A compound and a method for nucleic acid synthesis

Abstract

The application discloses a compound and a nucleic acid synthesis method. The compound is shown as formula A or B. The nucleic acid synthesis method uses a new protecting group to replace a DMT protecting group, and the deprotection reaction can be completed under alkaline conditions. The deprotection reaction can be efficiently and quantitatively completed to release 5'-OH for a coupling reaction in the next step. The deprotection reaction is an irreversible reaction. The protecting group is not sensitive to acid. The by-product after deprotection is a stable compound and does not react with each group on the oligo.

Description

Technical Field

[0001] This invention belongs to the field of biosynthesis and relates to a compound and a method for synthesizing nucleic acids. Specifically, it relates to a compound as shown in Formula A or a compound as shown in Formula B, its preparation method, the application of a compound as shown in Formula C in the synthesis of nucleic acids, a method for preparing a solid-phase support (SSEB-2'-dN-CPG) containing a starting nucleic acid molecule, and a method for synthesizing nucleic acids. Background Technology

[0002] In recent decades, with the rapid development of molecular biology and DNA-based in vitro diagnostic technologies, the application of oligonucleotides has become increasingly widespread. For example, in genome sequencing and PCR, artificially synthesized oligonucleotides serve as primers to guide the synthesis of complementary strands of templates; in quantitative real-time PCR, synthesized oligonucleotides can be used as probes for the detection, analysis, and quantification of target nucleic acids. In recent years, research on oligonucleotides as drug candidates has also made significant progress. For instance, antisense oligonucleotides (ASOs) can regulate gene expression by targeting specific RNAs and can also be used to treat systemic diseases; nucleic acid aptamers can specifically bind to targets such as small molecules, proteins, bacteria, viruses, cells, and tissues; small interfering RNAs (siRNAs) are used for gene silencing; and microRNAs (miRNAs) are used to block protein gene expression. Furthermore, DNA data storage technologies also require the application of oligonucleotide synthesis technology. With the development of these technologies, the demand for artificially synthesized oligonucleotides is increasing, thus greatly promoting the development of oligonucleotide synthesis technology.

[0003] Over the decades, various oligonucleotide synthesis methods have been reported for the synthesis of oligonucleotides, including H-phosphonic acid synthesis, phosphate diester synthesis, phosphate triester synthesis, and phosphoramidite triester synthesis. The most widely used oligonucleotide synthesis method currently is solid-phase phosphoramidite trimer synthesis (see Caruthers et al. US Pat. Nos. 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679 and 5,132,418; Koster et al. US Pat. No. Re. 34,069; McBride et al. (1983) Tetrahedron Letters 24:245-248; Sinha et al. (1983) Tetrahedron Letters 24:5843-5846). This technique uses nucleoside phosphoramidite as a monomer to synthesize the 3'-5' segments of oligonucleotides on a solid support. This method involves extending one base per cycle, and each cycle includes four steps: deprotection, coupling, capping, and oxidation. Figure 1).

[0004] (1) Deprotection. Before the start of each cycle, the terminal 5'-hydroxyl group on the CPG-oligonucleotide is protected by a dimethoxytriphenylmethyl (DMT) protecting group. The DMT is usually removed using a dichloromethane solution of trichloroacetic acid (TCA) to expose the 5'-hydroxyl group.

[0005] (2) Coupling. After deprotection, the corresponding oligonucleotide monomers and activators are added to the support to increase the number of bases. The product obtained by this reaction is a triphosphite.

[0006] (3) Capping. Use a capping reagent, such as the acetic anhydride / N-methylimidazole / pyridine / tetrahydrofuran system, to block the unreacted 5'-OH in the first step of coupling.

[0007] (4) Oxidation. The coupled triphosphite is oxidized to the corresponding phosphate ester using an oxidizing agent, such as an I2 / pyridine / tetrahydrofuran / water system.

[0008] The solid support used in this method can be controlled-pore glass (CPG) or microporous polystyrene (MPPS), with CPG being more commonly used. The oligo is linked to the 3'-position of the first base via a linker, and through multiple cycles, the 3'-5' synthesis of the oligo is achieved. After the target oligo is synthesized, it is cleaved from the solid support by ammonolysis with ammonia, simultaneously removing the protecting groups on the base and phosphate, thus obtaining the target oligo.

[0009] The above method has been widely used in laboratory oligo synthesis research and in commercially available micro- or large-scale custom oligonucleotide synthesis, for example, at a scale of 1 mol or more, for the synthesis of oligonucleotide drugs, primers, probes, etc. However, this method has inherent drawbacks, which are inherent to the DMT protecting group being a protecting group at the 5'-position hydroxyl group.

[0010] First, the DMT protecting group is an acid-sensitive group. In oligo synthesis, an acid is needed to complete deprotection and expose the 5'-hydroxyl group. A strong protic acid, such as trichloroacetic acid (TCA), is typically used. However, under strongly acidic conditions, oligonucleotides may undergo some side reactions, particularly depurination. Figure 2Depurination refers to the phenomenon where nucleic acids lose purine bases under acidic conditions, resulting in purine-free sites. However, using DMT as a protecting group inevitably exposes the oligonucleotide product to a strongly acidic environment in each cycle, making depurination a more likely side effect and impacting the quality of oligo synthesis. It is foreseeable that the earlier the purine bases are synthesized or the longer the oligo chain, the longer the purine bases are exposed to acidic conditions, and the more severe the depurination phenomenon will be.

[0011] Furthermore, after the DMT protecting group is removed under acidic conditions, it forms a DMT cation, while DMT... + It can react with hydroxyl groups, leading to the reprotection of the hydroxyl groups. Therefore, the acidic deprotection reaction of DMT protecting groups is a reversible reaction. Figure 3 This reversible reaction leads to incomplete deprotection. The unprotected sequence does not undergo chain elongation in subsequent coupling reactions, but can undergo deprotection in subsequent cycles to achieve chain elongation, thus yielding a base-deleted sequence. These base-deleted sequences are cleaved from the solid support along with the target sequence after synthesis, but their similar properties to the target sequence make separation difficult.

[0012] Third, the coupling reaction requires the use of an activator to activate the phosphorus amide group of the phosphorus amide monomer, which then reacts with the exposed 5'-hydroxyl group on the support to achieve chain elongation. However, the activators are all weak acids, which may cause some of the 5'-hydroxyl groups on the coupled phosphite triester to be removed, and then react with the activated phosphorus amide monomer, thereby producing a base insertion byproduct.

[0013] In addition, the acids and dichloromethane solvents used in the deprotection reaction also cause environmental pollution. Summary of the Invention

[0014] To address the aforementioned shortcomings of existing solid-phase phosphoramidite trimer methods, this application provides a novel nucleic acid synthesis method. This method uses a novel protecting group to replace the DMT protecting group, which allows for deprotection under alkaline conditions. This deprotection reaction is highly efficient and quantitative, releasing 5'-OH for subsequent coupling reactions. The deprotection reaction is irreversible. The protecting group is insensitive to acids. The deprotected byproduct is a stable compound that does not react with the groups on the oligo.

[0015] This invention provides compounds as shown in Formula A or as shown in Formula B.

[0016]

[0017] Among them, Base pgRefers to a base or a base with a protecting group;

[0018] R1 and R2 are each independently C 16 alkyl;

[0019] Or R1 and R2 can be connected to form C. 2-20 cycloalkyl, with one or more C 1-6 Alkyl-substituted C 2-20 The cycloalkyl group or heteroatom is selected from one, two, or three of N, O, and S, and the number of C heteroatoms is one, two, or three. 2-20 "Carbon heterocycle";

[0020] R3, R4, R5, and R6 are, independently, hydrogen, halogen, an amino group with a protecting group, a hydroxyl group with a protecting group, or C. 1-6 alkyl;

[0021] Alternatively, R3 and R4, R4 and R5, or R5 and R6 can be connected to form C. 2-20 cycloalkyl, with one or more C 1-6 Alkyl-substituted C 2-20 The cycloalkyl group or heteroatom is selected from one, two, or three of N, O, and S, and the number of C heteroatoms is one, two, or three. 2-20 "Carbon heterocycle";

[0022] R7 is a phosphate protecting group;

[0023] R8 and R9 are each independently C 1-6 alkyl;

[0024] Alternatively, R8 and R9 can be connected to form C. 2-20 cycloalkyl, with one or more C 1-6 Alkyl-substituted C 2-20 The cycloalkyl group or heteroatom is selected from one, two, or three of N, O, and S, and the number of C heteroatoms is one, two, or three. 2-20 "Carbon heterocycles".

[0025] In some embodiments, the protecting base is an A base with a phenylpropionyl group (Bz), a C base with an acetyl group (Ac), or an isobutyryl group (Bz). i The G base of Bu).

[0026] In some embodiments, R1 and R2 are independently methyl, ethyl or isopropyl, preferably methyl.

[0027] In some embodiments, R3, R4, R5, and R6 are hydrogen.

[0028] In some embodiments, R7 is β-cyanoethyl, methyl, benzyl, allyl, 2-(p-nitrophenyl)-1-ethyl, 4-methylthio-1-butyl, 2-(N-acetyl)-amino-1-ethyl or 2-naphthylcarbamoyloxy-1-ethyl, preferably β-cyanoethyl.

[0029] In some embodiments, R8 and R9 are independently methyl, ethyl, or isopropyl, preferably isopropyl.

[0030] In some embodiments, the compound as shown in Formula A is

[0031]

[0032] The present invention also provides the application of a compound of formula C in the synthesis of nucleic acids;

[0033]

[0034] The definitions of R1, R2, R3, R4, R5, and R6 are as described above.

[0035] In some embodiments, the compound as shown in Formula C is (SSEB).

[0036] This invention also provides a method for preparing a compound as shown in formula C, comprising the following steps: in a solvent, compound D reacts with trihydroxypropylphosphine as shown below to obtain compound C, where R is... R1, R2, R3, R4, R5, R6, R7, R8, R9, Base pg The definition is as stated above.

[0037]

[0038] In some embodiments, the solvent is one or more of nitrile solvents, alcohol solvents, and water.

[0039] In some embodiments, the nitrile solvent is acetonitrile.

[0040] In some embodiments, the alcohol solvent is methanol.

