A nucleoside trimer and its preparation method and application in oligonucleotide synthesis

Abstract

The present invention provides a nucleoside trimer and a preparation method thereof and an application in oligonucleotide synthesis, belonging to the technical field of DNA synthesis. The trimer nucleoside of the present invention uses pentavalent phosphate as the middle "joint", and the 5'-end and 3'-end are DMTr and phosphoramidite structures, respectively. The present invention is designed to obtain a trimer phosphoramidite structure, and in order to keep the trimer structure stable, the two middle phosphates are protected by methyl; in order to make the trimer nucleoside suitable for the oligonucleotide synthesis system and control the synthesis cost, the 3' end phosphate is protected by cyanoethyl. The nucleoside trimer provided by the present invention adopts a mixed skeleton of methyl phosphate and β-cyanoethyl phosphoramidite, and the trimer structure is stable, and can simplify the synthesis steps and reduce the synthesis error rate when used for oligonucleotide synthesis, and can also be used for gene library preparation, which has practical value.

Description

Technical Field

[0001] The invention relates to the technical field of DNA synthesis, and in particular to a nucleoside trimer and a preparation method thereof and application thereof in oligonucleotide synthesis. Background Art

[0002] As an emerging strategic international cutting-edge technology, synthetic biology has shown extensive application potential in the fields of biomedicine, energy, materials, etc., which is supported by DNA synthesis technology. As the underlying technology of synthetic biology, the existing DNA synthesis technology is difficult to meet the needs of synthetic biology in terms of synthesis length, accuracy and cost.

[0003] At present, there are two main factors that affect the results of DNA synthesis. First, when deprotected by trichloroformic acid, adenosine undergoes a side reaction of depurination, which becomes particularly prominent in the synthesis of long sequences. Second, as the number of couplings increases, the yield continues to decrease. Although the coupling efficiency of column synthesis can reach 99%, when synthesizing a 200 nt oligonucleotide sequence, the final theoretical yield is only 13%.

[0004] The use of trimer nucleoside modules to synthesize oligonucleotides reduces the number of synthesis steps and acid deprotection times to 1 / 3 of the original, which is expected to improve the quality of oligonucleotide synthesis. For example, a sequence that requires 90 synthesis of ordinary monomers can be completed only 30 times using trimers, which significantly reduces the number of reaction steps and the probability of errors. In addition, trimer nucleosides can also be used for gene-directed mutagenesis to achieve the preparation of protein libraries. The preparation of protein libraries is the key to protein engineering. There are currently two main synthesis strategies: through enzyme mutagenesis of the coding gene or through synthetic oligonucleotide amplification.

[0005] However, the existing methods for synthesizing trimer nucleosides are costly and difficult to synthesize, which limits their large-scale application. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a nucleoside trimer and a preparation method thereof and application in oligonucleotide synthesis. The nucleoside trimer provided by the present invention has a stable structure, simple synthesis and low cost. When used in oligonucleotide synthesis, it can simplify the synthesis steps and reduce the synthesis error rate. It can also be used for gene library preparation and has practical value.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a nucleoside trimer having a structure shown in Formula I:

[0009] Formula I;

[0010] In formula I, B1, B2 and B3 are independently selected from substituted or unsubstituted bases.

[0011] Preferably, B1, B2 and B3 are independently selected from the following groups, " represents the group connection position:

[0012] ;

[0013] ;

[0014] 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.

[0015] The present invention provides a method for preparing the above-mentioned nucleoside trimer, comprising the following steps:

[0016] In 1 H - In the presence of tetrazole, a nucleoside having a structure shown in Formula 1-1 and a 5ʹ-OH-3ʹ-O-TBDMS nucleoside having a structure shown in Formula 2 undergo a first coupling reaction to obtain a dimer precursor having a structure shown in Formula 3;

[0017] Formula 1-1; Formula 2; Formula 3;

[0018] The dimer precursor having the structure shown in Formula 3 is subjected to a first oxidation reaction with tert-butyl hydroperoxide to obtain a dipolyphosphate diester having the structure shown in Formula 4;

[0019] Formula 4;

[0020] The diphosphoric acid diester having the structure shown in Formula 4 is mixed with a resin, and a 5'-DMTr protecting group is removed to obtain a 5'-OH dimer having the structure shown in Formula 5;

[0021] Formula 5;

[0022] In 1 H - In the presence of tetrazole, the 5'-OH dimer having the structure shown in Formula 5 and the nucleoside having the structure shown in Formula 1-2 undergo a second coupling reaction to obtain a trimer precursor having the structure shown in Formula 6;

[0023] Formula 1-2; Formula 6;

[0024] The trimer precursor having the structure shown in Formula 6 is subjected to a second oxidation reaction with tert-butyl hydroperoxide to obtain a tripolyphosphate diester having the structure shown in Formula 7;

[0025] Formula 7;

[0026] The tripolyphosphate diester having the structure shown in Formula 7 is subjected to a TBDMS protecting group removal reaction to obtain a trimer precursor having the structure shown in Formula 8;

[0027] Formula 8;

[0028] In 1 H -tetrazolyl diisopropylammonium salt ammonium catalyst, the trimer precursor having the structure shown in formula 8 and 2-cyanoethyl- N,N,Nʹ,Nʹ -Tetraisopropylphosphorodiamidite is used for phosphoramidation reaction to obtain a nucleoside trimer having a structure shown in Formula I.

[0029] Preferably, the temperature of the first coupling reaction is room temperature, and the time is 8 to 14 h;

[0030] The temperature of the first oxidation reaction is room temperature and the time is 0.4~1h.

[0031] Preferably, the resin is Amberlyst 15 ion exchange resin;

[0032] The temperature of the 5'-DMTr protection group removal reaction is room temperature and the time is 8 to 14 h.

[0033] Preferably, the temperature of the second coupling reaction is room temperature, and the time is 8 to 14 h;

[0034] The temperature of the second oxidation reaction is room temperature and the time is 0.4~1h.

[0035] Preferably, the deprotection reagent used in the TBDMS protection group removal reaction includes Et3N and Et3N·3HF;

[0036] The time for the TBDMS protection group removal reaction is 6 to 14 h;

[0037] The time of the phosphoramidite reaction is 1 to 3 h.

[0038] Preferably, after the phosphoramidite reaction, the obtained phosphoramidite reaction solution is subjected to post-treatment, and the post-treatment comprises the following steps:

[0039] The phosphoramidite reaction solution is subjected to flash column separation and purification to obtain a purified product; the mobile phase used in the flash column separation and purification is an ethyl acetate-acetonitrile system, the ethyl acetate contains 1 vol% of triethylamine, and the acetonitrile contains 15 vol% of methanol;

[0040] 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 nucleoside trimer having a structure shown in Formula I.

[0041] The present invention provides application of the nucleoside trimer in oligonucleotide synthesis.

[0042] The present invention provides a method for synthesizing an oligonucleotide, comprising the following steps:

[0043] The nucleoside trimer is mixed with a coupling catalyst to carry out a coupling reaction to obtain a coupling reaction product;

[0044] The coupling reaction product is sequentially subjected to DMTr protecting group removal, aminolysis and desalting purification to obtain an oligonucleotide.

[0045] The present invention provides a nucleoside trimer having a structure shown in Formula I. The trimer nucleoside of the present invention uses pentavalent phosphate (P(V)) as an intermediate "linker", and the 5'-end and 3'-end are DMTr and phosphoramidite (P(III)) structures respectively. The synthesis process is to first synthesize a 5'-OH dimer nucleoside, then couple it with a 3'-OTBDM nucleoside to obtain a trimer precursor, and finally connect the phosphoramidite to obtain the corresponding trimer phosphoramidite. The present invention is designed to obtain a trimer phosphoramidite structure. In order to maintain the stability of the trimer structure, the two middle phosphates are protected by methyl groups; in order to make the trimer nucleoside suitable for an oligonucleotide synthesis system and control the synthesis cost, the 3'-end phosphate is protected by cyanoethyl groups. The new structure of the nucleoside trimer provided by the present invention uses a mixed skeleton of methyl phosphate and β-cyanoethyl phosphoramidite. The trimer structure is stable, and can simplify the synthesis steps and reduce the synthesis error rate when used for oligonucleotide synthesis. It can also be used for gene library preparation, and has practical value.

