CN118754922A - An allyl-cyanoethyl nucleoside dimer and its preparation method and application in oligonucleotide synthesis - Google Patents

Abstract

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

Description

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

Technical Field

The invention relates to the technical field of DNA synthesis, and in particular to an allyl-cyanoethyl nucleoside dimer and a preparation method thereof and application thereof in oligonucleotide synthesis.

Background Art

With the rapid development of synthetic biology, the demand for long-fragment, high-fidelity, and low-cost oligonucleotide synthesis is increasing. Due to the inherent limitations of chemical reactions, the length of commercially synthesized oligonucleotides is generally controlled below 100 nt. There are two main factors that affect 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 continues to decrease; second, side reactions such as depurination and cyanoethyl addition will further reduce the yield of oligonucleotides and lead to synthesis errors. Therefore, new chemical synthesis methods are needed to minimize side reactions and increase yields to obtain high-quality long-fragment oligonucleotides.

The use of dimer nucleoside modules to synthesize oligonucleotides reduces the number of synthesis steps and acid deprotection times by half. For example, a sequence that requires 100 times of synthesis with ordinary monomers can be completed with only 50 times using dimers, which significantly reduces the number of reaction steps and the probability of errors. In early studies, G. Kumar's research team successfully synthesized short oligonucleotides (about 20 nt) using dimer phosphoramidite modules. However, the reported dimer nucleosides have problems such as unstable structure, difficult purification, and complex synthesis, which cannot meet the needs of oligonucleotide synthesis based on modular strategies.

In the early stage, our research team invented a methyl-cyanoethyl mixed skeleton dimer, the structure of which is shown in Formula II:

Formula II.

The dimer structure is stable and easy to prepare, resulting in a high-purity, scalable, and easy-to-operate synthetic route to synthesize an oligonucleotide fragment of 100 nt in length. However, since the methyl group is difficult to deprotect using ammonia, the oligonucleotide sequence synthesized using this dimer structure requires stronger deprotection conditions to be completely deprotected, and needs to be immersed in ammonia water at 70°C for 12 hours. The extension of the aminolysis time increases uncertainty and may cause the breakage of the oligonucleotide chain and the generation of side reactions. In addition, this structure cannot be used to synthesize alkali-unstable compounds.

Summary of the invention

In view of this, the object of the present invention is to provide an allyl-cyanoethyl nucleoside dimer and a preparation method thereof 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.

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

The present invention provides an allyl-cyanoethyl nucleoside dimer having a structure shown in Formula I:

Formula I;

In Formula I, X is O or S; _B1 and _B2 are independently selected from substituted or unsubstituted bases.

Preferably, the _B1 is selected from any one of cytosine, guanine, adenine, thymine, uracil, hypoxanthine, xanthine, deazaadenine, deazaguanine, deazahypoxanthine, dihydrouracil and pseudouracil;

The _B2 is selected from any one of cytosine, guanine, adenine, thymine, uracil, hypoxanthine, xanthine, deazaadenine, deazaguanine, deazahypoxanthine, dihydrouracil and pseudouracil.

Preferably, B ₁ and B ₂ are independently selected from the following groups, " represents the 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 present invention provides a method for preparing the above-mentioned allyl-cyanoethyl nucleoside dimer, comprising the following steps:

(1) A 5ʹ-DMTr-3ʹ-O-allyl phosphoramidite nucleoside having a structure shown in Formula 1 is coupled with a 5ʹ-OH-3ʹ-O-TBDMS nucleoside having a structure shown in Formula 2 to obtain a dimer having a structure shown in Formula 3;

Formula 1; Formula 2; 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 S ₈ and N,O -bistrimethylsilylacetamide, and subjected to a sulfurization reaction to obtain a diphosphoric acid diester having the structure shown in Formula 4;

Formula 4;

(3) The diphosphodiester having the structure shown in Formula 4 is subjected to a TBDMS protecting group removal reaction to obtain a 3ʹ-OH dimer precursor having the structure shown in Formula 5;

Formula 5;

(4) Under the action of 1H -tetrazolyl diisopropylammonium salt catalyst, the 3ʹ-OH dimer precursor having the structure shown in Formula 5 is phosphorylated with 2-cyanoethyl- N,N,Nʹ,Nʹ -tetraisopropylphosphorodiamidite to obtain an allyl-cyanoethyl nucleoside dimer having the structure shown in Formula I.

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

The molar ratio of the 5ʹ-DMTr-3ʹ-O-allyl phosphoramidite nucleoside having the structure shown in Formula 1 to 1 H -tetrazolyl is (1-2):(0.5-1);

The coupling reaction time is 8 to 14 h.

Preferably, 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 S ₈ and N,O -bistrimethylsilylacetamide is 1:(1-2):(3-6);

The temperature of the vulcanization reaction is 40-60°C and the time is 20-50 min.

Preferably, the molar ratio of the 3ʹ-OH dimer precursor having the structure shown in Formula 5 to 2-cyanoethyl- N,N,Nʹ,Nʹ -tetraisopropylphosphorodiamidite is 1:1.4;

The molar ratio of the 3ʹ-OH dimer precursor having the structure shown in Formula 5 to the 1 H -tetrazolyl diisopropylammonium salt catalyst is 1:(1-3);

The phosphorylation reaction time is 1 to 3 h.

Preferably, 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.

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

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

The allyl-cyanoethyl nucleoside dimer is mixed with a coupling catalyst to carry out 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.