[0041] The present invention also provides a method for preparing a compound as shown in formula A, comprising the following steps: in a solvent, in the presence of a base, compound A-1 and... The reaction shown below yields compound A;

[0042]

[0043] R1, R2, R3, R4, R5, R6, R7, R8, R9, Base pg The definition is as described above.

[0044] In some embodiments, the solvent is a haloalkane solvent, preferably dichloromethane.

[0045] In some embodiments, the base is N,N-diisopropylethylamine.

[0046] In some embodiments, the The molar ratio with compound A-1 is 2:1.

[0047] In some embodiments, the molar ratio of the base to the compound A-1 is 4:1.

[0048] Preferably, the preparation method of the compound as shown in Formula A further includes the following steps: in a solvent, compound A-2 is reacted with Et3N·3HF as shown below to obtain compound A-1;

[0049]

[0050] R1, R2, R3, R4, R5, R6, Base pg The definition is as described above.

[0051] In some embodiments, the solvent is an ether solvent, preferably tetrahydrofuran.

[0052] In some embodiments, the molar ratio of Et3N·3HF to compound A-2 is 10:1.

[0053] Preferably, the method for preparing the compound as shown in Formula A further includes the following steps: in a solvent, in the presence of a base and a dehydrating agent, compound A-3 reacts with the compound as shown in Formula C as shown below to obtain compound A-2;

[0054]

[0055] R1, R2, R3, R4, R5, R6, Base pg The definition is as described above.

[0056] In some embodiments, the solvent is a haloalkane solvent, preferably dichloromethane.

[0057] In some embodiments, the base is 4-dimethylaminopyridine.

[0058] In some embodiments, the molar ratio of the base to the compound A-3 is 0.14:1.

[0059] In some embodiments, the dehydrating agent is dicyclohexylcarbodiimide.

[0060] In some embodiments, the molar ratio of the dehydrating agent to the compound A-3 is 1.1:1.

[0061] In some embodiments, the molar ratio of the compound as shown in Formula C to the compound A-3 is 1.1:1.

[0062] Preferably, the method for preparing the compound as shown in Formula A further includes the following step: in a solvent, in the presence of an acid, the compound A-4 undergoes the reaction shown below to obtain compound A-3;

[0063]

[0064] Base pg The definition is as described above.

[0065] In some embodiments, the solvent is a haloalkane solvent, preferably dichloromethane.

[0066] In some embodiments, the acid is trichloroacetic acid.

[0067] In some embodiments, the molar ratio of the acid to the compound A-4 is 10:1.

[0068] Preferably, the preparation method of the compound as shown in Formula A further includes the following steps: in a solvent, in the presence of imidazole, compound A-5 is reacted with TBS-Cl as shown below to obtain compound A-4;

[0069]

[0070] Base pg The definition is as described above.

[0071] In some embodiments, the solvent is an amide solvent, preferably N,N-dimethylformamide.

[0072] In some embodiments, the molar ratio of the imidazole to the compound A-5 is 2.5:1.

[0073] In some embodiments, the molar ratio of TBS-Cl to compound A-5 is 2:1.

[0074] Preferably, the method for preparing the compound as shown in Formula A further includes the following step: in a solvent, compound A-6 reacts with DMT-Cl as shown below to obtain compound A-5;

[0075]

[0076] Base pg The definition is as described above.

[0077] In some embodiments, the solvent is pyridine.

[0078] In some embodiments, the molar ratio of DMT-Cl to compound A-6 is 1.1:1.

[0079] This invention also provides a method for preparing a solid-phase support (SSEB-2'-dN-CPG) containing an initiating nucleic acid molecule, comprising the following steps:

[0080] In the presence of a dehydrating agent and a base, CPG-linker-NH2 reacts with compound E (SSEB-2'-dN) as shown below to obtain a solid-phase support (SSEB-2'-dN-CPG) containing the starting nucleic acid molecule. R1, R2, R3, R4, R5, R6, and Base... pg As defined above, dN represents any of dA, dC, dT, and dG, as well as other deoxynucleotides.

[0081]

[0082] In some embodiments, the CPG-linker-NH2 is

[0083] In some embodiments, compound E is

[0084] In some embodiments, the dehydrating agent is dicyclohexylcarbodiimide (DCC).

[0085] In some embodiments, the base is pyridine.

[0086] In some embodiments, the reaction further includes the following post-processing steps: washing and drying. The solvent used for washing is pyridine, dichloromethane, and acetonitrile.

[0087] In some embodiments, the method for preparing the solid-phase carrier containing the starting nucleic acid molecule further includes a capping step, for example, using capping reagent A and capping reagent B for capping.

[0088] The present invention also provides a method for nucleic acid synthesis, which includes the following steps:

[0089] (1) In a solvent, SSEB-2'-dN-CPG prepared by the method described above was subjected to a deprotection reaction with trihydroxypropylphosphine (THPP) to obtain... (dN-CPG);

[0090] (2) In the presence of an activator, the dN-CPG is reacted with a compound as shown in Formula A to generate a phosphite triester with a protecting group;

[0091] (3) Under the action of an oxidant, the phosphite with a protecting group is oxidized to obtain a phosphate with a protecting group.

[0092] (4) In a solvent, the phosphate ester with the protecting group is subjected to a deprotection reaction with trihydroxypropylphosphine to obtain the first extended product;

[0093] (5) Optionally, repeat steps (2), (3), and (4) to obtain the extended product;

[0094] (6) The extended product or the first extended product is reacted with the cleavage solvent to cleave CPG-Linker-NH2.

[0095] In some embodiments, in step (1), the solvent is one or more of nitrile solvents, alcohol solvents, and water.

[0096] In some embodiments, in step (1), the molar ratio of the trihydroxypropylphosphine to the SSEB-2'-dN-CPG is 20:1.

[0097] In some embodiments, in step (2), the activator is 5-ethimercaptotetrazole, preferably a 0.25 mol / L acetonitrile solution of 5-ethimercaptotetrazole.

[0098] In some embodiments, in step (3), the oxidant is an I2 / pyridine / tetrahydrofuran / water system, preferably a mixed solution of pyridine, tetrahydrofuran and water with a concentration of 0.02 mol / L.

[0099] In some embodiments, in step (4), the solvent is one or more of nitrile solvents, alcohol solvents, and water.

[0100] In some embodiments, in step (4), the molar ratio of the trihydroxypropylphosphine to the phosphate ester with the protecting group is 20:1.

[0101] In some embodiments, in step (6), the removal solvent is ammonia, preferably 1 mL of concentrated ammonia.

[0102] In some embodiments, step (2) further includes a capping step, for example, using capping reagent A and capping reagent B for capping.

[0103] In some embodiments, the method further includes the following steps: further deprotecting the system obtained in step (6) for, for example, 16 hours, and then concentrating it under vacuum. Preferably, the deprotection and vacuum concentration are carried out at, for example, 60°C.

[0104] The present invention also provides a kit containing a compound as shown in Formula A or a compound as shown in Formula B.

[0105] In some embodiments, the kit further contains one or more of the activator, oxidant, trihydroxypropylphosphine, excision solvent, capping reagent A, and capping reagent B as described above.

[0106] The positive and progressive effects of this invention are as follows:

[0107] This invention develops a novel method for oligonucleotide synthesis. This method uses a methyl perthioethyl benzoyl (SSEB) protecting group to replace the DMT protecting group to protect the 5'-OH of the nucleoside. In the solid-phase synthesis of oligonucleotides, trihydroxypropyl phosphorus (THPP) is used as a deprotecting agent to deprotect SSEB, exposing the hydroxyl group, which then couples with the next molecule of phosphorous acid monomer to achieve oligonucleotide chain elongation. The THPP deprotecting agent used in this method is a basic reagent, which avoids errors such as depurination, base deletion, and base insertion caused by using acidic reagents as deprotecting agents, thereby reducing the synthesis error rate, improving the quality of oligonucleotide synthesis, and reducing the difficulty of separation. Attached Figure Description

[0108] Figure 1 Synthesis of phosphorusamide via solid-phase phosphorusamide triester method

[0109] Figure 2 Depurinization reaction pathway

[0110] Figure 3 A reversible reaction for the acidic removal of DMT protecting groups.

[0111] Figure 4 Synthesizing T using CPG-A and CPG-B as carriers 15 Detailed Implementation

[0112] The present invention will be further illustrated by way of embodiments below, but the present invention is not limited to the scope of the embodiments described herein.

[0113] For experimental methods in the following examples where specific conditions are not specified, follow conventional methods and conditions, or select according to the product instructions.

[0114]

[0115] Example 1 Synthesis of 5'-DMT-2'-dT(2a):

[0116]

[0117] Deoxynucleoside 1a (2.42 g, 10 mmol) was placed in a 100 mL round-bottom flask, pyridine was added, and the mixture was rotary evaporated three times (3 x 20 mL) to remove water. 25 mL of dry pyridine was added to dissolve the pyridine, followed by the addition of DMTCl (3.72 g, 11 mmol). The mixture was stirred at room temperature for 2 hours. The system was then evaporated to dryness, and 20 mL of toluene was added. The mixture was then evaporated to dryness again to remove the remaining pyridine. The solution was passed through a column to obtain 4.90 g of 5'-DMT-2'-dT(2a), with a yield of 90%.

[0118] Example 2 Synthesis of 5'-DMT-3'-TBS-2'-dT(3a):

[0119]

[0120] Alcohol 2a (4.90 g, 9 mmol) was dissolved in 20 mL of dry DMF in a 100 mL round-bottom flask, followed by the addition of imidazole (1.53 g, 22.5 mmol) and TBSCl (2.7 g, 18 mmol). The mixture was stirred overnight at room temperature. The reaction was quenched with methanol (5 mL), and the pyridine was evaporated to dryness and dissolved in dichloromethane (50 mL). The solution was washed with saturated sodium carbonate and saturated brine. The organic phase was dried over anhydrous sodium sulfate and purified by column chromatography to give 5.56 g of 5'-DMT-3'-TBS-2'-dT(3a), with a yield of 94%.