[0046] The present invention provides a method for preparing the above-mentioned nucleoside trimer. The synthesis of the nucleoside trimer of the present invention adopts a "one-pot" method, and coupling-oxidation is carried out in the same container; further, the present invention adopts resin to remove the DMTr protecting group, the reaction is complete and the conditions are mild, no by-products are generated, and the post-treatment process is simplified. Only by filtering and removing the resin, a 5'-OH dimer having a structure shown in Formula 5 can be obtained. Then, a coupling-oxidation-deprotection "one-pot" reaction is carried out with phosphoramidite methyl nucleoside, and a trimer precursor having a structure shown in Formula 8 is obtained after the synthesis is completed. The purity of the trimer precursor can reach more than 97%. Finally, 2-cyanoethyl- N,N,Nʹ,Nʹ-Tetraisopropylphosphorodiamidite as phosphorylation reagent, 1 H -tetrazolyl diisopropylammonium salt is used as a catalyst for phosphoramidation reaction to obtain a high-purity nucleoside trimer, and its HPLC purity can reach more than 98.5%. The preparation method provided by the present invention overcomes the problems of high synthesis cost and difficulty in synthesis of nucleoside trimers. The synthetic route is simple to operate and can achieve large-scale preparation, laying a foundation for subsequent expansion of production. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The synthetic route of nucleoside trimers is shown in FIG.

[0048] Figure 2 is the mass spectrum of compound 8a;

[0049] Figure 3 is the liquid chromatogram of compound 9a;

[0050] Figure 4 is the mass spectrum of compound 9a;

[0051] Figure 5 is the mass spectrum of compound 8b;

[0052] Figure 6 is the liquid chromatogram of compound 9b;

[0053] Figure 7 is the mass spectrum of compound 9b;

[0054] Figure 8 is the mass spectrum of compound 8c;

[0055] Fig. 9 is the liquid chromatogram of compound 9c;

[0056] Fig.10 is the mass spectrum of compound 9c;

[0057] Fig.11 is the mass spectrum of compound 8d;

[0058] Fig.12 is the liquid chromatogram of compound 9d;

[0059] Fig.13 is the mass spectrum of compound 9d;

[0060] Fig.14 is the mass spectrum of compound 8e;

[0061] Fig.15 is the liquid chromatogram of compound 9e;

[0062] Fig.16 is the mass spectrum of compound 9e;

[0063] Fig.17 is the mass spectrum of compound 8f;

[0064] Fig.18 is the liquid chromatogram of compound 9f;

[0065] Fig.19 is the mass spectrum of compound 9f;

[0066] Fig. 20 is the mass spectrum of compound 8g;

[0067] Fig.21 is the liquid chromatogram of compound 9g;

[0068] Fig. 22 is the mass spectrum of compound 9g;

[0069] Fig.23 is the mass spectrum of compound 8h;

[0070] Fig.24 is the liquid chromatogram of compound 9h;

[0071] Fig.25 is the mass spectrum of compound 9h;

[0072] Fig.26 These are the mass spectrometry results of oligonucleotide sequences synthesized with trimers and monomers. DETAILED DESCRIPTION

[0073] The present invention provides a nucleoside trimer, characterized in that it has a structure shown in Formula I:

[0074] Formula I;

[0075] In formula I, B1, B2 and B3 are independently selected from substituted or unsubstituted bases.

[0076] In the present invention, B1, B2 and B3 are independently selected from the following groups, " represents the group connection position:

[0077] ;

[0078] ;

[0079] 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.

[0080] As a specific embodiment of the present invention, the nucleoside trimer has a structure shown in any one of formulas a to h:

[0081] .

[0082] The present invention provides a method for preparing the above-mentioned nucleoside trimer, comprising the following steps:

[0083] In 1 H - In the presence of tetrazole, a nucleoside having a structure shown in Formula 1-1 and a 5ʹ-OH-3ʹ-O-TBDMS nucleoside having a structure shown in Formula 2 undergo a first coupling reaction to obtain a dimer precursor having a structure shown in Formula 3;

[0084] Formula 1-1; Formula 2; Formula 3;

[0085] The dimer precursor having the structure shown in Formula 3 is subjected to a first oxidation reaction with tert-butyl hydroperoxide to obtain a dipolyphosphate diester having the structure shown in Formula 4;

[0086] Formula 4;

[0087] The diphosphoric acid diester having the structure shown in Formula 4 is mixed with a resin and subjected to a 5'-DMTr protecting group removal reaction to obtain a 5'-OH dimer having the structure shown in Formula 5;

[0088] Formula 5;

[0089] In 1 H - In the presence of tetrazole, the 5'-OH dimer having the structure shown in Formula 5 and the nucleoside having the structure shown in Formula 1-2 undergo a second coupling reaction to obtain a trimer precursor having the structure shown in Formula 6;

[0090] Formula 1-2; Formula 6;

[0091] The trimer precursor having the structure shown in Formula 6 is subjected to a second oxidation reaction with tert-butyl hydroperoxide to obtain a tripolyphosphate diester having the structure shown in Formula 7;

[0092] Formula 7;

[0093] The tripolyphosphate diester having the structure shown in Formula 7 is subjected to a TBDMS protecting group removal reaction to obtain a trimer precursor having the structure shown in Formula 8;

[0094] Formula 8;

[0095] In 1 H -tetrazolyl diisopropylammonium salt ammonium catalyst, the trimer precursor having the structure shown in formula 8 and 2-cyanoethyl- N,N,Nʹ,Nʹ -Tetraisopropylphosphorodiamidite is used for phosphoramidation reaction to obtain a nucleoside trimer having a structure shown in Formula I.

[0096] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0097] The present invention is H -tetrazolyl, a nucleoside having a structure as shown in Formula 1-1 and a 5ʹ-OH-3ʹ-O-TBDMS nucleoside having a structure as shown in Formula 2 are subjected to a first coupling reaction to obtain a dimer precursor having a structure as shown in Formula 3. In the present invention, the molar ratio of the nucleoside having a structure as shown in Formula 1-1 to the 5ʹ-OH-3ʹ-O-TBDMS nucleoside having a structure as shown in Formula 2 is preferably (1 to 1.5):1, more preferably 1.05:1; the nucleoside having a structure as shown in Formula 1-1 and 1 H The molar ratio of tetrazole is preferably (1-1.5):(0.6-1), more preferably 1.05:0.8.

[0098] In the present invention, the first coupling reaction is preferably carried out in an organic solvent, and the organic solvent used in the first coupling reaction is preferably dichloromethane. In the present invention, the first coupling reaction is preferably carried out under N2 protection; the temperature of the first coupling reaction is preferably room temperature, and the time is preferably 8 to 14 h, more preferably 12 h. After the first coupling reaction, the present invention does not perform post-treatment, and directly uses the obtained first coupling reaction liquid for subsequent reactions.

[0099] After obtaining the dimer precursor having the structure shown in Formula 3, the present invention conducts a first oxidation reaction between the dimer precursor having the structure shown in Formula 3 and tert-butyl hydroperoxide to obtain a dipolyphosphate diester having the structure shown in Formula 4. In the present invention, the molar ratio of the dimer precursor having the structure shown in Formula 3 to tert-butyl hydroperoxide is preferably 1:1. It was found in the experiment that when the oxidation step is performed, adding 2 times the amount of oxidant will release a large amount of heat, which brings uncertainty to the experimental safety and compound stability, so the oxidant is added in batches in the subsequent operation. Through monitoring of the experiment, it was found that the reaction can be completed by adding 1 times the amount of oxidant, which significantly reduces the amount of reagents used and simplifies the post-processing process.

[0100] In the present invention, the temperature of the first oxidation reaction is preferably room temperature, and the time is preferably 0.4 to 1 h, more preferably 0.5 h.

[0101] After the first oxidation reaction, the present invention preferably performs rotary concentration on the obtained first oxidation reaction to evaporate the solvent.

[0102] After obtaining the dipolyphosphate diester having the structure shown in Formula 4, the present invention mixes the dipolyphosphate diester having the structure shown in Formula 4 with a resin and performs a 5'-DMTr protecting group removal reaction to obtain a 5'-OH dimer having the structure shown in Formula 5. In the present invention, the resin is preferably Amberlyst 15 ion exchange resin.

[0103] The present invention preferably uses a solvent to dissolve the dipolyphosphate diester having the structure shown in Formula 4, and then adds the resin. In the present invention, the solvent is preferably methanol; the mass ratio of the dipolyphosphate diester having the structure shown in Formula 4 to the resin is preferably (2-4):1, more preferably 3:1.

