Use of silicon dioxide microspheres in synthesis of ultra-long oligonucleotide and method for synthesizing ultra-long oligonucleotide

Abstract

The present invention relates to the technical field of oligonucleotide synthesis. Disclosed are the use of silicon dioxide microspheres in the synthesis of an ultra-long oligonucleotide and a method for synthesizing an ultra-long oligonucleotide, wherein the silicon dioxide microspheres have a specific surface area of less than 1 m2 / g, and preferably, the silicon dioxide microspheres have modification groups on the surface. The method for synthesizing an ultra-long oligonucleotide comprises: performing a coupling reaction on a nucleoside phosphoramidite synthesis unit using the silicon dioxide microspheres having modification groups on the surface as carriers under coupling reaction conditions. The prepared carriers can be used to prepare an ultra-long oligonucleotide.

Description

Application of silica microspheres in synthesizing ultra-long oligonucleotides and method for synthesizing ultra-long oligonucleotides

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Chinese patent application No. 202311116401.9 filed on August 31, 2023, the contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the technical field of oligonucleotide synthesis, and in particular to the application of silica microspheres in synthesizing ultra-long oligonucleotides and a method for synthesizing ultra-long oligonucleotides. Background Art

[0004] Research on the chemical synthesis of oligonucleotides (DNA Oligo) began in the 1950s. In the 1980s, Marvin Caruthers first reported the synthesis of oligodinucleotides using the phosphodiester method. Currently, the most commonly used method for chemical synthesis of oligonucleotides is the solid-phase phosphoramidite method, which was developed by Beaucage and Caruthers and includes four steps: deprotection, coupling, capping, and oxidation.

[0005] Since the advent of the first chemical oligonucleotide synthesis method, improvements in the length and quality of synthesizable oligonucleotides have opened up new applications. The ability to synthesize oligonucleotides of 20-30 bases in length enabled the development of PCR and DNA sequencing, laying the foundation for recombinant DNA technology and molecular diagnostics. The ability to synthesize oligonucleotides of 50-100 bases in length has enabled new technologies for precise DNA manipulation, including site-directed mutagenesis and genetic engineering. However, due to inherent limitations, the length and quality of oligonucleotides produced have reached a plateau. Current synthesis processes, including deprotecting agents, coupling agents, and monomers, can lead to byproducts such as depurination, or incomplete capping, making the synthesis of ultra-long oligonucleotides difficult. However, there is a growing demand for ultra-long oligonucleotides, even gene-length synthetic DNA, for applications in cell and gene therapy, protein engineering, biomanufacturing, and basic life science research. Currently, longer DNA can only be produced by "splicing" shorter oligonucleotides together, but this process presents numerous challenges with specialized sequences, such as DNA secondary structures, repetitive sequences, and high or low GC content. In addition, many applications require ultra-long single-stranded DNA, which is currently very difficult to prepare. Therefore, a new or improved technology is needed to overcome the challenge of assembling these special sequences from shorter oligonucleotides and directly synthesize single ultra-long oligonucleotides.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to overcome the problem of short chain length of oligonucleotides prepared by chemical synthesis in the prior art, and to provide the application of silica microspheres in the synthesis of ultra-long oligonucleotides and a method for synthesizing ultra-long oligonucleotides.

[0008] In order to achieve the above-mentioned object, the first aspect of the present invention provides the use of silica microspheres in the synthesis of ultra-long oligonucleotides, wherein the specific surface area of ​​the silica microspheres is less than 1m 2 / g.

[0009] Preferably, the silica microspheres are solid silica microspheres, and the average particle size of the silica microspheres is 50-100 μm.

[0010] Preferably, the surface of the silica microspheres has a modified group;

[0011] Preferably, the structure of the silica microspheres having modified groups on the surface is as shown in formula (1);

[0012] Among them, A is silica microsphere, R 1 and R 3 Each independently selected from alkoxy, preferably C1-C 10 Alkoxy; R 2 Selected from C1-C 10 Alkylene-O-C1-C 10 Alkylene, -C1-C 10 Alkylene-(NH-C1-C 10 alkylene) n -、C1-C 10 Alkylene, n is an integer of 1-10; R 4 Selected from -NH-(CH2CH2O) m -C1-C 10 Alkylene-NH-, -(O-CH2CH2) p -O-, m and p are each independently an integer of 1-10; L1 is a linker group capable of undergoing a coupling reaction with nucleoside phosphoramidite.

[0013] Optionally, C1-C 10 Alkylene is C1-C 10 The present invention is substituted with at least one substituent selected from the group consisting of an alkyl ester group, a hydroxyl group, and an alkyloxy group.

[0014] A second aspect of the present invention provides a method for synthesizing ultralong oligonucleotides, comprising: under coupling reaction conditions, using the silica microspheres having modified groups on the surface as carriers to carry out a coupling reaction on nucleoside phosphoramidite synthesis units.

