CN117756872A - Method for preparing N-acetylgalactosamine oligonucleotide conjugate based on click chemistry technology - Google Patents
Method for preparing N-acetylgalactosamine oligonucleotide conjugate based on click chemistry technology Download PDFInfo
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- 108091034117 Oligonucleotide Proteins 0.000 title claims abstract description 43
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 29
- OVRNDRQMDRJTHS-CBQIKETKSA-N N-Acetyl-D-Galactosamine Chemical compound CC(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-CBQIKETKSA-N 0.000 title claims abstract description 25
- 238000005516 engineering process Methods 0.000 title claims abstract description 21
- 150000001875 compounds Chemical class 0.000 claims description 48
- 238000006243 chemical reaction Methods 0.000 claims description 32
- 239000003054 catalyst Substances 0.000 claims description 20
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 15
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 claims description 14
- 239000000178 monomer Substances 0.000 claims description 12
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 claims description 10
- 229910001431 copper ion Inorganic materials 0.000 claims description 10
- 150000008300 phosphoramidites Chemical class 0.000 claims description 10
- 235000010378 sodium ascorbate Nutrition 0.000 claims description 9
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 claims description 9
- 229960005055 sodium ascorbate Drugs 0.000 claims description 9
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- QMMFVYPAHWMCMS-UHFFFAOYSA-N dimethyl monosulfide Natural products CSC QMMFVYPAHWMCMS-UHFFFAOYSA-N 0.000 claims description 8
- 239000008213 purified water Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 229910021589 Copper(I) bromide Inorganic materials 0.000 claims description 6
- 239000003638 chemical reducing agent Substances 0.000 claims description 6
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 6
- 239000007791 liquid phase Substances 0.000 claims description 6
- 239000000758 substrate Substances 0.000 claims description 6
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 4
- 229960000643 adenine Drugs 0.000 claims description 4
- 239000007810 chemical reaction solvent Substances 0.000 claims description 4
- 229910052802 copper Inorganic materials 0.000 claims description 4
- 239000010949 copper Substances 0.000 claims description 4
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 4
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical group [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 4
- NKNDPYCGAZPOFS-UHFFFAOYSA-M copper(i) bromide Chemical group Br[Cu] NKNDPYCGAZPOFS-UHFFFAOYSA-M 0.000 claims description 4
- 229940104302 cytosine Drugs 0.000 claims description 4
- -1 hydroxyl compound Chemical class 0.000 claims description 4
- 239000003446 ligand Substances 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- WNWUMIPFLOKTEZ-MNVCMFJRSA-N n-[1-[(2r,3s,4r,5r)-5-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]-4-[2-cyanoethoxy-[di(propan-2-yl)amino]phosphanyl]oxy-3-methoxyoxolan-2-yl]-2-oxopyrimidin-4-yl]acetamide Chemical compound C([C@@H]1[C@@H](OP(OCCC#N)N(C(C)C)C(C)C)[C@@H]([C@@H](O1)N1C(N=C(NC(C)=O)C=C1)=O)OC)OC(C=1C=CC(OC)=CC=1)(C=1C=CC(OC)=CC=1)C1=CC=CC=C1 WNWUMIPFLOKTEZ-MNVCMFJRSA-N 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
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- 230000035484 reaction time Effects 0.000 claims description 4
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- 239000006227 byproduct Substances 0.000 claims description 3
- 238000010791 quenching Methods 0.000 claims description 3
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- 125000002103 4,4'-dimethoxytriphenylmethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)(C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H])C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H] 0.000 claims description 2
- 125000000304 alkynyl group Chemical group 0.000 claims description 2
- 125000003277 amino group Chemical group 0.000 claims description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 2
- 238000007664 blowing Methods 0.000 claims description 2
- 238000006555 catalytic reaction Methods 0.000 claims description 2
- 230000003647 oxidation Effects 0.000 claims description 2
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- VAKXPQHQQNOUEZ-UHFFFAOYSA-N 3-[4-[[bis[[1-(3-hydroxypropyl)triazol-4-yl]methyl]amino]methyl]triazol-1-yl]propan-1-ol Chemical compound N1=NN(CCCO)C=C1CN(CC=1N=NN(CCCO)C=1)CC1=CN(CCCO)N=N1 VAKXPQHQQNOUEZ-UHFFFAOYSA-N 0.000 claims 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 claims 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 abstract description 6
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- OVRNDRQMDRJTHS-KEWYIRBNSA-N N-acetyl-D-galactosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-KEWYIRBNSA-N 0.000 description 3
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- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 1
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- 108091032955 Bacterial small RNA Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- VMQMZMRVKUZKQL-UHFFFAOYSA-N Cu+ Chemical compound [Cu+] VMQMZMRVKUZKQL-UHFFFAOYSA-N 0.000 description 1
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- 241000700584 Simplexvirus Species 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a method for preparing N-acetylgalactosamine oligonucleotide conjugate based on click chemistry technology, which comprises the steps of firstly cutting oligonucleotide obtained by solid phase synthesis from a solid carrier, performing ultrafiltration ammonia removal operation, and then performing coupling reaction with N-acetylgalactosamine GalNAc fragment in homogeneous phase.