[0104] In the present invention, the temperature of the 5'-DMTr protection group removal reaction is preferably room temperature, and the time is preferably 8 to 14 h, more preferably 12 h.

[0105] After the 5'-DMTr protection group removal reaction, the present invention preferably sequentially filters the obtained 5'-DMTr protection group removal reaction solution, purifies by column chromatography, and rotary concentrates to evaporate the solvent to obtain a pure 5'-OH dimer having a structure shown in Formula 5. In the present invention, the mobile phase of the column chromatography separation is preferably DCM and MeOH, and the gradient volume concentration of the MeOH is preferably 0-10%. The present invention has no special requirements for the specific method of rotary concentration and evaporation of the solvent, and the above-mentioned operation well known in the art can be used.

[0106] After obtaining the 5'-OH dimer having the structure shown in Formula 5, the present invention H In the presence of tetrazole, the 5'-OH dimer having the structure shown in Formula 5 and the nucleoside having the structure shown in Formula 1-2 undergo a second coupling reaction to obtain a trimer precursor having the structure shown in Formula 6. In the present invention, the molar ratio of the 5'-OH dimer having the structure shown in Formula 5 to the nucleoside having the structure shown in Formula 1-2 is preferably 1:(1 to 1.5), more preferably 1:1.1; the 5'-OH dimer having the structure shown in Formula 5 and the nucleoside having the structure shown in Formula 1-2 are preferably 1:(1 to 1.5), more preferably 1:1.1; H The molar ratio of tetrazole is preferably 1:(0.6-1), more preferably 1:0.8.

[0107] In the present invention, the second coupling reaction is preferably carried out in an organic solvent, and the organic solvent is preferably dichloromethane.

[0108] In the present invention, the second coupling reaction is preferably carried out under N2 protection; the temperature of the second coupling reaction is preferably room temperature, and the time is preferably 8 to 14 h, more preferably 12 h. After the second coupling reaction, the present invention does not perform post-treatment, and the obtained second coupling reaction liquid is directly used for subsequent reactions.

[0109] The present invention conducts a second oxidation reaction between a trimer precursor having a structure shown in Formula 6 and tert-butyl hydroperoxide to obtain a tripolyphosphate diester having a structure shown in Formula 7. In the present invention, the molar ratio of the trimer precursor having a structure shown in Formula 6 to tert-butyl hydroperoxide is preferably 1:(1-4), more preferably 1:(2-3). In the present invention, the temperature of the second oxidation reaction is preferably room temperature, and the time is preferably 0.4-1 h, more preferably 0.5 h.

[0110] After the second oxidation reaction, the present invention does not perform post-treatment, and directly uses the obtained second oxidation reaction liquid for subsequent reactions.

[0111] In the present invention, the tripolyphosphate diester having the structure shown in Formula 7 is subjected to a TBDMS protecting group removal reaction to obtain a trimer precursor having the structure shown in Formula 8. In the present invention, the deprotection reagent used in the TBDMS protecting group removal reaction preferably includes Et3N and Et3N·3HF, and the molar ratio of Et3N to Et3N·3HF is preferably 2:3. In the present invention, the molar ratio of the tripolyphosphate diester having the structure shown in Formula 7 to Et3N is preferably 1:2.

[0112] In the present invention, the temperature of the TBDMS protection group removal reaction is preferably room temperature, and the time is preferably 6 to 14 h, more preferably 12 h.

[0113] After the TBDMS protection group removal reaction, the present invention preferably performs post-treatment on the obtained TBDMS protection group removal reaction solution, and the post-treatment preferably comprises the following steps:

[0114] The TBDMS protection group removal reaction solution is mixed with a saturated NaHCO3 solution until no bubbles are generated, the resulting mixture is extracted twice with dichloromethane and water, the organic phases are combined, and the resulting organic phases are washed, dried, filtered and concentrated to obtain a trimer precursor crude product having a structure shown in Formula 8;

[0115] The crude trimer precursor having the structure shown in Formula 8 is purified by column chromatography and the solvent is evaporated by rotary concentrator. The obtained residue is dissolved in anhydrous dichloromethane and added dropwise to methyl tert-butyl ether, filtered and dried to obtain a pure trimer precursor having the structure shown in Formula 8.

[0116] In the present invention, the mobile phase used in the column chromatography purification is preferably DCM / MeOH (0-10%).

[0117] After obtaining the trimer precursor having the structure shown in Formula 8, the present invention is H -tetrazolyl diisopropylammonium salt ammonium catalyst, the trimer precursor having the structure shown in formula 8 and 2-cyanoethyl- N,N,Nʹ,Nʹ -tetraisopropylphosphorodiamidite to undergo phosphoramidite reaction to obtain a nucleoside trimer having a structure shown in Formula I. In the present invention, the trimer precursor having a structure shown in Formula 8 is reacted with 2-cyanoethyl- N,N,Nʹ,Nʹ -tetraisopropylphosphorodiamidite is preferably in a molar ratio of 1:(1.2-2), more preferably 1:1.5; the trimer precursor having the structure shown in Formula 8 and 1 H The molar ratio of the tetrazole diisopropylammonium salt ammonium catalyst is preferably 1:(1.2-2), more preferably 1:1.5.

[0118] In the present invention, the phosphoramidite reaction is preferably carried out in an organic solvent, and the organic solvent is preferably dichloromethane. In the present invention, the phosphoramidite reaction is preferably carried out under N2 protection, the temperature of the phosphoramidite reaction is preferably room temperature, and the time is preferably 1 to 3 h, more preferably 2 h.

[0119] After the phosphoramidite reaction, the present invention preferably further comprises post-treatment of the obtained phosphoramidite reaction solution, wherein the post-treatment comprises the following steps:

[0120] The phosphoramidite reaction solution is subjected to flash column separation and purification to obtain a purified product;

[0121] 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 nucleoside trimer having a structure shown in Formula I.

[0122] In the present invention, the mobile phase used in the flash column separation and purification is preferably an ethyl acetate-acetonitrile (0-30%) system, wherein the ethyl acetate contains 1 vol% of triethylamine and the acetonitrile contains 15 vol% of methanol.

[0123] In the present invention, the organic solvent for dissolving the purified product is preferably anhydrous dichloromethane. In the present invention, the temperature at which the dissolving solution is added to methyl tert-butyl ether is preferably -78°C, and the adding method is preferably dropwise addition. In the present invention, the volume ratio of the methyl tert-butyl ether to the obtained dissolving solution is preferably 10:1.

[0124] As a specific embodiment of the present invention, the synthetic route of the nucleoside trimer is as follows Figure 1 shown. Figure 1 The reaction conditions and reagents are as follows: (a) 1 H-tetrazolyl, DCM, 12 h; (b) t-BuOOH, DCM, 0.5 h; (c) resin, MeOM, 10 h; (d) Et3N·3HF, Et3N, DCM, 12 h; (e) 2-cyanoethyl- N,N,N',N' -Tetraisopropylphosphorodiamidite, 1 H -tetrazolyl diisopropylammonium salt, DCM, 2 h.

[0125] The present invention provides application of the nucleoside trimer in oligonucleotide synthesis.

[0126] The present invention provides a method for synthesizing the above oligonucleotide, comprising the following steps:

[0127] The nucleoside trimer is mixed with a coupling catalyst to carry out a coupling reaction to obtain a coupling reaction product;

[0128] The coupling reaction product is sequentially subjected to DMTr protecting group removal, aminolysis and desalting purification to obtain an oligonucleotide.

[0129] The present invention preferably uses a 50 nmol synthesis column on a LK-192 DNA synthesizer to carry out the coupling reaction. In the present invention, the concentration of the nucleoside trimer is preferably 50-80 mg / mL, more preferably 70 mg / mL; in the present invention, the coupling catalyst is preferably 4,5-dicyanoimidazole (DCI). In the present invention, the temperature of the coupling reaction is preferably room temperature, and the time is preferably 120-160 s, more preferably 150 s.

[0130] In the present invention, the deprotection reagent used for the deprotection of the DMTr protecting group is preferably a DBLOCK reagent (trichloroacetic acid: dichloromethane = 3% (V / V)), and the time for the deprotection of the DMTr protecting group is preferably 40 to 70 s, more preferably 60 s. After the deprotection reaction of the DMTr protecting group, the present invention preferably performs another deprotection reaction of the DMTr protecting group according to the above conditions. After the deprotection of the DMTr protecting group, the present invention preferably uses acetonitrile to wash the obtained deprotection product of the DMTr protecting group.