[0015] Ultralong oligonucleotides (300-500 nt) can be prepared using the carrier prepared by the present invention. Using a preferred embodiment of the present invention, the carrier prepared by the present invention is used in combination with a combination coupling agent and a combination deprotecting agent to further increase the chain length of the oligonucleotide. Using a more preferred embodiment of the present invention, the carrier prepared by the present invention is used in combination with a combination coupling agent, a combination deprotecting agent, and a nucleoside phosphoramidite to not only further increase the chain length of the oligonucleotide, but also to improve synthesis efficiency and shorten synthesis time by approximately 40%. The method of the present invention can also reduce reagent consumption by approximately 50%. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG1 is a microscopic photograph of the solid non-porous silica microspheres used in Preparation Example 1;

[0017] FIG2 is a liquid chromatography (a)-mass spectrum (b) of the nucleoside phosphoramidite trimer of Preparation Example A1;

[0018] FIG3 is a PAGE gel image of the identification of the oligonucleotide chain in Example 1; wherein, lane A: single-stranded DNA ladder; lane C: oligonucleotide synthesized in Comparative Example 1; lane E: 329 nt oligonucleotide synthesized in Example 1;

[0019] FIG4 is a capillary electrophoresis peak diagram of the ultralong oligonucleotide chain after amplification in Example 1;

[0020] Figure 5 is a PAGE gel image of the identification of the oligonucleotide chain in Example 2; wherein, the first left lane: single-stranded DNA ladder; the second left lane: 300 nt, sample load 50 ng; the third left lane: 300 nt, sample load 100 ng; the fourth left lane: 350 nt, sample load 50 ng; the fifth left lane: 350 nt, sample load 100 ng; the sixth left lane: 400 nt, sample load 50 ng; the seventh left lane: 400 nt, sample load 100 ng;

[0021] FIG6 is a chemical synthesis route of silica microspheres (supports) having surface modification groups;

[0022] FIG7 is a schematic diagram of using surface-treated solid silica microspheres as carriers to prepare ultralong oligonucleotides. DETAILED DESCRIPTION

[0023] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0024] The first aspect of the present invention provides the use of silica microspheres in the synthesis of ultra-long oligonucleotides, wherein the specific surface area of ​​the silica microspheres is less than 1m 2 / g. In the present invention, the specific surface area of ​​the silica microspheres can be 0.9m 2 / g, 0.8m 2 / g, 0.7m 2 / g, 0.6m 2 / g, 0.5m 2 / g, 0.4m 2 / g, 0.3m 2 / g, 0.2m 2 / g, 0.1m 2 / g, 0.05m 2 / g, 0.04m 2 / g, 0.01m 2 / g, 0.001m 2 / g, and the range consisting of any two of the above points.

[0025] According to the present invention, preferably, the specific surface area of ​​the silica microspheres is less than 0.2 m 2 / g, more preferably less than 0.15m 2 / g, more preferably 0.03-0.05m 2 / g.

[0026] According to the present invention, preferably, the silica microspheres are solid silica microspheres, and the average particle size of the silica microspheres is 50-100 μm, for example, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, and a range consisting of any two of the above. In the present invention, unless otherwise specified, the specific surface area and particle size of the silica microspheres refer to the silica microspheres and do not include the surface modification groups of the silica microspheres, that is, they refer to the silica microsphere raw material before pretreatment.

[0027] According to the present invention, preferably, the surface of the silica microspheres has a modified group; more preferably, the structure of the silica microspheres having a modified group on the surface is as shown in formula (1);

[0028] Among them, A is silica microsphere, R 1 and R 3 Each independently selected from alkoxy, preferably C1-C 10 Alkoxy; R 2 Selected from C1-C 10 Alkylene-O-C1-C 10 Alkylene, -C1-C 10Alkylene-(NH-C1-C 10 alkylene) n -、C1-C 10 Alkylene, n is an integer of 1-10; R 4 Selected from -NH-(CH2CH2O) m -C1-C 10 Alkylene-NH-, -(O-CH2CH2) p -O-, m and p are each independently an integer of 1-10; L1 is a linker group capable of undergoing a coupling reaction with nucleoside phosphoramidite.

[0029] Optionally, R 2 、R 4 Any one of C1-C 10 Alkylene is C1-C 10 The present invention is substituted with at least one substituent selected from the group consisting of an alkyl ester group, a hydroxyl group, and an alkyloxy group.

[0030] In the present invention, C1-C 10 Alkoxy can be CH3O-, CH3CH2O-, CH3(CH2)2O-, CH3(CH2)3O-, CH3(CH2)4O-, CH3(CH2)5O-, CH3(CH2)6O-, CH3(CH2)7O-, CH3(CH2)8O-, CH3(CH2)9O-. 10 The alkoxy group may be a linear alkoxy group or a branched alkoxy group.

[0031] In the present invention, C1-C 10 Examples of the alkylene group include methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, and decylene. The alkylene group may be either a straight chain or a branched chain.

[0032] In the present invention, n, m, and p can each independently be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0033] According to the present invention, preferably, the silica microspheres having modified groups on the surface have a structure represented by formula (2) or formula (3):

[0034] In the present invention, the linker group can be provided by a conventional linker in the art that can undergo coupling reaction with nucleoside phosphoramidites. Preferably, the linker is selected from linker 1 to linker 31 described below in the present invention, and particularly preferably selected from linker 31.

[0035] The present invention also provides a method for preparing the above-mentioned silica microspheres having modified groups on the surface, the method comprising the following steps:

[0036] (1) Pre-treating silica microspheres to obtain silica microspheres rich in hydroxyl groups on the surface;

[0037] (2) using a silane reagent to perform a first grafting on the silica microspheres rich in hydroxyl groups on the surface;

[0038] (3) In the presence of a condensing agent and an alkylamine, the first grafted silica microspheres are subjected to a condensation reaction with the linker.

[0039] According to the present invention, preferably, the pretreatment conditions include: temperature of 20-50° C., time of 0.5-5 h, preferably 1-2 h.

[0040] According to the present invention, preferably, the pretreatment method includes pretreating the silica microspheres with a fluorine-containing solution.

[0041] According to the present invention, preferably, the amount of the fluorine-containing solution used is 0.5-1 mol in terms of fluorine element per 100 g of silica microspheres.

