An allyl-cyanoethyl nucleoside dimer, its preparation method and application in oligonucleotide synthesis

Abstract

The present invention provides an allyl-cyanoethyl nucleoside dimer, a preparation method thereof, and an application thereof in oligonucleotide synthesis, belonging to the technical field of DNA synthesis. The allyl-cyanoethyl nucleoside dimer provided by the present invention adopts a mixed backbone of allyl phosphate and β-cyanoethyl phosphoramidite. The methyl protecting group of the middle phosphoric acid of the dimer structural unit is replaced by an allyl protecting group. When the allyl group is used as a protecting group, it can be removed under ammonia conditions or mild non-basic conditions. In addition, the allyl protecting group has high selectivity for some functional groups, and can achieve the protection of target groups without affecting the reactions of other groups. In the present invention, the methyl protecting group of the middle phosphoric acid of the dimer structural unit is replaced by an allyl protecting group. The obtained allyl-cyanoethyl nucleoside dimer can avoid unnecessary decomposition or side reactions during the deprotection process, reduce the complexity of the purification steps, shorten the deprotection time, and improve the deprotection efficiency and the purity of the product.

Description

Technical Field

[0001] The present invention relates to the technical field of DNA synthesis, and particularly relates to an allyl-cyanoethyl nucleoside dimer, a preparation method thereof, and an application thereof in oligonucleotide synthesis. Background Art

[0002] With the rapid development of synthetic biology, the demand for the synthesis of long fragments, high-fidelity, and low-cost oligonucleotides is increasing day by day. Due to the inherent limitations of chemical reactions, the length of commercially synthesized oligonucleotides is generally controlled below 100 nt. There are mainly two factors affecting oligonucleotide synthesis: First, each cycle in oligonucleotide synthesis cannot react completely, and the coupling efficiency is less than 100%. As the oligonucleotide sequence grows, the total yield continuously decreases; Second, side reactions such as depurination and cyanoethyl addition will further reduce the yield of oligonucleotides and lead to the generation of synthesis errors. Therefore, new chemical synthesis methods are needed to minimize side reactions and improve the yield in order to obtain high-quality long-fragment oligonucleotides.

[0003] Using dimer nucleoside modules to synthesize oligonucleotides reduces the synthesis steps and the number of acid deprotection times by half. For example, for a sequence that requires 100 syntheses with ordinary monomers, only 50 syntheses are needed using dimers, significantly reducing the reaction steps and the probability of errors. The research team of G. Kumar successfully synthesized short oligonucleotides (about 20 nt) through dimer phosphoramidite modules in early research. However, the reported dimer nucleosides have problems such as unstable structure, difficult purification, and complex synthesis, and cannot meet the requirements of oligonucleotide synthesis based on modular strategies.

[0004] Previously, our research team invented a methyl-cyanoethyl mixed backbone dimer, and its structure is shown in Formula II:

[0005] Formula II.

[0006] This dimer has a stable structure and is easy to prepare, obtaining a synthetic route with high purity, scalability, and easy operation, and synthesizing an oligonucleotide fragment with a length of 100 nt. However, since it is difficult to deprotect the methyl group by ammonolysis with ammonia, for the oligonucleotide sequence synthesized using this dimer structure, stronger deprotection conditions are required to achieve complete deprotection. It is necessary to soak and ammonolyze with ammonia water at 70 °C for 12 hours. The extension of the ammonolysis time increases the uncertainty, which may cause the cleavage of the oligonucleotide chain and the generation of side reactions. In addition, this structure cannot be used to synthesize base-labile compounds. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide an allyl-cyanoethyl nucleoside dimer, its preparation method and application in oligonucleotide synthesis. The allyl-cyanoethyl nucleoside dimer provided by the present invention can shorten the deprotection time, improve the deprotection efficiency and the purity of the product.

[0008] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0009] The present invention provides an allyl-cyanoethyl nucleoside dimer having the structure shown in Formula I:

[0010] Formula I;

[0011] In Formula I, X is O or S; B1 and B2 are independently selected from substituted or unsubstituted bases.

[0012] Preferably, B1 is selected from any one of cytosine, guanine, adenine, thymine, uracil, hypoxanthine, xanthine, deazaadenine, deazaguanine, deazahypoxanthine, dihydrouracil and pseudouracil;

[0013] B2 is selected from any one of cytosine, guanine, adenine, thymine, uracil, hypoxanthine, xanthine, deazaadenine, deazaguanine, deazahypoxanthine, dihydrouracil and pseudouracil.

[0014] Preferably, B1 and B2 are independently selected from the following groups, " " represents the group connection position:

[0015]

[0016] ;

[0017] Among them, R', R'' and R''' are each independently selected from any one of hydrogen, amino, C1-C3 straight-chain or branched-chain alkyl, C1-C3 straight-chain or branched-chain alkyl acyl, phenyl and phenyl acyl.

[0018] The present invention provides a preparation method of the above-mentioned allyl-cyanoethyl nucleoside dimer, including the following steps:

[0019] (1) The 5ʹ-DMTr-3ʹ-O-allyl phosphoramidite nucleoside with the structure shown in Formula 1 is coupled with the 5ʹ-OH-3ʹ-O-TBDMS nucleoside with the structure shown in Formula 2 to obtain a dimer with the structure shown in Formula 3;

[0020] Formula 1; Formula 2; Formula 3;

[0021] (2) When X is O, the dimer with the structure shown in Formula 3 is mixed with tert-butyl hydroperoxide for an oxidation reaction to obtain a diphosphate diester with the structure shown in Formula 4;

[0022] When X is S, the dimer with the structure shown in Formula 3 is mixed with S8, N,O -bis(trimethylsilyl)acetamide for a sulfidation reaction to obtain a diphosphate diester with the structure shown in Formula 4;

[0023] Formula 4;

[0024] (3) The diphosphate diester with the structure shown in Formula 4 undergoes a reaction to remove the TBDMS protecting group to obtain a 3ʹ-OH dimer precursor with the structure shown in Formula 5;

[0025] Formula 5;

[0026] (4) Under the action of 1 H -tetrazole diisopropylammonium salt catalyst, the 3ʹ-OH dimer precursor with the structure shown in Formula 5 reacts with 2-cyanoethyl N,N,Nʹ,Nʹ -tetraisopropylphosphorodiamidite for a phosphorylation reaction to obtain an allyl-cyanoethyl nucleoside dimer with the structure shown in Formula I.

[0027] Preferably, the coupling reaction is carried out in the presence of 1 H -tetrazole, and the molar ratio of the 5ʹ-DMTr-3ʹ-O-allyl phosphoramidite nucleoside with the structure shown in Formula 1 to the 5ʹ-OH-3ʹ-O-TBDMS nucleoside with the structure shown in Formula 2 is (1~2):1;

[0028] The molar ratio of the 5ʹ-DMTr-3ʹ-O-allyl phosphoramidite nucleoside with the structure shown in Formula 1 to 1 H -tetrazole is (1~2):(0.5~1);

[0029] The time of the coupling reaction is 8~14 h.

[0030] Preferably, the molar ratio of the dimer with the structure shown in Formula 3 to tert-butyl hydroperoxide is 1:(1~6);

[0031] The time of the oxidation reaction is 0.4 - 1 h;

[0032] The molar ratio of the dimer with the structure shown in Formula 3 to S8, N,O -bis(trimethylsilyl)acetamide is 1:(1 - 2):(3 - 6);

[0033] The temperature of the sulfidation reaction is 40 - 60 °C, and the time is 20 - 50 min.

[0034] Preferably, the molar ratio of the 3'-OH dimer precursor with the structure shown in Formula 5 to 2-cyanoethyl- N,N,Nʹ,Nʹ -tetraisopropylphosphorodiamidite is 1:1.4;

[0035] The molar ratio of the 3'-OH dimer precursor with the structure shown in Formula 5 to 1 H -tetrazole diisopropylammonium salt catalyst is 1:(1 - 3);

[0036] The time of the phosphorylation reaction is 1 - 3 h.