The present invention provides an allyl-cyanoethyl nucleoside dimer having a structure shown in formula I. Compared with the methyl-cyanoethyl mixed skeleton dimer shown in formula II, the allyl-cyanoethyl nucleoside dimer provided by the present invention adopts a mixed skeleton of allyl phosphate and β-cyanoethyl phosphoramidite, and the methyl protecting group of the phosphoric acid in the middle of the dimer structural unit is replaced by allyl protection. When allyl is used as a protecting group, it can be removed under ammonia conditions or mild non-alkaline conditions. In addition, the allyl protecting group has high selectivity for some functional groups, and can achieve protection of the target group without affecting the reaction of other groups. The present invention replaces the methyl protecting group of the phosphoric acid in the middle of the dimer structural unit with allyl protection, and 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 step, shorten the deprotection time, and improve the deprotection efficiency and the purity of the product. In addition, the use of allyl groups can synthesize alkali-unstable modified nucleosides, such as acetylcytidine, which will further expand the scope of application of dimer synthesis. The synthesis results show that the aminolysis time of the oligonucleotide synthesized using the allyl-cyanoethyl dimer is significantly shorter than that of the methyl-cyanoethyl dimer. All protecting groups can be removed by aminolysis under ammonia conditions at 95°C for only 2 h, thus avoiding sequence errors caused by long-term aminolysis.

In addition, the present invention provides a method for preparing the above-mentioned allyl-cyanoethyl nucleoside dimer. Based on the basic principle of solid-phase phosphoramidite oligonucleotide synthesis, the present invention establishes a simple and scalable nucleoside module reagent synthesis method, and obtains a high-purity, new-structure allyl-cyanoethyl nucleoside dimer. The present invention adopts a "one-pot" synthesis method, coupling-oxidation-deprotection is carried out in the same container, the operation is simple, the cost is low, and it is easy to achieve industrial mass production.

In addition, the present invention optimizes and establishes an oligonucleotide synthesis method based on modular synthesis, optimizes the aminolysis conditions, and can obtain an oligonucleotide fragment of 80 nt in length. When the allyl-cyanoethyl nucleoside dimer provided by the present invention is used for oligonucleotide synthesis, the application range of the dimer can be broadened, the deprotection process can be simplified, and it is expected to further extend the length of dimer synthesis and reduce the synthesis error rate, which has practical value.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is the synthetic route diagram of allyl-cyanoethyl nucleoside dimer;

FIG2 is a liquid chromatogram of compound 5a;

FIG3 is a mass spectrum of compound 5a;

FIG4 is a liquid chromatogram of compound 6a;

FIG5 is a mass spectrum of compound 6a;

FIG6 is a liquid chromatogram of compound 5b;

FIG7 is a mass spectrum of compound 5b;

FIG8 is a liquid chromatogram of compound 5c;

FIG9 is a mass spectrum of compound 5c;

FIG10 is a liquid chromatogram of compound 5d;

FIG11 is a mass spectrum of compound 5d;

FIG12 is a liquid chromatogram of compound 5e;

FIG13 is a mass spectrum of compound 5e;

FIG14 is a liquid chromatogram of compound 6e;

FIG15 is a mass spectrum of compound 6e;

FIG16 is a liquid chromatogram of compound 5f;

FIG17 is a mass spectrum of compound 5f;

FIG18 is a liquid chromatogram of compound 6f;

FIG19 is a mass spectrum of compound 6f;

FIG20 is a liquid chromatogram of compound 5g;

FIG21 is a mass spectrum of compound 5g;

FIG22 is a liquid chromatogram of compound 6g;

FIG23 is a mass spectrum of compound 6g;

FIG24 is a mass spectrum of oligonucleotide aminolysis products synthesized from methyl dimer and allyl-cyanoethyl nucleoside dimer;

FIG. 25 is a mass spectrometry detection diagram of the 80 nt sequence synthesized from the allyl-cyanoethyl nucleoside dimer.

DETAILED DESCRIPTION

The present invention provides an allyl-cyanoethyl nucleoside dimer having a structure shown in Formula I:

Formula I;

In Formula I, X is O or S; _B1 and _B2 are independently selected from substituted or unsubstituted bases.

In the present invention, the _B1 is preferably selected from any one of cytosine, guanine, adenine, thymine, uracil, hypoxanthine, xanthine, deazaadenine, deazaguanine, deazahypoxanthine, dihydrouracil and pseudouracil;

The _B2 is preferably selected from any one of cytosine, guanine, adenine, thymine, uracil, hypoxanthine, xanthine, deazaadenine, deazaguanine, deazahypoxanthine, dihydrouracil and pseudouracil.

In the present invention, _B1 and _B2 are independently selected from the following groups, " represents the group connection position:

;

;

Wherein, R', R'' and R''' are 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.

Compared to the methyl-cyanoethyl mixed skeleton dimer shown in formula II, the present invention replaces the methyl protecting group of the phosphoric acid in the middle of the dimer with an allyl protecting group that is easier to remove, and the cyanoethyl protecting group at the 3'-OH end of the dimer is relatively easy to remove, and the cyanoethyl phosphoramidite synthetic raw material is cheaper and easier to obtain. The present invention uses allyl as a protecting group, which can be removed under ammonia conditions or mild non-alkaline conditions. In addition, the allyl protecting group has a higher selectivity for some functional groups, and the protection of the target group can be achieved without affecting the reaction of other groups. The present invention replaces the methyl protecting group of the phosphoric acid in the middle of the dimer structural unit with allyl protection, and 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 step, and shorten the deprotection time, improve the deprotection efficiency and the purity of the product. In addition, the use of allyl groups can synthesize alkali-unstable modified nucleosides, such as acetylcytidine, which will further expand the scope of application of dimer synthesis.

In the present invention, the allyl-cyanoethyl nucleoside dimer preferably has the structure shown below:

.

