WO2006095739A1 - Procede de deblocage des groupes 2'-hydroxyle des ribonucleosides - Google Patents

Procede de deblocage des groupes 2'-hydroxyle des ribonucleosides Download PDF

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Publication number
WO2006095739A1
WO2006095739A1 PCT/JP2006/304399 JP2006304399W WO2006095739A1 WO 2006095739 A1 WO2006095739 A1 WO 2006095739A1 JP 2006304399 W JP2006304399 W JP 2006304399W WO 2006095739 A1 WO2006095739 A1 WO 2006095739A1
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group
hydroxyl group
rna
hydroxyl
ribonucleoside
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PCT/JP2006/304399
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English (en)
Japanese (ja)
Inventor
Mitsuo Sekine
Hisao Saneyoshi
Kohji Seio
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Tokyo Institute Of Technology
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Publication of WO2006095739A1 publication Critical patent/WO2006095739A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/067—Pyrimidine radicals with ribosyl as the saccharide radical
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to a method for deprotecting the 2 ′ hydroxyl group of a ribonucleoside protected with a cyanoethyl group.
  • RNA is useful as a functional nucleic acid such as ribozyme, aptamer, siRNA, antisense nucleic acid, and nucleic acid pharmaceutical material (Nechiya Review Drug Discovery No. 3 318 2004).
  • ribozyme aptamer
  • siRNA siRNA
  • antisense nucleic acid and nucleic acid pharmaceutical material
  • RNA synthesis method currently in widespread use uses a quaternary butyldimethylsilyl group (TBDMS group) as a protecting group for the 2 'hydroxyl group (Journal of American Chemical Society, Reference No. 99, 7741 1977). .
  • TBDMS group quaternary butyldimethylsilyl group
  • this protecting group does not have sufficient chemical stability.
  • the TBDMS group is a bulky protecting group
  • the reaction that forms an internucleotide bond between the adjacent 3 'hydroxyl group and the 5' hydroxyl group of other nucleoside residues via a phosphorus atom is steric. And has problems such as a decrease in the yield of the internucleotide binding reaction and an increase in the required reaction time (Journal of American Chemical Society, Cited Reference No. 109, 7845 1987).
  • Non-patent Document 1 a 2'-O cyanoethyl nucleoside in which a cyanoethyl group is introduced into the 2 'hydroxyl group of a ribonucleoside.
  • Non-Patent Document 1 Nucleic Acid Research Symposium Series 48, 13-14 pages 2004
  • RNA synthesis using phosphoramidite method 5 Stable to the chemical reaction used in each process. 5) At the final stage of RNA synthesis, the base part, sugar part, phosphodiester part, etc. of the synthesized RNA are removed without significant damage. Conditions such as being able to be protected are required.
  • An object of the present invention is to provide a means for removing a cyanoethyl group from a 2 ′ O cyanonucleoside without greatly damaging the base part, sugar part, phosphodiester part and the like of RNA.
  • RNA containing 2'-O cyanoyl nucleoside As a result of intensive studies to solve the above-mentioned problems, the present inventor has found that tetra (n-butyl) ammonium full-fluid is required for deprotection of RNA containing 2'-O cyanoyl nucleoside. It has been found that deprotection without damaging the base portion, sugar portion, etc. of RNA can be achieved by using an olido and the present invention has been completed.
  • the present invention provides the following (1) to (4).
  • X represents a hydrogen atom or a hydroxyl-protecting group
  • Y represents a hydrogen atom, a hydroxyl-protecting group, or a general formula (III):
  • R represents the same or different alkyl group, or a group in which R 1 and R are bonded to each other to form a ring which may contain a hetero atom
  • R 3 represents a protecting group for a phosphate group.
  • B represents a nucleobase residue.
  • a ribonucleoside derivative protected with a 2 ′ hydroxyl group or an RNA derivative containing this ribonucleoside derivative as a constituent unit is reacted with a fluorine-containing compound or a basic compound to give a general formula (II):
  • RNA containing the ribonucleoside as a structural unit Or a RNA containing the ribonucleoside as a structural unit. 2) A method for deprotecting a hydroxyl group.
  • RNA having a hydroxyl group protected with a cyanoethyl group that does not damage the base part, sugar part, phosphodiester part, etc. of RNA can be deprotected. This enables efficient RNA synthesis.
  • FIG. 1 is a diagram showing reverse phase HPLC profiles of 2′-O-cyanethylated uridylate dimer (A), uridylate dimer (B), and uridin (C).
  • FIG. 2 is a graph showing the relationship between the protecting group of the 2′-hydroxyl group of phosphoramidite and the phosphate ester formation reaction rate.
  • FIG. 4 is a diagram showing the ion exchange HPLC profile of purified RNA oligomer (GACUGACUGACU).
  • FIG. 5 is a diagram showing an ion exchange HPLC profile of a purified RNA oligomer (uridine 30-mer).