[0037] Preferably, after the phosphorylation reaction, post-treatment of the obtained phosphorylation reaction solution is further included, and the post-treatment includes the following steps:

[0038] Subject the phosphorylation reaction solution to flash column separation and purification to obtain a purified product;

[0039] Dissolve the purified product with an organic solvent, add the obtained solution to methyl tert-butyl ether, and dry the obtained solid to obtain a pure product of allyl-cyanoethyl nucleoside dimer with the structure shown in Formula I.

[0040] The present invention provides the application of the above-mentioned allyl-cyanoethyl nucleoside dimer in oligonucleotide synthesis.

[0041] The present invention provides a method for synthesizing oligonucleotides, including the following steps:

[0042] Mix the above-mentioned allyl-cyanoethyl nucleoside dimer with a coupling catalyst and carry out a coupling reaction to obtain a coupling reaction product;

[0043] Successively carry out de-DMTr protecting group, ammonolysis and desalting purification on the coupling reaction product to obtain oligonucleotides.

[0044] The present invention provides an allyl-cyanoethyl nucleoside dimer with the structure shown in Formula I. Compared with the methyl-cyanoethyl mixed backbone dimer shown in Formula II, the allyl-cyanoethyl nucleoside dimer provided by the present invention adopts a mixed backbone of allyl phosphate and β-cyanoethyl phosphoramidite, and the methyl protecting group of the middle phosphate in the dimer structural unit is replaced by an allyl protecting group. When the allyl group is used as a protecting group, it can be removed under ammonia conditions or mild non-basic conditions. In addition, the allyl protecting group has high selectivity for some functional groups, and can achieve the protection of target groups without affecting the reactions of other groups. By replacing the methyl protecting group of the middle phosphate in the dimer structural unit with an allyl protecting group, the obtained allyl-cyanoethyl nucleoside dimer can avoid unnecessary decomposition or side reactions during the deprotection process, reduce the complexity of the purification steps, shorten the deprotection time, improve the deprotection efficiency and the purity of the product. In addition, the use of allyl can synthesize base-labile modified nucleosides, such as acetylcytidine, which will further expand the application scope of dimer synthesis. The synthesis results show that the ammonolysis time of the oligonucleotide synthesized using the allyl-cyanoethyl dimer is significantly shortened compared with that of the methyl-cyanoethyl dimer. Only by ammonolysis at 95 °C under ammonia conditions for 2 h can all the protecting groups be removed, avoiding sequence errors caused by long-term ammonolysis.

[0045] In addition, the present invention provides a preparation method of the above-mentioned allyl-cyanoethyl nucleoside dimer. Based on the basic principle of solid-phase phosphoramidite oligonucleotide synthesis, the present invention has established a synthesis method of nucleoside module reagents that is simple to operate and can be scaled up, and obtained an allyl-cyanoethyl nucleoside dimer with high purity and a new structure. The present invention adopts a "one-pot" synthesis method, in which coupling-oxidation-deprotection is carried out in the same container, with simple operation, low cost, and easy to realize industrial batch production.

[0046] In addition, the present invention has optimized and established an oligonucleotide synthesis method based on modular synthesis, optimized the ammonolysis conditions, and can obtain oligonucleotide fragments with a length of 80 nt. When the allyl-cyanoethyl nucleoside dimer provided by the present invention is used for oligonucleotide synthesis, it can broaden the application scope of the dimer, simplify the deprotection process, is expected to further extend the dimer synthesis length, reduce the synthesis error rate, and has practical value. Description of the Drawings

[0047] Figure 1 is the synthesis route diagram of the allyl-cyanoethyl nucleoside dimer;

[0048] Figure 2 is the liquid chromatogram of Compound 5a;

[0049] Figure 3 is the mass spectrum of Compound 5a;

[0050] Figure 4Liquid chromatogram of compound 6a;

[0051] Figure 5 Mass spectrum of compound 6a;

[0052] Figure 6 Liquid chromatogram of compound 5b;

[0053] Figure 7 Mass spectrum of compound 5b;

[0054] Figure 8 Liquid chromatogram of compound 5c;

[0055] Figure 9 Mass spectrum of compound 5c;

[0056] Figure 10 Liquid chromatogram of compound 5d;

[0057] Figure 11 Mass spectrum of compound 5d;

[0058] Figure 12 Liquid chromatogram of compound 5e;

[0059] Figure 13 Mass spectrum of compound 5e;

[0060] Figure 14 Liquid chromatogram of compound 6e;

[0061] Figure 15 Mass spectrum of compound 6e;

[0062] Figure 16 Liquid chromatogram of compound 5f;

[0063] Figure 17 Mass spectrum of compound 5f;

[0064] Figure 18 Liquid chromatogram of compound 6f;

[0065] Figure 19 Mass spectrum of compound 6f;

[0066] Figure 20 Liquid chromatogram of compound 5g;

[0067] Figure 21 Mass spectrum of compound 5g;

[0068] Figure 22 Liquid chromatogram of compound 6g;

[0069] Figure 23 Mass spectrum of compound 6g;

[0070] Figure 24 Mass spectrometry diagram of the oligonucleotide ammonolysis product synthesized from methyl dimer and allyl-cyanoethyl nucleoside dimer;

[0071] Figure 25 Mass spectrometry detection diagram of the 80 nt sequence synthesized from allyl-cyanoethyl nucleoside dimer. Specific implementation mode

[0072] The present invention provides an allyl-cyanoethyl nucleoside dimer with the structure shown in Formula I:

[0073] Formula I;

[0074] In Formula I, X is O or S; B1 and B2 are independently selected from substituted or unsubstituted bases.

[0075] In the present invention, B1 is preferably selected from any one of cytosine, guanine, adenine, thymine, uracil, hypoxanthine, xanthine, deazaadenine, deazaguanine, deazahypoxanthine, dihydrouracil, and pseudouracil;

[0076] B2 is preferably selected from any one of cytosine, guanine, adenine, thymine, uracil, hypoxanthine, xanthine, deazaadenine, deazaguanine, deazahypoxanthine, dihydrouracil, and pseudouracil.

[0077] In the present invention, B1 and B2 are independently selected from the following groups, " " represents the group connection position:

[0078] ;

[0079] ;

[0080] Among them, R', R'', and R''' are each independently selected from any one of hydrogen, amino, C1-C3 straight-chain or branched-chain alkyl, C1-C3 straight-chain or branched-chain alkyl acyl, phenyl, and phenyl acyl.

[0081] Compared with the methyl-cyanoethyl mixed backbone dimer shown in Formula II, in the present invention, the methyl protecting group of the middle phosphoric acid of the dimer is replaced with an allyl protecting group that is more easily removed. The cyanoethyl protecting group at the 3'-OH end of the dimer is relatively easy to remove, and the raw materials for synthesizing cyanoethyl phosphoramidite are cheaper and more readily available. In the present invention, using allyl as the protecting group can be removed under ammonia conditions or mild non-basic conditions. In addition, the allyl protecting group has high selectivity for some functional groups, and can achieve the protection of the target group without affecting the reactions of other groups. By replacing the methyl protecting group of the middle phosphoric acid in the dimer structural unit with allyl protection, the obtained allyl-cyanoethyl nucleoside dimer can avoid unnecessary decomposition or side reactions during the deprotection process, and can reduce the complexity of the purification steps, shorten the deprotection time, improve the deprotection efficiency and the purity of the product. In addition, the use of allyl can synthesize base-labile modified nucleosides, such as acetylcytidine, which will further expand the application scope of dimer synthesis.