The present invention provides a method for preparing the above-mentioned allyl-cyanoethyl nucleoside dimer, comprising the following steps:

(1) A 5ʹ-DMTr-3ʹ-O-allyl phosphoramidite nucleoside having a structure shown in Formula 1 is coupled with a 5ʹ-OH-3ʹ-O-TBDMS nucleoside having a structure shown in Formula 2 to obtain a dimer having a structure shown in Formula 3;

Formula 1; Formula 2; 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 S ₈ and N,O -bistrimethylsilylacetamide, and subjected to a sulfurization reaction to obtain a diphosphoric acid diester having the structure shown in Formula 4;

Formula 4;

(3) The diphosphodiester having the structure shown in Formula 4 is subjected to a TBDMS protecting group removal reaction to obtain a 3ʹ-OH dimer precursor having the structure shown in Formula 5;

Formula 5;

(4) Under the action of 1H -tetrazolyl diisopropylammonium salt catalyst, the 3ʹ-OH dimer precursor having the structure shown in Formula 5 is phosphorylated with 2-cyanoethyl- N,N,Nʹ,Nʹ -tetraisopropylphosphorodiamidite to obtain an allyl-cyanoethyl nucleoside dimer having the structure shown in Formula I.

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

In the present invention, 5ʹ-DMTr-3ʹ-O-allyl phosphoramidite nucleoside having a structure shown in Formula 1 is mixed with 5ʹ-OH-3ʹ-O-TBDMS nucleoside having a structure shown in Formula 2, and a coupling reaction is performed to obtain a dimer having a structure shown in Formula 3. In the present invention, the coupling reaction is preferably performed in the presence of 1H -tetrazole, and the molar ratio of 5ʹ-DMTr-3ʹ-O-allyl phosphoramidite nucleoside having a structure shown in Formula 1 to 1H -tetrazole is preferably (1-2):(0.5-1), and more preferably 1.05:0.8.

In the present invention, the molar ratio of the 5ʹ-DMTr-3ʹ-O-allyl phosphoramidite nucleoside having the structure shown in Formula 1 to the 5ʹ-OH-3ʹ-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.

In the present invention, the coupling reaction is preferably carried out under _N2 protection, the coupling reaction temperature is preferably room temperature, and the time is preferably 8 to 14 h, more preferably 10 to 13 h. After the coupling reaction, the present invention does not perform post-treatment, and the obtained coupling reaction solution is directly used for subsequent reactions.

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, performs an oxidation reaction, and obtains a diphosphoric acid diester having the structure shown in Formula 4. In the present invention, tert-butyl hydrogen peroxide has a fast oxidation speed and does not affect subsequent reactions, replacing the iodine oxidation method of oxidation in THF/lutidine/water solution according to phosphoramidite chemical standard conditions, avoiding the trouble caused by drying excess iodine aqueous solution.

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.

In the present invention, the oxidation reaction temperature is preferably room temperature, and the time is preferably 0.4 to 1 h, more preferably 0.5 to 0.8 h. After the oxidation reaction, the present invention does not perform post-treatment, and the obtained oxidation reaction liquid is directly used for subsequent reactions.

Alternatively, when X is S, the dimer having the structure shown in Formula 3 of the present invention is mixed with S ₈ and N,O -bistrimethylsilylacetamide and subjected to a sulfurization reaction to obtain a diphosphoric acid diester having the structure shown in Formula 4.

In the present invention, the molar ratio of the dimer having the structure shown in Formula 3 to S ₈ and N,O -bistrimethylsilylacetamide is 1:(1-2):(4-6), more preferably 1:1.5:5; the temperature of the sulfurization reaction is preferably 40-60°C, more preferably 50°C, and the time is preferably 20-50 min, more preferably 30 min. After the sulfurization reaction, the present invention vacuum concentrates to obtain a sulfurized product, and the obtained sulfurized product is directly used for subsequent reactions.

After obtaining the dipolyphosphate diester having the structure shown in Formula 4, the present invention performs a TBDMS protection group removal reaction on the dipolyphosphate diester having the structure shown in Formula 4 to obtain a 3ʹ-OH dimer precursor having the structure shown in Formula 5. In the present invention, the deprotection reagent used in the TBDMS protection group removal reaction preferably includes Et ₃ N and Et ₃ N·3HF. In the present invention, the molar ratio of the dipolyphosphate diester having the structure shown in Formula 4 to Et ₃ N is preferably 1:(1-3), more preferably 1:2; the molar ratio of the dipolyphosphate diester having the structure shown in Formula 4 to Et ₃ N·3HF is preferably 1:(2-4), more preferably 1:3.

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.

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:

The TBDMS protection group removal reaction solution was mixed with a saturated NaHCO ₃ solution until no bubbles were generated, the resulting mixture was extracted twice with dichloromethane and water, the organic phases were combined, and the resulting organic phases were washed, dried, filtered and concentrated to obtain a crude 3ʹ-OH dimer precursor;

The crude 3ʹ-OH dimer precursor is purified by column chromatography and the solvent is evaporated by rotary concentrator. The obtained residue is dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether, filtered and dried to obtain a pure 3ʹ-OH dimer precursor having a structure shown in Formula 5.

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

After obtaining a 3ʹ-OH dimer precursor having a structure shown in Formula 5, the present invention performs a phosphorylation reaction on the 3ʹ-OH dimer precursor having a structure shown in Formula 5 and 2-cyanoethyl-N, N ,Nʹ,Nʹ -tetraisopropylphosphodiamidite under the action of a 1H-tetrazolyl diisopropylammonium salt catalyst to obtain an allyl-cyanoethyl nucleoside dimer having a structure shown in Formula I. The present invention uses a 1H -tetrazolyl diisopropylammonium salt catalyst as a catalyst and 2-cyanoethyl- N,N,Nʹ,Nʹ -tetraisopropylphosphodiamidite as a phosphorylation reagent. Since the acidity of 1H -tetrazole is similar to acetic acid, 1H -tetrazolyl diisopropylammonium salt is used as a catalyst to avoid a DMTr removal side reaction that may occur during a longer coupling time.

