CN117088913A - Pseudo uridine phosphoramidite monomer protected by tertiary butyl dimethyl silicon, preparation method and application thereof - Google Patents
Pseudo uridine phosphoramidite monomer protected by tertiary butyl dimethyl silicon, preparation method and application thereof Download PDFInfo
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- CN117088913A CN117088913A CN202310649180.5A CN202310649180A CN117088913A CN 117088913 A CN117088913 A CN 117088913A CN 202310649180 A CN202310649180 A CN 202310649180A CN 117088913 A CN117088913 A CN 117088913A
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- tert
- phosphoramidite monomer
- compound
- protected
- pseudouridine
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- 229930185560 Pseudouridine Natural products 0.000 title claims abstract description 46
- PTJWIQPHWPFNBW-UHFFFAOYSA-N Pseudouridine C Natural products OC1C(O)C(CO)OC1C1=CNC(=O)NC1=O PTJWIQPHWPFNBW-UHFFFAOYSA-N 0.000 title claims abstract description 46
- WGDUUQDYDIIBKT-UHFFFAOYSA-N beta-Pseudouridine Natural products OC1OC(CN2C=CC(=O)NC2=O)C(O)C1O WGDUUQDYDIIBKT-UHFFFAOYSA-N 0.000 title claims abstract description 46
- -1 Pseudo uridine phosphoramidite Chemical class 0.000 title claims abstract description 37
- 239000000178 monomer Substances 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- HHSARRMUXPDGJD-UHFFFAOYSA-N butyl(dimethyl)silicon Chemical group CCCC[Si](C)C HHSARRMUXPDGJD-UHFFFAOYSA-N 0.000 title description 2
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims abstract description 15
- 230000005847 immunogenicity Effects 0.000 claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 7
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 7
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 7
- 150000001875 compounds Chemical class 0.000 claims description 25
- 238000000034 method Methods 0.000 claims description 25
- 238000006243 chemical reaction Methods 0.000 claims description 16
- 239000003153 chemical reaction reagent Substances 0.000 claims description 14
- PTJWIQPHWPFNBW-GBNDHIKLSA-N pseudouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1C1=CNC(=O)NC1=O PTJWIQPHWPFNBW-GBNDHIKLSA-N 0.000 claims description 12
- 239000003054 catalyst Substances 0.000 claims description 11
- 238000010511 deprotection reaction Methods 0.000 claims description 10
- 230000035484 reaction time Effects 0.000 claims description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical group [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 claims description 9
- 239000007810 chemical reaction solvent Substances 0.000 claims description 9
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 8
- 229910052698 phosphorus Inorganic materials 0.000 claims description 8
- 239000011574 phosphorus Substances 0.000 claims description 8
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 7
- XGDRLCRGKUCBQL-UHFFFAOYSA-N 1h-imidazole-4,5-dicarbonitrile Chemical compound N#CC=1N=CNC=1C#N XGDRLCRGKUCBQL-UHFFFAOYSA-N 0.000 claims description 6
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 6
- UXGNZZKBCMGWAZ-UHFFFAOYSA-N dimethylformamide dmf Chemical group CN(C)C=O.CN(C)C=O UXGNZZKBCMGWAZ-UHFFFAOYSA-N 0.000 claims description 6
- BCNZYOJHNLTNEZ-UHFFFAOYSA-N tert-butyldimethylsilyl chloride Chemical compound CC(C)(C)[Si](C)(C)Cl BCNZYOJHNLTNEZ-UHFFFAOYSA-N 0.000 claims description 6
- JBWYRBLDOOOJEU-UHFFFAOYSA-N 1-[chloro-(4-methoxyphenyl)-phenylmethyl]-4-methoxybenzene Chemical compound C1=CC(OC)=CC=C1C(Cl)(C=1C=CC(OC)=CC=1)C1=CC=CC=C1 JBWYRBLDOOOJEU-UHFFFAOYSA-N 0.000 claims description 5
- DEZRYPDIMOWBDS-UHFFFAOYSA-N dcm dichloromethane Chemical compound ClCCl.ClCCl DEZRYPDIMOWBDS-UHFFFAOYSA-N 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- 239000002994 raw material Substances 0.000 claims description 4
- RKVHNYJPIXOHRW-UHFFFAOYSA-N 3-bis[di(propan-2-yl)amino]phosphanyloxypropanenitrile Chemical group CC(C)N(C(C)C)P(N(C(C)C)C(C)C)OCCC#N RKVHNYJPIXOHRW-UHFFFAOYSA-N 0.000 claims description 3
- KPUWHANPEXNPJT-UHFFFAOYSA-N disiloxane Chemical compound [SiH3]O[SiH3] KPUWHANPEXNPJT-UHFFFAOYSA-N 0.000 claims description 3
- GRJJQCWNZGRKAU-UHFFFAOYSA-N pyridin-1-ium;fluoride Chemical compound F.C1=CC=NC=C1 GRJJQCWNZGRKAU-UHFFFAOYSA-N 0.000 claims description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 2
- 125000002883 imidazolyl group Chemical group 0.000 claims description 2
- FPGGTKZVZWFYPV-UHFFFAOYSA-M tetrabutylammonium fluoride Chemical compound [F-].CCCC[N+](CCCC)(CCCC)CCCC FPGGTKZVZWFYPV-UHFFFAOYSA-M 0.000 claims description 2