[0131] In the present invention, the aminolysis is preferably ammonia ammonolysis. In the present invention, before the aminolysis, the present invention preferably pre-treats the obtained DMTr protection group-removed product, and the pre-treatment preferably includes: washing and drying the DMTr protection group-removed product.

[0132] In the present invention, the reagent used for washing is preferably acetonitrile with a volume concentration of 70%; and the drying is preferably vacuum drying.

[0133] In the present invention, the ammonolysis is preferably carried out in an ammonolysis rack, and the conditions for the ammonolysis preferably include: the ammonolysis temperature is preferably 90-95°C; the ammonia pressure is preferably 470-520 kPa, more preferably 500 kPa; the ammonolysis time is preferably 1-3 h, more preferably 2 h.

[0134] In the present invention, the desalting and purification method preferably comprises the following steps:

[0135] The product after aminolysis is mixed with acetonitrile solution and subjected to a first centrifugation; pure water is then added and subjected to a second centrifugation.

[0136] In the present invention, the volume concentration of the acetonitrile solution is preferably 90%; the speed of the first centrifugation is preferably 500 r / min, and the time is preferably 2 min.

[0137] In the present invention, the volume ratio of the acetonitrile solution to pure water is preferably 1:1; the second centrifugation preferably includes first centrifuging at 500 r / min for 2 min, and then centrifuging at 2000 r / min for 2 min.

[0138] The nucleoside trimer provided by the present invention, its preparation method and its application in oligonucleotide synthesis are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0139] In the following examples, 1a-1d were purchased from Beijing Ruiboao Biotechnology Co., Ltd., and 2a-2d were purchased from Wuhu Huaren Technology Co., Ltd. The resin was Amberlyst 15 ion exchange resin.

[0140] Example 1

[0141]

[0142] Reaction conditions and reagents for the synthesis of trimer CAA-Amidite: (a) 1 H -tetrazolyl, DCM, 12 h; (b) t -BuOOH, DCM, 0.5 h; (c) resin, MeOH, 12 h; (d) Et3N·3HF, Et3N, DCM, 12 h; (e) 2-cyanoethyl- N,N,N', N' -Tetraisopropylphosphorodiamidite, 1H-tetrazolyldiisopropylammonium salt, DCM, 2 h.

[0143] N 4 -Benzoyl- P -methyl-5'- O -Dimethoxytrityl-2'-deoxycytidine-(3'→5')-N 6 -Benzoyl-2'-deoxyadenosine-(3'→5')- N 6 -Synthesis of benzoyl-2'-deoxyadenosine (8a):

[0144] 1a (10.00 g, 1.05 eq) and 2a (5.46 g, 1.00 eq) were dissolved in dichloromethane and acetonitrile to remove water, and then dissolved in anhydrous dichloromethane. 1 H -tetrazolyl (0.66 g, 0.80 eq) was stirred at room temperature for 12 hours under N2 protection to obtain compound 3a. Anhydrous t-BuOOH (1.05 g, 1.00 eq) was directly added to the mixture and stirred at room temperature for 0.5 hours to obtain 4a. The solvent was evaporated by rotary concentrator, then dissolved in methanol, added to resin, and stirred at room temperature for 12 hours to obtain crude compound 5a. After filtration, column chromatography purification (DCM / MeOH (0-10%)) was performed, and the solvent was evaporated by rotary concentrator to obtain compound 5a.

[0145] Weigh 5a (5.00 g, 1.00 eq) and 1b (4.85 g, 1.10 eq) in a 200 mL round-bottom flask, add dichloromethane and acetonitrile to remove water. Then dissolve in anhydrous dichloromethane and add 1 H -tetrazolyl (0.31 g, 0.80 eq), stirred at room temperature for 12 hours under N2 protection to obtain compound 6a. Anhydrous t-BuOOH (0.50 g, 1.00 eq) was directly added to the mixture and stirred at room temperature for 0.5 hours to obtain compound 7a. Et3N (1.12 g, 2.00 eq) and Et3N·3HF (2.69 g, 3.00 eq) were added and stirred at room temperature for 12 hours. After the reaction was complete, saturated NaHCO3 was added until no bubbles were generated. The mixture was extracted twice with dichloromethane and water. The combined organic layer was washed with saturated brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo to obtain crude compound 8a. Purification by column chromatography (DCM / MeOH (0-10%)), and then rotary concentrated to evaporate the solvent. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether, filtered, and dried under vacuum to obtain compound 8a, 5.72 g of white solid, with a purity of 98.5% and a yield of 68.87%. ESI-MS: m / z calcd for C 73 H 71 N 13 O 19 P2[M / 2+H] + 748.73, found:748.85.

[0146] The mass spectrum of compound 8a is shown in Figure 2 shown.

[0147] N 4 -Benzoyl- P -methyl-5'- O -Dimethoxytrityl-2'-deoxycytidine-(3'→5')- N 6 -Benzoyl-3'- O -[(N,N-diisopropylamino)-cyanoethylphosphino]-2'-deoxyadenosine-(3'→5')- N 6 - Synthesis of benzoyl-2'-deoxyadenosine (9a):

[0148] Compound 8a (1.30 g, 1.00 eq) was dissolved in dichloromethane and toluene and evaporated to dryness under reduced pressure to remove water from the raw material. Then, it was dissolved in 10 mL of anhydrous dichloromethane and cyanoethyl phosphoramidite (0.39 g, 1.50 eq) and 1 H -tetrazolyl diisopropylammonium salt (0.22 g, 1.50 eq) was reacted at room temperature for 2 h under N2 protection. After the reaction was complete, it was purified by column chromatography (EA (1% Et3N) -ACN (15% MeOH)), and then the solvent was evaporated by rotary concentrator. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether at -78°C, filtered, and vacuum dried to obtain compound 9a, 1.13 g of white solid, purity 99.4%, yield 76.7%. ESI-MS: m / z calcd for C 82 H 88 N 15 O 20 P3[MH] - 1694.55, found:1694.55.

[0149] The liquid chromatogram of compound 9a is shown in Figure 3 As shown in the mass spectrum Figure 4 shown.

[0150] Example 2

[0151]

[0152] Reaction conditions and reagents for the synthesis of trimer TAA-Amidite: (a) 1 H -tetrazolyl, DCM, 12 h; (b) t -BuOOH, DCM, 0.5 h; (c) resin, MeOH, 12 h; (d) Et3N·3HF, Et3N, DCM, 12 h; (e) 2-cyanoethyl- N,N,N', N' -Tetraisopropylphosphorodiamidite, 1H -tetrazolyl diisopropylammonium salt, DCM, 2 h.

[0153] P -methyl-5'- O -Dimethoxytrityl-thymidine-(3'→5')- N 6 -Benzoyl-2'-deoxyadenosine-(3'→5')- N 6 - Synthesis of benzoyl-2'-deoxyadenosine (8b):

[0154] 1a (10.00 g, 1.05 eq) and 2a (5.46 g, 1.00 eq) were dissolved in dichloromethane and acetonitrile to remove water, and then dissolved in anhydrous dichloromethane. 1 H -tetrazolyl (0.66 g, 0.80 eq) was stirred at room temperature for 12 hours under N2 protection to obtain compound 3a. Anhydrous t-BuOOH (1.05 g, 1.00 eq) was directly added to the mixture and stirred at room temperature for 0.5 hours to obtain 4a. The solvent was evaporated by rotary concentrator, then dissolved in methanol, added to resin, and stirred at room temperature for 12 hours to obtain crude compound 5a. After filtration, column chromatography purification (DCM / MeOH (0-10%)) was performed, and the solvent was evaporated by rotary concentrator to obtain compound 5a.

[0155] Weigh 5a (5.0 g, 1.00 eq) and 1d (4.31 g, 1.00 eq) in a 200 mL round-bottom flask, add dichloromethane and acetonitrile to remove water. Then dissolve in anhydrous dichloromethane and add 1 H-tetrazolyl (0.33 g, 0.80 eq), stirred at room temperature for 12 hours under N2 protection to obtain compound 6b. Anhydrous t-BuOOH (0.50 g, 1.00 eq) was directly added to the mixture and stirred at room temperature for 0.5 hours to obtain compound 7b. Et3N (1.12 g, 2.00 eq) and Et3N·3HF (2.69 g, 3.00 eq) were added and stirred at room temperature for 12 hours. After the reaction was complete, saturated NaHCO3 was added until no bubbles were generated. The mixture was extracted twice with dichloromethane and water. The combined organic layer was washed with saturated brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo to obtain crude compound 8b. Purification by column chromatography (DCM / MeOH (0-10%)), and then rotary concentrated to evaporate the solvent. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether, filtered, and dried under vacuum to obtain compound 8b, 5.56 g of white solid, with a purity of 98.3% and a yield of 71.18%. ESI-MS: m / z calcd for C 67 H 68 N 12 O 19 P2[M / 2+H] + 704.21, found:704.48.