[0082] In the present invention, the allyl-cyanoethyl nucleoside dimer preferably has the following structure:

[0083] 。

[0084] The present invention provides a preparation method of the above-mentioned allyl-cyanoethyl nucleoside dimer, comprising the following steps:

[0085] (1) Coupling reaction of 5ʹ-DMTr-3ʹ-O-allyl phosphoramidite nucleoside having the structure shown in Formula 1 and 5ʹ-OH-3ʹ-O-TBDMS nucleoside having the structure shown in Formula 2 to obtain a dimer having the structure shown in Formula 3;

[0086] Formula 1; Formula 2; Formula 3;

[0087] When X is O, the dimer having the structure shown in Formula 3 is mixed with tert-butyl hydroperoxide for an oxidation reaction to obtain a diphosphate diester having the structure shown in Formula 4;

[0088] When X is S, the dimer having the structure shown in Formula 3 is mixed with S8, N,O -bis(trimethylsilyl)acetamide for a sulfidation reaction to obtain a diphosphate diester having the structure shown in Formula 4;

[0089] Formula 4;

[0090] (3) The diphosphate diester having the structure shown in Formula 4 is subjected to a reaction for removing the TBDMS protecting group to obtain a 3ʹ-OH dimer precursor having the structure shown in Formula 5;

[0091] Formula 5;

[0092] (4) In the presence of 1 H -tetrazole diisopropylammonium salt catalyst, the 3'-OH dimer precursor having the structure shown in Formula 5 reacts with 2-cyanoethyl- N,N,Nʹ,Nʹ -tetraisopropylphosphorodiamidite to carry out a phosphorylation reaction to obtain an allyl-cyanoethyl nucleoside dimer having the structure shown in Formula I.

[0093] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0094] In the present invention, the 5'-DMTr-3'-O-allylphosphoramidite nucleoside having the structure shown in Formula 1 is mixed with the 5'-OH-3'-O-TBDMS nucleoside having the structure shown in Formula 2, and a coupling reaction is carried out to obtain a dimer having the structure shown in Formula 3. In the present invention, the coupling reaction is preferably carried out in the presence of 1 H -tetrazole, and the molar ratio of the 5'-DMTr-3'-O-allylphosphoramidite nucleoside having the structure shown in Formula 1 to 1 H -tetrazole is preferably (1~2):(0.5~1), more preferably 1.05:0.8.

[0095] In the present invention, the molar ratio of the 5'-DMTr-3'-O-allylphosphoramidite nucleoside having the structure shown in Formula 1 to the 5'-OH-3'-O-TBDMS nucleoside having the structure shown in Formula 2 is preferably (1~2):1, more preferably 1.05:1; in the present invention, the coupling reaction is preferably carried out in an organic solvent, and the organic solvent used in the coupling reaction is preferably dichloromethane.

[0096] In the present invention, the coupling reaction is preferably carried out under N2 protection, the temperature of the coupling reaction is preferably room temperature, and the time is preferably 8~14 h, more preferably 10~13 h. After the coupling reaction, no post-treatment is carried out in the present invention, and the obtained coupling reaction solution is directly used for the subsequent reaction.

[0097] After obtaining the dimer having the structure shown in Formula 3, when X is O, the present invention mixes the dimer having the structure shown in Formula 3 with tert-butyl hydroperoxide to carry out an oxidation reaction to obtain a diphosphate diester having the structure shown in Formula 4. In the present invention, tert-butyl hydroperoxide has a fast oxidation rate and will not affect the subsequent reaction, replacing the iodine oxidation method for oxidation under the standard conditions of phosphoramidite chemistry in a THF / lutidine / water solution, and avoiding the trouble caused by drying the excessive iodine aqueous solution.

[0098] In the present invention, the molar ratio of the dimer having the structure shown in Formula 3 to tert-butyl hydroperoxide is preferably 1:(1 - 6), more preferably 1:2.

[0099] In the present invention, the oxidation reaction temperature is preferably room temperature, and the time is preferably 0.4 - 1 h, more preferably 0.5 - 0.8 h. After the oxidation reaction, no post-treatment is carried out in the present invention, and the obtained oxidation reaction solution is directly used for the subsequent reaction.

[0100] Alternatively, when X is S, the dimer having the structure shown in Formula 3 in the present invention is mixed with S8, N,O -bis(trimethylsilyl)acetamide and subjected to a sulfidation reaction to obtain a diphosphoric acid diester having the structure shown in Formula 4.

[0101] In the present invention, the molar ratio of the dimer having the structure shown in Formula 3 to S8, N,O -bis(trimethylsilyl)acetamide is 1:(1 - 2):(4 - 6), more preferably 1:1.5:5; the temperature of the sulfidation reaction is preferably 40 - 60 °C, more preferably 50 °C, and the time is preferably 20 - 50 min, more preferably 30 min. After the sulfidation reaction, the present invention concentrates under vacuum to obtain a sulfidation product, and the obtained sulfidation product is directly used for the subsequent reaction.

[0102] After obtaining the diphosphoric acid diester having the structure shown in Formula 4, the present invention subjects the diphosphoric acid diester having the structure shown in Formula 4 to a reaction for removing the TBDMS protecting group to obtain a 3ʹ-OH dimer precursor having the structure shown in Formula 5. In the present invention, the deprotecting reagent used in the reaction for removing the TBDMS protecting group preferably includes Et3N and Et3N·3HF. In the present invention, the molar ratio of the diphosphoric acid diester having the structure shown in Formula 4 to Et3N is preferably 1:(1 - 3), more preferably 1:2; the molar ratio of the diphosphoric acid diester having the structure shown in Formula 4 to Et3N·3HF is preferably 1:(2 - 4), more preferably 1:3.

[0103] In the present invention, the temperature of the reaction for removing the TBDMS protecting group is preferably room temperature, and the time is preferably 6 - 14 h, more preferably 12 h.

[0104] After the reaction for removing the TBDMS protecting group, the present invention preferably performs post-treatment on the obtained reaction solution for removing the TBDMS protecting group, and the post-treatment preferably includes the following steps:

[0105] Mix the reaction solution for removing the TBDMS protecting group with a saturated NaHCO3 solution until no bubbles are generated, extract the obtained mixture twice with dichloromethane and water, combine the organic phases, wash, dry, filter, and concentrate the obtained organic phase to obtain a crude product of the 3ʹ-OH dimer precursor;

[0106] The crude 3ʹ-OH dimer precursor is purified by column chromatography, the solvent is rotary concentrated and evaporated to dryness, the obtained residue is dissolved in anhydrous dichloromethane, dropped into methyl tert-butyl ether, filtered and dried to obtain the pure 3ʹ-OH dimer precursor with the structure shown in Formula 5.

[0107] In the present invention, the mobile phase used for the column chromatography purification is preferably DCM / MeOH (0-10%). In the present invention, the moving speed of the 3ʹ-OH dimer precursor on the silica gel column is significantly slower than that of the residual 3ʹ-O-methyl phosphoramidite derivative, and it is easy to separate and obtain a high-purity compound.

[0108] After obtaining the 3ʹ-OH dimer precursor with the structure shown in Formula 5, in the present invention, under the action of 1H-tetrazole diisopropylammonium salt catalyst, the 3ʹ-OH dimer precursor with the structure shown in Formula 5 reacts with 2-cyanoethyl- H -tetraisopropylphosphorodiamide to carry out a phosphorylation reaction to obtain the allyl-cyanoethyl nucleoside dimer with the structure shown in Formula I. In the present invention, 1H-tetrazole diisopropylammonium salt catalyst is used as the catalyst, and 2-cyanoethyl- N,N,Nʹ,Nʹ -tetraisopropylphosphorodiamide is used as the phosphorylation reagent. Since the acidity of 1H-tetrazole is similar to that of acetic acid, 1H-tetrazole diisopropylammonium salt is used as the catalyst to avoid the possible side reaction of DMTr deprotection during the long coupling time. H -tetrazole diisopropylammonium salt catalyst, and 2-cyanoethyl- N,N,Nʹ,Nʹ -tetraisopropylphosphorodiamide as the phosphorylation reagent. Since the acidity of 1H-tetrazole is similar to that of acetic acid, 1H-tetrazole diisopropylammonium salt is used as the catalyst to avoid the possible side reaction of DMTr deprotection during the long coupling time. H -tetrazole is similar to acetic acid in acidity, so 1H-tetrazole diisopropylammonium salt is used as the catalyst to avoid the possible side reaction of DMTr deprotection during the long coupling time. H -tetrazole diisopropylammonium salt as the catalyst to avoid the possible side reaction of DMTr deprotection during the long coupling time.