- WHRNULOCNSKMGB-UHFFFAOYSA-N tetrahydrofuran thf Chemical compound C1CCOC1.C1CCOC1 WHRNULOCNSKMGB-UHFFFAOYSA-N 0.000 claims description 2
- 150000003536 tetrazoles Chemical class 0.000 claims description 2
- NTJBWZHVSJNKAD-UHFFFAOYSA-N triethylazanium;fluoride Chemical compound [F-].CC[NH+](CC)CC NTJBWZHVSJNKAD-UHFFFAOYSA-N 0.000 claims description 2
- 125000001889 triflyl group Chemical group FC(F)(F)S(*)(=O)=O 0.000 claims description 2
- ILMRJRBKQSSXGY-UHFFFAOYSA-N tert-butyl(dimethyl)silicon Chemical compound C[Si](C)C(C)(C)C ILMRJRBKQSSXGY-UHFFFAOYSA-N 0.000 claims 1
- 230000002708 enhancing effect Effects 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 2
- 125000001981 tert-butyldimethylsilyl group Chemical group [H]C([H])([H])[Si]([H])(C([H])([H])[H])[*]C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 abstract description 2
- 239000007858 starting material Substances 0.000 description 10
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 108020004999 messenger RNA Proteins 0.000 description 5
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 4
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 4
- 229940045145 uridine Drugs 0.000 description 4
- 108091034117 Oligonucleotide Proteins 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 239000002777 nucleoside Substances 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- UVBYMVOUBXYSFV-XUTVFYLZSA-N 1-methylpseudouridine Chemical compound O=C1NC(=O)N(C)C=C1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 UVBYMVOUBXYSFV-XUTVFYLZSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 108020004459 Small interfering RNA Proteins 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- YMWUJEATGCHHMB-UHFFFAOYSA-N dichloromethane Natural products ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 150000003833 nucleoside derivatives Chemical class 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 239000003223 protective agent Substances 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 1
- 229940022005 RNA vaccine Drugs 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 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
- 238000004458 analytical method Methods 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- HXLVDKGPVGFXTH-UHFFFAOYSA-N butyl(dimethyl)silane Chemical group CCCC[SiH](C)C HXLVDKGPVGFXTH-UHFFFAOYSA-N 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 108700021021 mRNA Vaccine Proteins 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000002715 modification method Methods 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 108091008104 nucleic acid aptamers Proteins 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 150000004713 phosphodiesters Chemical group 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 229910000077 silane Inorganic materials 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- IPGXXWZOPBFRIZ-UHFFFAOYSA-N tert-butyl(silyl)silane Chemical compound CC(C)(C)[SiH2][SiH3] IPGXXWZOPBFRIZ-UHFFFAOYSA-N 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
- C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
Abstract
The invention discloses a tert-butyl disilicide protected pseudo-uridine phosphoramidite monomer, a preparation method and application thereof, and relates to the technical field of organic synthesis. The tert-butyl disilicide protected pseudo-uridine phosphoramidite monomer has a structural formula shown in the following formula (I):
Description
Technical Field
The invention relates to the technical field of organic synthesis, in particular to a tert-butyl disilyl protected pseudouridine phosphoramidite monomer, a preparation method and application thereof.
Background
In recent years, with the development of nucleic acid chemical synthesis techniques, a series of chemically modified oligonucleotide derivatives have been reported in academia and industry. Chemical modifications of oligonucleotides include modifications to the sugar ring, to the base site, to the phosphodiester backbone, or to replace natural nucleosides with chemical structures having specific functions, and the like. The modification method and the reported oligonucleotide derivative provide an important material basis for research and development of nucleic acid bionic medicaments such as antisense nucleotide, nucleic acid aptamer, small interfering RNA and the like and for nucleic acid-based biomedical diagnosis.