[0156] The mass spectrum of compound 8b is shown in Figure 5 shown.

[0157] P -methyl-5'- O -Dimethoxytrityl-thymidine-(3'→5')- N 6 -Benzoyl-2'-deoxyadenosine-(3'→5')- N 6 -Benzoyl-3'- O Synthesis of -[(N,N-diisopropylamino)-cyanoethylphosphino]-2'-deoxyadenosine (9b):

[0158] Compound 8b (1.54 g, 1.00 eq) was dissolved in dichloromethane and toluene and evaporated to dryness under reduced pressure to remove water from the raw material. Then it was dissolved in 10 mL of anhydrous dichloromethane, and cyanoethyl phosphoramidite (0.50 g, 1.50 eq) and 1 H-tetrazolyl diisopropylammonium salt (0.28 g, 1.50 eq) was reacted at room temperature for 2 h under N2 protection. After the reaction was complete, it was purified by column chromatography (EA (1% Et3N) -ACN (15% MeOH)), and then the solvent was evaporated by rotary concentrator. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether at -78°C, filtered, and vacuum dried to obtain compound 9b, 1.16 g of white solid, purity 98.6%, yield 65.9%. ESI-MS: m / z calcd for C 76 H 85 N 14 O 20 P3[MH] - 1605.52, found:1605.52.

[0159] The liquid chromatogram of compound 9b is as follows Figure 6 As shown in the mass spectrum Figure 7 shown.

[0160] Example 3

[0161]

[0162] Reaction conditions and reagents for the synthesis of trimer CAG-Amidite: (a) 1 H -tetrazolyl, DCM, 12 h; (b) t -BuOOH, DCM, 0.5 h; (c) resin, MeOH, 12 h; (d) Et3N·3HF, Et3N, DCM, 12 h; (e) 2-cyanoethyl- N,N,N', N' -Tetraisopropylphosphorodiamidite, 1H -tetrazolyl diisopropylammonium salt, DCM, 2 h.

[0163] N 4 -Benzoyl- P -methyl-5'- O -Dimethoxytrityl-2'-deoxycytidine-(3'→5')- N 6 -Benzoyl-2'-deoxyadenosine-(3'→5')- N 2 -Synthesis of isobutyryl-2'-deoxyguanosine (8c):

[0164] 1a (18.05 g, 1.05 eq) and 2c (9.48 g, 1.00 eq) were dissolved in anhydrous dichloromethane and 1 H-tetrazolyl (1.18 g, 0.80 eq) was stirred at room temperature for 12 hours under N2 protection to obtain compound 3b. Anhydrous t-BuOOH (1.90 g, 1.00 eq) was directly added to the mixture and stirred at room temperature for 0.5 hours to obtain 4b. The solvent was evaporated by rotary concentrator, then dissolved in methanol, added to resin, and stirred at room temperature for 12 hours to obtain crude compound 5b. After filtration, column chromatography purification (DCM / MeOH (0-10%)) was performed, and the solvent was evaporated by rotary concentrator to obtain compound 5b.

[0165] Weigh 5b (5.0 g, 1.00 eq) and 1b (4.95 g, 1.00 eq) in a 200 mL round-bottom flask, add dichloromethane and acetonitrile to remove water. Then dissolve in anhydrous dichloromethane and add 1 H -tetrazolyl (0.32 g, 0.80 eq), stirred at room temperature for 12 hours under N2 protection to obtain compound 6c. Anhydrous t-BuOOH (0.51 g, 1.00 eq) was directly added to the mixture and stirred at room temperature for 0.5 hours to obtain compound 7c. Et3N (1.15 g, 2.00 eq) and Et3N·3HF (2.74 g, 3.00 eq) were added and stirred at room temperature for 12 hours. After the reaction was complete, saturated NaHCO3 was added until no bubbles were generated. The mixture was extracted twice with dichloromethane and water. The combined organic layer was washed with saturated brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo to obtain crude compound 8c. Purification by column chromatography (DCM / MeOH (0-10%)), and then rotary concentrated to evaporate the solvent. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether, filtered, and dried under vacuum to obtain compound 8c, 5.12 g of white solid, with a purity of 98.7% and a yield of 61.15%. ESI-MS: m / z calcd for C 70 H 73 N 13 O 20 P2[M / 2+Na] + 761.73 found:761.93.

[0166] The mass spectrum of compound 8c is shown in Figure 8 shown.

[0167] N 4 -Benzoyl- P -methyl-5'- O -Dimethoxytrityl-2'-deoxycytidine-(3'→5')- N 6 -Benzoyl-2'-deoxyadenosine-(3'→5')- N2 -isobutyryl-3'- O Synthesis of -[(N,N-diisopropylamino)-cyanoethylphosphino]-2'-deoxyguanosine (9c):

[0168] Compound 8c (1.60 g, 1.00 eq) was dissolved in dichloromethane and toluene and evaporated to dryness under reduced pressure to remove water from the raw material. Then it was dissolved in 10 mL of anhydrous dichloromethane, and cyanoethyl phosphoramidite (0.65 g, 2.00 eq) and 1 H -tetrazolyl diisopropylammonium salt (0.37 g, 2.00 eq) was reacted at room temperature for 2 h under N2 protection. After the reaction was complete, it was purified by column chromatography (EA (1% Et3N) -ACN (15% MeOH)), and then the solvent was evaporated by rotary concentrator. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether at -78°C, filtered, and dried in vacuo to obtain compound 9c, 1.19 g of white solid, purity 99.4%, yield 65.5%. ESI-MS: m / z calcd for C 79 H 90 N 15 O 21 P3[MH] - 1676.56, found:1676.55.

[0169] The liquid chromatogram of compound 9c is shown in Fig. 9 As shown in the mass spectrum Fig.10 shown.

[0170] Example 4

[0171]

[0172] Reaction conditions and reagents for the synthesis of trimer TAG-Amidite: (a) 1 H -tetrazolyl, DCM, 12 h; (b) t -BuOOH, DCM, 0.5 h; (c) resin, MeOH, 12 h; (d) Et3N·3HF, Et3N, DCM, 12 h; (e) 2-cyanoethyl- N,N,N', N' -Tetraisopropylphosphorodiamidite, 1H -tetrazolyl diisopropylammonium salt, DCM, 2 h.

[0173] P -methyl-5'- O -Dimethoxytrityl-thymidine-(3'→5')- N 6 -Benzoyl-2'-deoxyadenosine-(3'→5')-N 2 -Synthesis of isobutyryl-2'-deoxyguanosine (8d):

[0174] 1a (18.05 g, 1.05 eq) and 2c (9.48 g, 1.00 eq) were dissolved in anhydrous dichloromethane and 1 H -tetrazolyl (1.18 g, 0.80 eq) was stirred at room temperature for 12 hours under N2 protection to obtain compound 3b. Anhydrous t-BuOOH (1.90 g, 1.00 eq) was directly added to the mixture and stirred at room temperature for 0.5 hours to obtain 4b. The solvent was evaporated by rotary concentrator, then dissolved in methanol, added to resin, and stirred at room temperature for 12 hours to obtain crude compound 5b. After filtration, column chromatography purification (DCM / MeOH (0-10%)) was performed, and the solvent was evaporated by rotary concentrator to obtain compound 5b.

[0175] Weigh 5b (6.0 g, 1.00 eq) and 1d (5.28 g, 1.00 eq) in a 200 mL round-bottom flask, add dichloromethane and acetonitrile to remove water. Then dissolve in anhydrous dichloromethane and add 1 H -tetrazolyl (0.38 g, 0.80 eq), stirred at room temperature for 12 hours under N2 protection to obtain compound 6d. Anhydrous t-BuOOH (0.62 g, 1.00 eq) was directly added to the mixture and stirred at room temperature for 0.5 hours to obtain compound 7d. Et3N (1.38 g, 2.00 eq) and Et3N·3HF (3.29 g, 3.00 eq) were added and stirred at room temperature for 12 hours. After the reaction was complete, saturated NaHCO3 was added until no bubbles were generated. The mixture was extracted twice with dichloromethane and water. The combined organic layer was washed with saturated brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo to obtain crude compound 8d. Purification by column chromatography (DCM / MeOH (0-10%)), and then rotary concentrated to evaporate the solvent. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether, filtered, and dried under vacuum to obtain compound 8d, 6.09 g of white solid, with a purity of 100.00% and a yield of 64.50%. ESI-MS: m / z calcd for C 64 H 70 N 12 O 20 P21388.43, found: 1387.42.