Description
Technical Field
The invention belongs to the technical field of chemical biological medicine, and particularly relates to a method for preparing N-acetylgalactosamine oligonucleotide conjugate based on click chemistry technology.
Background
With the continued advancement of science and technology, nucleic acid pharmaceuticals have been attracting attention as a novel and potential method. The nucleic acid medicine has wide application range and may be used in treating various diseases, including cancer, genetic diseases, infectious diseases, etc. However, nucleic acid drugs themselves have problems of poor stability, high immunogenicity, low cellular uptake efficiency, and the like, which limit their development in medical applications. To overcome these challenges, researchers have begun looking at nucleic acid drug delivery systems.
Nucleic acid drug delivery systems refer to a technique for delivering nucleic acid drugs into target cells. Early studies have used viruses primarily as vectors for delivering nucleic acids, including adenoviruses, adeno-associated viruses, lentiviruses, herpes simplex viruses, and the like. However, the use of viruses as vectors presents some undesirable properties, such as potential carcinogenicity and high immunogenicity, which limit their progress in medical applications. Thus, researchers have begun looking for alternative delivery systems.
N-acetylgalactosamine (GalNAc) oligonucleotide coupling modification is one of the more commonly used nucleic acid drug delivery systems in the prior art, and the N-acetylgalactosamine oligonucleotide conjugate (GalNAc-siRNA conjugate) obtained by the GalNAc based on the RNA interference technology is an oligonucleotide drug and has wide medical application prospect.
RNA interference is a mechanism that inhibits gene expression by specific small RNA molecules. siRNA (Small interfering RNA) is a short stretch of double stranded RNA molecules, typically consisting of 20 to 25 nucleobases. When the siRNA enters the cell, it can interact with the RNA-induced silencing complex (RISC) and introduce one of the strands (the dominant strand) into the RISC. RISC can then recognize and bind to a specific mRNA sequence that matches the dominant strand, resulting in degradation or translational inhibition of that mRNA, thereby achieving the effect of inhibiting expression of a specific gene.
Compared with the traditional siRNA, the GalNAc-siRNA is characterized in that the charged part of the oligonucleotide molecule has GalNAc modification. Such modification allows GalNAc-siRNA to selectively bind to and be recognized and taken up by GalNAc receptors on the surface of liver cells, thereby increasing the enrichment of the drug in the liver.
GalNAc-siRNA has great potential in the treatment of liver diseases due to drug enrichment in the liver. For example, hepatitis virus infection, hereditary metabolic disease, lipid metabolism disorder, and the like can be treated by interfering with the expression of specific genes. Traditional therapies may have limitations, whereas GalNAc-siRNA may achieve therapeutic effects by targeted inhibition of specific mRNA.
In the prior art methods, the preparation of GalNAc-siRNA conjugates usually uses a diimide coupling agent such as DCC, DIC, EDCI or a phosphonium salt such as PyBOP, requires the additional use of an organic base, and is typically run in a polar aprotic solvent such as DMF or DMSO, requiring to be performed under liquid phase conditions. However, the by-products produced in the aforementioned technical methods are difficult to purify and separate, and are limited to liquid phase conditions, and are suitable for laboratory-scale synthesis and are not well suited for economical and commercial mass production.