[0109] In the present invention, the molar ratio of the 3ʹ-OH dimer precursor with the structure shown in Formula 5 to 2-cyanoethyl- N,N,Nʹ,Nʹ -tetraisopropylphosphorodiamide is preferably 1:1.4. In the present invention, the molar ratio of the 3ʹ-OH dimer precursor with the structure shown in Formula 5 to 1H-tetrazole diisopropylammonium salt catalyst is preferably 1:(1-3), more preferably 1:1.4. H -tetrazole diisopropylammonium salt catalyst is preferably 1:(1-3), more preferably 1:1.4.

[0110] In the present invention, the phosphorylation reaction is preferably carried out in an organic solvent, and the organic solvent is preferably dichloromethane.

[0111] In the present invention, the phosphorylation reaction is preferably carried out under N2 protection, the temperature of the phosphorylation reaction is preferably room temperature, and the time is preferably 1-3 h, more preferably 2 h.

[0112] After the phosphorylation reaction, the present invention also preferably includes post-treatment of the obtained phosphorylation reaction solution, and the post-treatment includes the following steps:

[0113] The phosphorylation reaction solution is subjected to flash column separation purification to obtain a purified product;

[0114] Dissolve the purified product in an organic solvent, add the resulting solution to methyl tert-butyl ether, and dry the resulting solid to obtain a pure product of allyl-cyanoethyl nucleoside dimer having the structure shown in Formula I.

[0115] In the present invention, the mobile phase for flash column separation and purification is preferably a dichloromethane-methanol system, and the dichloromethane preferably contains 0.4 vol% of triethylamine. In the present invention, in the dichloromethane-methanol system, the volume content of methanol is 0 to 10%. In the present invention, using dichloromethane (0.4 vol% triethylamine)-methanol in the purification system can better separate the product and maintain the stability of the product.

[0116] In the present invention, the organic solvent for dissolving the purified product is preferably anhydrous dichloromethane. In the present invention, the temperature for adding the solution to methyl tert-butyl ether is preferably -78 °C, and the addition method is preferably dropwise addition. In the present invention, the volume ratio of methyl tert-butyl ether to the resulting solution is preferably 10:1.

[0117] In the present invention, the solid is preferably obtained by centrifugation, and the drying method is preferably vacuum drying. In the present invention, methyl tert-butyl ether has a lower solubility for phosphoramidite dimer and a higher solubility for impurities, better meeting the requirements for separation and purification of the dimer.

[0118] As a specific embodiment of the present invention, the synthesis route of the allyl-cyanoethyl nucleoside dimer is as Figure 1 shown. Figure 1 In, the reaction conditions and reagents are as follows: (a) 1 H -tetrazole, DCM, 8 - 14 h; (b) t-BuOOH, DCM, 0.4 - 1 h; (c) Et3N·3HF, Et3N, DCM, 12 h; (d) 2-cyanoethyl -N,N,N',N' -tetraisopropylphosphorodiamidite, 1 H -tetrazole diisopropylammonium salt, DCM, 1 - 3 h.

[0119] The preparation method of the allyl-cyanoethyl nucleoside dimer provided by the present invention realizes the preparation of a nucleoside dimer with high purity, and the HPLC purity can reach more than 95%, which can meet the requirements for high-fidelity DNA synthesis.

[0120] The present invention provides the application of allyl-cyanoethyl nucleoside dimer in oligonucleotide synthesis.

[0121] The present invention provides a method for synthesizing oligonucleotides, comprising the following steps:

[0122] Mix the above allyl-cyanoethyl nucleoside dimer with a coupling catalyst and conduct a coupling reaction to obtain a coupling reaction product;

[0123] Successively perform de-DMTr protecting group, ammonolysis, and desalting purification on the coupling reaction product to obtain an oligonucleotide.

[0124] Preferably, in the present invention, the coupling reaction is carried out on an LK-192 DNA synthesizer using a 50 nmol synthesis column. In the present invention, the concentration of the propyl-cyanoethyl nucleoside dimer is preferably 30 - 60 mg / mL, more preferably 50 mg / mL; in the present invention, the coupling catalyst is preferably 4,5-dicyanoimidazole. In the present invention, the temperature of the coupling reaction is preferably room temperature, and the time is preferably 80 - 150 s, more preferably 100 - 120 s.

[0125] In the present invention, the deprotecting reagent used for de-DMTr protecting group is preferably a DBLOCK reagent, and the time for de-DMTr protecting group is preferably 40 - 70 s, more preferably 60 s. After the de-DMTr protecting group reaction, preferably, the de-DMTr protecting group reaction is carried out again under the above conditions. After the de-DMTr protecting group, preferably, the obtained de-DMTr protecting group product is washed with acetonitrile.

[0126] In the present invention, the ammonolysis is preferably ammonia ammonolysis. In the present invention, before the ammonolysis, preferably, the obtained de-DMTr protecting group product is pretreated, and the pretreatment preferably includes:

[0127] Washing and drying the de-DMTr protecting group product.

[0128] In the present invention, the reagent used for washing is preferably acetonitrile with a concentration of 70 vol%; the drying is preferably drying by suction.

[0129] In the present invention, the ammonolysis is preferably ammonia ammonolysis or 2-mercaptoethanol ammonolysis. When using ammonia ammonolysis, the ammonolysis is preferably carried out in an ammonolysis rack, and the conditions for the ammonolysis preferably include:

[0130] The ammonolysis temperature is preferably 90 - 95 °C, more preferably 92 - 94 °C;

[0131] The ammonia pressure is preferably 470 - 520 kPa, more preferably 500 kPa;

[0132] The ammonolysis time is preferably 1 - 3 h, more preferably 2 h.

[0133] When ammonolysis is carried out using 2-mercaptoethanol, the reagent for ammonolysis is preferably an aqueous solution of concentrated ammonium hydroxide containing 2-mercaptoethanol. In the present invention, in the aqueous solution of concentrated ammonium hydroxide containing 2-mercaptoethanol, the volume fraction of 2-mercaptoethanol is preferably 1 to 5%, more preferably 2 to 4%; the concentration of ammonium hydroxide is preferably 20 to 40%, more preferably 30%.

[0134] The conditions for ammonolysis include:

[0135] The ammonolysis temperature is 50 to 60 °C, more preferably 55 °C;

[0136] The ammonolysis time is 12 to 18 h, more preferably 14 to 16 h.

[0137] In the present invention, the method for desalting and purification preferably includes the following steps:

[0138] Mix the product after ammonolysis with an acetonitrile solution and perform the first centrifugation; then add pure water and perform the second centrifugation.

[0139] In the present invention, the concentration of the acetonitrile solution is preferably 90%; the rate of the first centrifugation is preferably 500 r / min and the time is preferably 2 min.

[0140] In the present invention, the volume ratio of the acetonitrile solution to pure water is preferably 1:1; the second centrifugation preferably includes centrifuging at 500 r / min for 2 min first and then at 2000 r / min for 2 min.

