 1 M TEAB and pure water. The fraction was dried and then purified by reverse phase preparative purification and lyophilized to obtain 21 mg of compound U8.

[0271] Characterization data of compound U9: 1 H NMR (400MHz, DMSO-d6): δ (ppm) 2.03-2.11 (m, 2H), 3.48 (m, 2H), 4.14 (m, 1H), 4.20 -4.36(m,6H),4.75(s,1H),5.50(d,1H),5.91(d,1H),7.65(d,1H),10.04(s,1H).

[0272] Figure 1The present invention shows a process for enzymatic DNA synthesis using the isocyanate reversibly blocked nucleotide (R2 is -H) provided by the present invention, which is specifically as follows: 1) a starting sequence is chemically cleaved and bonded to a solid support; 2) a nucleotide having a 3' hydroxyl group with a reversible end (i.e., R1 in Formula I of the present invention) blocked is added, and extension is carried out in the presence of TdT enzyme; 3) a reagent for cleaving the reversible end bonded to the 3' hydroxyl group is added, and elution is performed to expose the 3' hydroxyl group; 4) steps 2) and 3 are repeated in a cycle to synthesize a DNA sequence; 5) after sequence synthesis is completed, the DNA chain is cleaved from the solid support.

[0273] The compounds used in the following application examples are as follows, and the following compounds are purchased from Shenzhen Tanmi Technology Co., Ltd.

[0274]

[0275] Application Example 1

[0276] This application example provides a TdT enzymatic DNA extension assay:

[0277] (1) Experimental materials

[0278] 1. Oligo DNA sequence: 5'-GCAGA TAATA CGACT CACTA TAGGG ATTTA GACTA CCCCAAAAC GAAAGG GGACT AAAAC-3' (60nt, SEQ ID NO.1);

[0279] 2. TdT (0.12 mg / mL)

[0280] 3. TdT reaction buffer: 10× TdT reaction buffer, 10× CoCl2 (2.5 mM) (NEB, B0315s), of which 1× TdT buffer: 20 mM Tris acetate, 50 mM potassium acetate, 10 mM magnesium acetate, pH 7.9 @ ​​25°C;

[0281] 4. Substrate: compound T6 (10 mM), compound T9 (10 mM), compound Cold dTTP (10 mM), dNTP mix (10 mM);

[0282] 5. Solvent: ddH2O.

[0283] (2) Experimental operation

[0284] (1) Configure five TdT enzymatic DNA extension reaction systems:

[0285] The oligo DNA sequence (5 μL, 2.5 mM, 60 nt), 10× TdT reaction buffer (5 μL), 2.5 mM CoCl2 solution (5 μL), 8.3 μL TdT (0.12 mg / mL), and the corresponding nucleotide substrate were added to a microcentrifuge tube (1.5 mL), and the mixture was diluted to 50 μL with ddH2O;

[0286] Substrate and system Volume / μL Isocyanurate blocking T6 0.5 Isocyanurate blocking T9 0.5 Azidomethylene blocking Cold dTTP 0.5 No blocking dNTP 4 blank blank \

[0287] (2) Dilute the mixture to 50 μL with ddH2O. Prepare three replicates for each of the T6, T9, and Cold dTTP systems, and divide the groups according to the subsequent incubation time.

[0288] (3) The mixture was incubated at 37°C for 1 h, 3 h, or 5 h depending on the substrate:

[0289] (4) Place 50 μL of the mixture on a 3% agarose gel (2.4 g of solute plus 80 mL of 1× TBE for gel preparation) and perform gel electrophoresis.

[0290] (5) For T6, T9, and Cold dTTP systems, after incubation, 2 μL of dNTPs were added to each system without inactivation. After incubation for 3 h, gel electrophoresis was performed to detect whether the oligo DNA in the system had reacted completely.

[0291] (3) Experimental results

[0292] The experimental results are as follows Figure 2 As shown by Figure 2 It can be seen that the T6 and T9 substrates provided by the present invention can complete oligo DNA extension within 1 hour, while the control Cold dTTP requires 3 hours.