Therefore, the field needs to develop a new method for preparing N-acetylgalactosamine oligonucleotide conjugate, which has the advantages of simple operation, economy, environmental protection, mild reaction conditions, good substrate conversion rate and high stability, and is beneficial to the large-scale production of the N-acetylgalactosamine oligonucleotide conjugate GalNAc-siRNA.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for preparing N-acetylgalactosamine oligonucleotide conjugate based on Click chemistry technology, and azide/alkyne 1, 3-dipolar cycloaddition (Click chemistry) provides simple reaction conditions for small molecules and biomolecules, and has become the first choice method for bioconjugate in pharmaceutical chemistry. The strategy of finding new, efficient syntheses of large numbers of analogs makes "click chemistry" a widely used concept in the fields of pharmaceutical chemistry and drug discovery. Copper (I) -catalyzed Huisgen 1, 3-dipolar cycloaddition of azides to terminal alkynes is probably one of the most widely used click reactions because of its high specificity and efficiency in linking two different molecular entities.
The application provides a method for preparing N-acetylgalactosamine oligonucleotide conjugate based on click chemistry technology, which comprises the steps of firstly cutting oligonucleotide obtained by solid phase synthesis from immobilization, performing ultrafiltration ammonia removal operation, and performing coupling reaction with N-acetylgalactosamine GalNAc fragment in homogeneous phase, wherein the method comprises the following steps:
step one: under the condition of liquid phase, a compound of a formula VI and a compound of a formula VII are used as substrates, sodium ascorbate is added as a reducing agent under the catalysis of a copper ion catalyst, and the compound of a formula V is obtained through reaction;
the structure of the compound of the formula VI is as follows:in the compound of formula VI, R group is a long chain compound with hydroxy or amino which is removed from immobilization, and the R group is a hydroxyl or amino group>The group is a hydroxyl compound with three alkynyl groups and protecting groups, the Oligo is an oligonucleotide chain, the oligonucleotide chain is a mer with the sequence length of 20-23 mer, and the oligonucleotide chain consists of a plurality of phosphoramidite monomers;
the structure of the compound of the formula VII is as follows:Linker-N in the compound of formula VII 3 The chain is PEG chain, and the length of the PEG chain is 3-5 PEG molecules;
the structure of the compound of formula V is:
step two: quenching after the reaction is finished, and removing copper after the reaction is quenched.
Specifically, the copper ion catalyst is cuprous bromide dimethyl sulfide complex.
Specifically, the copper ion catalyst is copper sulfate and a catalyst ligand, and the catalyst ligand is tri- (3-hydroxypropyl triazolylmethyl) amine.
Specifically, the reaction solvent in the liquid phase condition is a mixture of N, N-dimethylformamide and purified water or a mixture of purified water and tertiary butanol.
Specifically, in the reaction process of the first step, nitrogen is blown in from the bottom of the reactor, and oxygen in the solution can be removed by blowing in the nitrogen, so that the reaction is carried out under the anaerobic condition, and the generation of excessive oxidation byproducts is reduced.
Specifically, the phosphoramidite monomer is selected from the following 8 phosphoramidite monomers, which are respectively: n6-benzoyl-5 ' -ODMT-2' -OMe-adenine ribophosphoramidite with CAS number 110782-31-5, N4-acetyl-5 ' -ODMT-2' -OMe-cytosine phosphoramidite with CAS number 199593-09-4, N2-isobutyryl-5 ' -ODMT-2' -OMe-guanine ribophosphoramidite with CAS number 150780-67-9, 5' -O-DMT-2' -OMe-uracil ribophosphoramidite with CAS number 110764-79-9, N6-benzoyl-5 ' -ODMT-2' -fluoro-adenine ribophosphoramidite with CAS number 136834-22-5, N4-acetyl-5 ' -ODMT-2' -fluoro-cytosine deoxyribophosphoramidite with CAS number 159414-99-0, N2-isobutyryl-5 ' -ODMT-2' -fluoro-guanine ribophosphoramidite with CAS number 144089-97-4, and 5' -O-DMT-2' -fluoro-ribophosphoramidite with CAS number 5649-22-5 ' -fluoro-ribophosphoramidite with CAS number 146954-8-9.
Specifically, the phosphoramidite monomers are connected by a phosphoric acid skeleton.
In particular, in the compound of formula VI, the protecting group is 4,4' -dimethoxytrityl.
Specifically, in the first step, the concentration of the compound of the formula VI is 21-1021 OD/mL.
Preferably, the concentration of the compound of formula VI is 21OD/mL, 105OD/mL or 210OD/mL.