Pseudouridine is the most abundant modified nucleoside on RNA, also known as the "fifth nucleoside" of RNA. In 2005, katalin Karik et al found that the introduction of pseudouridine into RNA reduced its immunogenicity, and that the immunogenicity of RNA decreased with increasing proportion of pseudouridine introduced. In 2008, katalin Karik et al also found that complete replacement of uridine mRNA with pseudouridine not only greatly reduced mRNA immunogenicity, but also improved mRNA stability and enhanced translation ability. In 2015, oli wia Andries et al found that complete replacement of uridine with N1-methyl pseudouridine reduced mRNA immunogenicity and enhanced mRNA protein expression capacity more than complete replacement of uridine with pseudouridine. These studies suggest that the introduction of pseudouridine or N1-methyl pseudouridine into RNA may be effective in reducing the immunogenicity of RNA vaccines, enhancing the stability of RNA, and enhancing its protein expression capacity.
The tert-butyl disilicide protected pseudo-uridine phosphoramidite monomer has the effects of increasing stability, reducing immunogenicity and enhancing protein expression capability when used for replacing uridine in siRNA synthesis. However, a method for preparing the pseudo uridine phosphoramidite monomer protected by tertiary butyl disilyl is not reported in literature.
Therefore, it is an urgent need to provide a synthesis method of tert-butyldimethylsilyl-protected pseudouridine phosphoramidite monomer, which is easy to operate and has high yield.
Disclosure of Invention
The invention provides a tert-butyl disilicide-protected pseudo-uridine phosphoramidite monomer, a preparation method and application thereof, and aims to solve the problems in the background technology.
In order to achieve the technical purpose, the invention mainly adopts the following technical scheme:
in a first aspect, the invention provides a tert-butyl disilyl protected pseudouridine phosphoramidite monomer having the structural formula shown in the following formula (I):
in a second aspect, the invention provides a preparation method of the tert-butyl disilyl protected pseudo-uridine phosphoramidite monomer according to the first aspect, which comprises the following synthesis process:
the method comprises the following steps:
(1) The silane protecting group is used for selectively protecting hydroxyl groups at the 3,5 positions on the raw material of the pseudo uridine SM to obtain a compound SM1;
(2) Reacting compound SM1 with tert-butyldimethylchlorosilane and a catalyst to obtain compound SM2;
(3) Removing silane protecting groups at positions 3 and 5 selectively from the compound SM2 by using a deprotection reagent to obtain a compound SM3;
(4) Reacting compound SM3 with DMTrCl to obtain compound SM4;
(5) And (3) reacting the compound SM4 with a phosphorus reagent and a catalyst to obtain a final product tert-butyl disilyl protected pseudouridine phosphoramidite monomer.
In a preferred embodiment of the present invention, in step (1), the silane-based protecting group is bis (trifluoromethanesulfonyl) di-t-butyl-silyl ester or 1,3 dichloro-1, 3-tetraisopropyl disiloxane.
Preferably, bis (trifluoromethanesulfonyl) di-t-butyl-silyl ester is used as the silane protecting group, because the use of 1,3 dichloro-1, 3-tetraisopropyl disiloxane in step 3 to remove the silicon protecting agent results in removal of a portion of the t-butyldimethylsilyl chloride protecting agent and reduced yields.
Further, in the step (1), the molar ratio of the pseudouridine to the silane protecting group is 1:1.1-2.5, the reaction temperature is room temperature, and the reaction time is 0.5-3h.
In a preferred embodiment of the present invention, in the step (2), the molar ratio of the compound SM1, the catalyst and the tert-butyldimethylchlorosilane is 1:1-2:1-2, the reaction solvent is N, N-dimethylformamide DMF, the reaction temperature is room temperature to 60 ℃, and the reaction time is 3-8h.
Further, in the step (2), the catalyst is imidazole.
In the preferred embodiment of the present invention, in the step (3), the deprotection reagent is one or two of pyridine hydrofluoric acid, triethylamine hydrofluoric acid, TBAF, ethanol or methanol, the molar ratio of the compound SM2 to the deprotection reagent is 1:1-3, in the deprotection process, the reaction solvent is any one of dichloromethane DCM, tetrahydrofuran THF or N, N-dimethylformamide DMF, the reaction temperature is room temperature, and the reaction time is 2-4h.
Preferably, the deprotection reagent is pyridine hydrofluoric acid, as other deprotection reagents also tend to remove the tertiary butyldimethylsilane at the 3-position back to the pseudouridine starting material.
Preferably, the reaction solvent is dichloromethane DCM.