[0042] According to the present invention, preferably, the concentration of the fluorine-containing solution is 0.5-3 mol / L, and the solute in the fluorine-containing solution includes at least one of hydrogen fluoride, ammonium fluoride, tetrabutylammonium fluoride, tetraethylammonium fluoride, triethylamine hydrogen fluoride and pyridine hydrogen fluoride.

[0043] According to the present invention, preferably, the conditions for the first grafting include: a temperature of 25-50° C., and a time of 1-24 h, preferably 16-18 h.

[0044] According to the present invention, preferably, the amount of the silane reagent used is 50-100 g per 100 g of silica microspheres.

[0045] According to the present invention, in order to obtain a carrier with a smaller steric hindrance effect, preferably, the silane reagent includes at least one of 5,6-epoxyhexyltriethoxysilane, 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane, [8-(epoxypropyloxy)-n-octyl]trimethoxysilane, 3-glycidyloxypropyltriethoxysilane, 11-(triethoxysilyl)undecane-1-amine and 11-aminoundecyltrimethoxysilane. In the present invention, the silane reagent can be contacted with the silica microspheres having a rich surface hydroxyl group in the form of a solution to perform the first grafting. The content of the silane reagent in the silane reagent solution can be 1-50% by weight, and the solvent in the silane reagent solution can be acetonitrile.

[0046] According to the present invention, in order to further reduce the steric hindrance effect of the carrier, preferably, the preparation method further comprises performing a second grafting on the silica microspheres after the first grafting, and then performing a condensation reaction with the linker.

[0047] According to the present invention, preferably, the conditions for the second grafting include: a temperature of 25-50° C., and a time of 1-24 h, preferably 16-18 h.

[0048] According to the present invention, preferably, the amount of the grafting agent used in the second grafting is 0.5-2 g per 100 g of silica microspheres.

[0049] According to the present invention, preferably, the grafting agent used in the second grafting (hereinafter referred to as the second grafting agent) is a long-chain molecule containing an active functional group, wherein the active functional group is selected from a hydroxyl group, and the number of carbon atoms in the long-chain molecule is 2-50 (preferably 5-50); more preferably, the long-chain molecule containing an active functional group includes at least one of pentaethylene glycol, hexaethylene glycol and polyethylene glycol (e.g., PEG-800).

[0050] According to the present invention, preferably, the condensation reaction conditions include: temperature of 20-50° C., time of 1-24 h, preferably 16-18 h.

[0051] According to the present invention, preferably, with respect to every 100 g of silica microspheres, the amount of the linker is 0.5-2 g, the amount of the condensing agent is 1-4 g, and the amount of the alkylamine is 0.5-2 g.

[0052] According to the present invention, preferably, the linker is selected from at least one of the following compounds:

[0053] DMTr (dimethoxytrityl) is a protecting group. DMTr can also be replaced with MMTr (paramethoxytrityl) or Tr (trityl). The carboxyl groups in the linker react with the amino or hydroxyl groups in the second grafted silica microspheres through a condensation reaction.

[0054] According to the present invention, preferably, the condensing agent includes at least one of dicyclohexylcarbodiimide, diisopropylcarbodiimide, N-ethyl-N'-3-dimethylaminopropylcarbodiimide or its hydrochloride, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, 1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazole-3-oxide hexafluorophosphate and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

[0055] According to the present invention, preferably, the alkylamine is an alkylamine having 2 to 10 carbon atoms, preferably N,N-diisopropylethylamine.

[0056] In the present invention, the linker, condensing agent, and alkylamine can be contacted with the first grafted silica microspheres in the form of a solution to produce a condensation reaction. The concentrations of the linker solution, condensing agent solution, and alkylamine solution can each independently be 1-5 mmol / L, and the solvent in the solution can be acetonitrile.

[0057] The inventors have further discovered that when using silica microspheres with modified groups on the surface prepared from long-chain molecules with active functional groups being amine groups as carriers for oligonucleotide synthesis, ultra-long oligonucleotides can also be prepared, and the chain length of the oligonucleotide can reach more than 300 nt. Therefore, the active functional group in the long-chain molecules containing active functional groups of the present invention can also be an amine group. Preferably, the long-chain molecules containing active functional groups are Here, q is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0058] In the present invention, when the grafting agent used in the second grafting contains a long chain molecule with an active functional group selected from When the second grafting is performed, a protecting group is required to protect the amino group (NH2) at one end of the long chain molecule, for example, The grafting agent used for the second grafting is used to perform the second grafting on the silica microspheres after the first grafting.

[0059] According to the present invention, preferably, the present invention also provides a method of A method for preparing silica microspheres having a structure shown in formula (2) and a surface modified group for a second grafting reagent: (1) pre-treating the silica microspheres with a fluorine-containing aqueous solution to obtain silica microspheres rich in hydroxyl groups on the surface; (2) performing a first grafting on the silica microspheres rich in hydroxyl groups using a silane reagent; (3) performing a second grafting on the silica microspheres after the first grafting using a second grafting reagent in the presence of an alkylamine, then removing the protecting group in the second grafting reagent, and then allowing the silica microspheres after the second grafting to undergo a condensation reaction with a linker in the presence of a condensing agent; the preparation method can refer to the flow chart shown in Figure 6. Step (3) can also include reacting acetic anhydride with the hydroxyl groups in the silica microspheres after the second grafting to prevent the linker from reacting with the hydroxyl groups. The inventors further found that compared with using hydroxyl groups as active functional groups, using amine groups as active functional groups can further increase the chain length of ultra-long oligonucleotides.