In the present invention, the molar ratio of the 3ʹ-OH dimer precursor having the structure shown in Formula 5 to 2-cyanoethyl- N,N,Nʹ,Nʹ -tetraisopropylphosphorodiamidite is preferably 1:1.4. In the present invention, the molar ratio of the 3ʹ-OH dimer precursor having the structure shown in Formula 5 to the 1H -tetrazolyl diisopropylammonium salt catalyst is preferably 1:(1~3), more preferably 1:1.4.

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

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 to 3 h, more preferably 2 h.

After the phosphorylation reaction, the present invention preferably further comprises post-processing the obtained phosphorylation reaction solution, wherein the post-processing 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.

In the present invention, the mobile phase of the flash column separation and purification is preferably a dichloromethane-methanol system, and the dichloromethane preferably contains 0.4 vol% triethylamine. In the present invention, the volume content of methanol in the dichloromethane-methanol system is 0-10%. In the present invention, the purification system uses dichloromethane (0.4 vol% triethylamine)-methanol, which can better separate the product and keep the product stable.

In the present invention, the organic solvent of 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.

The present invention preferably adopts centrifugation to obtain the solid, and the drying method is preferably draining. In the present invention, methyl tert-butyl ether has a low solubility for phosphoramidite dimers and a high solubility for impurities, which better meets the dimer separation and purification requirements.

As a specific embodiment of the present invention, the synthesis route of the allyl-cyanoethyl nucleoside dimer is shown in Figure _1. In Figure 1, the reaction conditions and reagents are as follows: (a) 1H -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, 1H-tetrazole diisopropylammonium salt, DCM, 1~3 h.

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

The invention provides application of allyl-cyanoethyl nucleoside dimer in oligonucleotide synthesis.

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

The allyl-cyanoethyl nucleoside dimer is mixed with a coupling catalyst to carry out 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.

The present invention preferably uses a 50 nmol synthesis column on an LK-192 DNA synthesizer to carry out the coupling reaction. 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.

In the present invention, the deprotection reagent used for removing the DMTr protecting group is preferably a DBLOCK reagent, and the time for removing the DMTr protecting group is preferably 40 to 70 s, more preferably 60 s. After the DMTr protecting group removal reaction, the present invention preferably performs another DMTr protecting group removal reaction according to the above conditions. After the DMTr protecting group removal, the present invention preferably uses acetonitrile to wash the obtained DMTr protecting group removal product.

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:

The product from which the DMTr protection group is removed is washed and dried.

In the present invention, the reagent used for washing is preferably 70 vol% acetonitrile; and the drying is preferably vacuum drying.

In the present invention, the aminolysis is preferably ammonia ammonolysis or 2-mercaptoethanol ammonolysis. When ammonia ammonolysis is used, the aminolysis is preferably carried out in an ammonolysis rack, and the conditions of the aminolysis preferably include:

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

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

The ammonolysis time is preferably 1 to 3 h, more preferably 2 h.

When 2-mercaptoethanol is used for ammonolysis, the reagent for ammonolysis is preferably a concentrated aqueous ammonium hydroxide solution containing 2-mercaptoethanol. In the present invention, in the concentrated aqueous ammonium hydroxide solution containing 2-mercaptoethanol, the volume fraction of 2-mercaptoethanol is preferably 1-5%, more preferably 2-4%; the concentration of ammonium hydroxide is preferably 20-40%, more preferably 30%.

The conditions of the aminolysis include:

The ammonolysis temperature is 50-60°C, more preferably 55°C;

The time for aminolysis is 12 to 18 h, more preferably 14 to 16 h.

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

The product after aminolysis was mixed with acetonitrile solution and subjected to the first centrifugation; then pure water was added and subjected to the second centrifugation.

In the present invention, the 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.

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.

The allyl-cyanoethyl nucleoside dimer provided by the present invention and its preparation method and 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.

In the following examples, 1a-1d was purchased from Beijing Ruiboao Biotechnology Co., Ltd., and 2a-2d was purchased from Wuhu Huaren Technology Co., Ltd.

Example 1

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

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

1a (10.00 g, 1.05 eq) and 2a (5.02 g, 1.00 eq) were dissolved in dichloromethane and acetonitrile, and the mixture was dried to remove water. Then, the mixture was dissolved in anhydrous dichloromethane, 1H -tetrazole (0.63 g, 0.80 eq) was added, and the mixture was stirred at room temperature for 13 hours under _N2 protection to obtain compound 3a. Anhydrous t-BuOOH (2.03 g, 2.00 eq) was directly added to the mixture, and the mixture was stirred at room temperature for 0.5 hours to obtain 4a. _Et3N (2.28 g, 2.00 eq) and _Et3N ·3HF (5.45 g, 3.00 eq) were added, and the mixture was stirred at room temperature for 12 hours. After the reaction is complete, saturated NaHCO ₃ is added until no bubbles are generated. The mixture is extracted twice with dichloromethane and water. The organic layers are combined and washed with saturated brine, dried over anhydrous MgSO ₄ , filtered, and concentrated in vacuo to obtain crude compound 5a. Column chromatography purification (DCM/MeOH (0-10%)) was performed, and then the solvent was evaporated by rotary concentration. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether, filtered, and dried in vacuo to obtain 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 N ₈ O ₁₃ P [M+H] ⁺ 1091.36, found: 1091.19.

The liquid chromatogram of compound 5a is shown in FIG2 , and the mass spectrum is shown in FIG3 .