Specifically, in the first step, the molar ratio of the compound of formula VI to the compound of formula VII is 1: (3-9).
Preferably, in step one, the molar ratio of the compound of formula VI to the compound of formula VII is 1:4, 1:5, 1:6, 1:7 or 1:8.
Specifically, in the first step, the molar ratio of the copper ion catalyst to the compound of formula vi is 1: (3-6).
Preferably, in step one, the molar ratio of copper ion catalyst to compound of formula VI is 1:3.5, 1:4, 1:4.5, 1:5 or 1:5.5.
Specifically, in the first step, the molar ratio of sodium ascorbate to the compound of formula vi is 1: (20-100).
Preferably, in step one, the molar ratio of sodium ascorbate to the compound of formula VI is 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90.
Specifically, in the first step, the reaction temperature is 15-50 ℃ and the reaction time is 1-16 h.
Preferably, in the first step, the reaction temperature is 20 ℃, 30 ℃ or 40 ℃, and the reaction time is preferably 1 to 8 hours, more preferably 3 to 7 hours.
In the application, the solid phase synthesis adopts the conventional technical means in the field, such as solid phase organic synthesis principle and application guide, and 4 steps of reaction cycles are carried out on resin for multiple times: deprotection, coupling, oxo or thio and capping to finally obtain the target long chain.
The beneficial effects of this application include:
1. in the preparation method provided by the application, the oligonucleotide obtained by solid phase synthesis is firstly cut off from the solid carrier, ultra-filtration ammonia operation is carried out, and then coupling reaction is carried out with the N-acetylgalactosamine GalNAc fragment in a homogeneous phase.
2. The catalyst system provided by the application is used for synthesizing the GalNAc-siRNA conjugate, the used reagent is low in cost and easy to obtain, and the conversion rate of the substrate is high, so that the yield of the product and the production efficiency are improved, and reference experience is provided for the subsequent modification modes of oligonucleotides with different sequences.
3. The preparation method provided by the application is simple to operate, mild in reaction condition, low in cost, high in yield and obviously reduced in key impurities, so that the product is high in purity, good in stability, environment-friendly and pollution-free, and can be successfully applied to industrial scale-up production.
Drawings
FIG. 1 is a technical scheme showing the preparation of N-acetylgalactosamine oligonucleotide conjugate in example 1 of the present application.
Detailed Description
The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. The experimental materials involved in the invention are all commercial reagents unless specified, and are available from commercial sources.
Example 1
1. Referring to the technical scheme shown in FIG. 1, a cuprous bromide dimethyl sulfide complex (CuBr. Me) 2 S) as a catalyst, preparing a compound of formula V:
100mmol of the compound of formula VI and 900mmol of the compound of formula VII are taken as substrates, and added into a reaction kettle, and cuprous bromide dimethyl sulfide complex (CuBr. Me) 2 S) as catalyst. Wherein the compound of formula VI comprises an oligonucleotide sequence consisting of 22 monomers, the hydroxyl resin is removed from immobilization, and the oligonucleotide sequence consists of phosphoramidite monomers selected from the group consisting of: n6-benzoyl-5 '-ODMT-2' -OMe-adenine ribophosphoramidite with CAS number 110782-31-5, N4-acetyl-5 '-ODMT-2' -OMe-cytosine phosphoramidite with CAS number 199593-09-4, N2-isobutyryl-5 '-ODMT-2' -OMe-guanine ribophosphoramidite with CAS number 150780-67-9, 5'-O-DMT-2' -OMe-uracil ribophosphoramidite with CAS number 110764-79-9Amine, N6-benzoyl-5 '-ODMT-2' -fluoro-adenine ribophosphoramidite with CAS number 136834-22-5, N4-acetyl-5 '-ODMT-2' -fluoro-cytosine deoxyribophosphoramidite with CAS number 159414-99-0, N2-isobutyryl-5 '-ODMT-2' -fluoro-guanine deoxyribophosphoramidite with CAS number 144089-97-4, and 5'-O-DMT-2' -fluoro-uracil deoxyribophosphoramidite with CAS number 146954-75-8.