In a preferred embodiment of the present invention, in the step (4), the molar ratio of the compound SM3 to DMTrCl is 1:1.1-2.5, the reaction solvent is N, N-dimethylformamide DMF or pyridine Py, the reaction temperature is 0-35 ℃, and the reaction time is 3-4h.
In a preferred embodiment of the present invention, in the step (5), the phosphorus reagent is bis (diisopropylamino) (2-cyanoethoxy) phosphine, the catalyst is any one of 4, 5-dicyanoimidazole or tetrazole, the molar ratio of the compound SM4, the phosphorus reagent and the 4, 5-dicyanoimidazole is 1:1-2:0.5-1, the reaction solvent is dichloromethane DCM, the reaction temperature is room temperature, and the reaction time is 8-48h.
Preferably, the catalyst is 4, 5-dicyanoimidazole.
In a third aspect, the present invention provides the use of a tert-butyldimethylsilyl-protected pseudouridine phosphoramidite monomer according to the first aspect for the preparation of a pharmaceutical or health product having reduced immunogenicity and enhanced protein expression.
Compared with the prior art, the invention has the following beneficial effects:
the preparation method of the tert-butyl disilicide protected pseudo-uridine phosphoramidite monomer is simple to operate and convenient to purify, and well solves the problem that the cross is difficult to separate when TBDMS is arranged at the 2',3' -position;
the tert-butyl disilicide protected pseudouridine phosphoramidite monomer prepared by the method has higher purity, has the functions of reducing immunogenicity and enhancing protein expression capacity, and can be used for preparing medicines or health-care products.
Drawings
FIG. 1 is a hydrogen spectrum of a tert-butyldimethylsilyl-protected pseudouridine phosphoramidite monomer prepared according to the present invention;
FIG. 2 is a phosphorus spectrum of a tert-butyldimethylsilyl-protected pseudouridine phosphoramidite monomer prepared according to the present invention;
FIG. 3 is a HPLC detection chart of the tert-butyldimethylsilyl-protected pseudouridine phosphoramidite monomer prepared by the present invention.
Detailed Description
The following description of the preferred embodiments of the present invention refers to the accompanying drawings, which make the technical contents thereof more clear and easy to understand. The present invention may be embodied in many different forms of embodiments and the scope of the present invention is not limited to only the embodiments described herein.
A preparation method of a tert-butyl disilyl protected pseudouridine phosphoramidite monomer comprises the following synthesis processes:
the preparation method comprises the following steps:
1. 50g of pseudouridine SM is added with 150ml of DMF and stirred uniformly, 150g of di (trifluoromethanesulfonyl acid) di-tert-butyl silicon ester is added dropwise, stirring is carried out for 30min, TLC detection is carried out, the point of the raw material disappears, a new point is formed on the upper part of the raw material, and the compound SM1 is obtained and used for the next reaction directly without treatment.
2. 70g of imidazole and 80g of TBDMS-Cl were added to the reaction solution, and the mixture was stirred at room temperature for 5 hours, and the reaction solution was checked by TLC to eliminate the starting material, and a new point was formed on the upper part of the starting material. Adding 30mL of water for quenching, stirring the sample with silica gel, and purifying by column chromatography to obtain the product 100gSM with the yield of 98% and the purity of 98.3%.
3. 100g of SM2 was dissolved in 350Ml of DCM (350 mL) under stirring, 78.7g of HF. Py solution was added dropwise and reacted at room temperature for 3 hours, as detected by TLC, the starting material had disappeared and a new spot was formed in the lower part of the starting material. The reaction solution was washed with saturated brine, and a large amount of white solid was produced, and 65g of SM3 was obtained by filtration and drying, with a purity of 99.5%.
4. 65g of SM3 was added to 650mL of pyridine solution, 64.2g of DMTrCl was added in 3 portions over 30min, stirred at room temperature for 3-4 hours, and TLC was performed to detect disappearance of starting material spots, which had a new spot on top of the starting material. The reaction was washed with saturated sodium bicarbonate (200 ml x 2), saturated brine (200 ml x 2), dried over anhydrous sodium sulfate, and the organic phase was concentrated and purified by column (DCM/MeOH) to give 96g of SM4, 98.8% purity, 80% yield.
5. 96g of SM4 were dissolved in 1LDCM, 13.74g of 4,5 dicyanoimidazole and 61.32g of bis (diisopropylamino) (2-cyanoethoxy) phosphine were added and stirred at room temperature for 24 hours with < 0.5% of starting material remaining in the HPLC. The reaction solution was washed with saturated sodium bicarbonate (100 ml x 2) and saturated saline (100 ml x 2), the organic phase was dried over anhydrous sodium sulfate and concentrated, and purified by silica gel column to obtain 96g of white solid a, i.e. tert-butyldisilyl-protected pseudouridine phosphoramidite monomer, whose hydrogen and phosphorus spectra are shown in fig. 1 and 2, respectively. The purity of the prepared tert-butyldisilane protected pseudouridine phosphoramidite monomer (comprising two isomers) was 98.6% by HPLC, as shown in FIG. 3.