[0293] Application Example 2

[0294] This application example provides a deprotection experiment

[0295] (1) Experimental materials

[0296] Deprotection reagents: THPP buffer solution (purchased from Shenzhen Carbon Dioxide Technology Co., Ltd.) was used for Cold dTTP substrate, and 80 mM pyridine tetrazine solution (adjusted to pH = 10 with 50 mM sodium bicarbonate) was used for T6 and T9 substrates;

[0297] (2) Experimental operation

[0298] (1) Configure four TdT enzymatic DNA extension reaction systems:

[0299] Oligo DNA (15 μL, 2.5 mM, 60 nt), 10× TdT reaction buffer (15 μL), 2.5 mM CoCl2 solution (15 μL), 16.6 μL TdT (0.12 mg / mL), and the corresponding nucleotide substrate were added to a microcentrifuge tube (1.5 mL), and the mixture was diluted to 150 μL with ddH2O;

[0300] Substrate and system Volume / μL Isocyanurate blocking T6 1.5 Isocyanurate blocking T9 1.5 Azidomethylene blocking Cold dTTP 1.5 No blocking dNTP 12 blank blank \

[0301] (2) The mixture was incubated at 37°C for 1 h, 3 h, or 5 h depending on the substrate:

[0302] (3) The reaction solution was removed and 3 μL of THPP buffer solution was added to the Cold dTTP system, and 80 mM pyridine tetrazine solution (adjusted to pH 10 with 50 mM sodium bicarbonate) was added to the T6 and T9 systems;

[0303] (4) Heating to 50°C and reacting for 3 min for deprotection;

[0304] (5) Purify the product using a DNA purification column, collect the eluate, and concentrate it;

[0305] (6) 10× TdT reaction buffer (15 μL), 2.5 mM CoCl2 solution (15 μL), dNTP (10 mM, 12 μL), and 16.6 μL TdT (0.12 mg / mL) were added to the concentrated T6, T9, and Cold dTTP systems, respectively, and then incubated at 37°C for 1.5 h. The mixture was diluted to 150 μL with ddH2O.

[0306] (7) Place 50 μL of the mixture on a 3% agar gel (2.4 g of solute plus 80 mL of 1×TEB for gel preparation) for detection.

[0307] (3) Experimental results

[0308] The experimental results are as follows Figure 3 As shown by Figure 3 It can be seen that the T6 and T9 substrates provided by the present invention can complete orthogonal deprotection by reacting at 50°C for 3 minutes.

[0309] Application Example 3

[0310] This application example provides a PUP enzymatic RNA extension assay.

[0311] (1) Experimental materials

[0312] 1. Oligo RNA sequence: 5'-GCAUA UUACA CAACU GAGUA UAGCG AUCUA GAGUA CCGGAAAUGC GAGCGG GGACU AAAAC-3' (60nt, SEQ ID NO.2);

[0313] 2. PUP (0.12 mg / mL)

[0314] 3. PUP reaction buffer: 10× PUP reaction buffer, 1mM DTT NEBuffer 2, of which 1× PUP buffer: 10mM Tris hydrochloride, 50mM NaCl, 10mM MgCl2, pH 7.9@25℃;

[0315] 4. Substrates: Compound U6 (10 mM), Compound U9 (10 mM), Compound Cold TTP (10 mM), NTP mix (10 mM);

[0316] 5. Solvent: ddH2O;

[0317] (2) Experimental operation

[0318] (1) Configure five PUP enzymatic RNA extension reaction systems:

[0319] Add oligo RNA (5 μL, 2.5 mM, 60 nt), 10× PUP reaction buffer (5 μL), RNase Inhibitor* (6 μL, 40 units / μL), UTP (0.5 mM), 8.3 μL PUP (0.12 mg / mL), and the corresponding nucleotide substrate to a microcentrifuge tube (1.5 mL), and dilute the mixture to 50 μL with ddH2O;

[0320] Substrate and system Volume / μL Isocyanurate blocking U6 0.5 Isocyanurate blocking U9 0.5 Azidomethylene blocking Cold TTP 0.5 No blocking NTP 4 blank blank \

[0321] (2) Dilute the mixture to 50 μL with ddH2O. Make three replicates for each of the U6, U9, and Cold TTP systems, and group them according to the subsequent incubation time.

[0322] (3) The mixture was incubated at 37°C for 0.5 h, 1 h, or 5 h depending on the substrate:

[0323] (4) Place 50 μL of the mixture on a 3% agarose gel (2.4 g of solute plus 80 mL of 1× TBE for gel preparation) and perform gel electrophoresis.

[0324] (5) For the U6, U9, and Cold TTP systems, after incubation, 2 μL of NTP was added to each system without inactivation. After incubation for 3 h, gel electrophoresis was performed to detect whether the oligo RNA in the system had reacted completely.

[0325] (3) Experimental results

[0326] The experimental results are as follows Figure 4 As shown by Figure 4 It can be seen that the U6 and U9 substrates provided by the present invention can complete oligo RNA extension within 1 hour, while the control Cold TTP requires 5 hours.