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

Compound 5a (4.00 g, 1.00 eq) was dissolved in dichloromethane and toluene, and evaporated to dryness 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 1H -tetrazolyl 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 being DCM (1% _Et3N v/v)-MeOH (0-10%), and the mixture was collected and dried. Then, it was dissolved in 5 mL of anhydrous dichloromethane and dripped into 45 mL of methyl tert-butyl ether at -78°C. The supernatant was discarded by centrifugation and the lower solid was placed in a vacuum box to dry 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/zcalcd for C ₆₆ H ₇₂ N ₁₀ O ₁₄ P ₂ [M+H] ⁺ 1291.47, found:1291.48.

The liquid chromatogram of compound 6a is shown in FIG4 , and the mass spectrum is shown in FIG5 .

Example 2

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

Synthesis of N ⁶ -benzoyl- p -allyl 5'- O -dimethoxytrityl-2'-deoxyadenosyl-(3'→5')-thymidine (5b):

1a (10.00 g, 1.05 eq) and 2b (4.02 g, 1.00 eq) were dissolved in dichloromethane and acetonitrile, and the mixture was dried to remove water. Then, the mixture was dissolved in anhydrous dichloromethane, 1H -tetrazole (0.63 g, 0.80 eq) was added, and the mixture was stirred at room temperature for 12 hours under _N2 protection to obtain compound 3b. Anhydrous t-BuOOH (2.03 g, 2.00 eq) was directly added to the mixture, and the mixture was stirred at room temperature for 0.5 hours to obtain 4b. _Et3N (2.28 g, 2.00 eq) and _Et3N ·3HF (5.45 g, 3.00 eq) were added, and the mixture was stirred at room temperature for 14 hours. After the reaction is complete, saturated NaHCO ₃ is added until no bubbles are generated. The mixture is extracted twice with dichloromethane and water. The organic layers are combined and washed with saturated brine, dried over anhydrous MgSO ₄ , filtered, and concentrated in vacuo to obtain crude compound 5b. Column chromatography purification (DCM/MeOH (0-10%)) was performed, and then the solvent was evaporated by rotary concentration. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether, filtered, and dried in vacuo to obtain 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 N ₇ O ₁₃ P [M+H] ⁺ 1002.34, found: 1002.18.

The liquid chromatogram of compound 5b is shown in FIG6 , and the mass spectrum is shown in FIG7 .

Example 3

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

Synthesis of N ⁴ -benzoyl- p -allyl-5'- O -dimethoxytrityl-2'-deoxycytidine-(3'→5')- N ⁶ -benzoyl-2'-deoxyadenosine (5c):

1b (10.00 g, 1.05 eq) and 2c (5.45 g, 1.00 eq) were dissolved in dichloromethane and acetonitrile, and the mixture was dried to remove water. Then, the mixture was dissolved in anhydrous dichloromethane, 1H -tetrazole (0.65 g, 0.80 eq) was added, and the mixture was stirred at room temperature for 12 hours under _N2 protection to obtain compound 3c. Anhydrous t-BuOOH (1.57 g, 1.50 eq) was directly added to the mixture, and the mixture was stirred at room temperature for 0.5 hours to obtain 4c. _Et3N (2.35 g, 2.00 eq) and _Et3N ·3HF (5.61 g, 3.00 eq) were added, and the mixture was stirred at room temperature for 12 hours. After the reaction is complete, saturated NaHCO ₃ is added until no bubbles are generated. The mixture is extracted twice with dichloromethane and water. The organic layers are combined and washed with saturated brine, dried over anhydrous MgSO ₄ , filtered, and concentrated in vacuo to obtain crude compound 5c. Column chromatography purification (DCM/MeOH (0-10%)) was performed, and then the solvent was evaporated by rotary concentration. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether, filtered, and dried in vacuo to obtain 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 N ₈ O ₁₃ P [M+H] ⁺ 1091.36, found: 1091.20.

The liquid chromatogram of compound 5c is shown in FIG8 , and the mass spectrum is shown in FIG9 .

Example 4

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

Synthesis of N ⁴ -benzoyl- p -allyl-5'- O -dimethoxytrityl-2'-deoxycytidine-(3'→5')-thymidine (5d):

1b (20.00 g, 1.05 eq) and 2d (8.27 g, 1.00 eq) were dissolved in dichloromethane and acetonitrile, and the mixture was dried to remove water. Then, the mixture was dissolved in anhydrous dichloromethane, 1H -tetrazole (1.30 g, 0.80 eq) was added, and the mixture was stirred at room temperature for 11 hours under _N2 protection to obtain compound 3d. Anhydrous t-BuOOH (2.09 g, 1.00 eq) was directly added to the mixture, and the mixture was stirred at room temperature for 0.5 hours to obtain 4d. _Et3N (4.70 g, 2.00 eq) and Et3N·3HF (11.23 g, 3.00 eq) were added, and the mixture was stirred at room temperature for 13 hours. After the reaction is complete, saturated NaHCO ₃ is added until no bubbles are generated. The mixture is extracted twice with dichloromethane and water. The organic layers are combined and washed with saturated brine, dried over anhydrous MgSO ₄ , filtered, and concentrated in vacuo to obtain crude compound 5d. Column chromatography purification (DCM/MeOH (0-10%)) was performed, and then the solvent was evaporated by rotary concentrator. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether, filtered, and dried in vacuo to obtain 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 N ₅ O ₁₄ P [MH] ^- 976.32, found: 976.10.

The liquid chromatogram of compound 5d is shown in FIG10 , and the mass spectrum is shown in FIG11 .