[0176] The mass spectrum of compound 8d is shown in Fig.11 shown.

[0177] P -methyl-5'- O-Dimethoxytrityl-thymidine-(3'→5')- N 6 -Benzoyl-2'-deoxyadenosine-(3'→5')- N 2 -isobutyryl-3'- O Synthesis of -[(N,N-diisopropylamino)-cyanoethylphosphino]-2'-deoxyguanosine (9d):

[0178] Compound 8d (1.85 g, 1.00 eq) was dissolved in dichloromethane and toluene and evaporated to dryness under reduced pressure to remove the water in the raw material. Then it was dissolved in 30 mL of anhydrous dichloromethane, and cyanoethyl phosphoramidite (0.80 g, 2.00 eq) and 1 H -tetrazolyl diisopropylammonium salt (0.46 g, 2.00 eq) was reacted at room temperature for 2 h under N2 protection. After the reaction was complete, it was purified by column chromatography (EA (1% Et3N) -ACN (15% MeOH)), and then the solvent was evaporated by rotary concentrator. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether at -78°C, filtered, and dried in vacuo to obtain compound 9d, 1.51 g of white solid, purity 98.9%, yield 71.3%. ESI-MS: m / z calcd for C 73 H 87 N 14 O 21 P3[MH] - 1587.53, found:1587.53.

[0179] The liquid chromatogram of compound 9d is as follows Fig.12 As shown in the mass spectrum Fig.13 shown.

[0180] Example 5

[0181]

[0182] Reaction conditions and reagents for the synthesis of trimer CGA-Amidite: (a) 1 H -tetrazolyl, DCM, 12 h; (b) t -BuOOH, DCM, 0.5 h; (c) resin, MeOH, 12 h; (d) Et3N·3HF, Et3N, DCM, 12 h; (e) 2-cyanoethyl- N,N,N', N' -Tetraisopropylphosphorodiamidite, 1H -tetrazolyl diisopropylammonium salt, DCM, 2 h.

[0183] N 4-Benzoyl- P -methyl-5'- O -Dimethoxytrityl-2'-deoxycytidine-(3'→5')- N 2 -isobutyryl-2'-deoxyguanosine-(3'→5')- N 6 - Synthesis of benzoyl-2'-deoxyadenosine (8e):

[0184] 1c (15.00 g, 1.05 eq) and 2a (8.37 g, 1.00 eq) were dissolved in anhydrous dichloromethane and 1 H -tetrazolyl (1.00 g, 0.80 eq) was stirred at room temperature for 12 hours under N2 protection to obtain compound 3c. Anhydrous t-BuOOH (1.61 g, 1.00 eq) was directly added to the mixture and stirred at room temperature for 0.5 hours to obtain 4c. The solvent was evaporated by rotary concentrator, then dissolved in methanol, added to resin, and stirred at room temperature for 12 hours to obtain crude compound 5c. After filtration, column chromatography purification (DCM / MeOH (0-10%)) was performed, and the solvent was evaporated by rotary concentrator to obtain compound 5c.

[0185] Weigh 5c (4.00 g, 1.00 eq) and 1b (3.96 g, 1.10 eq) in a 200 mL round-bottom flask, add dichloromethane and acetonitrile to remove water. Then dissolve in anhydrous dichloromethane and add 1 H -tetrazolyl (0.25 g, 0.80 eq), stirred at room temperature for 12 hours under N2 protection to obtain compound 6e. Anhydrous t-BuOOH (0.41 g, 1.00 eq) was directly added to the mixture and stirred at room temperature for 0.5 hours to obtain compound 7e. Et3N (0.92 g, 2.00 eq) and Et3N·3HF (2.19 g, 3.00 eq) were added and stirred at room temperature for 14 hours. After the reaction was complete, saturated NaHCO3 was added until no bubbles were generated. The mixture was extracted twice with dichloromethane and water. The combined organic layer was washed with saturated brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo to obtain crude compound 8e. Purification by column chromatography (DCM / MeOH (0-10%)), and then rotary concentrated to evaporate the solvent. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether, filtered, and dried under vacuum to obtain compound 8e, 4.56 g of white solid, with a purity of 98.8% and a yield of 68.08%. ESI-MS: m / z calcd for C 70 H 73 N 13 O 20 P2[M / 2+Na] +761.73, found:762.16.

[0186] The mass spectrum of compound 8e is shown in Fig.14 shown.

[0187] N 4 -Benzoyl- P -methyl-5'- O -Dimethoxytrityl-2'-deoxycytidine-(3'→5')-N 2 -isobutyryl-2'-deoxyguanosine-(3'→5')- N 6 -Benzoyl-3'- O Synthesis of -[(N,N-diisopropylamino)-cyanoethylphosphino]-2'-deoxyadenosine (9e):

[0188] Compound 8e (3.00 g, 1.00 eq) was dissolved in dichloromethane and toluene and evaporated to dryness under reduced pressure to remove water from the raw material. Then it was dissolved in 20 mL of anhydrous dichloromethane, and cyanoethyl phosphoramidite (1.22 g, 2.00 eq) and 1 H -tetrazolyl diisopropylammonium salt (0.70 g, 2.00 eq) was reacted at room temperature for 2 h under N2 protection. After the reaction was complete, it was purified by column chromatography (EA (1% Et3N) -ACN (15% MeOH)), and then the solvent was evaporated by rotary concentrator. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether at -78°C, filtered, and dried in vacuo to obtain compound 9e, 2.68 g of white solid, purity 98.5%, yield 78.7%. ESI-MS: m / z calcd for C 79 H 90 N 15 O 21 P3[M+H] + 1678.56, found:1678.55.

[0189] The liquid chromatogram of compound 9e is shown in Fig.15 As shown in the mass spectrum Fig.16 shown.

[0190] Example 6

[0191]

[0192] Reaction conditions and reagents for the synthesis of trimer TGA-Amidite: (a) 1 H -tetrazolyl, DCM, 12 h; (b) t-BuOOH, DCM, 0.5 h; (c) resin, MeOH, 12 h; (d) Et3N·3HF, Et3N, DCM, 12 h; (e) 2-cyanoethyl- N,N,N', N' -Tetraisopropylphosphorodiamidite, 1H -tetrazolyl diisopropylammonium salt, DCM, 2 h.

[0193] P -methyl-5'- O -Dimethoxytrityl-thymidine-(3'→5')- N 2 -isobutyryl-2'-deoxyguanosine-(3'→5')- N 6 - Synthesis of benzoyl-2'-deoxyadenosine (8f):

[0194] 1c (15.00 g, 1.05 eq) and 2a (8.37 g, 1.00 eq) were dissolved in anhydrous dichloromethane and 1 H -tetrazolyl (1.00 g, 0.80 eq) was stirred at room temperature for 12 hours under N2 protection to obtain compound 3c. Anhydrous t-BuOOH (1.61 g, 1.00 eq) was directly added to the mixture and stirred at room temperature for 0.5 hours to obtain 4c. The solvent was evaporated by rotary concentrator, then dissolved in methanol, added to resin, and stirred at room temperature for 12 hours to obtain crude compound 5c. After filtration, column chromatography purification (DCM / MeOH (0-10%)) was performed, and the solvent was evaporated by rotary concentrator to obtain compound 5c.

[0195] Weigh 5c (7.50 g, 1.00 eq) and 1d (6.59 g, 1.10 eq) in a 200 mL round-bottom flask, add dichloromethane and acetonitrile to remove water. Then dissolve in anhydrous dichloromethane and add 1 H-tetrazole (0.48 g, 0.80 eq), stirred at room temperature for 12 hours under N2 protection to obtain compound 6f. Anhydrous t-BuOOH (0.77 g, 1.00 eq) was directly added to the mixture and stirred at room temperature for 0.5 hours to obtain compound 7f. Et3N (1.72 g, 2.00 eq) and Et3N·3HF (4.11 g, 3.00 eq) were added and stirred at room temperature for 12 hours. After the reaction was complete, saturated NaHCO3 was added until no bubbles were generated. The mixture was extracted twice with dichloromethane and water. The combined organic layer was washed with saturated brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo to obtain crude compound 8f. Purification by column chromatography (DCM / MeOH (0-10%)), and then rotary concentrated to evaporate the solvent. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether, filtered, and dried under vacuum to obtain compound 8f as a white solid (9.21 g), with a purity of 97.6% and a yield of 78.04%. ESI-MS: m / z calcd for C 64 H 70 N 12 O 20 P2[M / 2+Na] + 717.22, found:717.43.