[0327] Application Example 4

[0328] This application example provides a deprotection experiment

[0329] (1) Experimental materials

[0330] Deprotection reagents: THPP buffer solution (purchased from Shenzhen Carbon Mi Technology Co., Ltd.) was used for Cold TTP substrate, and 80 mM pyridine tetrazine solution (adjusted to pH = 10 with 50 mM sodium bicarbonate) was used for U6 and U9 substrates;

[0331] (2) Experimental operation

[0332] (1) Configure four PUP enzymatic RNA extension reaction systems:

[0333] Mix oligo RNA (15μL, 2.5mM, 60nt), 10×PUP reaction buffer (15μL), 16.6μL PUP

[0334] (0.12 mg / mL) and the corresponding nucleotide substrate were added to a microcentrifuge tube (1.5 mL), and the mixture was diluted to 150 μL with ddH O;

[0335] Substrate and system Volume / μL Isocyanurate blocking U6 1.5 Isocyanurate blocking U9 1.5 Azidomethylene blocking Cold TTP 1.5 No blocking NTP 12 blank blank \

[0336] (2) The mixture was incubated at 37°C for 0.5 h, 1 h, or 4 h depending on the substrate:

[0337] (3) The reaction solution was taken out, 3 μL of THPP buffer solution was added to the Cold TTP system, and 80 mM pyridine tetrazine solution (adjusted to pH 10 with 50 mM sodium bicarbonate) was added to the U6 and U9 systems;

[0338] (4) Heating to 50°C and reacting for 3 min for deprotection;

[0339] (5) Purify the product using an RNA purification column, collect the eluate, and concentrate it;

[0340] (6) 10× PUP reaction buffer (15 μL), NTP (10 mM, 12 μL), and 16.6 μL PUP (0.12 mg / mL) were added to the concentrated U6, U9, and Cold TTP systems, respectively, and then incubated at 37°C for 1 h. The mixture was diluted to 150 μL with ddH2O.

[0341] (7) Place 50 μL of the mixture on a 3% agar gel (2.4 g of solute plus 80 mL of 1×TEB for gel preparation) for detection.

[0342] (3) Experimental results

[0343] The experimental results are as follows Figure 5 As shown by Figure 5 It can be seen that the U6 and U9 substrates provided by the present invention can complete orthogonal deprotection by reacting at 50°C for 4 minutes.

[0344] The applicant declares that the present invention uses the above-described embodiments to illustrate the detailed process equipment and process flow of the present invention. However, the present invention is not limited to the above-described detailed process equipment and process flow, and does not necessarily rely on the above-described detailed process equipment and process flow in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for the raw materials of the present invention's products, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

Claims

1. An isocyanate reversibly blocked nucleotide, characterized in that The isocyanate reversible blocking nucleotide has a structure as shown in the following formula I: Among them, R1 represents The wavy lines indicate the junction sites; R2 is -H or -OH; The base group represents a C3-C20 heteroaryl group.

2. The isocyanate reversible blocking nucleotide according to claim 1, characterized in that The base group represents any one of the following groups: The dotted lines indicate the connection sites.

3. The isocyanate reversible blocking nucleotide according to claim 1 or 2, characterized in that The isocyanate reversibly blocked nucleotides include the following compounds:

4. A method for removing a reversible blocking group in an isocyanate reversibly blocked nucleotide according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: After the isocyanide reversible blocking nucleotide reacts with the tetrazine compound, deprotection is performed to complete the removal of the reversible blocking group in the isocyanide reversible blocking nucleotide.

5. The method according to claim 4, characterized in that The tetrazine compounds include any one or a combination of at least two of 3,6-di(2-pyridyl)-1,2,4,5-tetrazine, 3,6-dimethyl-1,2,4,5-tetrazine, 3,6-dimethoxy-1,2,4,5-tetrazine, bis(methylthio)-1,2,4,5-tetrazine, 3,6-di-p-tolyl-1,2,4,5-tetraazabenzene or 3,6-di(3,5-dimethyl-1H-pyrazol-1-yl)-1,2,4,5-tetrazine.

6. The method according to claim 4 or 5, characterized in that The molar ratio of the isocyanate reversible blocking nucleotide to the tetrazine compound is 1:(10-50).

7. The method according to any one of claims 4 to 6, characterized in that: The reaction temperature is 45-55°C.

8. The method according to any one of claims 4 to 7, characterized in that: The reaction time is 3 to 5 minutes.

9. The method according to any one of claims 4 to 8, characterized in that The reaction is carried out at a pH of 9.5 to 10.

5.

10. The method according to any one of claims 4 to 9, characterized in that: The reaction is carried out in the presence of a solvent, and the solvent includes any one or a combination of at least two of DMF, methanol, DMSO, THF, acetonitrile or water.

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