Example 5

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

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

1c (20.00 g, 1.05 eq) and 2b (10.26 g, 1.00 eq) were dissolved in dichloromethane and acetonitrile, and the mixture was dried to remove water. Then, the mixture was dissolved in anhydrous dichloromethane, 1H -tetrazole (1.29 g, 0.80 eq) was added, and the mixture was stirred at room temperature for 12 hours under _N2 protection to obtain compound 3e. Anhydrous t-BuOOH (3.12 g, 1.50 eq) was directly added to the mixture, and the mixture was stirred at room temperature for 0.5 hours to obtain 4e. _Et3N (4.66 g, 2.00 eq) and _Et3N ·3HF (11.15 g, 3.00 eq) were added, and the mixture was stirred at room temperature for 15 hours. After the reaction is complete, saturated NaHCO ₃ is added until no bubbles are generated. The mixture is extracted twice with dichloromethane and water. The organic layers are combined and washed with saturated brine, dried over anhydrous MgSO ₄ , filtered, and concentrated in vacuo to obtain crude compound 5e. Column chromatography purification (DCM/MeOH (0-10%)) was performed, and then the solvent was evaporated by rotary concentration. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether, filtered, and dried in vacuo to obtain 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 N ₈ O ₁₄ P [MH] ^- 1071.37, found: 1071.00.

The liquid chromatogram of compound 5e is shown in FIG12 , and the mass spectrum is shown in FIG13 .

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

Compound 5e (2.40 g, 1.00 eq) was dissolved in dichloromethane and toluene, and evaporated to dryness under reduced pressure to remove water. Then, it was dissolved in 15 mL of anhydrous dichloromethane, and cyanoethyl phosphoramidite (1.01 g, 1.50 eq) and 1H -tetrazolyl diisopropylammonium salt (0.57 g, 1.50 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, and the mobile phase was DCM (1% Et3N v/v)-MeOH (0-10%), and the mixture was collected and dried. Then, it was dissolved in 3 mL of anhydrous dichloromethane, and dripped dropwise into 30 mL of methyl tert-butyl ether at -78°C, and the supernatant was discarded by centrifugation. The lower layer of solid was placed in a vacuum box and dried to obtain compound 6e, 2.11 g of white powdery solid, with a purity of 97.4% and a yield of 74.15%. ESI-MS: m/z calcd for C ₆₄ H ₇₄ N ₁₀ O ₁₅ P ₂ [M+H] ⁺ 1272.48, found:1273.50.

The liquid chromatogram of compound 6e is shown in FIG14 , and the mass spectrum is shown in FIG15 .

Example 6

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

Synthesis of N ² -isobutyryl- P -allyl-5'- O -dimethoxytrityl-2'-deoxyguanosine-(3'→5')-thymidine (5f):

1c (15.00 g, 1.05 eq) and 2d (6.16 g, 1.00 eq) were dissolved in dichloromethane and acetonitrile, and the mixture was dried to remove water. Then, the mixture was dissolved in anhydrous dichloromethane, 1H -tetrazole (0.97 g, 0.80 eq) was added, and the mixture was stirred at room temperature for 12 hours under _N2 protection to obtain compound 3f. Anhydrous t-BuOOH (1.56 g, 1.00 eq) was directly added to the mixture, and the mixture was stirred at room temperature for 0.5 hours to obtain 4f. _Et3N (3.50 g, 2.00 eq) and _Et3N ·3HF (8.36 g, 3.00 eq) were added, and the mixture was stirred at room temperature for 12 hours. After the reaction is complete, saturated NaHCO ₃ is added until no bubbles are generated. The mixture is extracted twice with dichloromethane and water. The organic layers are combined and washed with saturated brine, dried over anhydrous MgSO ₄ , filtered, and concentrated in vacuo to obtain crude compound 5f. Column chromatography purification (DCM/MeOH (0-10%)) was performed, and then the solvent was evaporated by rotary concentration. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether, filtered, and dried in vacuo to obtain 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 N ₇ O ₁₄ P [M+Et ₃ N] ⁺ 1084.54, found: 1085.20.

The liquid chromatogram of compound 5f is shown in FIG16 , and the mass spectrum is shown in FIG17 .

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

Compound 5f (4.00 g, 1.00 eq) was dissolved in dichloromethane and toluene, and evaporated to dryness under reduced pressure to remove water. Then, it was dissolved in 20 mL of anhydrous dichloromethane, and cyanoethyl phosphoramidite (1.59 g, 1.00 eq) and 1H -tetrazolyl diisopropylammonium salt (0.91 g, 1.00 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, and the mobile phase was DCM (1% Et3N v/v)-MeOH (0-10%), and the mixture was collected and dried. Then, it was dissolved in 5 mL of anhydrous dichloromethane, and dripped dropwise into 45 mL of methyl tert-butyl ether at -78°C, and the supernatant was discarded by centrifugation. The lower layer of solid was placed in a vacuum box and dried 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 N ₉ O ₁₅ P ₂ [MH] ^- 1182.45, found: 1182.22.

The liquid chromatogram of compound 6f is shown in FIG18 , and the mass spectrum is shown in FIG19 .