[0141] The allyl-cyanoethyl nucleoside dimer provided by the present invention, its preparation method and its application in oligonucleotide synthesis will be described in detail below with reference to examples, but they should not be construed as limiting the scope of protection of the present invention.

[0142] In the following examples, 1a~1d were purchased from Beijing Ribaoao Biotechnology Co., Ltd., and 2a~2d were purchased from Wuhu Huaren Technology Co., Ltd.

[0143] Example 1

[0144]

[0145] Reaction conditions and reagents for the synthesis of dimer AC-Amidite: (a) 1 H -tetrazole, DCM, 13 h; (b) t -BuOOH, DCM, 0.5 h; (c) Et3N·3HF, Et3N, DCM, 12 h; (d) 2-cyanoethyl- N,N,N',N' -tetraisopropylphosphorodiamidite, 1 H -tetrazole diisopropylammonium salt, DCM, 2 h.

[0146] N 6 -benzoyl- P -allyl-5'- O -dimethoxytrityl-2'-deoxyadenosine-(3'→5')- N 4 Synthesis of -benzoyl-2'-deoxycytidine (5a):

[0147] Dissolve 1a (10.00 g, 1.05 eq) and 2a (5.02 g, 1.00 eq) in dichloromethane and acetonitrile, spin-dry to remove water, then dissolve in anhydrous dichloromethane, add 1 H -tetrazole (0.63 g, 0.80 eq), stir at room temperature for 13 h under N2 protection to obtain compound 3a. Directly add anhydrous t-BuOOH (2.03 g, 2.00 eq) to the mixture and stir at room temperature for 0.5 h to obtain 4a. Add Et3N (2.28 g, 2.00 eq), Et3N·3HF (5.45 g, 3.00 eq), and stir at room temperature for 12 h. After the reaction is complete, add saturated NaHCO3 until no bubbles are generated, extract the mixture twice with dichloromethane and water, combine the organic layers, wash with saturated brine, dry over anhydrous MgSO4, filter, concentrate in vacuo to obtain the crude compound 5a. Purify by column chromatography (DCM / MeOH(0 - 10%)), then rotary evaporate to dryness. Dissolve the residue in anhydrous dichloromethane, dropwise add it to methyl tert-butyl ether, filter, and dry in vacuo to obtain the dimer precursor compound 5a, 8.89 g of white solid, purity 99.2%, yield 72.3%. ESI-MS: m / z calcd for C 57 H 55 N8O 13 P [M+H] + 1091.36, found: 1091.19.

[0148] The liquid chromatogram of compound 5a is as Figure 2 shown, and the mass spectrum is as Figure 3 shown.

[0149] N 6 -benzoyl- P -allyl-5'- O -dimethoxytrityl-2'-deoxyadenosine-(3'→5')- N 4 -benzoyl-3'- O Synthesis of -benzoyl-3'-[(N,N-diisopropylamino)-cyanoethylphosphino]-2'-deoxycytidine (6a):

[0150] Compound 5a (4.00 g, 1.00 eq) was dissolved in dichloromethane and toluene, and the solvent was evaporated under reduced pressure to remove water. Then it was dissolved in 20 mL of anhydrous dichloromethane, and 2-cyanoethyl- N,N,N',N' -tetraisopropylphosphorodiamidite (1.55 g, 1.40 eq) and 1 H -tetrazolium diisopropylammonium salt (0.88 g, 1.40 eq) were added, and the reaction was carried out under N2 protection for 2 h. After the reaction was complete, it was purified by column chromatography with the mobile phase of DCM(1%Et3N v / v)-MeOH (0-10%), collected and evaporated to dryness. Then it was dissolved in 5 mL of anhydrous dichloromethane, and at -78 °C, it was slowly dropped into 45 mL of methyl tert-butyl ether. After centrifugation, the supernatant was discarded, and the solid in the lower layer was dried in a vacuum oven to obtain compound 6a, 3.84 g of white powdery solid, with a purity of 95.71% and a yield of 81.11%. ESI-MS: m / z calcd for C 66 H 72 N 10 O 14 P2[M+H] + 1291.47, found: 1291.48.

[0151] The liquid chromatogram of compound 6a is as Figure 4 shown, and the mass spectrum is as Figure 5 shown.

[0152] Example 2

[0153]

[0154] Reaction conditions and reagents for the synthesis of dimer AT-Amidite: (a) 1 H -tetrazole, DCM, 12 h; (b) t -BuOOH, DCM, 0.5 h; (c) Et3N·3HF, Et3N, DCM, 14 h; (d) 2-cyanoethyl- N,N,N',N' -tetraisopropylphosphorodiamidite, 1 H -tetrazolium diisopropylammonium salt, DCM, 2 h.

[0155] N 6 -benzoyl- P -allyl 5'- O -dimethoxytrityl-2'-deoxyadenosine-(3'→5')-thymidine (5b) synthesis:

[0156] Dissolve 1a (10.00 g, 1.05 eq) and 2b (4.02 g, 1.00 eq) in dichloromethane and acetonitrile, spin-dry to remove water, then dissolve in anhydrous dichloromethane, add 1 H -tetrazole (0.63 g, 0.80 eq), stir at room temperature for 12 h under N2 protection to obtain compound 3b. Directly add anhydrous t-BuOOH (2.03 g, 2.00 eq) to the mixture and stir at room temperature for 0.5 h to obtain 4b. Add Et3N (2.28 g, 2.00 eq), Et3N·3HF (5.45 g, 3.00 eq), and stir at room temperature for 14 h. After the reaction is complete, add saturated NaHCO3 until no bubbles are generated. Extract the mixture twice with dichloromethane and water, combine the organic layers, wash with saturated brine, dry over anhydrous MgSO4, filter, concentrate in vacuo to obtain crude compound 5b. Purify by column chromatography (DCM / MeOH(0 - 10%)), then rotary evaporate to dryness. Dissolve the residue in anhydrous dichloromethane, dropwise add to methyl tert-butyl ether, filter, and dry in vacuo to obtain the dimer precursor compound 5b, 8.65 g of white solid, purity 99.2%, yield 76.6%. ESI-MS: m / z calcd for C 51 H 52 N7O 13 P [M+H] + 1002.34, found: 1002.18.

[0157] The liquid chromatogram of compound 5b is as shown in Figure 6 and the mass spectrum is as shown in Figure 7 .

[0158] Example 3

[0159]

[0160] Reaction conditions and reagents for the synthesis of dimer CA-Amidite: (a) 1 H -tetrazole, DCM, 12 h; (b) t -BuOOH, DCM, 0.5 h; (c) Et3N·3HF, Et3N, DCM, 12 h; (d) 2-cyanoethyl- N,N,N',N' -tetraisopropylphosphorodiamidite, 1 H -tetrazole diisopropylammonium salt, DCM, 2 h.

[0161] N 4 -benzoyl- P -allyl-5'- O-Dimethoxytrityl-2'-deoxycytidine-(3'→5')- N 6 Synthesis of -Benzoyl-2'-deoxyadenosine (5c):

[0162] Dissolve 1b (10.00 g, 1.05 eq) and 2c (5.45 g, 1.00 eq) in dichloromethane and acetonitrile, spin-dry to remove water, then dissolve in anhydrous dichloromethane, add 1 H -tetrazole (0.65 g, 0.80 eq), stir at room temperature for 12 h under N2 protection to obtain compound 3c. Directly add anhydrous t-BuOOH (1.57 g, 1.50 eq) to the mixture and stir at room temperature for 0.5 h to obtain 4c. Add Et3N (2.35 g, 2.00 eq), Et3N·3HF (5.61 g, 3.00 eq), and stir at room temperature for 12 h. After the reaction is complete, add saturated NaHCO3 until no bubbles are generated, extract the mixture twice with dichloromethane and water, combine the organic layers, wash with saturated brine, dry over anhydrous MgSO4, filter, and concentrate in vacuo to obtain the crude compound 5c. Purify by column chromatography (DCM / MeOH(0-10%)), then rotary evaporate to dry the solvent. The residue is dissolved in anhydrous dichloromethane, dropped into methyl tert-butyl ether, filtered, and dried in vacuo to obtain the dimer precursor compound 5c, 10.22 g of white solid, purity 99.2%, yield 80.74%. ESI-MS: m / z calcd for C 57 H 55 N8O 13 P [M+H] + 1091.36, found: 1091.20.