[0121] Example 3 Synthesis of 3'-TBS-2'-dT(4a)

[0122]

[0123] Compound 3a (5.26 g, 8 mmol) was dissolved in 50 mL of dichloromethane in a 250 mL round-bottom flask. Trichloroacetic acid (13.7 g, 80 mmol) was added with stirring in an ice bath. After the addition was complete, the mixture was stirred in an ice bath for 10 minutes. The pH of the system was adjusted to alkaline with saturated sodium carbonate. The aqueous phase was extracted with dichloromethane, and the organic phases were combined. The mixture was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, evaporated to dryness, and passed through a column to give 2.74 g of 3'-TBS-2'-dT(4a), with a yield of 96%.

[0124] Example 4 Synthesis of 5'-SSEB-3'-TBS-2'-dT(5a)

[0125]

[0126] Alcohol 4a (2.13 g, 6 mmol) and 2-(1-methyldithio)ethylbenzoic acid (1.5 g, 6.6 mmol) were dissolved in 30 mL of dichloromethane. DCC (1.37 g, 6.6 mmol) and DMAP (100 mg) were added, and the mixture was stirred for 3 h after the addition was complete. The mixture was filtered, and the residue was washed with dichloromethane. The filtrate was evaporated to dryness and passed through a column to give 3.12 g of 5'-SSEB-3'-TBS-2'-dT(5a), with a yield of 92%. Product 5a was a mixture of two epimeric derivatives. 1 H NMR(400MHz, CDCl3)δ9.08(brs,0.5H),9.06(brs,0.5H),7.81-7.76(m,1H),7.65-7.59(m,1H),7.56-7.50 (m,1H),7.32-7.28(m,1H),7.25-7.22(m,1H),6.33-6.26(m,1H),5.32-5.25(m,1H),4.60(dd,J=12.3,4.0 Hz,0.5H),4.51-4.41(m,2.5H),4.20-4.16(m,1H),2.40-2.28(m,1H),2.21-2.13(m,0.5H),2.11(s,1.5H) ,2.10-2.05(m,0.5H),2.03(s,1.5H),1.72-1.67(m,4.5H),1.60(s,1.5H),0.89(s,9H),0.11-0.08(m,6H); 13 C NMR (100MHz, CDCl3) δ167.2,167.1,163.8,163.8,150.3,144.5,144.4, 135.2, 135.2, 132.6, 132.6, 129.9, 129.8, 129.3, 129.1, 128.1, 128.1, 127.2, 111.3, 111.3, 85.2, 8 5.185.0,84.6,72.1,71.8,64.1,64.0,43.7,43.5,41.1,41.1,34.0,25.8,25.8u,25. 7,25.0.23.7,23.6,20.5,20.4,18.0,12.5,12.4,-4.5,-4.7,-4.7.MS(ESI)calcd.for C 26 H 39 N₂O₆S₂Si(M+H) + :567.2,found:567.2.

[0127] Example 5 Synthesis of 5'-SSEB-2'-dT(6a)

[0128]

[0129] Compound 5a (2.26 g, 4 mmol) was dissolved in 20 mL of THF in a 100 mL round-bottom flask, followed by the addition of Et3N·3HF (2.15 g, 40 mmol HF). The mixture was stirred overnight at room temperature. The pH of the system was adjusted to alkaline with saturated sodium carbonate. The THF was evaporated to dryness, and the aqueous phase was extracted with DCM. The organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, evaporated to dryness, and passed through a column to give 1.56 g of 5'-SSEB-2'-dT(6a), with a yield of 86%. Product 6a was a mixture of epimers. 1 H NMR(400MHz, CDCl3)δ10.13(brs,0.5H),9.74(brs,0.5H),7.80-7.68(m,1H),7.62- 7.40(m,2H),7.32-7.14(m,2H),6.37-6.25(m,1H),5.29-5.20(m,0.5H),5.14-4.88( m,0.5H),4.72-4.40(m,3H),4.32-4.22(m,1H),3.51(brs,1H),2.54-2.42(m,1H),2. 41(s,1.5H),2.25-2.10(m,1H),2.08(s,0.75H),2.04(s,0.75H),1.70-1.40(s,6H); 13 C NMR (100MHz, CDCl3) δ167.4,164.4,164.2,164.1,151.0,150.9,150.8,150.7, 144.4,144.2,135.8,135.4,132.6,130.0,129.9,129.2,129.1,128.1,127.2, 127.2,111.6,111.4,85.3,85.1,84.6,84.4,71.7,71.5,64.3,43.9,43.6,40. 5,40.5,23.7,23.6,22.3,21.4,21.3,21.1,20.5,20.412.4;MS(ESI)calcd.for C 20 H 25 N₂O₆S₂(M+H) + :453.5,found:453.6.

[0130] Example 6 Synthesis of 5'-SSEB-2'-dT-phosphoramidite (7a)

[0131]

[0132] Compound 6a (1.36 g, 3 mmol) was placed in a 100 mL round-bottom flask, sealed, and purged with argon. 20 mL of dry dichloromethane was added to dissolve the compound. The system was cooled in an ice bath, and DIPEA (1.98 mL, 12 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.34 mL, 6 mmol) were added dropwise. After the addition was complete, the mixture was stirred in an ice bath for 30 min. The reaction system was diluted with 20 mL of dichloromethane, washed successively with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, and filtered through a HEX / EA / TEA column. 1.50 g of 5'-SSEB-2'-dT-phosphoramidite (7a) was obtained, with a yield of 77%. The product was a mixture of four epimers. 1 H NMR(400MHz, CDCl3)δ9.50(brs,1H),7.82-7.78(m,1H),7.62-7.58(m,1H),7.54-7.49(m,1H),7.29(t ,J=7.6Hz,1H),7.24-7.19(m,1H),6.35-6.30(m,1H),5.30-5.24(m,1H),4.68-4.48(m,3H),4.38-4.27 (m,1H),3.90-3.81(m,1H),3.76-3.69(m,1H),3.65-3.55(m,2H),2.66-2.60(m,2H),2.59-2.44(m,1H) ,2.25-2.15(m,1H),2.10-1.97(m,3H),1.69-1.65(m,4.5H),1.59-1.57(m,1.5H),1.19-1.15(m,12H); 13C NMR (100MHz, CDCl3) δ167.1, 167.0, 166.9, 166.9, 163.9, 150.5, 150.4, 144.3, 144.3, 144.3, 135.1, 135.1, 135.1, 132.5, 129. 9.129.8 .0,64.2,64.1,64.0,63.9,58.3,58.2,58.1,58.0,58.0,45.3,45.3,43.6,43.5,43.4,43.4,43.3,43.3,39.6,39.6,24.7,24.6,24.6,24.6,24.5,24.5,23.6,23.5,23.5,23.4,23,0,23.0,22.9,22.9,20.5,20.5,20.4,20.4,20.3,20.3,12.4,12.3,12.2; 31 P NMR(162MHz, CDCl3)δ149.4,149.3,149.2,149.1; MS(ESI)calcd.for C 29 H 42 N4O7PS2(M+H) + :653.8,found:653.8.

[0133] Example 7 Synthesis of 5'-DMT-2'-dA(Bz)(2b)

[0134]

[0135] Deoxynucleoside 1b (3.55 g, 10 mmol) was placed in a 100 mL round-bottom flask. Pyridine was added and evaporated three times (3 x 20 mL) to remove water. 25 mL of dry pyridine was added to dissolve the pyridine, followed by the addition of DMTCl (3.72 g, 11 mmol). The mixture was stirred at room temperature for 2 hours. The system was evaporated to dryness, and 20 mL of toluene was added. The mixture was then evaporated to dryness again to remove the remaining pyridine. The solution was passed through a column to give 6.04 g of 5'-DMT-2'-dA(Bz)(2b), with a yield of 92%.

[0136] Example 8 Synthesis of 5'-DMT-3'-TBS-2'-dA(Bz)(3b)

[0137]

[0138] 5.91 g (9 mmol) of alcohol 2b was dissolved in 20 mL of dry DMF in a 100 mL round-bottom flask, followed by the addition of imidazole (1.53 g, 22.5 mmol) and TBSCl (2.7 g, 18 mmol). The mixture was stirred overnight at room temperature. The reaction was quenched with methanol (5 mL), and the pyridine was evaporated to dryness and dissolved in dichloromethane (50 mL). The solution was washed with saturated sodium carbonate and saturated brine. The organic phase was dried over anhydrous sodium sulfate and purified by column chromatography to give 6.38 g of 5'-DMT-3'-TBS-2'-dA(Bz)(3b), with a yield of 92%.

[0139] Example 9 Synthesis of 3'-TBS-2'-dA(Bz)(4b)

[0140]

[0141] Compound 3b (6.17 g, 8 mmol) was dissolved in 50 mL of dichloromethane in a 250 mL round-bottom flask. Trichloroacetic acid (13.7 g, 80 mmol) was added with stirring in an ice bath. After the addition was complete, the mixture was stirred in an ice bath for 10 minutes. The pH of the system was adjusted to alkaline with saturated sodium carbonate. The mixture was separated, and the aqueous phase was extracted with dichloromethane. The organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, evaporated to dryness, and passed through a column to give 3.28 g of 3'-TBS-2'-dA(Bz)(4b), with a yield of 88%.

[0142] Example 10 Synthesis of 5'-SSEB-3'-TBS-2'-dA(Bz)(5b)

[0143]

[0144] Alcohol 4b (2.84 g, 6 mmol) and 2-(1-methyldithio)ethylbenzoic acid (1.5 g, 6.6 mmol) were dissolved in 30 mL of dichloromethane. DCC (1.37 g, 6.6 mmol) and DMAP (100 mg) were added, and the mixture was stirred for 3 h. The mixture was filtered, and the residue was washed with dichloromethane. The filtrate was evaporated to dryness and passed through a column to give 3.59 g of 5'-SSEB-3'-TBS-2'-dA(Bz)(5b), with a yield of 88%. Product 5b was a mixture of two epimeric derivatives. 1H NMR(400MHz, CDCl3)δ9.27(brs,1H),8.72(s,0.5H),8.71(s,0.5H),8.12(s,1H),8.02-7.96 (m,2H),7.75-7.67(m,1H),7.59-7.51(m,2H),7.50-7.42(m,3H),7.28-7.20(m,1H),6.47-6 .39(m,1H),5.29-5.21(m,1H),4.78-4.71(m,1H),4.58-4.45(m,2H),4.32-4.26(m,1H),3.0 3-2.93(m,1H),2.54-2.44(m,1H),2.00(s,1.5H),1.97(s,1.5H),1.64(d,J=7.0Hz,1.5H),1 .63(d,J=7.0Hz,1.5H),0.91(s,9H),0.13-0.09(m,6H);167.1,167.0,164.8,152.6,151.4, 151.3,149.7,144.3,144,2,141.9,141.8,133.7,132.8,132.4,130.2,130.1,129.1,129.0 ,128.9,128.0,127.8,127.1,127.0,123.8,123.7,85.3,85.1,85.1,85.0,72.5,72.4,64.2 ,64.0,43.6,40.4,40.2,25.8,23.5,20.5,20.4,18.0,-4.6,-4.7,-4.7;MS(ESI)calcd.forC 33 H 42 N5O5S2Si(M+H) + :680.9,found:680.8.