[0196] The mass spectrum of compound 8f is shown in Fig.17 shown.

[0197] P -methyl-5'- O -Dimethoxytrityl-thymidine-(3'→5')- N 2 -isobutyryl-2'-deoxyguanosine-(3'→5')- N 6 -Benzoyl-3'- O Synthesis of -[(N,N-diisopropylamino)-cyanoethylphosphino]-2'-deoxyadenosine (9f):

[0198] Compound 8f (4.86 g, 1.00 eq) was dissolved in dichloromethane and toluene and evaporated to dryness under reduced pressure to remove the water in the raw material. Then it was dissolved in 30 mL of anhydrous dichloromethane, and cyanoethyl phosphoramidite (1.56 g, 2.00 eq) and 1 H-tetrazolyl diisopropylammonium salt (0.89 g, 2.00 eq) was reacted at room temperature for 2 h under N2 protection. After the reaction was complete, it was purified by column chromatography (EA (1% Et3N) -ACN (15% MeOH)), and then the solvent was evaporated by rotary concentrator. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether at -78°C, filtered, and vacuum dried to obtain compound 9f, 3.29 g of white solid, purity 98.5%, yield 79.9%. ESI-MS: m / z calcd for C 73 H 87 N 14 O 21 P3[MH] - 1587.53, found:1587.50.

[0199] The liquid chromatogram of compound 9f is as follows Fig.18 As shown in the mass spectrum Fig.19 shown.

[0200] Example 7

[0201]

[0202] Reaction conditions and reagents for the synthesis of trimer CGG-Amidite: (a) 1 H -tetrazolyl, DCM, 12 h; (b) t -BuOOH, DCM, 0.5 h; (c) resin, MeOH, 12 h; (d) Et3N·3HF, Et3N, DCM, 12 h; (e) 2-cyanoethyl- N,N,N', N' -Tetraisopropylphosphorodiamidite, 1H -tetrazolyl diisopropylammonium salt, DCM, 2 h.

[0203] N 4 -Benzoyl- P -methyl-5'- O -Dimethoxytrityl-2'-deoxycytidine-(3'→5')-N2-isobutyryl-2'-deoxyguanosine-(3'→5')- N 2 -Synthesis of isobutyryl-2'-deoxyguanosine (8 g):

[0204] Dissolve 1c (18.00 g, 1.05 eq) and 2c (9.66 g, 1.00 eq) in anhydrous dichloromethane and add 1 H-tetrazolyl (1.20 g, 0.80 eq) was stirred at room temperature for 12 hours under N2 protection to obtain compound 3d. Anhydrous t-BuOOH (1.93 g, 1.00 eq) was directly added to the mixture and stirred at room temperature for 0.5 hours to obtain 4d. The solvent was evaporated by rotary concentrator, then dissolved in methanol, added to resin, and stirred at room temperature for 12 hours to obtain crude compound 5d. After filtration, column chromatography purification (DCM / MeOH (0-10%)) was performed, and the solvent was evaporated by rotary concentrator to obtain compound 5d.

[0205] Weigh 5d (6.00 g, 1.00 eq) and 1b (6.07 g, 1.10 eq) in a 200 mL round-bottom flask, add dichloromethane and acetonitrile to remove water. Then dissolve in anhydrous dichloromethane and add 1 H -tetrazolyl (0.39 g, 0.80 eq), stirred at room temperature for 12 hours under N2 protection to obtain compound 6g. Anhydrous t-BuOOH (0.63 g, 1.00 eq) was directly added to the mixture and stirred at room temperature for 0.5 hours to obtain compound 7g. Et3N (1.40 g, 2.00 eq) and Et3N·3HF (3.36 g, 3.00 eq) were added and stirred at room temperature for 12 hours. After the reaction was complete, saturated NaHCO3 was added until no bubbles were generated. The mixture was extracted twice with dichloromethane and water. The combined organic layer was washed with saturated brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo to obtain crude compound 8g. Purification by column chromatography (DCM / MeOH (0-10%)), and then rotary concentrated to evaporate the solvent. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether, filtered, and dried under vacuum to obtain 8 g of compound, 7.33 g of white solid, with a purity of 98.2% and a yield of 72.35%. ESI-MS: m / z calcd for C 67 H 75 N 13 O 21 P21459.47, found: 1458.41.

[0206] The mass spectrum of compound 8f is shown in Fig. 20 shown.

[0207] N 4 -Benzoyl- P -methyl-5'- O -Dimethoxytrityl-2'-deoxycytidine-(3'→5')- N 2 -isobutyryl-2'-deoxyguanosine-(3'→5')- N 2 -isobutyryl-3'-O Synthesis of -[(N,N-diisopropylamino)-cyanoethylphosphino]-2'-deoxyguanosine (9 g):

[0208] Dissolve compound 8g (3.00 g, 1.00 eq) in dichloromethane and toluene, evaporate to dryness under reduced pressure to remove water from the raw material. Then dissolve in 20 mL of anhydrous dichloromethane, add cyanoethyl phosphoramidite (1.24 g, 2.00 eq) and 1 H -Tetrazole diisopropylammonium salt (0.79 g, 2.00 eq), react at room temperature for 2 h under N2 protection. After the reaction is complete, column chromatography purification (DCM (0.4% Et3N) / MeOH (0-10%)), then rotary concentration and evaporation of the solvent. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether at -78°C, filtered, and vacuum dried to obtain compound 9g, 2.51 g white solid, purity 98.7%, yield 73.6%. ESI-MS: m / z calcd for C 76 H 92 N 15 O 22 P3[M+H] + 1660.57, found:1660.58.

[0209] The liquid chromatogram of compound 9g is as follows Fig.21 As shown in the mass spectrum Fig. 22 shown.

[0210] Example 8

[0211]

[0212] Reaction conditions and reagents for the synthesis of trimer TGG-Amidite: (a) 1 H -tetrazolyl, DCM, 12 h; (b) t -BuOOH, DCM, 0.5 h; (c) resin, MeOH, 12 h; (d) Et3N·3HF, Et3N, DCM, 12 h; (e) 2-cyanoethyl- N,N,N', N' -Tetraisopropylphosphorodiamidite, 1H -tetrazolyl diisopropylammonium salt, DCM, 2 h.

[0213] P -methyl-5'- O -Dimethoxytrityl-thymidine-(3'→5')- N 2 -isobutyryl-2'-deoxyguanosine-(3'→5')-N 2-Synthesis of isobutyryl-2'-deoxyguanosine (8h):

[0214] Dissolve 1c (18.00 g, 1.05 eq) and 2c (9.66 g, 1.00 eq) in anhydrous dichloromethane and add 1 H -tetrazolyl (1.20 g, 0.80 eq) was stirred at room temperature for 12 hours under N2 protection to obtain compound 3d. Anhydrous t-BuOOH (1.93 g, 1.00 eq) was directly added to the mixture and stirred at room temperature for 0.5 hours to obtain 4d. The solvent was evaporated by rotary concentrator, then dissolved in methanol, added to resin, and stirred at room temperature for 12 hours to obtain crude compound 5d. After filtration, column chromatography purification (DCM / MeOH (0-10%)) was performed, and the solvent was evaporated by rotary concentrator to obtain compound 5d.