Example 7

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

Synthesis of P -allyl-5'- O -dimethoxytrityl-thymidine-(3' ^→ 5')- N6 -benzoyl-2'-deoxyadenosine (5 g):

1d (20.00 g, 1.05 eq) and 2a (12.22 g, 1.00 eq) were dissolved in dichloromethane and acetonitrile, and the mixture was dried to remove water. Then, the mixture was dissolved in anhydrous dichloromethane, 1H -tetrazole (1.46 g, 0.80 eq) was added, and the mixture was stirred at room temperature for 12 hours under _N2 protection to obtain compound 3g. Anhydrous t-BuOOH (2.35 g, 1.00 eq) was directly added to the mixture, and the mixture was stirred at room temperature for 0.5 hours to obtain 4g. _Et3N (5.27 g, 2.00 eq) and Et3N·3HF (12.60 g, 3.00 eq) were added, and the mixture was stirred at room temperature for 12 hours. After the reaction is complete, saturated NaHCO ₃ is added until no bubbles are generated. The mixture is extracted twice with dichloromethane and water. The organic layers are combined and washed with saturated brine, dried over anhydrous MgSO ₄ , filtered, and concentrated in vacuo to obtain 5 g of crude compound. Purification by column chromatography (DCM/MeOH (0-10%)) was performed by rotary concentration to evaporate the solvent. The residue was dissolved in anhydrous dichloromethane, added dropwise to methyl tert-butyl ether, filtered, and dried in vacuo to obtain 5 g of dimer precursor compound, 17.79 g of white solid, purity 99.2%, yield 68.21%. ESI-MS: m/z calcd for C ₅₁ H ₅₂ N ₇ O ₁₃ P [M+H] ⁺ 1002.34, found: 1002.00.

The liquid chromatogram of compound 5g is shown in FIG20 , and the mass spectrum is shown in FIG21 .

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

Compound 5g (4.00 g, 1.00 eq) was dissolved in dichloromethane and toluene, and evaporated to dryness under reduced pressure to remove water. Then, it was dissolved in 20 mL of anhydrous dichloromethane, and cyanoethyl phosphoramidite (1.44 g, 1.00 eq) and 1H -tetrazolyl diisopropylammonium salt (0.82 g, 1.00 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, and the mobile phase was DCM (1% Et3N v/v)-MeOH (0-10%), and the mixture was collected and dried. Then, it was dissolved in 5 mL of anhydrous dichloromethane, and dripped into 45 mL of methyl tert-butyl ether dropwise at -78°C, and the supernatant was discarded by centrifugation. The lower layer of solid was placed in a vacuum box and dried to obtain compound 6g, 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 N ₉ O ₁₄ P ₂ [M+H] ⁺ 1202.44, found:1202.07.

The liquid chromatogram of compound 6g is shown in FIG22 , and the mass spectrum is shown in FIG23 .

Structural characterization

(1) The present invention successfully prepared seven 3'-OH dimer precursors 5 with a total yield of more than 70% and a synthesis scale of 20 g, and characterized them by HPLC-MS. The liquid phase purity and mass spectrometry identification results of the obtained allyl dimer precursors 5a~5g are shown in Table 1.

Table 1 Liquid phase purity and mass spectrometry identification results of allyl dimer precursors 5a-g

HPLC analysis showed that all dimer precursors 5 were diastereoisomers with a purity greater than 96%.

(2) According to the liquid chromatography and mass spectra of compounds 6a~6g, it can be seen that the present invention achieves the preparation of high-purity nucleoside dimers, and the HPLC purity can reach more than 95%, which can meet the requirements of high-fidelity DNA synthesis. The synthesis experiment of the present invention successfully synthesized allyl-cyanoethyl dimer, verifying the feasibility of the synthetic route. The synthetic route is simple to operate and can achieve large-scale preparation, laying a foundation for subsequent expansion of production.

Application Example 1 Experiment on the synthesis of oligonucleotide fragments using allyl-cyanoethyl nucleoside dimers

In order to verify whether the oligonucleotide synthesized by allyl-cyanoethyl nucleoside dimer is easy to deprotect, the present invention selects two allyl-cyanoethyl nucleoside dimers to perform a 20 nt sequence synthesis experiment. On the LK-192 DNA synthesizer, a 50nmol synthesis column, a dimer concentration of 50 mg/mL, a coupling catalyst of 4,5-dicyanoimidazole, and a coupling time of 120 s were used. The single cycle synthesis program of the synthesizer is shown in Table 2.

Table 2 Synthesizer single cycle synthesis program

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.

Under the same conditions, methyl dimer (TA, GC) (structure as shown in Formula II) and allyl-cyanoethyl nucleoside dimer (TA, GC) were used to synthesize a 20 nt sequence after 10 cycles, as shown in SEQ ID NO.1, specifically: TA GC TA GC TA GC TA GC TA GC. Under the same conditions, the comparison of the mass spectra of the oligonucleotide aminolysis products synthesized by methyl dimer and allyl-cyanoethyl nucleoside dimer is shown in Figure 24. In Figure 24, (a) is the mass spectrum of the oligonucleotide synthesized by methyl dimer, and (b) is the mass spectrum of the oligonucleotide synthesized by allyl-cyanoethyl nucleoside dimer.

The results showed that the oligonucleotide synthesized by the methyl dimer had more impurities after aminolysis, with impurity peaks of molecular weight +14 and +28, and more fragment peaks, and the product purity was low; while the mass spectrometry identification results of the oligonucleotide synthesized by the allyl-cyanoethyl nucleoside dimer of the present invention showed ESI-MS (m/z) 6115.1, theoretical molecular weight: 6116.89; there were fewer by-products after aminolysis, the product purity was high, the deprotection was complete, and there was no obvious impurity peak in the mass spectrum; this shows that the allyl-cyanoethyl nucleoside dimer exhibits better deprotection characteristics and can be used as an alternative structure to the methyl dimer.

Application Example 2

In gene synthesis, multiple segments of 60 nt to 80 nt are usually synthesized and then spliced. In order to further verify the feasibility of using allyl-cyanoethyl nucleoside dimers for oligonucleotide synthesis, the present invention synthesizes 80 nt segments. Using two allyl-cyanoethyl nucleoside dimers (TA, AC), an 80 nt sequence was synthesized after 40 cycles according to Table 2, 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 AC. For the aminolysis conditions, see Application Example 1.