[0145] Example 11 Synthesis of 5'-SSEB-2'-dA(Bz)(6b)

[0146]

[0147] Compound 5b (2.76 g, 4 mmol) was dissolved in 20 mL of THF in a 100 mL round-bottom flask, followed by the addition of Et3N·3HF (2.15 g, 40 mmol HF). The mixture was stirred overnight at room temperature. The pH of the system was adjusted to alkaline with saturated sodium carbonate. The THF was evaporated to dryness, and the aqueous phase was extracted with DCM. The organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, evaporated to dryness, and purified by column chromatography to give 1.99 g of 5'-SSEB-2'-dA(Bz)(6b), with a yield of 88%. Product 6b was a mixture of epimers.1 H NMR(400MHz, CDCl3)δ9.45(brs,1H,),8.71(s,0.5H),8.69(s,0.5H),8.20-8.14(m,2H),8.03-7.96(m,2H ),7.72-7.76(m,1H),7.58-7.50(m,2H),7.49-7.41(m,3H),7.25-7.18(m,1H),6.46(t,J=6.4Hz,1H),5.2 1(dd,J=13.9,6.9Hz,0.5H),5.20(dd,J=13.9,7.0Hz,0.5H),4.85-4.79(m,1H),4.63-4.50(m,2H),4.40- 4.34(m,1H),2.97-2.85(m,1H),2.65-2.55(m,1H),1.96(s,1.5H),1.95(s,1.5H),1.62(d,J=7.0Hz,3H); 13 C NMR (100MHz, CDCl3) δ167.3,165.2,152.5,152.3,149.6,144.1,144.0,141.9,141.8,133.6,132.9,132.4,130.2,129.1,129.1,128.9,128 .1,127.8,127.1127.0,123.4,85.1,85.0,84.9,84.8,71.3,71.6,64.6,64.4,53.5,43.8,43.7,40.0,23.6,23.5,20.4; MS(ESI)calcd.for C 27 H 28 N5O5S2(M+H) + :566.7,found:566.6.

[0148] Example 12 Synthesis of 5'-SSEB-2'-dA(Bz)-phosphoramidite(7b)

[0149]

[0150] Compound 6b (1.70 g, 3 mmol) was placed in a 100 mL round-bottom flask, sealed, and purged with argon. 20 mL of dry dichloromethane was added to dissolve the compound. The system was cooled in an ice bath, and DIPEA (1.98 mL, 12 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.34 mL, 6 mmol) were added dropwise. After the addition was complete, the mixture was stirred in an ice bath for 30 min. The reaction system was diluted with 20 mL of dichloromethane, washed successively with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, and filtered through a HEX / EA / TEA column. 1.56 g of 5'-SSEB-2'-dA(Bz)-phosphoramidite (7b) was obtained, with a yield of 68%. The product was a mixture of four epimers. 1 H NMR(400MHz, CDCl3)δ9.36(brs,1H),8.12(s,1H),8.01-7.95(m,2H),7.77-7.70(m,1H),7.57 -7.50(m,2H),7.48-7.42(m,3H),7.27-7.20(m,1H),6.48-6.40(m,1H),5.28-5.20(m,1H),4.9 4-4.83(m,1H),4.68-4.40(m,3H),3.92-3.82(m,1H),3.80-3.71(m,1H),3.68-3.58(m,2H),3. 12-2.96(m,1H),2.74-2.58(m,3H),2.04-1.90(m,3H),1.66-1.58(m,3H),1.24-1.12(m,12H); 13C NMR (100MHz, CDCl3) δ167.0,167.0,167.0,166.9,152.5,151.4,151.4,151.3,151.3,149.7,144.1,144.1,141.9,141 ,8,133.7,132.7,132.3,130.2,129.1,129.0,129.0,128.8,127.9,127.7,127.0,127.0,123.7,117.6,85.0,84.9,84. 3,84.3,84.2,84.2,84.1,84.0,83.9,83.9,73.8,73.6,73.2,73.2,73.0,73.0,64.3,64.2,64.1,64.0,58.5,58.3,58. 3,58.1,46.1,43.6,43.4,43.4,43.3,43.2,39.0,39.0,38.9,24.7,24.6,24.6,23.5,23.4,23.4,20.5,20.4,20.4; 31P NMR(162MHz, CDCl3)δ149.2,149.2,149.1,149.1; MS(ESI)calcd.for C36H45N7O6PS2(M+H) + :766.9,found:766.8.

[0151] Example 13 Synthesis of 5'-DMT-2'-dG(iBu)(2c)

[0152]

[0153] Deoxynucleoside 1c (3.37 g, 10 mmol) was placed in a 100 mL round-bottom flask, pyridine was added, and the mixture was rotary evaporated three times (3 x 20 mL) to remove water. 25 mL of dry pyridine was added to dissolve the pyridine, followed by the addition of DMTCl (3.72 g, 11 mmol). The mixture was stirred at room temperature for 2 hours. The system was then evaporated to dryness, and 20 mL of toluene was added. The mixture was then evaporated to dryness again to remove the remaining pyridine. The solution was passed through a column to obtain 5.90 g of 5'-DMT-2'-dG(iBu)(2c), with a yield of 93%.

[0154] Example 14 Synthesis of 5'-DMT-3'-TBS-2'-d G(iBu)(3c)

[0155]

[0156] 5.75 g (9 mmol) of alcohol 2c was dissolved in 20 mL of dry DMF in a 100 mL round-bottom flask, followed by the addition of imidazole (1.53 g, 22.5 mmol) and TBSCl (2.7 g, 18 mmol). The mixture was stirred overnight at room temperature. The reaction was quenched with methanol (5 mL), and the pyridine was evaporated to dryness and dissolved in dichloromethane (50 mL). The solution was washed with saturated sodium carbonate and saturated brine. The organic phase was dried over anhydrous sodium sulfate and purified by column chromatography to give 5.90 g of 5'-DMT-3'-TBS-2'-dG(iBu)(3c), with a yield of 87%.

[0157] Example 15 Synthesis of 3'-TBS-2'-dG(iBu)(4c)

[0158]

[0159] Compound 3c (5.42 g, 8 mmol) was dissolved in 50 mL of dichloromethane in a 250 mL round-bottom flask. Trichloroacetic acid (13.7 g, 80 mmol) was added with stirring in an ice bath. After the addition was complete, the mixture was stirred in an ice bath for 10 minutes. The pH of the system was adjusted to alkaline with saturated sodium carbonate. The aqueous phase was extracted with dichloromethane, and the organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, evaporated to dryness, and passed through a column to give 3.24 g of 3'-TBS-2'-dG(iBu)(4c), with a yield of 90%.

[0160] Example 16 Synthesis of 5'-SSEB-3'-TBS-2'-dG(iBu)(5c)

[0161]

[0162] Alcohol 4c (2.71 g, 6 mmol) and 2-(1-methyldithio)ethylbenzoic acid (1.5 g, 6.6 mmol) were dissolved in 30 mL of dichloromethane. DCC (1.37 g, 6.6 mmol) and DMAP (100 mg) were added, and the mixture was stirred for 3 h. The mixture was filtered, and the residue was washed with dichloromethane. The filtrate was evaporated to dryness and passed through a column to give 3.50 g of 5'-SSEB-3'-TBS-2'-dG(iBu)(5c), with a yield of 88%. Product 5c was a mixture of two epimeric derivatives. 11H NMR (400 MHz, CDCl3) δ 12.11 (brs, 0.5H), 12.09 (brs, 0.5H), 9.49 (brs, 0.5H), 9.43 (brs, 0.5H), 7.78 - 7.76 (m, 1H), 7.75 - 7.68 (m, 1H), 7.59 - 7.55 (m, 1H), 7.49 (t, J = 7.6 Hz, 0.5H), 7.49 (t, J = 7.6 Hz, 0.5H), 7.28 (t, J = 7.5 Hz, 1H), 6.27 - 6.21 (m, 1H), 5.25 - 5.18 (m, 1H), 4.78 (dd, J = 11.4, 5.9, Hz, 0.5H), 4.75 (dd, J = 11.7, 5.5, Hz, 0.5H), 4.60 - 4.47 (m, 2H), 4.36 - 4.28 (m, 1H), 2.92 - 2.71 (m, 2H), 2.38 - 2.30 (m, 1H), 1.95 (s, 1.5H), 1.92 (s, 1.5H), 1.64 (d, J = 7.0 Hz, 1.5H), 1.62 (d, J = 7.0 Hz, 1.5H), 1.25 - 1.20 (m, 6H), 0.88 (s, 4.5H), 0.87 (s, 4.5H), 0.10 - 0.07 (m, 6H); 13 13C NMR (100 MHz, CDCl3) δ 179.2, 179.1, 168.2, 167.8, 155.7, 147.9, 147.8, 147.6, 147.5, 144.1, 144.0, 138.2, 137.8, 132.6, 132.5, 130.3, 130.2, 129.1, 128.9, 127.9, 127.9, 127.2, 122.4, 122.1, 85.9, 85.2, 73.1, 72.9, 64.6, 64.2, 43.9, 43.7, 39.9, 39.6, 36.4, 36.4, 34.0, 33.9, 25.8, 23.7, 23.6, 20.4, 20.3, 19.2, 19.1, 18.9, 18.0, -4.6, -4.6, -4.7, -4.8; MS (ESI) calcd. for C 30 H 44 N5O6S2Si (M + H) + : 662.9, found: 662.9. [[ID=E9]]