[0163] The HPLC chromatogram of compound 5c is as Figure 8 shown, and the mass spectrum is as Figure 9 shown.

[0164] Example 4

[0165]

[0166] Reaction conditions and reagents for the synthesis of dimer CT-Amidite: (a) 1 H -tetrazole, DCM, 11 h; (b) t -BuOOH, DCM, 0.5 h; (c) Et3N·3HF, Et3N, DCM, 13 h; (d) 2-cyanoethyl- N,N,N',N' -tetraisopropylphosphorodiamidite, 1 H -tetrazole diisopropylammonium salt, DCM, 2 h.

[0167] N 4 -Benzoyl- P -Allyl-5'- O Synthesis of -Dimethoxytrityl-2'-deoxycytidine-(3'→5')-thymidine (5d):

[0168] Dissolve 1b (20.00 g, 1.05 eq) and 2d (8.27 g, 1.00 eq) in dichloromethane and acetonitrile, spin-dry to remove water, then dissolve in anhydrous dichloromethane, add 1 H -tetrazole (1.30 g, 0.80 eq), stir at room temperature for 11 h under N2 protection to obtain compound 3d. Directly add anhydrous t-BuOOH (2.09 g, 1.00 eq) to the mixture and stir at room temperature for 0.5 h to obtain 4d. Add Et3N (4.70 g, 2.00 eq), Et3N·3HF (11.23 g, 3.00 eq), and stir at room temperature for 13 h. After the reaction is complete, add saturated NaHCO3 until no bubbles are generated, extract the mixture twice with dichloromethane and water, combine the organic layers, wash with saturated brine, dry over anhydrous MgSO4, filter, concentrate in vacuo to obtain the crude compound 5d. Purify by column chromatography (DCM / MeOH(0-10%)), then rotary evaporate to dryness. Dissolve the residue in anhydrous dichloromethane, dropwise add it to methyl tert-butyl ether, filter, and dry in vacuo to obtain the dimer precursor compound 5d, 15.56 g of white solid, purity 99.2%, yield 68.57%. ESI-MS: m / z calcd for C 50 H 52 N5O 14 P [M-H] - 976.32, found: 976.10.

[0169] The liquid chromatogram of compound 5d is as Figure 10 shown, and the mass spectrum is as Figure 11 shown.

[0170] Example 5

[0171]

[0172] Reaction conditions and reagents for the synthesis of dimer GC-Amidite: (a) 1 H -tetrazole, DCM, 12 h; (b) t -BuOOH, DCM, 0.5 h; (c) Et3N·3HF, Et3N, DCM, 15 h; (d) 2-cyanoethyl- N,N,N',N' -tetraisopropylphosphorodiamidite, 1H -tetrazolium diisopropylammonium salt, DCM, 2 h.

[0173] N 2 -isobutyryl- P -allyl-5'- O -dimethoxytrityl-2'-deoxyguanosine-(3'→5')- N 4 Synthesis of -benzoyl-2'-deoxycytidine (5e):

[0174] Dissolve 1c (20.00 g, 1.05 eq) and 2b (10.26 g, 1.00 eq) in dichloromethane and acetonitrile, spin-dry to remove water, then dissolve in anhydrous dichloromethane, add 1 H -tetrazole (1.29 g, 0.80 eq), stir at room temperature for 12 h under N2 protection to obtain compound 3e. Directly add anhydrous t-BuOOH (3.12 g, 1.50 eq) to the mixture and stir at room temperature for 0.5 h to obtain 4e. Add Et3N (4.66 g, 2.00 eq), Et3N·3HF (11.15 g, 3.00 eq), and stir at room temperature for 15 h. After the reaction is complete, add saturated NaHCO3 until no bubbles are generated, extract the mixture twice with dichloromethane and water, combine the organic layers, wash with saturated brine, dry over anhydrous MgSO4, filter, and concentrate in vacuo to obtain the crude compound 5e. Purify by column chromatography (DCM / MeOH(0-10%)), then rotary evaporate to dryness. Dissolve the residue in anhydrous dichloromethane, dropwise add to methyl tert-butyl ether, filter, and dry in vacuo to obtain the dimer precursor compound 5e, 16.56 g of white solid, purity 99.2%, yield 67.0%. ESI-MS: m / z calcd for C 54 H 57 N8O 14 P [M-H] - 1071.37, found: 1071.00.

[0175] The liquid chromatogram of compound 5e is as Figure 12 shown, and the mass spectrum is as Figure 13 shown.

[0176] N 2 -isobutyryl- P -allyl-5'- O -dimethoxytrityl-2'-deoxyguanosine-(3'→5')-3'- O -[(N,N-diisopropylamino)-cyanoethylphosphino]- N4 Synthesis of -benzoyl-2'-deoxycytidine (6e):

[0177] Dissolve compound 5e (2.40 g, 1.00 eq) in dichloromethane and toluene, and evaporate to dryness under reduced pressure to remove water. Then dissolve it in 15 mL of anhydrous dichloromethane, add cyanoethyl phosphoramidite (1.01 g, 1.50 eq), and H 1 64 H 74 N 10 O 15 P2[M+H] + 1272.48, found: 1273.50.

[0178] The liquid chromatogram of compound 6e is as Figure 14 shown, and the mass spectrum is as Figure 15 shown.

[0179] Example 6

[0180]

[0181] Reaction conditions and reagents for the synthesis of dimer GT-Amidite: (a) 1 H -tetrazole, DCM, 12 h; (b) t -BuOOH, DCM, 0.5 h; (c) Et3N·3HF, Et3N, DCM, 12 h; (d) 2-cyanoethyl- N,N,N',N' -tetraisopropylphosphorodiamidite, 1 H -tetrazolium diisopropylammonium salt, DCM, 2 h.

[0182] N 2 -isobutyryl- P -allyl-5'- O -dimethoxytrityl-2'-deoxyguanosine-(3'→5')-thymidine (5f) synthesis:

[0183] Dissolve 1c (15.00 g, 1.05 eq) and 2d (6.16 g, 1.00 eq) in dichloromethane and acetonitrile, spin-dry to remove water, then dissolve in anhydrous dichloromethane, add 1 H -tetrazole (0.97 g, 0.80 eq), stir at room temperature for 12 h under N2 protection to obtain compound 3f. Directly add anhydrous t-BuOOH (1.56 g, 1.00 eq) to the mixture and stir at room temperature for 0.5 h to obtain 4f. Add Et3N (3.50 g, 2.00 eq), Et3N·3HF (8.36 g, 3.00 eq), and stir at room temperature for 12 h. After the reaction is complete, add saturated NaHCO3 until no bubbles are generated. Extract the mixture twice with dichloromethane and water, combine the organic layers, wash with saturated brine, dry over anhydrous MgSO4, filter, and concentrate in vacuo to obtain the crude compound 5f. Purify by column chromatography (DCM / MeOH(0 - 10%)), then rotary evaporate to dryness. Dissolve the residue in anhydrous dichloromethane, dropwise add it to methyl tert-butyl ether, filter, and dry in vacuo to obtain the dimer precursor compound 5f, 12.67 g of white solid, purity 99.2%, yield 74.53%. ESI-MS: m / z calcd for C 48 H 54 N7O 14 P [M+Et3N] + 1084.54, found: 1085.20.