The mass spectrometry detection diagram of the 80 nt sequence synthesized by allyl-cyanoethyl nucleoside dimer is shown in Figure 25. The mass spectrometry identification results showed ESI-MS (m/z) 24337.7, theoretical molecular weight: 24334.04, and the target sequence was obtained. The successful synthesis of the 80 nt fragment shows that the allyl-protected dimer of the present invention can be used for oligonucleotide synthesis, and has certain advantages. The oligonucleotide synthesized by allyl-cyanoethyl nucleoside dimer has greatly improved the difficulty of deprotection, and the reaction can be completed by using only ammonia reaction for 2h, avoiding the uncertainty of the oligonucleotide sequence caused by the long-term high temperature alkaline environment. The present invention verifies the idea of designing allyl-cyanoethyl nucleoside dimer, and the use of the dimer is expected to further improve the quality of oligonucleotide synthesis, improve the purity of the product, and reduce synthesis errors.

In summary, the present invention further optimizes the structure of dimer nucleoside, synthesizes 7 dimer precursors, 4 allyl-cyanoethyl nucleoside dimers, and determines a high-purity, high-yield, easy-to-operate synthesis route by optimizing the reaction conditions. And it is used in the synthesis of oligonucleotide fragments, and the aminolysis comparison experiment is carried out with the dimer protected by methyl. The results show that the oligonucleotide deprotection rate synthesized by the allyl-cyanoethyl nucleoside dimer is significantly improved, and there are fewer by-products. The new structure of the allyl-cyanoethyl nucleoside dimer obtained by the present invention can be used for the synthesis of oligonucleotides, and it is expected to broaden the scope of application of the dimer nucleoside and achieve high-quality synthesis.

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 (10)

1. An allyl-cyanoethyl nucleoside dimer, characterized in that it has a structure shown in formula I: Formula I; In Formula I, X is O or S; _B1 and _B2 are independently selected from substituted or unsubstituted bases. 2. The allyl-cyanoethyl nucleoside dimer according to claim 1, wherein the _B1 is selected from any one of cytosine, guanine, adenine, thymine, uracil, hypoxanthine, xanthine, deazaadenine, deazaguanine, deazahypoxanthine, dihydrouracil and pseudouracil; The _B2 is selected from any one of cytosine, guanine, adenine, thymine, uracil, hypoxanthine, xanthine, deazaadenine, deazaguanine, deazahypoxanthine, dihydrouracil and pseudouracil. 3. The allyl-cyanoethyl nucleoside dimer according to claim 1 or 2, characterized in that B ₁ and B ₂ are independently selected from the following groups, " " represents the 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. 4. The method for preparing an allyl-cyanoethyl nucleoside dimer according to any one of claims 1 to 3, characterized in that the method comprises the following steps: (1) A 5ʹ-DMTr-3ʹ-O-allyl phosphoramidite nucleoside having a structure shown in Formula 1 is coupled with a 5ʹ-OH-3ʹ-O-TBDMS nucleoside having a structure shown in Formula 2 to obtain a dimer having a structure shown in Formula 3; Formula 1; Formula 2; 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 S ₈ and N,O -bistrimethylsilylacetamide, and subjected to a sulfurization reaction to obtain a diphosphoric acid diester having the structure shown in Formula 4; Formula 4; (3) The diphosphodiester having the structure shown in Formula 4 is subjected to a TBDMS protecting group removal reaction to obtain a 3ʹ-OH dimer precursor having the structure shown in Formula 5; Formula 5; (4) Under the action of 1H -tetrazolyl diisopropylammonium salt catalyst, the 3ʹ-OH dimer precursor having the structure shown in Formula 5 is phosphorylated with 2-cyanoethyl- N,N,Nʹ,Nʹ -tetraisopropylphosphorodiamidite to obtain an allyl-cyanoethyl nucleoside dimer having the structure shown in Formula I. 5. The preparation method according to claim 4, characterized in that the coupling reaction is carried out in the presence of 1H -tetrazole, and the molar ratio of the 5ʹ-DMTr-3ʹ-O-allyl phosphoramidite nucleoside having the structure shown in Formula 1 to the 5ʹ-OH-3ʹ-O-TBDMS nucleoside having the structure shown in Formula 2 is (1-2): 1; The molar ratio of the 5ʹ-DMTr-3ʹ-O-allyl phosphoramidite nucleoside having the structure shown in Formula 1 to 1 H -tetrazolyl is (1-2):(0.5-1); The coupling reaction time is 8 to 14 h. 6. The preparation method according to claim 4, 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 S ₈ 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 min. 7. The preparation method according to claim 4, characterized in that the molar ratio of the 3ʹ-OH dimer precursor having the structure shown in Formula 5 to 2-cyanoethyl- N,N,Nʹ,Nʹ -tetraisopropylphosphorodiamidite is 1:1.4; The molar ratio of the 3ʹ-OH dimer precursor having the structure shown in Formula 5 to the 1 H -tetrazolyl diisopropylammonium salt catalyst is 1:(1-3); The phosphorylation reaction time is 1 to 3 h. 8. The preparation method according to claim 4 or 7, 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. 9. Use of the allyl-cyanoethyl nucleoside dimer described in any one of claims 1 to 3 or the allyl-cyanoethyl nucleoside dimer prepared by the preparation method described in any one of claims 4 to 8 in oligonucleotide synthesis. 10. A method for synthesizing an oligonucleotide, comprising the following steps: The allyl-cyanoethyl nucleoside dimer according to any one of claims 1 to 3 or the allyl-cyanoethyl nucleoside dimer prepared by the preparation method according to any one of claims 4 to 8 is mixed with a coupling catalyst to carry out 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.