[0163] Example 17 Synthesis of 5'-SSEB-2'-dG(iBu) (6c)

[0164]

[0165] Compound 5c (2.65 g, 4 mmol) was dissolved in 20 mL of THF in a 100 mL round-bottom flask, followed by the addition of Et3N·3HF (2.15 g, 40 mmol HF). The mixture was stirred overnight at room temperature. The pH of the system was adjusted to alkaline with saturated sodium carbonate. The THF was evaporated to dryness, and the aqueous phase was extracted with DCM. The organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, evaporated to dryness, and purified by column chromatography to give 1.75 g of 5'-SSEB-2'-dG(iBu)(6c), with a yield of 80%. Product 6c was a mixture of epimers. 1 H NMR(400MHz, CDCl3)δ12.43(brs,1H),10.51(brs,0.5H),10.43(brs,0.5H),7.95-7.91(m,1H),7.70(t ,J=7.6Hz,1H),7.51(d,J=7.9Hz,1H),7.43(t,J=7.6Hz,1H),7.21(t,J=7.5Hz,1H),6.29-6.20(m,1H), 5.20-5.13(m,1H),5.01-4.93(m,1H),4.68-4.48(m,2H),4.42-4.35(m,1H),2.97-2.87(m,1H),2.82-2 .70(m,1H),2.60-2.50(m,1H),1.93(s,1.5H),1.90(s,1.5H),1.60(d,J=7.0Hz,3H),1.29-1.23(m,6H); 13 C NMR (100MHz, CDCl3) δ180.4,168.0,167.7,156.0,148.3,148.2,148.2,14 4.0,144.0,138.8,138.6,132.4,130.4,129.4,129.2,127.8,127.1,121. 4,121.2,85.7,85.4,85.0,71.5,71.4,65.3,65.0,53.6,43.9,43.8,39.8 ,36.4,36.3,23.6,23.5,20.4,20.4,19.2,19.2,19.1;MS(ESI)calcd.for C 24 H 30 N5O6S2(M+H) + :548.6,found:548.5.

[0166] Example 18 Synthesis of 5'-SSEB-2'-dG(iBu)-phosphoramidite(7c)

[0167]

[0168] Compound 6c (1.64 g, 3 mmol) was placed in a 100 mL round-bottom flask, sealed, and purged with argon. 20 mL of dry dichloromethane was added to dissolve the compound. The system was cooled in an ice bath, and DIPEA (1.98 mL, 12 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.34 mL, 6 mmol) were added dropwise. After the addition was complete, the mixture was stirred in an ice bath for 30 min. The reaction system was diluted with 20 mL of dichloromethane, washed successively with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, and filtered through a HEX / EA / TEA column. 1.36 g of 5'-SSEB-2'-dG(iBu)-phosphoramidite (7c) was obtained, with a yield of 60%. The product was a mixture of four epimers. 1H NMR(400MHz,CDCl3)δ9.35(brs,1H),7.84-7.60(m,2H),7.59-7.42(m,2H),7.29-7.21(m,1H),6.24-6.16(m,1H),5.26-5.18(m,1H),4.96-4.82(m,1H),4.77-4.66(m,1H),4.62-4.52(m,1H),4.50-4.38(m,1H),3.96-3.84(m,1H)3.77-3.69(m,1H),3.67-3.54(m,2H),3.01-2.50(m,5H),1.99-1.94(m,3H),1.67-1.60(m,3H),1.25-1.15(m,18H);179.5,179.5,179.4,179.3,179.3,170.3,167.9,167.8,167.6,167.4,155.7,152.5,147.8,147.7,147.7,147.6,147.6,144.0,144.0,143.9,138.4,138.2,137.9,137.6,133.4,132.4,132.4,130.6,130.5,130.3,130.2,129.2,129.1,128.9,128.1,127.8,127.7,127.1,127.0,124.9,123.6,118.0,117.9,85.9,85.7,85.4,85.0,84.3,84.2,84.1,84.0,83.8,74.6,74.4,73.6,73.5,64.9,64.6,64.4,64.3,57.9,57.8,57.7,57.4,45.3,43.8,43.6,43.4,43.4,43.3,43.2,39.3,38.9,38.8,36.2,36.1,36.0,35.9,34.5,24.7,24.6,24.6,24.6,24.5,23.5,20.7,20.6,20.6,20.4,20.3,20.3,19.1,19.1,19.1,18.9,18.9; 31 PNMR(162MHz,CDCl3)δ149.2,149.1,148.4,148.3;MS(ESI)calcd.for C 33 H 47 N7O7PS2(M+H) + :748.9,found:748.8.

[0169] Example 19 Synthesis of 5'-DMT-2'-dC(Ac)(2d)

[0170]

[0171] Deoxynucleoside 1d (2.69 g, 10 mmol) was placed in a 100 mL round-bottom flask, pyridine was added, and the mixture was rotary evaporated three times (3 x 20 mL) to remove water. 25 mL of dry pyridine was added to dissolve the pyridine, followed by the addition of DMTCl (3.72 g, 11 mmol). The mixture was stirred at room temperature for 2 hours. The system was then evaporated to dryness, 20 mL of toluene was added, and the mixture was evaporated again to dryness to remove the remaining pyridine. The solution was passed through a column to give 5.20 g of 5'-DMT-2'-dC(Ac)(2d), with a yield of 91%.

[0172] Example 20 Synthesis of 5'-DMT-3'-TBS-2'-dC(Ac)(3d)

[0173]

[0174] 5.13 g (9 mmol) of ethanol was dissolved in 20 mL of dry DMF in a 100 mL round-bottom flask, followed by the addition of imidazole (1.53 g, 22.5 mmol) and TBSCl (2.7 g, 18 mmol). The mixture was stirred overnight at room temperature. The reaction was quenched with methanol (5 mL), and the pyridine was evaporated to dryness and dissolved in dichloromethane (50 mL). The solution was washed with saturated sodium carbonate and saturated brine. The organic phase was dried over anhydrous sodium sulfate and purified by column chromatography to give 5.56 g of 5'-DMT-3'-TBS-2'-dC(Ac)(3d), with a yield of 90%.

[0175] Example 21 Synthesis of 3'-TBS-2'-dC(Ac)(4d)

[0176]

[0177] Compound 3d (5.49 g, 8 mmol) was dissolved in 50 mL of dichloromethane in a 250 mL round-bottom flask. Trichloroacetic acid (13.7 g, 80 mmol) was added with stirring in an ice bath. After the addition was complete, the mixture was stirred in an ice bath for 10 minutes. The pH of the system was adjusted to alkaline with saturated sodium carbonate. The aqueous phase was extracted with dichloromethane, and the organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, evaporated to dryness, and passed through a column to give 2.60 g of 3'-TBS-2'-dC(Ac)(4d), with a yield of 85%.

[0178] Example 22 Synthesis of 5'-SSEB-3'-TBS-2'-dC(Ac)(5d)

[0179]

[0180] 2.30 g (6 mmol) of alcohol 4d and 1.5 g (6.6 mmol) of 2-(1-methyldithio)ethylbenzoic acid were dissolved in 30 mL of dichloromethane. DCC (1.37 g, 6.6 mmol) and DMAP (100 mg) were added, and the mixture was stirred for 3 h. The mixture was filtered, and the residue was washed with dichloromethane. The filtrate was evaporated to dryness and passed through a column to give 3.14 g of 5'-SSEB-3'-TBS-2'-dC(Ac)(5d), with a yield of 88%. Product 5d was a mixture of two epimers. 1 H NMR (400MHz, CDCl3) δ10.06(brs,1H),8.08(t,J=8.0Hz,1H),7.76-7.71(m,1H),7.64-7.60(m,1H),7.58-7.53(m,1H),7.37-7.32(m, 1H),7.30(d,J=7.6Hz,0.5H),7.21(d,J=7.5Hz,0.5H),6.24-6.16(m,1H),5.31-5.24(m,1H),4.61(dd,J=12.4,4.0Hz,0.5H),4.54(d, J=4.0Hz,1H),4.51(dd,J=12.4,3.0Hz,0.5H),4.39-4.31(m,1H),4.26-4.21(m,1H),2.62-2.52(m,1H),2.27(s,1.5H),2.26(s,1.5H) ,2.20-2.10(m,1H),2.06(s,1.5H),1.98(s,1.5H),1.69(d,J=7.0Hz,1.5H),1.69(d,J=7.0Hz,1.5H),0.87(s,9H),0.08-0.04(m,6H); 13 C NMR (100MHz, CDCl3) δ171.0,167.1,167.0,162.9,154.8,144.3,144.3,144. 0,143.9,132.7,132.7,129.8,129.8,129.2,129.0,128.1,127.2,96.7,87. 3,87.2,85.3,84.9,71.2,70.6,63.5,63.4,43.7,43.6,42.2,42.0,25.8,25 .0,23.6,23.6,20.4,20.4,18.0,-4.5,-4.5,-4.8,-4.8;MS(ESI)calcd.for C 27 H 39 N₂O₇S₂Si(M+H) +:594.8,found:594.6.

[0181] Example 23 Synthesis of 5'-SSEB-2'-dC(Ac)(6d)

[0182]

[0183] Compound 5d (2.38 g, 4 mmol) was dissolved in 20 mL of THF in a 100 mL round-bottom flask, followed by the addition of Et3N·3HF (2.15 g, 40 mmol HF). The mixture was stirred overnight at room temperature. The pH of the system was adjusted to alkaline with saturated sodium carbonate. The THF was evaporated to dryness, and the aqueous phase was extracted with DCM. The organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, evaporated to dryness, and passed through a column to give 1.70 g of 5'-SSEB-2'-dC(Ac)(6d), with a yield of 89%. Product 6d was a mixture of epimers. 1 H NMR(400MHz, CDCl3)δ9.51(brs,1H),8.09(t,J=7.5Hz,1H),7.77-7.73(m,1H),7.62-7.57(m,1H),7 .52(t,J=7.8Hz,1H),7.35-7.27(m,2H),6.28(t,J=5.9Hz,1H),5.29-5.21(m,1H),4.71(dd,J=4.4, 12.4Hz,0.5H),4.64-4.50(m,1.5H),4.50-4.42(m,1H),4.40-4.34(m,1H),2.88-2.76(m,1H),2.24 (s,1.5H),2.23(s,1.5H),2.20-2.10(m,1H),2.03(s,1.5H),1.99(s,1.5H),1.68(d,J=7.0Hz,3H); 13 C NMR (100MHz, CDCl3) δ170.9, 170.8, 167.5, 167.4, 162.6, 155.5, 144.2, 144.1, 132.6, 130.0, 129.2, 129.1, 128.0, 127.3, 12 7.2,97.0,96.9,87.6,85.1,84.9,70.9,70.9,64.4,64.1,43.8,43.7,41.7,25.0,23.7,23.6,20.4,20.3; MS(ESI)calcd.for C 21 H 25 N₂O₇S₂(M+H) + :480.6,found:480.5.