In the reaction system, the addition amount of the catalyst is 450mmol, sodium ascorbate (Sodium ascorbate) is used as a reducing agent, the consumption of the reducing agent is 5000mmol, and the reaction solvent is N, N-Dimethylformamide (DMF) and purified water (H) 2 O), N, N-Dimethylformamide (DMF) and purified water (H) 2 O) is 1:3, and N is blown from the bottom of the kettle in the whole reaction process 2 Stirring and reacting for 5 hours at 20-25 ℃, wherein copper ions can cause long chain breakage of the oligonucleotide so as to generate sequencing impurities, so that the oligonucleotide is quenched in time after the reaction is finished and copper removal operation is carried out by adopting a reversed phase chromatographic column so as to keep the stability of the product. The product is subjected to ion pair chromatography IPC detection, and the result shows that the compound of the formula VI is 0%, the catalytic conversion rate is 100%, and the purity of the obtained compound of the formula V reaches 75%.
2. Referring to the technical scheme as shown in fig. 1, copper sulfate (CuSO 4 ) As catalyst, preparing a compound of formula v:
100mmol of the compound of formula VI and 900mmol of the compound of formula VII are taken as substrates, and are added into a reaction kettle, copper sulfate (CuSO 4 ) As a catalyst. Wherein the compound of formula VI is an oligonucleotide sequence containing 22 monomers, the hydroxyl resin is removed from immobilization, and the oligonucleotide sequence consists of the following phosphoramidite monomers: n6-benzoyl-5 '-ODMT-2' -OMe-adenine ribophosphoramidite with CAS number 110782-31-5, N4-acetyl-5 '-ODMT-2' -OMe-cytosine phosphoramidite with CAS number 199593-09-4, N2-isobutyryl-5 '-ODMT-2' -OMe-guanine ribophosphoramidite with CAS number 150780-67-9, 5'-O-DMT-2' -OMe-uracil ribophosphoramidite with CAS number 110764-79-9, N6-benzoyl-5 '-ODMT-2' -fluoro-adenine ribophosphoramidite with CAS number 136834-22-5, N4-acetyl-5 '-ODMT-2' -fluoro-cytosine deoxyribophosphoramidite with CAS number 159414-99-0, CAS number 144089-97-4N 2-isobutyryl-5 '-ODMT-2' -fluoro-guanine deoxyribophosphoramidite and 5'-O-DMT-2' -fluoro-uracil deoxyribophosphoramidite with CAS number 146954-75-8.
In the reaction system, the addition amount of the catalyst is 450mmol, sodium ascorbate (Sodium ascorbate) is used as a reducing agent, the use amount of the reducing agent is 5000mmol, and the reaction solvent is tert-butyl alcohol (t-BuOH) and purified water (H) 2 O), t-butanol (t-BuOH) and purified water (H) 2 O) is 1:3, and the whole reaction process is carried out from the bottom of the kettle to the drum N 2 Stirring and reacting for 5h at 20-25 ℃, and quenching in time and adopting a reversed phase chromatographic column to perform copper removal operation at the end of the reaction so as to keep the stability of the product because of the generation of sequencing impurities in the reaction. The product is subjected to ion pair chromatography IPC detection, and the result shows that the compound of the formula VI is 0%, the catalytic conversion rate is 100%, and the purity of the obtained compound of the formula V reaches 70.4%.
The reaction time was adjusted and the purity of the obtained product was as shown in table 1 below:
TABLE 1
As can be seen from the results of Table 1, the optimal reaction conditions of the preparation method provided by the embodiment are 20-25 ℃ for 5 hours, and the method is used for preparing the compound of formula V, so that the method is simple to operate, mild in conditions, and capable of realizing better conversion rate after 5 hours at room temperature while ensuring the purity of the product, and the synthesis efficiency of the N-acetylgalactosamine oligonucleotide conjugate is improved, thereby being beneficial to industrial amplified production.
In summary, the above embodiments are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, but any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (13)
1. A method for preparing an N-acetylgalactosamine oligonucleotide conjugate based on click chemistry technology, characterized in that said method comprises the steps of:
step one: under the condition of liquid phase, a compound of a formula VI and a compound of a formula VII are used as substrates, sodium ascorbate is added as a reducing agent under the catalysis of a copper ion catalyst, and the compound of a formula V is obtained through reaction;
the structure of the compound of the formula VI is as follows:in the compound of formula VI, R group is a long chain compound with hydroxy or amino which is removed from immobilization, and the R group is a hydroxyl or amino group>The group is a hydroxyl compound with three alkynyl groups and protecting groups, the Oligo is an oligonucleotide chain, the oligonucleotide chain is a mer with the sequence length of 20-23 mer, and the oligonucleotide chain consists of a plurality of phosphoramidite monomers;
the structure of the compound of the formula VII is as follows:Linker-N in the compound of formula VII 3 The chain is PEG chain, and the length of the PEG chain is 3-5 PEG molecules;
the structure of the compound of formula V is:
step two: quenching after the reaction is finished, and removing copper after the reaction is quenched.