[0184] The liquid chromatogram of compound 5f is as shown in Figure 16 and the mass spectrum is as shown in Figure 17 .

[0185] N 2 -isobutyryl- P -allyl-5'- O -dimethoxytrityl-2'-deoxyguanosine-(3'→5')-3'- O -[(N,N-diisopropylamino)-cyanoethylphosphino]-thymidine (6f) synthesis:

[0186] Dissolve compound 5f (4.00 g, 1.00 eq) in dichloromethane and toluene, evaporate under reduced pressure to remove water. Then dissolve it in 20 mL of anhydrous dichloromethane, add cyanoethylphosphoramidite (1.59 g, 1.00 eq), and 1 H-tetrazole diisopropylammonium salt (0.91 g, 1.00 eq), reacted for 2 h under N2 protection. After the reaction was complete, it was purified by column chromatography, and the mobile phase was DCM(1%Et3N v / v)-MeOH (0-10%), collected and concentrated by rotary evaporation. Then it was dissolved in 5 mL of anhydrous dichloromethane, and was slowly added dropwise to 45 mL of methyl tert-butyl ether at -78 °C. After centrifugation, the supernatant was discarded, and the solid in the lower layer was dried in a vacuum oven to obtain compound 6f, 3.53 g of white powdery solid, with a purity of 99.43% and a yield of 73.33%. ESI-MS: m / z calcd for C 51 H 71 N9O 15 P2[M-H] - 1182.45, found: 1182.22.

[0187] The liquid chromatogram of compound 6f is as shown in Figure 18 shown, and the mass spectrum is as shown in Figure 19 shown.

[0188] Example 7

[0189]

[0190] Reaction conditions and reagents for the synthesis of dimer TA-Amidite: (a) 1 H -tetrazole, DCM, 12 h; (b) t -BuOOH, DCM, 0.5 h; (c) Et3N·3HF, Et3N, DCM, 12 h; (d) 2-cyanoethyl- N,N,N',N' -tetraisopropylphosphorodiamidite, 1 H -tetrazole diisopropylammonium salt, DCM, 2 h.

[0191] P -allyl-5'- O -dimethoxytrityl-thymidine-(3'→5')- N 6 -benzoyl-2'-deoxyadenosine (5 g) synthesis:

[0192] 1d (20.00 g, 1.05 eq) and 2a (12.22 g, 1.00 eq) were dissolved in dichloromethane and acetonitrile and concentrated by rotary evaporation to remove water, then dissolved in anhydrous dichloromethane, and 1 H-Tetrazole (1.46 g, 0.80 eq) was stirred at room temperature for 12 h under N2 protection to obtain compound 3g. Anhydrous t-BuOOH (2.35 g, 1.00 eq) was directly added to the mixture and stirred at room temperature for 0.5 h to obtain 4g. Et3N (5.27 g, 2.00 eq) and Et3N·3HF (12.60 g, 3.00 eq) were added and stirred at room temperature for 12 h. After the reaction was complete, saturated NaHCO3 was added until no bubbles were generated. The mixture was extracted twice with dichloromethane and water. The organic layers were combined, washed with saturated brine, dried over anhydrous MgSO4, filtered, concentrated in vacuo to obtain the crude compound 5g. Purification by column chromatography (DCM / MeOH(0-10%)) was followed by rotary evaporation to remove the solvent. The residue was dissolved in anhydrous dichloromethane and added dropwise to methyl tert-butyl ether, filtered, and dried in vacuo to obtain the dimer precursor compound 5g, 17.79 g of white solid, purity 99.2%, yield 68.21%. ESI-MS: m / z calcd for C 51 H 52 N7O 13 P [M+H] + 1002.34, found: 1002.00.

[0193] The liquid chromatogram of compound 5g is as shown in Figure 20 and the mass spectrum is as shown in Figure 21 as follows.

[0194] P -allyl-5'- O -dimethoxytrityl-thymidine-(3'→5')- N 6 -benzoyl-3'- O -[(N,N-diisopropylamino)-cyanoethylphosphino]-2'-deoxyadenosine (6g) synthesis:

[0195] Compound 5g (4.00 g, 1.00 eq) was dissolved in dichloromethane and toluene and evaporated under reduced pressure to remove water. Then it was dissolved in 20 mL of anhydrous dichloromethane, and cyanoethylphosphoramidite (1.44 g, 1.00 eq) and 1 H- Tetrazole diisopropylammonium salt (0.82 g, 1.00 eq), react for 2 h under N2 protection. After the reaction is complete, purify by column chromatography, the mobile phase is DCM(1%Et3N v / v)-MeOH (0 - 10%), collect and rotary evaporate to dryness. Then dissolve in 5 mL of anhydrous dichloromethane, and dropwise add it into 45 mL of methyl tert-butyl ether at -78 °C. Centrifuge and discard the supernatant, and dry the solid in the lower layer in a vacuum oven to obtain 6 g of the compound, 3.76 g of white powdery solid, with a purity of 99.36% and a yield of 78.34%. ESI-MS: m / z calcd for C 69 H 69 N9O 14 P2[M+H] + 1202.44, found: 1202.07.

[0196] The liquid chromatogram of compound 6g is as shown in Figure 22 shown, and the mass spectrum is as shown in Figure 23 shown.

[0197] Structure Characterization

[0198] (1) Seven 3'-OH dimer precursors 5 were successfully prepared in this invention, with a total yield of more than 70%, a synthesis scale of 20 g, and characterized by HPLC-MS. The liquid phase purity and mass spectrum identification results of the obtained allyl dimer precursors 5a - 5g are shown in Table 1.

[0199] Table 1 Liquid Phase Purity and Mass Spectrum Identification Results of Allyl Dimer Precursors 5a - g

[0200]

[0201] HPLC analysis shows that all dimer precursors 5 are diastereoisomers with a purity of more than 96%.

[0202] (2) From the liquid chromatograms and mass spectra of compounds 6a - 6g, it can be seen that high-purity nucleoside dimers were prepared in this invention, and the HPLC purity can reach more than 95%, which can meet the requirements of high-fidelity DNA synthesis. The allyl-cyanoethyl dimer was successfully synthesized in the synthesis experiment of this invention, verifying the feasibility of the synthesis route. This synthesis route is simple to operate and can achieve large-scale preparation, laying a foundation for subsequent scale-up production.

[0203] Application Example 1 Experiment on the Synthesis of Oligonucleotide Fragments from Allyl-Cyanoethyl Nucleoside Dimers

[0204] To verify whether the oligonucleotides synthesized from allyl-cyanoethyl nucleoside dimers are prone to deprotection, the present invention selected two allyl-cyanoethyl nucleoside dimers for a 20 nt sequence synthesis experiment. On the LK-192 DNA synthesizer, a 50 nmol synthesis column was used, the dimer concentration was 50 mg / mL, the coupling catalyst was 4,5-dicyanoimidazole, and the coupling time was 120 s. The single-cycle synthesis program of the synthesizer is shown in Table 2.

[0205] Table 2 Single-cycle synthesis program of the synthesizer

[0206]

[0207] After synthesis, ammonia ammonolysis was carried out. The ammonolysis was carried out at 95 °C for 2 h in an ammonolysis instrument, and the ammonia pressure was 470 - 520 kPa. After ammonolysis, desalting and purification were carried out. First, 200 μL of 90% acetonitrile was added to the synthesis column, and centrifuged at 500 r / min for 2 min. Then, 200 μL of pure water was added to each synthesis column, centrifuged at 500 r / min for 2 min, and then centrifuged at 2000 r / min for 2 min to obtain an aqueous solution of oligonucleotides.