[0184] Example 24 Synthesis of 5'-SSEB-2'-dC(Ac)-phosphoramidite(7d)

[0185]

[0186] Compound 6d (1.44 g, 3 mmol) was placed in a 100 mL round-bottom flask, sealed, and purged with argon. 20 mL of dry dichloromethane was added to dissolve the compound. The system was cooled in an ice bath, and DIPEA (1.98 mL, 12 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.34 mL, 6 mmol) were added dropwise. After the addition was complete, the mixture was stirred in an ice bath for 30 min. The reaction system was diluted with 20 mL of dichloromethane, washed successively with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, and filtered through a HEX / EA / TEA column. 1.34 g of 5'-SSEB-2'-dC(Ac)-phosphoramidite (7d) was obtained, with a yield of 66%. The product was a mixture of four epimers. 1 H NMR(400MHz, CDCl3)δ9.86(brs,1H),8.08-7.98(m,1H),7.78-7.72(m,1H),7.64-7.58(m,1H),7.57-7.50 (m,1H),7.36-7.20(m,2H),6.28-6.18(m,1H),5.31-5.22(m,1H),4.70-4.49(m,3H),4.47-4.36(m,1H),3. 90-3.80(m,1H),3.78-3.68(m,1H),3.66-3.54(m,2H),2.82-2.68(m,1H),2.66-2.58(m,2H),2.25(s,1.5H ),2.24(s,1.5H),2.05(s,0.75H),2.04(s,0.75H),1.99(s,1.5H),1.72-1.67(m,3H),1.19-1.14(m,12H); 13C NMR (100MHz, CDCl3) δ171.0,167.1,167.0,167.0,166.9,162.9,162.9,155.0,144.3,144.2,143 .8,132.6,129.9,129.2,129.1,129.0,129.0,128.1,128.0,127.2,117.6,96.8,96.8,96.8.87. 4,87.3,87.2,84.6,84.2,84.2,83.8,72.7,72.5,72.2,63.8,63.7,58.4,58.2,46.2,43.7,43.6,43.5,43.5,43.4,43.4,41.0,41.0,40.9,40.9,25.0,24.7,24.7,24.6,23.6,23.6,20.4,20.4; 31 P NMR(162MHz, CDCl3)δ149.6,149.5,149.5,149.4; MS(ESI)calcd.for C 30 H 43 N5O7PS2(M+H) + :680.8,found:680.8.

[0187] Example 25 Synthesis of 5'-SSEB-2'-dT-CPG

[0188] (1) Synthesis of 5'-SSEB-3'-succinic acid-2'-dT(8a)

[0189]

[0190] Alcohol 6a (904 mg, 2 mmol) and DMAP (366 mg, 3 mmol) were placed in a 100 mL round-bottom flask. 50 mL of dry pyridine was added to dissolve the pyridine, followed by the addition of succinic anhydride (300 mg, 3 mmol). The reaction was stirred at room temperature for 48 h, and the reaction was quenched with 3 mL of water. The pyridine was evaporated to dryness, dissolved in toluene, and evaporated to dryness again to remove the remaining pyridine. The solution was then purified by column chromatography using dichloromethane / methanol to give approximately 972 mg of acid 8a, in 88% yield. 8a is a mixture of epimers. 1H NMR (400MHz, CDCl3) δ10.05(brs,1H),7.81-7.75(m,1H),7.65-7.60(m,1H),7.56-7.60(m ,1H),7.34-7.28(m,1H),7.26-7.24(m,1H),6.36-6.29(m,1H),5.41-5.34(m,1H),5.31-5. 22(m,1H),4.70-4.48(m,2H),4.43-4.38(m,1H),2.74-2.62(m,4H),2.59-2.48(m,1H),2. 26-2.15(m,1H),2.13(s,1.5H),2.06(s,1.5H),1.72-1.65(m,4.5H),1.56-1.53(m,1.5H); 13 C NMR (100MHz, CDCl3) δ176.2,172.0,171.9,167.0,166.9,164.6,150.6,144.6,135.2,132.7,129.8,129.7,129.1,129.0,128.2, 128.2,127.2,111.9,111.7,84.8,82.0,74.7,74.5,64.6,64.4,43.6,43.4,37.7,37.6,29.3,29.0,23.6,20.5,20.3,12.3,12.3.

[0191] (2) Synthesis of 5'-SSEB-2'-dT-CPG

[0192]

[0193] 500 mg of CPG-linker-NH2 and 156 mg (0.3 mmol) of compound 8a were placed in a 100 mL Erlenmeyer flask. 10 mL of pyridine was added, followed by DCC (550 mg, 2.64 mmol). The system was sealed and shaken at room temperature for 48 h. The system was then filtered, and the resulting CPG powder was washed successively with pyridine, dichloromethane, and acetonitrile, and dried. The obtained CPG powder was placed in a 100 mL dry Erlenmeyer flask, and 5 mL each of commercially available capping reagent A (Cap A) and capping reagent B (Dinacinco) were added. The system was sealed and shaken for 48 h, filtered, and the resulting CPG powder was washed with acetonitrile and dried to obtain 500 mg of CPG-dT-SSEB 9 (CPG-A).

[0194] SSEB content determination: 10 mg of CPG-dT-SSEB 9 was weighed and treated with 0.5 mL of 20 mM THPP aqueous solution. The treated CPG was then washed with 0.5 mL of acetonitrile. The two were combined and analyzed by HPLC. The results were compared with a standard curve (using the product after SSEB removal as the standard, gradient concentrations of 1.5 mg / mL, 750 μg / mL, 250 μg / mL, 150 μg / mL, 75 μg / mL, and 15 μg / mL were prepared, and the standard curve was plotted with peak area as the ordinate and corresponding concentration as the abscissa). The SSEB content of the synthesized CPG was approximately 29.5 μmol / g.

[0195] In the following examples, the oxidizing agent is 0.02 M I2 of pyridine / tetrahydrofuran / aqueous solution.

[0196] Example 26 Synthesis of oligonucleotide T4

[0197] Oligonucleotide T4 was synthesized on an ABI 3400 oligonucleotide synthesizer using CPG-A (SSEB-2'-dT-CPG) as the solid-phase support at a synthesis volume of 0.2 μmol. The synthesis followed the standard 0.2 μmol synthesis program set on the synthesizer, employing DMT-OFF synthesis mode. SSEB was removed after the synthesis of the last base. The 5-ethimercaptotetrazole activator, oxidizing agent, Cap A, and Cap B reagents used in the synthesis were all commercially available reagents purchased from Dynacin. The deprotecting agent was a 20 mM THPP aqueous solution. The monomer was an acetonitrile solution of SSEB-2'-dT-phosphoramidite (7a) at a concentration of 50 mg / mL. After synthesis, the CPG support was treated with 1 mL of concentrated ammonia for 1 h to cleave the product from the CPG. The resulting ammonia solution was then deprotected at 60 °C for 16 h. The system was concentrated with ammonia under vacuum at 60 °C to obtain the crude product T4. This crude product was dissolved in 200 μL of deionized water and analyzed by HPLC, with the chromatogram compared to the target sequence. ESI-MS analysis (calculated m / z for T4 1154.1, found 1153.1 [MH]) + ] - ,576[M-2H + ] 2- .

[0198] Example 27 Oligonucleotide T 10 Synthesis

[0199] Oligonucleotide T 10The synthesis was performed on an ABI 3400 oligonucleotide synthesizer using CPG-A (SSEB-2'-dT-CPG) as the solid support at a synthesis volume of 0.2 μmol. The synthesis followed the standard 0.2 μmol synthesis program set on the synthesizer, employing the DMT-OFF synthesis mode. SSEB was removed after the synthesis of the last base. The 5-ethimercaptotetrazole activator, oxidizing agent, Cap A, and Cap B reagents used in the synthesis were all commercially available reagents purchased from Dynacin. The deprotection agent was a 20 mM THPP aqueous solution, and the monomer was an acetonitrile solution of SSEB-2'-dT-phosphoramidite (7a) at a concentration of 50 mg / mL. After synthesis, the CPG support was treated with 1 mL of concentrated ammonia for 1 h to cleave the product from the CPG. The resulting ammonia solution was then deprotected at 60 °C for 16 h. The system was then concentrated with ammonia under vacuum at 60 °C to obtain T. 10 The crude product was dissolved in 200 μL of deionized water, detected by HPLC, and compared with the HPLC chromatogram of the target sequence. ESI-MS analysis (calculated m / z for T) 10 2980.0,744.0[M-2H + ] 2- ,595.3[M-5H + ] 5- .

[0200] Example 28 Oligonucleotide T 15 Synthesis

[0201] Oligonucleotide T 15 The synthesis was performed on an ABI 3400 oligonucleotide synthesizer using CPG-A (SSEB-2'-dT-CPG) as the solid support at a synthesis volume of 0.2 μmol. The synthesis followed the standard 0.2 μmol synthesis program set on the synthesizer, employing the DMT-OFF synthesis mode. SSEB was removed after the synthesis of the last base. The 5-ethimercaptotetrazole activator, oxidizing agent, Cap A, and Cap B reagents used in the synthesis were all commercially available reagents purchased from Dynacin. The deprotection agent was a 20 mM THPP aqueous solution, and the monomer was an acetonitrile solution of SSEB-2'-dT-phosphoramidite (7a) at a concentration of 50 mg / mL. After synthesis, the CPG support was treated with 1 mL of concentrated ammonia for 1 h to cleave the product from the CPG. The resulting ammonia solution was then deprotected at 60 °C for 16 h. The system was then concentrated with ammonia under vacuum at 60 °C to obtain T. 15The crude product was dissolved in 200 μL of deionized water, detected by HPLC, and compared with the HPLC chromatogram of the target sequence. ESI-MS analysis (calculated m / z for T) 15 4501.0, found 1502.0 [M-3H] + ] 3- ,1124.0[M-4H + ] 4- 898.2[M-5H + ] 5- 748.4 [M-6H] + ] 6- .