[0060] A second aspect of the present invention provides a method for synthesizing ultralong oligonucleotides, comprising: under coupling reaction conditions, using the silica microspheres having modified groups on the surface as carriers to carry out a coupling reaction on nucleoside phosphoramidite synthesis units.

[0061] As is known in the art, oligonucleotides are usually prepared under anhydrous and anaerobic conditions. Therefore, the present invention also prepares oligonucleotides under anhydrous and anaerobic conditions commonly used in the art.

[0062] According to the present invention, preferably, the method further comprises: removing the protecting groups of the silica microspheres by using a deprotecting agent, and then performing the coupling reaction.

[0063] According to the present invention, preferably, the deprotecting agent comprises at least one of trichloroacetic acid, dichloroacetic acid, and 3,5-dinitrobenzoic acid. More preferably, the deprotecting agent comprises trichloroacetic acid, dichloroacetic acid, and 3,5-dinitrobenzoic acid. Further preferably, the deprotecting agent consists of trichloroacetic acid, dichloroacetic acid, and 3,5-dinitrobenzoic acid.

[0064] According to the present invention, preferably, the weight ratio of trichloroacetic acid, dichloroacetic acid and 3,5-dinitrobenzoic acid is 1:1-10:1-10. In the present invention, the weight ratio of trichloroacetic acid, dichloroacetic acid and 3,5-dinitrobenzoic acid can be 1:1:1, 1:2:2, 1:3:3, 1:4:4, 1:5:5, 1:6:6, 1:7:7, 1:8:8, 1:9:9, 1:10:10, and ranges consisting of any two of the above.

[0065] According to the present invention, preferably, the temperature for removing the protecting groups of the silica microspheres is 25-50°C.

[0066] According to the present invention, preferably, the coupling reaction is carried out in the presence of a coupling agent, wherein the coupling agent is at least one of 5-benzylthio-1H-tetrazole, N-methylimidazole, and 4,5-dicyanoimidazole. More preferably, the coupling agent comprises 5-benzylthio-1H-tetrazole, N-methylimidazole, and 4,5-dicyanoimidazole. Further preferably, the coupling agent consists of 5-benzylthio-1H-tetrazole, N-methylimidazole, and 4,5-dicyanoimidazole.

[0067] According to the present invention, preferably, the weight ratio of 5-benzylthio-1H-tetrazole, N-methylimidazole and 4,5-dicyanoimidazole is 5-15:0.5-3:1. In the present invention, the weight ratio of 5-benzylthio-1H-tetrazole, N-methylimidazole and 4,5-dicyanoimidazole can be 5:0.5:1, 6:0.5:1, 7:0.5:1, 8:0.5:1, 9:0.5:1, 10:0.5:1, 11:0.5:1, 12:0.5:1, 13:0.5:1, 14:0.5:1, 15:0.5:1, 9:2:1, 11:2:1, and ranges consisting of any two of the above.

[0068] According to the present invention, preferably, the nucleoside phosphoramidite synthesis unit comprises a nucleoside phosphoramidite monomer and / or a nucleoside phosphoramidite multimer, and preferably, the nucleoside phosphoramidite multimer comprises at least one of a nucleoside phosphoramidite dimer, a nucleoside phosphoramidite trimer and a nucleoside phosphoramidite tetramer.

[0069] According to the present invention, preferably, the nucleoside phosphoramidite dimer has a structure shown in formula (1), and the nucleoside phosphoramidite trimer has a structure shown in formula (2);

[0070] Wherein, B1, B2 and B3 are each independently a base or a base with a protecting group, R is selected from H or -O-hydroxy protecting group, and Pg is a protecting group of phosphoramidite.

[0071] According to the present invention, preferably, the base is selected from at least one of adenine, thymine, cytosine and guanine.

[0072] According to the present invention, preferably, the protecting group of the base is selected from substituted or unsubstituted C1-C10 acyl groups, substituted or unsubstituted amidino groups; more preferably, the C1-C10 acyl groups are selected from acetyl, benzoyl, isobutyryl, phenoxyacetyl, methoxyacetyl, and the amidino group is selected from dimethylformamidino.

[0073] According to the present invention, preferably, the hydroxyl protecting group in R is selected from triisopropylsiloxymethyl, tert-butyldimethylsilyl, tert-butyldisulfide, 2-cyanoethoxymethyl, pivaloyloxymethyl, bis-(2-acetoxyethoxy)methyl), thiocarbamate, iminooxymethylpropionic acid ethyl ester, 2-cyano-2,2-dimethylethylimine-N-oxymethyl, p-nitrobenzyloxymethyl, cyanoethylation, 2-(4-toluenesulfonyl)ethoxymethyl, acetal levulinate.

[0074] According to the present invention, preferably, Pg is selected from alkyl, -CH2CH2CN, -CH2CH=CHCH2CN, p-CH2C6H4CH2CN, -(CH2)2-N(H)COCF3, -CH2CH2Si(C6H5)2CH3, -CH2CH2N(CH3)COCF3.

[0075] In the present invention, the nucleoside phosphoramidite dimer can be obtained by condensing two nucleoside phosphoramidite monomers, or can be synthesized by referring to other methods in the prior art, or can be directly purchased. For example, the nucleoside phosphoramidite dimer is selected from AA, AC, AT, AG, CA, CT, CC, CG, TG, TT, TA, TC, GA, GC, GT, and GG.

[0076] According to the present invention, the nucleoside phosphoramidite trimer can be obtained by condensing two nucleoside phosphoramidite monomers, or can be synthesized by referring to other methods in the prior art, or can be directly purchased. For example: TTC, AAA, AAC, ACT, ATC, ATG, CAG, CAT, CCG, CGT, CTG, GAA, GAC, GCT, GGT, GTT, TAC, TCT, TGC, TGG, TTC.