The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention without requiring creative effort by one of ordinary skill in the art. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.
Claims (10)
1. The tert-butyl disilicide protected pseudo-uridine phosphoramidite monomer is characterized by having a structural formula shown in the following formula (I):
2. a method for preparing a tert-butyl dimethylsilane protected pseudouridine phosphoramidite monomer according to claim 1, which is characterized in that the synthesis process is as follows:
the method comprises the following steps:
(1) The silane protecting group is used for selectively protecting hydroxyl groups at the 3,5 positions on the raw material of the pseudo uridine SM to obtain a compound SM1;
(2) Reacting compound SM1 with tert-butyldimethylchlorosilane and a catalyst to obtain compound SM2;
(3) Removing silane protecting groups at positions 3 and 5 selectively from the compound SM2 by using a deprotection reagent to obtain a compound SM3;
(4) Reacting compound SM3 with DMTrCl to obtain compound SM4;
(5) And (3) reacting the compound SM4 with a phosphorus reagent and a catalyst to obtain a final product tert-butyl disilyl protected pseudouridine phosphoramidite monomer.
3. The method for preparing the tert-butyldimethylsilyl-protected pseudouridine phosphoramidite monomer according to claim 2, wherein the method comprises the following steps: in the step (1), the silane protecting group is di-tert-butyl silyl bis (trifluoromethanesulfonyl acid) or 1,3 dichloro-1, 3-tetraisopropyl disiloxane.
4. The method for preparing the tert-butyldimethylsilyl-protected pseudouridine phosphoramidite monomer according to claim 2, wherein the method comprises the following steps: in the step (1), the molar ratio of the pseudouridine to the silane protecting group is 1:1.1-2.5, the reaction temperature is room temperature, and the reaction time is 0.5-3h.
5. The method for preparing the tert-butyldimethylsilyl-protected pseudouridine phosphoramidite monomer according to claim 2, wherein the method comprises the following steps: in the step (2), the mol ratio of the compound SM1 to the catalyst to the tert-butyldimethyl chlorosilane is 1:1-2:1-2, the reaction solvent is N, N-dimethylformamide DMF, the reaction temperature is room temperature to 60 ℃, and the reaction time is 3-8h.
6. The method for preparing the tert-butyldimethylsilyl-protected pseudouridine phosphoramidite monomer according to claim 2, wherein the method comprises the following steps: in the step (2), the catalyst is imidazole.
7. The method for preparing the tert-butyldimethylsilyl-protected pseudouridine phosphoramidite monomer according to claim 2, wherein the method comprises the following steps: in the step (3), the deprotection reagent is one or two of pyridine hydrofluoric acid, triethylamine hydrofluoric acid, TBAF, ethanol or methanol, the molar ratio of the compound SM2 to the deprotection reagent is 1:1-3, in the deprotection process, the reaction solvent is any one of dichloromethane DCM, tetrahydrofuran THF or N, N-dimethylformamide DMF, the reaction temperature is room temperature, and the reaction time is 2-4h.
8. The method for preparing the tert-butyldimethylsilyl-protected pseudouridine phosphoramidite monomer according to claim 2, wherein the method comprises the following steps: in the step (4), the molar ratio of the compound SM3 to DMTrCl is 1:1.1-2.5, the reaction solvent is N, N-dimethylformamide DMF or pyridine Py, the reaction temperature is 0-35 ℃, and the reaction time is 3-4h.
9. The method for preparing the tert-butyldimethylsilyl-protected pseudouridine phosphoramidite monomer according to claim 2, wherein the method comprises the following steps: in the step (5), the phosphorus reagent is bis (diisopropylamino) (2-cyanoethoxy) phosphine, the catalyst is any one of 4, 5-dicyanoimidazole or tetrazole, the molar ratio of the compound SM4 to the phosphorus reagent to the 4, 5-dicyanoimidazole is 1:1-2:0.5-1, the reaction solvent is dichloromethane DCM, the reaction temperature is room temperature, and the reaction time is 8-48h.
10. Use of a tert-butyldimethylsilyl-protected pseudouridine phosphoramidite monomer according to claim 1 for the preparation of a pharmaceutical or health product having reduced immunogenicity and enhanced protein expression.
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