[0208] Under the same conditions, 20 nt sequences were synthesized using methyl dimers (TA, GC) (structure shown in Formula II) and allyl-cyanoethyl nucleoside dimers (TA, GC) through 10 cycles, as shown in SEQ ID NO.1, specifically: TA GC TA GC TAGC TA GC TA GC. Under the same conditions, the mass spectrometry comparison of the ammonolysis products of oligonucleotides synthesized from methyl dimers and allyl-cyanoethyl nucleoside dimers is as Figure 24 shown. Figure 24 Among them, (a) is the mass spectrometry diagram of the oligonucleotides synthesized from methyl dimers, and (b) is the mass spectrometry diagram of the oligonucleotides synthesized from allyl-cyanoethyl nucleoside dimers.

[0209] The results show that there are more impurities in the oligonucleotides synthesized from methyl dimers after ammonolysis, with impurity peaks of molecular weight +14 and +28, and more fragment peaks, and the product purity is low; while the mass spectrometry identification results of the oligonucleotides synthesized from allyl-cyanoethyl nucleoside dimers of the present invention show ESI-MS (m / z) 6115.1, theoretical molecular weight: 6116.89; there are fewer by-products after its ammonolysis, the product purity is high, the deprotection is complete, and there are no obvious impurity peaks in the mass spectrometry; this shows that allyl-cyanoethyl nucleoside dimers exhibit good deprotection characteristics and can be used as an alternative structure to methyl dimers.

[0210] Application Example 2

[0211] In gene synthesis, multiple segments of 60 nt to 80 nt are usually synthesized and then spliced. To further verify the feasibility of allyl-cyanoethyl nucleoside dimers for oligonucleotide synthesis, the present invention synthesizes an 80 nt fragment. Using two allyl-cyanoethyl nucleoside dimers (TA, AC), an 80 nt sequence is synthesized according to Table 2 after 40 cycles, as shown in SEQ ID NO.2, specifically: TA AC TA AC TA AC TA AC TA AC TA AC TA AC TA AC TA AC TA AC TA AC TA AC TA AC TA AC TA AC TA AC TA AC TA AC TA AC TA AC. The ammonolysis conditions are referred to Application Example 1.

[0212] The mass spectrometry detection chart of the 80 nt sequence synthesized by allyl-cyanoethyl nucleoside dimers is as Figure 25 shown. The mass spectrometry identification result shows ESI-MS (m / z) 24337.7, and the theoretical molecular weight: 24334.04, obtaining the target sequence. The successful synthesis of the 80 nt fragment indicates that the allyl-protected dimers of the present invention can be used for oligonucleotide synthesis and have certain advantages. The oligonucleotides synthesized by allyl-cyanoethyl nucleoside dimers have been greatly improved in terms of the ease of deprotection. The reaction can be completed only by reacting with ammonia gas for 2 h, avoiding the uncertainty of the oligonucleotide sequence caused by the long-term high-temperature alkaline environment. The present invention verifies the concept when designing allyl-cyanoethyl nucleoside dimers. The use of this dimer is expected to further improve the quality of oligonucleotide synthesis, increase the product purity, and reduce synthesis errors.

[0213] In summary, the present invention further optimizes the structure of the dimer nucleoside, synthesizes 7 dimer precursors and 4 allyl-cyanoethyl nucleoside dimers. By optimizing the reaction conditions, a synthesis route with high purity, high yield and easy operation is determined. And it is used in the synthesis of oligonucleotide fragments, and an ammonolysis comparison experiment is carried out with the methyl-protected dimer. The results show that the deprotection rate of the oligonucleotides synthesized by allyl-cyanoethyl nucleoside dimers is significantly increased and the by-products are less. The new structure of the allyl-cyanoethyl nucleoside dimers obtained by the present invention can be used for the synthesis of oligonucleotides, is expected to broaden the applicable range of dimer nucleosides, and achieve high-quality synthesis.

[0214] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing an allyl-cyanoethyl nucleoside dimer, characterized in that: The allyl-cyanoethyl nucleoside dimer has a structure shown in Formula I: In formula I, X is O or S; B1 and B2 are independently selected from the following groups, Representative group connection position: Wherein, the R', R" and R'" are each independently selected from any one of hydrogen, amino, C1-C3 straight chain or branched alkyl, C1-C3 straight chain or branched alkyl acyl, phenyl and phenyl acyl; The preparation method of the allyl-cyanoethyl nucleoside dimer comprises the following steps: (1) a 5′-DMTr-3′-O-allyl phosphoramidite nucleoside having a structure shown in Formula 1 is coupled with a 5′-OH-3′-O-TBDMS nucleoside having a structure shown in Formula 2 to obtain a dimer having a structure shown in Formula 3; (2) When X is O, the dimer having the structure shown in Formula 3 is mixed with tert-butyl hydroperoxide and subjected to an oxidation reaction to obtain a dipolyphosphate diester having the structure shown in Formula 4; When X is S, the dimer having the structure shown in Formula 3 is mixed with S8 and N,O-bistrimethylsilylacetamide, and subjected to a sulfurization reaction to obtain a dipolyphosphate diester having the structure shown in Formula 4; (3) the diphosphodiester having the structure shown in Formula 4 is subjected to a TBDMS protecting group removal reaction to obtain a 3′-OH dimer precursor having the structure shown in Formula 5; (4) Under the action of 1H-tetrazolyl diisopropylammonium salt catalyst, the 3′-OH dimer precursor having the structure shown in Formula 5 is phosphorylated with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite to obtain an allyl-cyanoethyl nucleoside dimer having the structure shown in Formula I.

2. The preparation method according to claim 1, characterized in that: The coupling reaction is carried out in the presence of 1H-tetrazole, and the molar ratio of the 5′-DMTr-3′-O-allyl phosphoramidite nucleoside having the structure shown in Formula 1 to the 5′-OH-3′-O-TBDMS nucleoside having the structure shown in Formula 2 is (1-2):1; The molar ratio of the 5′-DMTr-3′-O-allyl phosphoramidite nucleoside having the structure shown in Formula 1 to 1H-tetrazole is (1-2):(0.5-1); The coupling reaction time is 8 to 14 hours.

3. The preparation method according to claim 1, characterized in that: The molar ratio of the dimer having the structure shown in Formula 3 to tert-butyl hydroperoxide is 1:(1-6); The oxidation reaction time is 0.4 to 1 h; The molar ratio of the dimer having the structure shown in Formula 3 to S8 and N,O-bistrimethylsilylacetamide is 1:(1-2):(4-6); The temperature of the vulcanization reaction is 40-60° C. and the time is 20-50 minutes.

4. The preparation method according to claim 1, characterized in that: The molar ratio of the 3′-OH dimer precursor having the structure shown in Formula 5 to 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite is 1:1.4; The molar ratio of the 3′-OH dimer precursor having the structure shown in Formula 5 to the 1H-tetrazolyl diisopropylammonium salt catalyst is 1:(1-3); The phosphorylation reaction time is 1 to 3 hours.

5. The preparation method according to claim 1 or 4, characterized in that: After the phosphorylation reaction, the phosphorylation reaction solution is subjected to post-treatment, and the post-treatment comprises the following steps: The phosphorylation reaction solution is subjected to flash column separation and purification to obtain a purified product; The purified product is dissolved in an organic solvent, the obtained solution is added to methyl tert-butyl ether, and the obtained solid is dried to obtain a pure product of allyl-cyanoethyl nucleoside dimer having a structure shown in Formula I.

6. Use of the allyl-cyanoethyl nucleoside dimer prepared by the preparation method according to any one of claims 1 to 5 in the synthesis of oligonucleotides.

7. A method for synthesizing an oligonucleotide, comprising the following steps: Mixing the allyl-cyanoethyl nucleoside dimer prepared by the preparation method according to any one of claims 1 to 5 with a coupling catalyst, and performing a coupling reaction to obtain a coupling reaction product; The coupling reaction product is sequentially subjected to DMTr protecting group removal, aminolysis and desalting purification to obtain an oligonucleotide.

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