[0202] Example 29 Oligonucleotide T 20 Synthesis

[0203] Oligonucleotide T 20 The synthesis was performed on an ABI 3400 oligonucleotide synthesizer using CPG-A (SSEB-2'-dT-CPG) as the solid support at a synthesis volume of 0.2 μmol. The synthesis followed the standard 0.2 μmol synthesis program set on the synthesizer, employing the DMT-OFF synthesis mode. SSEB was removed after the synthesis of the last base. The 5-ethimercaptotetrazole activator, oxidizing agent, Cap A, and Cap B reagents used in the synthesis were all commercially available reagents purchased from Dynacin. The deprotection agent was a 20 mM THPP aqueous solution, and the monomer was an acetonitrile solution of SSEB-2'-dT-phosphoramidite (7a) at a concentration of 50 mg / mL. After synthesis, the CPG support was treated with 1 mL of concentrated ammonia for 1 h to cleave the product from the CPG. The resulting ammonia solution was then deprotected at 60 °C for 16 h. The system was then concentrated with ammonia under vacuum at 60 °C to obtain T. 20 The crude product was dissolved in 200 μL of deionized water, detected by HPLC, and compared with the HPLC chromatogram of the target sequence. ESI-MS analysis (calculated m / z for T) 20 6021.1, 1504.3 [M-4H] + ] 4- ,1023.2[M-5H + ] 5- ,1002.5[M-6H + ] 6- ,859.1[M-7H + ] 7- ,668.0[M-9H + ] 9- .

[0204] Example 30 Synthesis of oligonucleotide TA4

[0205] Oligonucleotide TA4 was synthesized on an ABI 3400 oligonucleotide synthesizer using CPG-A (SSEB-2'-dT-CPG) as the solid-phase support at a synthesis volume of 0.2 μmol. The synthesis followed the standard 0.2 μmol synthesis program set on the synthesizer, employing DMT-OFF synthesis mode. SSEB was removed after the synthesis of the last base. The 5-ethimercaptotetrazole activator, oxidizing agent, Cap A, and Cap B reagents used in the synthesis were all commercially available reagents purchased from Dynacin. The deprotecting agent was a 20 mM THPP aqueous solution. The monomer was an acetonitrile solution of SSEB-2'-dA(Bz)-phosphoramidite(7b) at a concentration of 100 mg / mL. After synthesis, the CPG support was treated with 1 mL of concentrated ammonia for 1 h to cleave the product from the CPG. The resulting ammonia solution was then deprotected at 60 °C for 16 h. The system was concentrated with ammonia under vacuum at 60 °C to obtain crude TA4. This crude product was dissolved in 200 μL of deionized water and analyzed by HPLC, with the chromatogram compared to that of the target sequence. ESI-MS analysis (calculated m / z for TA4 2913.0, found 970.0 [M-3H]) + ] 3- 727.4 [M-4H] + ] 4- .

[0206] Example 31 Synthesis of oligonucleotide TC4

[0207] Oligonucleotide TC4 was synthesized on an ABI 3400 oligonucleotide synthesizer using CPG-A (SSEB-2'-dT-CPG) as the solid-phase support at a synthesis volume of 0.2 μmol. The synthesis followed the standard 0.2 μmol synthesis program set on the synthesizer, employing DMT-OFF synthesis mode. SSEB was removed after the synthesis of the last base. The 5-ethimercaptotetrazole activator, oxidizing agent, Cap A, and Cap B reagents used in the synthesis were all commercially available reagents purchased from Dynacin. The deprotecting agent was a 20 mM THPP aqueous solution. The monomer was an acetonitrile solution of SSEB-2'-dC(Ac)-phosphoramidite (7d) at a concentration of 100 mg / mL. After synthesis, the CPG support was treated with 1 mL of concentrated ammonia for 1 h to cleave the product from the CPG. The resulting ammonia solution was then deprotected at 60 °C for 16 h. The system was concentrated with ammonia under vacuum at 60 °C to obtain crude TC4. This crude product was dissolved in 200 μL of deionized water and analyzed by HPLC, with the chromatogram compared to that of the target sequence. ESI-MS analysis (calculated m / z for TC4 1399.0, found 1397.8 [MH]) + ] - ,698.4[M-2H + ] 2- .

[0208] Example 32 Synthesis of oligonucleotide TGTG

[0209] The oligonucleotide TGTG was synthesized on an ABI 3400 oligonucleotide synthesizer using CPG-A (SSEB-2'-dT-CPG) as the solid-phase support at a synthesis volume of 0.2 μmol. The synthesis followed the standard 0.2 μmol synthesis program set on the synthesizer, employing DMT-OFF synthesis mode. SSEB was removed after the synthesis of the last base. The 5-ethimercaptotetrazole activator, oxidizing agent, Cap A, and Cap B reagents used in the synthesis were all commercially available reagents purchased from Dynacin. The deprotecting agent was a 20 mM THPP aqueous solution. The monomers used were an acetonitrile solution of SSEB-2'-dT-phosphoramidite (7a) at a concentration of 50 mg / mL and an acetonitrile solution of SSEB-2'-dG(iBu)-phosphoramidite (7c) at a concentration of 100 mg / mL. After synthesis, the CPG support was treated with 1 mL of concentrated ammonia for 1 h to cleave the product from the CPG. The resulting ammonia solution was then deprotected at 60 °C for 16 h. The system was concentrated with ammonia under vacuum at 60 °C to obtain crude TGTG, which was dissolved in 200 μL of deionized water. HPLC analysis was performed, and the results were compared with the HPLC chromatogram of the target sequence. ESI-MS analysis (calculated m / z for TGTG 1204.9, found 1204.0 [MH]) + ] - 601.5[M-2H + ] 2- .

[0210] The single-cycle yields of the oligonucleotides synthesized in this invention are shown in the table below, all of which are above 98%.

[0211] Table 1. Single-cycle yield of synthesized oligonucleotides

[0212] sequence Single-cycle yield T4 99％ T10 98.5％ T15 98.5％ T20 98.5％ TAAAA 98％ TCCCC 98.5％ TGTG 98.5％

[0213] Example 33 Screening of deprotected solvents

[0214]

[0215] The removal efficiency of THPP solutions prepared with different solvents was investigated using the synthesized 5'-SSEB-3'-TBS-2'-dT(5a) as a substrate. A 1 mM acetonitrile solution of 5'-SSEB-3'-TBS-2'-dT was prepared, then mixed 1:1 with the 20 mM THPP solution to be tested. After standing for 1 min, the mixture was analyzed by HPLC as a control. The test structures are shown in the table below.

[0216] Table 2. Deprotection efficiency of 20mM THPP in different solvents

[0217]

[0218] As shown in the table above, the deprotection efficiency is worst when using pure organic solvents such as acetonitrile and methanol. In this case, the raw material partially decomposes, and the system mainly consists of intermediates before ring closure, with very little product. Using a pure aqueous solution of 20 mM THPP yields the best results, and deprotection can be basically completed within 1 minute.

[0219] Example 34 Stability of the Protecting Base (SSEB)

[0220] During oligo synthesis, the SSEB protecting group is present at the oligo terminus in each of the three steps of coupling, oxidation, and capping in each cycle, thus being exposed to the coupling activator, oxidizing agent, and capping agent for an extended period. Therefore, it is necessary to investigate whether this group can remain stable in these reagents.

[0221] 5'-SSEB-3'-TBS-2'-dT(5a) was used as a model to investigate the stability of SSEB in these reagents. A 1 mM acetonitrile solution of 5'-SSEB-3'-TBS-2'-dT(5a) was prepared and mixed with an equal volume of commercially available activator (0.25 M 5-ethimercaptotetrazole in acetonitrile), oxidizing agent (0.02 M I2 of pyridine / tetrahydrofuran / aqueous solution), capping reagent A (CAP A), capping reagent B (CAP B), and CAP A / CAP B (1:1, v:v). After 24 hours, HPLC analysis showed no significant changes. Therefore, it is believed that commercially available reagents for synthesizing oligos can be used in SSEB-protected systems.

[0222] Example 35 Effect of deprotecting agent (THPP) on amino protection

[0223] Except for the T base, the bases on monomers A, C, and G all have active amino groups, which can affect the reactions during the synthesis of phosphorusamide and oligo. Therefore, the amino groups of bases A, C, and G need to be protected before the synthesis of phosphorusamide. In this example, the A base is protected with benzoyl (Bz), the C base with acetyl (Ac), and the G base with isobutyryl (Bz). i Bu) protection.

[0224] Therefore, this invention synthesizes the corresponding 5'-DMT-3'-TBS-2'-dA(Bz)(5b), 5'-DMT-3'-TBS-2'-dG( iBu)(5c), 5'-DMT-3'-TBS-2'-dC(Ac)(5d), and prepared into a 1mM acetonitrile solution, were mixed with an equal volume of 20mM THPP. After 24 hours, HPLC analysis showed no change. Therefore, it can be inferred that THPP does not affect the corresponding protecting group on the amino group. Thus, in this invention, the amino group of monomer A is protected with benzoyl (Bz), the amino group of monomer C is protected with acetyl (Ac), and the amino group of monomer G is protected with isobutyryl (Bz). i Bu) protection.

[0225] Example 36: Screening of the linker between the solid support and the oligo

[0226] (1) Using commercially available DMT-protected general-purpose CPG An attempt was made to synthesize polyT. First, the DMT protecting group was manually removed using TCA before the product was placed on a synthesizer. The complete set of reagents of this invention (SSEB-2'-dT-phosphoramide monomer solution, THPP deprotection reagent and capping reagent, oxidizing reagent) was used to synthesize polyT. The result was that almost no product was generated.