[0077] In the present invention, in the nucleoside phosphoramidite synthesis unit, A is adenine ribonucleoside, T is thymine ribonucleoside, C is cytosine ribonucleoside, and G is guanine ribonucleoside.

[0078] According to the present invention, preferably, the temperature of the coupling reaction is 20-40°C.

[0079] According to a specific embodiment of the present invention, the method for synthesizing ultralong oligonucleotides (as shown in FIG7 ) comprises the following steps:

[0080] (a) removing the protecting groups on the linker of the carrier under the action of a deprotecting agent;

[0081] (b) subjecting the nucleoside phosphoramidite synthesis unit to a coupling reaction in the presence of a coupling agent and the support obtained in step (a);

[0082] (c) subjecting the product of the coupling reaction to a first capping and oxidation step;

[0083] (d) Repeat the process of steps (a)-(c).

[0084] According to the present invention, preferably, step (c) further comprises performing a second capping on the oxidation product. More preferably, the capping reagent used in the second capping can be the same as the first capping reagent.

[0085] According to the present invention, preferably, in step (a), the deprotecting agent is used in the form of a solution, and the single dosage of the deprotecting agent solution is 10-40 μL relative to 20 mg of the carrier. The deprotecting agent can be formulated as a solution in toluene or dichloromethane, and the content of the deprotecting agent in the solution can be 1-5% by weight. The content of the deprotecting agent in the solution refers to the total weight of trichloroacetic acid, dichloroacetic acid, and 3,5-dinitrobenzoic acid in the solution.

[0086] According to the present invention, preferably, in step (a), the coupling agent is used in the form of a solution, and the single dosage of the coupling agent solution is 5-20 μL relative to 20 mg of the carrier. The coupling agent can be formulated into a solution of acetonitrile, and the concentration of the coupling agent in the solution can be 0.1-0.5 mol / L. Wherein, the concentration of the coupling agent in the solution refers to the sum of the concentrations of 5-benzylthio-1H-tetrazole, N-methylimidazole and 4,5-dicyanoimidazole in the solution.

[0087] According to the present invention, preferably, in step (a), the nucleoside phosphoramidite synthesis unit is used in the form of a solution (for example, the deprotecting agent is prepared as an acetonitrile solution), and the single dosage of the nucleoside phosphoramidite synthesis unit solution is 5-20 μL relative to 20 mg of the carrier.

[0088] According to the present invention, the first capping step is to block sites where the coupling reaction failed to successfully occur, thereby preventing the coupling reaction from occurring in the next cycle and introducing a deletion sequence. The first capping step can be performed using conventional capping reagents in the art. Preferably, the first capping reagent is selected from at least one of acetic anhydride (acetic anhydride), pyridine, and N-methylimidazole. More preferably, the first capping reagent is selected from acetic anhydride, pyridine, and N-methylimidazole, wherein the weight ratio of acetic anhydride, pyridine, and N-methylimidazole is 1:0.5-2:70-90.

[0089] According to the present invention, the oxidation is to oxidize the trivalent phosphine after the coupling reaction to a pentavalent phosphine, thereby improving the stability of the oligonucleotide chain; the oxidation can use a conventional oxidizing agent in the art, preferably, the oxidizing agent is selected from iodine, more preferably a mixed solution of iodine, pyridine and water, wherein the concentration of iodine in the mixed solution is 0.01-0.1 mol / L, and the volume ratio of pyridine to water is 15-20:1.

[0090] According to the present invention, preferably, the method further comprises sequentially performing cleavage deprotection, desalting, and purification on the product of the coupling reaction to obtain the ultralong oligonucleotide. The cleavage deprotection, desalting, and purification can be performed using methods commonly used in the art.

[0091] The present invention will be described in detail below by way of examples.

[0092] Normal temperature is about 25-30℃.

[0093] Preparation Example A1

[0094] This preparation example is used to illustrate the preparation method of nucleoside phosphoramidite trimer

[0095] Preparation process of nucleoside phosphoramidite trimer TTC

[0096] Specific experimental steps:

[0097] (1) Compound 1 (50 g, 78.9 mmol) was added to a 2 L eggplant-shaped single-necked flask, a magnetic stirrer was added, and then 800 ml of dry pyridine was added. The mixture was stirred evenly with a magnetic stirrer to fully dissolve Compound 1. The reaction system was filled with nitrogen and placed in an ice bath to maintain the temperature at 0°C. Tert-butyldimethylsilyl chloride (14.27 g, 94.68 mmol) was added to the pyridine solution of Compound 1, and the reaction was stirred thoroughly under a nitrogen atmosphere. After 30 minutes, the ice bath was removed and the reaction was allowed to proceed at room temperature. After 4 hours, a sample was taken for ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis, which showed that the reaction of the raw material Compound 1 (MW: 633.70) was complete, yielding the product Compound 2 (MW: 747.96). Saturated sodium bicarbonate (400 ml) at 0°C was added to the reaction mixture, followed by extraction with dichloromethane (600 ml x 2). The aqueous phase was analyzed for residual product by UPLC-MS. The organic phases from the two extractions were combined, dried over anhydrous sodium sulfate, filtered, and the dichloromethane solvent was removed by rotary evaporation under reduced pressure to yield a brown oil. The crude product was redissolved in 400 ml of dichloromethane, and pyridine was washed with 2% aqueous citric acid and separated by extraction. After repeated washings, a sample was taken for UPLC-MS analysis, indicating that the pyridine was largely removed. The dichloromethane solution was dried over anhydrous sodium sulfate, filtered, and the dichloromethane solvent was removed by rotary evaporation under reduced pressure to yield a brown oil. The crude product was used in the next reaction without further processing.