2. The method for preparing an N-acetylgalactosamine oligonucleotide conjugate based on click chemistry technology according to claim 1, characterized in that said copper ion catalyst is cuprous bromide dimethyl sulfide complex.
3. The method for preparing an N-acetylgalactosamine oligonucleotide conjugate based on click chemistry technology according to claim 1, characterized in that said copper ion catalyst is copper sulfate and catalyst ligand, said catalyst ligand is tris- (3-hydroxypropyl triazolylmethyl) amine.
4. The method for preparing an N-acetylgalactosamine oligonucleotide conjugate based on click chemistry technology according to claim 1, characterized in that said reaction solvent under liquid phase condition is a mixture of N, N-dimethylformamide and purified water or a mixture of purified water and tert-butanol.
5. The method for preparing N-acetylgalactosamine oligonucleotide conjugate based on click chemistry technology according to claim 1, wherein nitrogen is blown from the bottom of the reactor during the reaction of said step one, and oxygen in the solution can be removed by blowing nitrogen, so that the reaction is performed under anaerobic condition, and the generation of excessive oxidation byproducts is reduced.
6. The method for preparing an N-acetylgalactosamine oligonucleotide conjugate based on click chemistry technology according to claim 1, characterized in that said phosphoramidite monomer is selected from the following 8 phosphoramidite monomers, respectively: n6-benzoyl-5 ' -ODMT-2' -OMe-adenine ribophosphoramidite with CAS number 110782-31-5, N4-acetyl-5 ' -ODMT-2' -OMe-cytosine phosphoramidite with CAS number 199593-09-4, N2-isobutyryl-5 ' -ODMT-2' -OMe-guanine ribophosphoramidite with CAS number 150780-67-9, 5' -O-DMT-2' -OMe-uracil ribophosphoramidite with CAS number 110764-79-9, N6-benzoyl-5 ' -ODMT-2' -fluoro-adenine ribophosphoramidite with CAS number 136834-22-5, N4-acetyl-5 ' -ODMT-2' -fluoro-cytosine deoxyribophosphoramidite with CAS number 159414-99-0, N2-isobutyryl-5 ' -ODMT-2' -fluoro-guanine ribophosphoramidite with CAS number 144089-97-4, and 5' -O-DMT-2' -fluoro-ribophosphoramidite with CAS number 5649-22-5 ' -fluoro-ribophosphoramidite with CAS number 146954-8-9.
7. The method for preparing an N-acetylgalactosamine oligonucleotide conjugate based on click chemistry technology according to claim 1, wherein said phosphoramidite monomers are linked by a phosphate backbone.
8. The method for preparing N-acetylgalactosamine oligonucleotide conjugate based on click chemistry technology according to claim 1, characterized in that in compound of formula VI, said protecting group is 4,4' -dimethoxytrityl.
9. The method for preparing N-acetylgalactosamine oligonucleotide conjugate based on click chemistry technology according to claim 1, characterized in that in said step one, the concentration of compound of formula VI is 21-1021 OD/mL.
10. The method of preparing an N-acetylgalactosamine oligonucleotide conjugate based on click chemistry technology according to claim 1, characterized in that in said step one, the molar ratio of compound of formula vi and compound of formula vii is 1: (3-9).
11. The method of preparing an N-acetylgalactosamine oligonucleotide conjugate based on click chemistry technology according to claim 1, characterized in that in said step one, the molar ratio of copper ion catalyst to compound of formula vi is 1: (3-6).
12. The method of preparing an N-acetylgalactosamine oligonucleotide conjugate based on click chemistry technology according to claim 1, characterized in that in said step one, the molar ratio of sodium ascorbate to compound of formula vi is 1: (20-100).
13. The method for preparing N-acetylgalactosamine oligonucleotide conjugate based on click chemistry technology according to claim 1, wherein in said step one, the reaction temperature is 15-50 ℃ and the reaction time is 1-16 h.
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