[0227] (2) 5'-SSEB-2'-dT(6a) was reacted with succinic anhydride to obtain the product 5'-SSEB-3'-succinic acid-2'-dT(8a), which was then reacted with CPG-Linker-NH2. Unreacted amino groups were quenched using a capping agent, resulting in two SSEB-2'-dT-CPGs with different linkers: CPG A and CPG B. CPG A has a long carbon chain linker, while CPG B's linker is similar to that of general-purpose CPGs. Instrumental testing revealed that CPG A is well-suited for the SSEB protection-deprotection synthesis system of this invention, while CPG B, similar to general-purpose CPGs, is similar to commercially available general-purpose CPGs after DMT removal and is not suitable for this system. For example, using CPG A and CPG B respectively in our system to synthesize T... 15 Similar to the general CPG synthesis, it was found that CPG B failed to yield the reaction product, while CPG A successfully synthesized T15. The results are as follows: Figure 4 As shown (Note: Figure 4 (The presence of superimposed images does not affect the interpretation of the technical solution / effect.)

[0228]

Claims

1. A compound represented by Formula A, ， wherein Base pg Refers to a base or a base with a protecting group; R1and R2are each independently C 1-6 alkyl; R3, R4, R5, R6 are each independently hydrogen; R7 is a β-cyanoethyl group; R8and R9are each independently C 1-6 alkyl.

2. The compound of claim 1, according to Formula A, wherein one or more of the following conditions: (1) the base with a protecting group is an A base with a phenylpropionyl group, a C base with an acetyl group, or a G base with an isobutyryl group; (2) R1 and R2 are each independently methyl, ethyl, or isopropyl; (3) R8 and R9 are each independently methyl, ethyl, or isopropyl.

3. The compound of claim 1, having the formula A, ###0001### A one or two of the following conditions: (1) R1 and R2 are each independently methyl; (2) R8 and R9 are each independently isopropyl.

4. The compound of claim 1, according to Formula A, wherein The compound as shown in Formula A is , , , , , or .

5. A process for the preparation of a compound of formula A as claimed in claim 1, comprising the step of reacting a compound A-1 with ###00003### A-1 in the presence of a base in a solvent to obtain compound A, the reaction scheme of said step being represented as follows: ###00004### A-1 A by carrying out a reaction as represented below to obtain compound A, the reaction scheme of said step being represented as follows: ###00005### ； wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, Base pg As defined in claim 1 for a compound of formula A.

6. The production method according to claim 5, wherein one or more of the following conditions: (1) the solvent is a halogenated alkane solvent; (2) the base is N,N-diisopropylethylamine; (3) said molar ratio of 2: 1 with the compound A-1; (4) the molar ratio of the base to the compound A-1 is 4:

1.

7. The production method according to claim 5, wherein the solvent is dichloromethane.

8. The production method according to any one of claims 5 to 7, wherein The method for preparing the compound as shown in formula A further comprises the following step: reacting compound A-2 with Et3N 3HF to obtain compound A-1, and the reaction formula of the step is as shown in the following: 。 9. The production method according to claim 8, wherein one or two of the following conditions: (1) the solvent is an ether solvent; (2) said Et3N The molar ratio of 3HF to said compound A-2 is 10:

1.

10. The production method according to claim 8, wherein the solvent is tetrahydrofuran.

11. The production method according to claim 8, wherein The preparation method of the compound represented by Formula A further comprises the following step: in a solvent, compound A-3 and a compound represented by Formula C are subjected to the following reaction in the presence of a base and a dehydrating agent to obtain compound A-2, and the reaction formula of the step is as follows: 。 12. The production method according to claim 11, wherein one or more of the following conditions: (1) the solvent is a halogenated alkane solvent; (2) the base is 4-dimethylaminopyridine; (3) the molar ratio of the base to the compound A-3 is 0.14:1; (4) the dehydrating agent is dicyclohexyl carbodiimide; (5) the molar ratio of the dehydrating agent to the compound A-3 is 1.1:1; (6) the molar ratio of the compound represented by Formula C to the compound A-3 is 1.1:

1.

13. The production method according to claim 11, wherein the solvent is dichloromethane.

14. The production method according to claim 11, wherein The preparation method of the compound represented by Formula A further comprises the following step: in a solvent, the compound A-4 is subjected to the following reaction in the presence of an acid to obtain compound A-3, and the reaction formula of the step is as follows: 。 15. The production method according to claim 14, wherein one or more of the following conditions: (1) the solvent is a halogenated alkane solvent; (2) the acid is trichloroacetic acid; (3) the molar ratio of the acid to the compound A-4 is 10:

1.

16. The production method according to claim 14, wherein the solvent is dichloromethane.

17. The production method according to claim 14, wherein The preparation method of the compound represented by Formula A further comprises the following step: in a solvent, compound A-5 and TBS-Cl are subjected to the following reaction in the presence of imidazole to obtain compound A-4, and the reaction formula of the step is as follows: 。 18. The production method according to claim 17, wherein one or more of the following conditions: (1) the solvent is an amide solvent; (2) the molar ratio of the imidazole to the compound A-5 is 2.5:1; (3) the molar ratio of the TBS-Cl to the compound A-5 is 2:

1.

19. The production method according to claim 17, wherein the solvent is N,N-dimethylformamide.

20. The production method according to claim 17, wherein The preparation method of the compound represented by Formula A further comprises the following step: in a solvent, compound A-6 and DMT-Cl are subjected to the following reaction to obtain compound A-5, and the reaction formula of the step is as follows: 。 21. The production method according to claim 20, wherein one or both of the following conditions is met: (1) the solvent is pyridine; (2) the molar ratio of the DMT-Cl to the compound A-6 is 1.1:

1.

22. A method of preparing a compound of Formula C, comprising the step of reacting compound D with trihydroxypropylphosphine in a solvent as shown below to obtain compound C, R is , or , R1, R2, R3, R4, R5, R6, R7, R8, R9, Base pg as defined in claim 1, said step comprising the reaction as shown below: 。 23. The production method according to claim 22, wherein the solvent is selected from one or more of a nitrile solvent, an alcohol solvent, and water.

24. The production method according to claim 23, wherein one or both of the following conditions is met: (1) the nitrile solvent is acetonitrile; (2) the alcohol solvent is methanol.

25. A method for preparing a solid support containing a starting nucleic acid molecule SSEB-2'-dN-CPG, comprising the following steps: CPG-linker-NH2is reacted with compound E SSEB-2'-dN in the presence of a dehydrating agent and a base as shown below to give a solid support SSEB-2'-dN-CPG containing a starting nucleic acid molecule, R1, R2, R3, R4, R5, R6, Base pg as defined in claim 1, dN represents any one of dA, dC, dT and dG and other deoxynucleotides, and the reaction of the step is shown below: ， wherein The CPG-linker-NH2 is .

26. The production method according to claim 25, wherein one or more of the following conditions is met: (1) the compound E is , , , , , or ; (2) the dehydrating agent is dicyclohexyl carbodiimide; (3) the base is pyridine; (4) the reaction further comprises the following post-treatment steps: washing and drying; (5) the method for preparing the solid support containing a starting nucleic acid molecule further comprises a capping step.

27. The production method according to claim 26, wherein one or both of the following conditions is met: (1) the solvent used for washing is pyridine, dichloromethane, and acetonitrile; (2) the method for preparing the solid support containing a starting nucleic acid molecule further comprises capping using a capping reagent.

28. A method for synthesizing a nucleic acid, comprising the following steps: (1) SSEB-2'-dN-CPG prepared by the method of claim 12 is subjected to a deprotection reaction with trihydroxypropyl phosphine in a solvent to obtain dN-CPG; (2) reacting the dN-CPG with the compound of formula A as claimed in claim 1 in the presence of an activating agent to form a protected phosphite triester; (3) oxidizing the protected phosphite triester in the presence of an oxidizing agent to obtain a protected phosphate ester; (4) deprotecting the protected phosphate ester with trihydroxypropyl phosphine in a solvent to obtain a first extension product; (5) optionally, repeating steps (2), (3), (4) to obtain an extension product; (6) reacting the extension product or the first extension product with a cleaving solvent to cleave the CPG-Linker-NH2.

29. The nucleic acid synthesis method of claim 28, wherein, one or more of the following conditions is met: (1) in step (1), the solvent is one or more of a nitrile solvent, an alcohol solvent, and water; (2) in step (1), the molar ratio of the trihydroxypropyl phosphine to the SSEB-2'-dN-CPG is 20:1; (3) in step (2), the activating agent is 5-ethylmercapto tetrazole; (4) in step (3), the oxidizing agent is a mixture of I2, pyridine, tetrahydrofuran, and water; (5) in step (4), the solvent is one or more of a nitrile solvent, an alcohol solvent, and water; (6) in step (4), the molar ratio of the trihydroxypropyl phosphine to the protected phosphate ester is 20:1; (7) in step (6), the cleaving solvent is ammonia; (8) the step (2) further comprises a capping step; (9) further comprising the following step: further deprotecting the system obtained in step (6) and vacuum concentrating.

30. The method of nucleic acid synthesis of claim 29, wherein, one or more of the following conditions is met: (1) in step (1), the nitrile solvent is acetonitrile; (2) in step (1), the alcohol solvent is methanol; (3) in step (2), the activating agent is 0.25 mol / L 5-ethylmercapto tetrazole in acetonitrile; (4) In step (3), the oxidizing agent is a mixture of 0.02 mol / L I2, pyridine, tetrahydrofuran and water; (5) In step (4), the nitrile solvent is acetonitrile; (6) In step (4), the alcohol solvent is methanol; (7) In step (6), the cleavage solvent is 1 mL of concentrated ammonia; (8) The step (2) further comprises capping using a capping reagent; (9) Further comprising the step of further deprotecting the system obtained in step (6) for 16 h; (10) Further comprising the step of further deprotecting the system obtained in step (6) and vacuum concentration, wherein the deprotecting and vacuum concentration are performed at 60 °C.

31. A kit comprising the compound of formula A as defined in claim 1.

32. The kit of claim 31, wherein which satisfies the following conditions: the kit further comprises the activating agent, the oxidizing agent, the trihydroxypropyl phosphine and the cleavage solvent as defined in the nucleic acid synthesis method of claim 28, the capping reagent as defined in the nucleic acid synthesis method of claim 30.

Images