[0098] (2) Compound 2 (59.02 g, 78.91 mmol) was dissolved in 800 ml of dry dichloromethane and cooled to 0°C in an ice bath. Dodecanethiol (25.55 g, 126.25 mmol, 30.24 ml) was added with magnetic stirring, and the temperature was maintained at 0°C. After 30 minutes, trifluoroacetic acid (44.99 g, 394.54 mmol, 30.19 ml) was slowly added. Half an hour later, a sample was taken for UPLC-MS analysis. The reaction of the starting compound 2 (MW: 747.96) was complete, yielding the product compound 3 (MW: 445.59). A saturated aqueous sodium bicarbonate solution at 0°C was added to the reaction system to neutralize excess trifluoroacetic acid. The product was then extracted with dichloromethane (600ml*3). The aqueous phase was analyzed by UPLC-MS. No compound 3 remained in the aqueous phase. The organic phase solutions were combined, dried over anhydrous sodium sulfate, and filtered to remove the solvent dichloromethane by rotary evaporation under reduced pressure to obtain a crude oil. The crude product was dissolved in 2000ml of methyl tert-butyl ether / n-hexane (V:V=1:1) and stirred thoroughly with a magnetic stirrer to obtain a white suspended solid. The white filter cake was filtered. The filter cake was rotary evaporated and dried at an oil pump to obtain a white powder. A sample was taken for UPLC-MS analysis, and the product purity was >95%.

[0099] (3) Compound 3 (10 g, 22.44 mmol) was dissolved in 200 ml of dry dichloromethane (DCM). DCI (4,5-dicyanoimidazole, 5.3 g, 2 eq) was then added under magnetic stirring to clarify the solution. Compound (I) (20.09 g, 1.2 eq) was then added. The reaction system was stirred at room temperature under nitrogen for 2 hours. Samples were taken for UPLC-MS analysis. After the raw material was completely converted into the intermediate product, the reaction system was cooled to 0°C in an ice bath. 70% tert-butyl peroxide (7.22 g, 2.5 eq) was added and the reaction system was maintained at 0°C. After 2 hours, samples were taken for UPLC-MS analysis. After the oxidation of the intermediate product was complete, dodecanethiol (7.26 g, 1.6 eq) was added. The mixture was stirred at 0°C for 10 min, and trifluoroacetic acid (12.79 g, 5 eq) was slowly added dropwise. After 30 minutes, the ice bath was removed and a sample was taken for UPLC-MS analysis. The 5'-DMTr protecting group was completely removed. The reaction system was placed in an ice bath at 0°C and saturated sodium bicarbonate aqueous solution was added to neutralize the excess trifluoroacetic acid. Extraction and washing were then performed. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the dichloromethane solvent was removed by rotary evaporation. The crude product was purified using a thin layer silica gel column (0-10 wt% ethanol in dichloromethane). The components were concentrated and dried at an oil pump to obtain 16.4 g of white powder. A sample was taken for UPLC-MS analysis. The product purity was >95%.

[0100] (4) Compound 4 (1.93 g, 2.61 mmol) was dissolved in 20 ml of dry dichloromethane. DCI (4,5-dicyanoimidazole, 615.38 mg, 2 eq) and imidazole (2.06 g, 15 eq) were then added under magnetic stirring. After dissolution and clarification, compound dT-phosphoramidite (2.339 g, 1.2 eq) was added. The reaction system was stirred at room temperature under nitrogen for 2 hours. Samples were taken for UPLC-MS analysis. After the raw materials were completely converted into the intermediate products, the reaction system was cooled to 0°C in an ice bath. 70% tert-butyl peroxide (587 mg, 2.5 eq) was added and the reaction system was maintained at 0°C. After 2 hours, samples were taken for UPLC-MS analysis. The oxidation of the intermediate products was complete. Py.HF (pyridine hydrofluoride, 521.24 mg, 10 eq) was then added dropwise slowly at 0°C. After 30 minutes, the ice bath was removed and the reaction was allowed to proceed at room temperature for 1 hour. Samples were taken for UPLC-MS analysis and detection, indicating that the 3'-TDDMS protecting group was completely removed. The reaction system was placed in an ice bath at 0°C and excess trifluoroacetic acid was neutralized by adding a saturated aqueous sodium bicarbonate solution. Extraction and washing were then performed, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the dichloromethane solvent was removed by rotary evaporation. The crude product was purified using a thin layer silica gel column (0-10 wt% ethanol in dichloromethane). The components were concentrated and dried at an oil pump to obtain 16.4 g of white powder. A sample was taken for UPLC-MS analysis, and the product purity was >95%.

[0101] (5) The dried compound 5 (2 g, 1.48 mmol) was dissolved in 20 ml of dry dichloromethane, and then DCI (4,5-dicyanoimidazole, 350.39 mg, 2.97 mmol, 2 eq) was added. The mixture was stirred under nitrogen until it was clear. The phosphorus reagent (668.5 mg, 2.23 mmol, 1.5 eq) was weighed and added to the reaction system under nitrogen atmosphere. The mixture was reacted at room temperature for half an hour, and then a sample was taken for UPLC-MS analysis. The reaction was complete. The reaction system was added dropwise to 100 ml of stirred n-hexane. A white precipitate was precipitated. The mixture was filtered and the filtrate was removed to obtain a white solid compound 6. The mixture was dried by rotary evaporation at low temperature and the product was sent to UPLC-MS. 31 P-NMR analysis was performed. UPLC-MS analysis results are shown in Figure 2, where Figure 2(a) shows the ultra-high performance liquid chromatography (UPLC) and Figure 2(b) shows the mass spectrum. The molecular weight of the white solid compound 6 was determined from the LC and mass spectrometry. The phosphorus spectrum showed that the product had a purity of >95% and could be used for subsequent oligonucleotide synthesis reactions.