[0215] Weigh 5d (4.00 g, 1.00 eq) and 1d (3.59 g, 1.10 eq) in a 200 mL round-bottom flask, add dichloromethane and acetonitrile to remove water. Then dissolve in anhydrous dichloromethane and add 1 H -tetrazolyl (0.26 g, 0.80 eq), stirred at room temperature for 12 hours under N2 protection to obtain compound 6h. Anhydrous t-BuOOH (0.42 g, 1.00 eq) was directly added to the mixture and stirred at room temperature for 0.5 hours to obtain compound 7h. Et3N (0.94 g, 2.00 eq) and Et3N·3HF (2.24 g, 3.00 eq) were added and stirred at room temperature for 12 hours. After the reaction was complete, saturated NaHCO3 was added until no bubbles were generated. The mixture was extracted twice with dichloromethane and water. The combined organic layer was washed with saturated brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo to obtain crude compound 8h. Purification by column chromatography (DCM / MeOH (0-10%)), and then rotary concentrated to evaporate the solvent. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether, filtered, and dried under vacuum to obtain compound 8h as a white solid (4.47 g), with a purity of 97.1% and a yield of 70.48%. ESI-MS: m / z calcd for C 61 H 72 N 12 O 21 P21370.44, found: 1369.41.

[0216] The mass spectrum of compound 8h is shown in Fig.23 shown.

[0217] P -methyl-5'- O -Dimethoxytrityl-thymidine-(3'→5')-N 2 -isobutyryl-2'-deoxyguanosine-(3'→5')- N 2 -isobutyryl-3'- O Synthesis of -[(N,N-diisopropylamino)-cyanoethylphosphino]-2'-deoxyguanosine (9h):

[0218] Compound 8h (3.00 g, 1.00 eq) was dissolved in dichloromethane and toluene, and evaporated to dryness under reduced pressure to remove the water in the raw material. Then it was dissolved in 30 mL of anhydrous dichloromethane, and cyanoethyl phosphoramidite (1.32 g, 2.00 eq) and 1 H -tetrazolyl diisopropylammonium salt (0.75 g, 2.00 eq) was reacted at room temperature for 2 h under N2 protection. After the reaction was complete, it was purified by column chromatography (EA (1% Et3N) -ACN (15% MeOH)), and then the solvent was evaporated by rotary concentrator. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether at -78°C, filtered, and dried in vacuo to obtain compound 9h, 2.67 g of white solid, purity 98.7%, yield 77.7%. ESI-MS: m / z calcd for C 70 H 89 N 14 O 22 P3[M+H] + 1570.52, found:1570.54.

[0219] The liquid chromatogram of compound 9h is as follows Fig.24 As shown in the mass spectrum Fig.25 shown.

[0220] Structural characterization

[0221] (1) The liquid phase purity and mass spectrometry identification results of trimer precursors 8a~8h are shown in Table 1.

[0222] Table 1 Liquid phase purity and mass spectrometry identification results of trimer precursors 8a~8h

[0223]

[0224] As can be seen from Table 1, the purity of the nucleoside trimer precursor obtained by the present invention can reach more than 97%.

[0225] (2) The liquid phase purity and mass spectrometry identification results of nucleoside trimers 9a~9h are shown in Table 2.

[0226] Table 2 Liquid phase purity and mass spectrometry identification results of nucleoside trimers 9a~9h

[0227]

[0228] As can be seen from Table 2, the HPLC purity of the nucleoside trimer obtained by the present invention can reach up to 98.5% or more, which can meet the requirements of high-fidelity DNA synthesis. The synthesis experiment of the new skeleton nucleoside trimer overcomes the problems of high cost and difficulty in synthesizing the trimer, verifies the feasibility of the synthetic route, and the synthetic route is simple to operate and can achieve large-scale preparation, laying the foundation for subsequent expansion of production.

[0229] Application Example 1

[0230] The present invention further uses the trimer for the synthesis of oligonucleotide sequences. On a LK-192 DNA synthesizer, a 50 nmol synthesis column is used, the trimer concentration is 70 mg / mL, the coupling catalyst is 4,5-dicyanoimidazole, and the coupling time is 150 s. The synthesizer single cycle synthesis program is shown in Table 3.

[0231] Table 3 Synthesizer single cycle synthesis program

[0232]

[0233] A 40 nt fragment was synthesized by using a trimer (CAG) and a common monomer mixed synthesis method. The sequence is shown in SEQ ID NO.1, specifically: CAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGT.

[0234] After the synthesis is completed, ammonia is used for ammonolysis at 95°C for 2 h in an ammonolysis instrument with an ammonia pressure of 470-520 kPa. After the ammonolysis is completed, desalting and purification are performed. First, 200 μL of 90% acetonitrile is added to the synthesis column and centrifuged at 500 r / min for 2 min. Then, 200 μL of pure water is 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 oligonucleotide aqueous solution.

[0235] After the synthesis is completed, the mass spectrometry results of the obtained trimer and monomer combined with the synthesized oligonucleotide sequence are as follows: Fig.26 As shown. It can be seen that the target sequence is obtained in the present invention, and the molecular weight is accurate, ESI-MS (m / z) 12295.3, calculated MS: 12295.74. It is proved that the nucleoside trimer obtained in the present invention can be used for oligonucleotide synthesis, and the feasibility of the oligonucleotide synthesis method is also proved.

[0236] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a nucleoside trimer having a structure of any one of formulas a to h, characterized in that: The following steps are involved: In the presence of 1H-tetrazole, a nucleoside having a structure shown in Formula 1-1 and a 5′-OH-3′-O-TBDMS nucleoside having a structure shown in Formula 2 undergo a first coupling reaction to obtain a dimer precursor having a structure shown in Formula 3; The dimer precursor having the structure shown in Formula 3 is subjected to a first oxidation reaction with tert-butyl hydroperoxide to obtain a dipolyphosphate diester having the structure shown in Formula 4; The diphosphoric acid diester having the structure shown in Formula 4 is mixed with Amberlyst 15 ion exchange resin, and a 5'-DMTr protecting group is removed to obtain a 5'-OH dimer having the structure shown in Formula 5; In the presence of 1H-tetrazole, the 5'-OH dimer having the structure shown in Formula 5 and the nucleoside having the structure shown in Formula 1-2 undergo a second coupling reaction to obtain a trimer precursor having the structure shown in Formula 6; The trimer precursor having the structure shown in Formula 6 is subjected to a second oxidation reaction with tert-butyl hydroperoxide to obtain a tripolyphosphate diester having the structure shown in Formula 7; The tripolyphosphate diester having the structure shown in Formula 7 is subjected to a TBDMS protecting group removal reaction to obtain a trimer precursor having the structure shown in Formula 8; the deprotection reagent used in the TBDMS protecting group removal reaction includes Et3N and Et3N·3HF; Under the action of 1H-tetrazolyl diisopropylammonium salt ammonium salt catalyst, a trimer precursor having a structure shown in Formula 8 is subjected to a phosphoramidite reaction with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite to obtain a nucleoside trimer having a structure of any one of Formulas a to h; The definitions of B1, B2 and B3 in Formula 1-1, Formula 2, Formula 3, Formula 4, Formula 5, Formula 1-2, Formula 6, Formula 7 and Formula 8 are the same as those of B1, B2 and B3 in Formulas a to h.

2. The preparation method according to claim 1, characterized in that: The temperature of the first coupling reaction is room temperature and the time is 8 to 14 hours; The temperature of the first oxidation reaction is room temperature, and the time is 0.4 to 1 hour.

3. The preparation method according to claim 1, characterized in that: The temperature of the 5'-DMTr protection group removal reaction is room temperature, and the time is 8 to 14 hours.

4. The preparation method according to claim 1, characterized in that: The temperature of the second coupling reaction is room temperature and the time is 8 to 14 hours; The temperature of the second oxidation reaction is room temperature, and the time is 0.4 to 1 hour.

5. The preparation method according to claim 1, characterized in that: The time for the TBDMS protection group removal reaction is 6 to 14 hours; The time of the phosphoramidite reaction is 1 to 3 hours.

6. The preparation method according to claim 1 or 5, characterized in that: After the phosphoramidite reaction, the obtained phosphoramidite reaction solution is subjected to post-treatment, and the post-treatment comprises the following steps: The phosphoramidite reaction solution is subjected to flash column separation and purification to obtain a purified product; the mobile phase used in the flash column separation and purification is an ethyl acetate-acetonitrile system, the ethyl acetate contains 1% triethylamine by volume, and the acetonitrile contains 15% methanol by volume; 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 nucleoside trimer having a structure represented by any one of formulas a to h.

7. A method for synthesizing an oligonucleotide, comprising the following steps: (1) preparing a nucleoside trimer according to the preparation method according to any one of claims 1 to 6; (2) mixing the nucleoside trimer with a coupling catalyst to perform a coupling reaction to obtain a coupling reaction product; (3) The coupling reaction product is sequentially subjected to removal of the DMTr protecting group, aminolysis and desalting purification to obtain an oligonucleotide.

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