[0102] Preparation Example A2-20

[0103] Nucleoside phosphoramidite trimers were prepared by referring to the method of Preparation Example A1: AAA, AAC, ACT, ATC, ATG, CAG, CAT, CCG, CGT, CTG, GAA, GAC, GCT, GGT, GTT, TAC, TCT, TGC, TGG, TTC.

[0104] Preparation Example 1

[0105] This preparation example is used to illustrate the preparation method of the vector for chemically synthesizing ultra-long oligonucleotides.

[0106] (1) 100 g of solid non-porous silica microspheres (the microscope image of solid non-porous silica microspheres is shown in Figure 1, the average particle size of solid non-porous silica microspheres is about 60 μm, and the specific surface area is about 0.045 m 2 / g) was added to 500 mL of an aqueous solution of ammonium fluoride (concentration of 2 mol / L), stirred and ultrasonicated at room temperature for 1 hour, then washed with deionized water and dried. The stirring speed was 60 r / min.

[0107] (2) Adding a 10% w / w solution of 3-glycidyloxypropyltriethoxysilane to the silica microspheres dried in step (1) for a first grafting reaction. The first grafting reaction was performed at room temperature for 16 hours. The amount of silane reagent used was 80 g per 100 g of silica microspheres.

[0108] (3) Pentaethylene glycol was added to the silica microspheres after the first grafting for a second grafting, followed by washing with deionized water and drying. The second grafting conditions included a temperature of 30° C. and a time of 16 hours. The amount of grafting agent used in the second grafting was 2 g per 100 g of silica microspheres.

[0109] To the second grafted silica microspheres, an acetonitrile solution of a linker (2 mM), an acetonitrile solution of a condensing agent (3 mM), and an acetonitrile solution of DIEA (4 mM) were added for a condensation reaction to prepare silica microspheres with surface modification groups. The condensation reaction conditions included a temperature of 25°C and a reaction time of 16 hours. For every 100 g of silica microspheres, the amount of linker used was 1 g, the amount of condensing agent used was 2 g, and the amount of DIEA used was 1 g. The linker was unylinker, and the condensing agent was O-(benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

[0110] Example 1

[0111] 20 mg of the carrier prepared in Preparation Example 1 was loaded into an empty synthesis column of a solid phase synthesizer, and then the ultra-long oligonucleotide was automatically synthesized according to the set program. The synthesis process (performed at room temperature) included:

[0112] (a) adding a toluene solution of a combined deprotecting agent (the content of the combined deprotecting agent in the toluene solution is 3%, w / w) to a synthesis column to remove the protecting groups on the linker of the support; then cleaning with a cleaning agent; wherein the combined deprotecting agent is composed of trichloroacetic acid, dichloroacetic acid, and 3,5-dinitrobenzoic acid in a weight ratio of 1:1:2;

[0113] (b) adding an acetonitrile solution of a combined coupling agent (the combined coupling agent concentration in the acetonitrile solution of the combined coupling agent is 0.2 mol / L) and an acetonitrile solution of a nucleoside phosphoramidite trimer prepared in Preparation Example A1-20 to a synthesis column to couple the activated nucleoside phosphoramidite trimer with a small amount of nucleoside phosphoramidite monomers (T, C, G, A); wherein the combined coupling agent is composed of 5-benzylthio-1H-tetrazole, N-methylimidazole, and 4,5-dicyanoimidazole in a weight ratio of 10:1:1;

[0114] (c) subjecting the product of the coupling reaction to a first capping step, an oxidation step, and a second capping step in sequence;

[0115] (d) Repeat the process of steps (a)-(c).

[0116] The types and amounts of each reagent are shown in Table 1.

[0117] Table 1

[0118] Note: Cap A is a mixed solution of 10 wt% acetic anhydride / 10 wt% 2,6-lutidine / 80 wt% tetrahydrofuran; Cap B is a tetrahydrofuran solution of 16 wt% N-methylimidazole; the oxidant is a 0.05 mol / L I2 solution (the solvent consists of pyridine and water in a volume ratio of 95:5).

[0119] After the synthesis process is complete, the carrier containing the ultralong oligonucleotide is cleaved and deprotected by treating it with 30% ammonia at 80°C for 2 hours. Desalting and purification are then performed using a C18 desalting column, eluting with deionized water and then recovering it with a 60% acetonitrile aqueous solution to obtain the ultralong oligonucleotide. The crude synthesized ultralong oligonucleotide product is analyzed using a polyacrylamide gel. The recovered product is then recovered on the PAGE gel, and the purified ultralong oligonucleotide is then amplified by PCR.

[0120] The chain length of ultra-long oligonucleotides was measured using a capillary electrophoresis instrument (Qsep, Taiwan Guangding) using the Qsep Standard Cartridge Kit. The test results are shown in Figure 3. Lane E shows the electropherogram of the oligonucleotide prepared in Example 1. The figure shows that the full-length product is clearly visible at the 329 nt product band of the oligonucleotide prepared in Example 1.

[0121] The oligonucleotide synthesis sequence prepared in Example 1 is the Herceptin VLκ gene sequence:

[0122] The capillary electrophoresis peak diagram of the oligonucleotide chain prepared in Example 1 after amplification (test method: Standard Cartridge Kit of Qsep equipment) is shown in Figure 4. As can be seen from Figure 4, the nucleotide chain length after amplification by primers containing flanking regions on both sides is 361 bp.

[0123] Comparative Example 1

[0124] The method of Example 1 was followed, except that the solid silica microsphere carrier was replaced with commercially available microporous glass (CPG), which had an average particle size of 80 μm and a specific surface area of ​​30 m 2 / g, the loading capacity is 40 μmol / g, and the average pore size is 1000 angstroms; the combined deprotecting agent is replaced by trichloroacetic acid; the combined coupling agent is replaced by 5-ethylthio-1H-tetrazole; and the nucleoside phosphoramidite trimer is replaced by a nucleoside phosphoramidite monomer.

[0125] The test results are shown in FIG3 , from which it can be seen that the full-length product cannot be observed at the target band position of 329 nt in lane C (electrophoresis diagram of oligonucleotide prepared in Comparative Example 1).

[0126] Example 2

[0127] The method of Example 1 was followed, except that the nucleoside phosphoramidite trimer prepared in Preparation Example A1-20 was replaced with a nucleoside phosphoramidite trimer of TTT, and a small amount of C, G, and A monomers were replaced with T monomers. The crude synthesized ultralong oligonucleotide product was analyzed using PAGE (polyacrylamide gel) identification gel, and the test results are shown in Figure 5. In Figure 5, the first lane on the left: single-stranded DNA ladder; the second lane on the left: 300 nt, 50 ng loading; the third lane on the left: 300 nt, 100 ng loading; the fourth lane on the left: 350 nt, 50 ng loading; the fifth lane on the left: 350 nt, 100 ng loading; the sixth lane on the left: 400 nt, 50 ng loading; and the seventh lane on the left: 400 nt, 100 ng loading.

[0128] As can be seen from Figure 5, three lengths of ultra-long oligonucleotides were selected for this synthesis, and the three synthetic sequence examples were 300 consecutive Ts, 350 consecutive Ts, and 400 consecutive Ts.

[0129] Compared with Example 1, when the nucleoside phosphoramidite trimer of TTT is used, the method of the present invention can synthesize oligonucleotides with longer chain lengths.

[0130] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. Application of silica microspheres in the synthesis of ultra-long oligonucleotides, characterized in that: The specific surface area of ​​the silica microspheres is less than 1m 2 / g.

2. The use according to claim 1, wherein: The silica microspheres are solid silica microspheres, and the average particle size of the silica microspheres is 50-100 μm.

3. The use according to claim 1 or 2, wherein: The surface of the silica microspheres has a modified group; Preferably, the structure of the silica microspheres having modified groups on the surface is as shown in formula (1); Among them, A is silica microsphere, R 1 and R 3 Each independently selected from alkoxy, preferably C1-C 10 Alkoxy; R 2 Selected from C1-C 10 Alkylene-O-C1-C 10 Alkylene, -C1-C 10 Alkylene-(NH-C1-C 10 alkylene) n -、C1-C 10 Alkylene, n is an integer of 1-10; R 4 Selected from -NH-(CH2CH2O) m -C1-C 10 Alkylene-NH-, -(O-CH2CH2) p -O-, m and p are each independently an integer of 1-10; L1 is a linker group capable of undergoing a coupling reaction with nucleoside phosphoramidite.

4. A method for synthesizing ultra-long oligonucleotides, characterized in that: The method comprises: under coupling reaction conditions, using the silica microspheres described in claim 3 as carriers to carry out coupling reaction on the nucleoside phosphoramidite synthesis unit.

5. The method according to claim 4, wherein: The method further comprises: removing the protecting groups of the silica microspheres by using a deprotecting agent, and then performing a coupling reaction; Preferably, the deprotecting agent comprises at least one of trichloroacetic acid, dichloroacetic acid and 3,5-dinitrobenzoic acid; More preferably, the deprotecting agent includes trichloroacetic acid, dichloroacetic acid and 3,5-dinitrobenzoic acid.

6. The method according to claim 5, wherein: The weight ratio of trichloroacetic acid, dichloroacetic acid and 3,5-dinitrobenzoic acid is 1:1-10:1-10.

7. The method according to claim 4, wherein: The coupling reaction is carried out in the presence of a coupling agent, wherein the coupling agent is at least one of 5-benzylthio-1H-tetrazole, N-methylimidazole and 4,5-dicyanoimidazole; More preferably, the coupling agent includes 5-benzylthio-1H-tetrazole, N-methylimidazole and 4,5-dicyanoimidazole.

8. The method according to claim 7, wherein: The weight ratio of 5-benzylthio-1H-tetrazole, N-methylimidazole and 4,5-dicyanoimidazole is 5-15:0.5-3:

1.

9. The method according to any one of claims 4 to 8, wherein: The nucleoside phosphoramidite synthesis unit includes a nucleoside phosphoramidite monomer and / or a nucleoside phosphoramidite polymer. Preferably, the nucleoside phosphoramidite polymer includes at least one of a nucleoside phosphoramidite dimer, a nucleoside phosphoramidite trimer and a nucleoside phosphoramidite tetramer.

10. The method according to claim 4, wherein: The method comprises the following steps: (a) removing the protecting groups on the linker of the carrier under the action of a deprotecting agent; (b) subjecting the nucleoside phosphoramidite synthesis unit to a coupling reaction in the presence of a coupling agent and the carrier obtained in step (a); (c) subjecting the product of the coupling reaction to a first capping and oxidation; (d) Repeat the process of steps (a)-(c).

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