  • a ribonucleoside derivative protected with a 2 ′ hydroxyl group or an RNA derivative containing this ribonucleoside derivative as a constituent unit is reacted with a fluorine-containing compound or a basic compound to give a general formula (II):
  • RNA containing this ribonucleoside as a structural unit Or a RNA containing this ribonucleoside as a structural unit.
  • X in the general formulas (I) and (II) represents a hydrogen atom or a protecting group for a hydroxyl group.
  • a general protecting group used for protecting the 5′-hydroxyl group or the 3′-hydroxyl group of the nucleoside can be used.
  • a silyl group which may have a substituent for example, tert-butyl diphenyl). -Rusilyl group
  • 4-methoxytrityl group 4,4'-dimethoxytrityl group and the like
  • X may be combined with Y to form a protecting group. Examples of such protecting groups include 1, 1, 3, 3-tetraisopropyldisiloxa-lide. And cyclic silyl groups such as di (t-butyl) silane diyl.
  • Y in the general formulas (I) and (II) represents a hydrogen atom, a hydroxyl-protecting group, or the general formula (III): [0026] [Chemical Formula 6] D 2
  • R 2 represents the same or different alkyl group, or a group in which R 1 and R 2 are bonded to each other to form a ring which may contain a hetero atom
  • R 3 represents a protecting group for a phosphate group.
  • R 2 includes an isopropyl group
  • R 3 includes, but is not limited to, a 2-cyanoethyl group.
  • nucleobase refers to all nucleobases found in naturally occurring nucleic acids such as chromosomal DNA, plasmid DNA, messenger RNA, ribosomal RNA, transfer RNA, and small nuclear RNA of organisms. And all heteroaromatic rings optionally having substituents that can be used for nucleic acid synthesis.
  • Representative nucleobases include, but are not limited to, adenine, guanine, cytosine, uracil, thymine and the like. Nucleobases also include those having a protecting group.
  • RNA derivative containing a ribonucleoside derivative with a protected 2 'hydroxyl group represented by general formula (I) as a constituent unit is a formula obtained by removing X and Y in general formula (I). It means that the residue of the represented nucleoside derivative is contained in the RNA derivative.
  • RNA containing a ribonucleoside represented by the general formula (II) as a structural unit has the same meaning.
  • the fluorine-containing compound is not particularly limited as long as it can remove the cyanoethyl group.
  • Tetramethylammonium fluoride, tetraethylammonium fluoride, tetra (n-butyl) ammonium- Examples include tetraalkyl ammonium fluoride such as um fluoride. Tetraalkyl ammonium fluoride contains tetra (n —Butyl) ammonium fluoride (TBAF) is particularly preferred.
  • the amount of the fluorine-containing compound used in the reaction is not particularly limited.
  • the basic compound is not particularly limited as long as the cyanoethyl group can be eliminated, and examples thereof include ammonia, primary amine, secondary amine, tertiary amine, amidine compound, and the like. . Of these compounds, ammonia is preferred.
  • the amount of the basic compound used in the reaction is also not particularly limited.
  • amount of the basic compound used in the reaction is also not particularly limited.
  • ammonia about 1 to 3000 moles with respect to 1 mole of the ribonucleoside derivative represented by the general formula (I), etc. It is preferred to use.
  • the above reaction is performed in an organic solvent which may contain an aqueous solvent.
  • the volume ratio of the organic solvent and the aqueous solvent is not particularly limited, but is preferably 0: 100 to 100: 0.
  • the organic solvent is not particularly limited as long as it does not inhibit the reaction, and a mixed organic solvent obtained by mixing a plurality of organic solvents that do not inhibit the reaction at an arbitrary volume ratio may be used.
  • As the aqueous solvent not only water but also any buffer solution can be used, and a mixed buffer solution in which a plurality of buffer solutions are mixed at an arbitrary concentration may be used.
  • the reaction temperature is not particularly limited, but is preferably in the range of -78 ° C to 100 ° C.
  • a method for synthesizing 2'-O-cyanoethyl nucleoside by introducing a cyanoethyl group into the 2 'hydroxyl group of ribonucleoside is, for example, Nucleic Acid Research Symposium Series No. 48, pages 13-14. 2004 It is described in!
  • the lyophilized 2,1 O-cyanethylated uridylic acid dimer (0.5 micromolar) was placed in a tetrahydrofuran solution of TBAF (1M, 2001) and dissolved by vortexing. After standing at room temperature for 24 hours, it was diluted with 0.1 M ammonium acetate buffer (3 ml) and the organic solvent was distilled off under reduced pressure. The residue of the reagent was removed from the obtained residue using OASIS MCX Cartridge. The deprotected uridylate dimer is analyzed by reverse phase HPLC and the Thereafter, phosphodiesterase 1 (0.1 U) and alkaline phosphatase (1 U) were added and subjected to enzymatic degradation at 37 ° C. for 24 hours to obtain uridine.
  • FIG. 1B shows a reverse-phase HPLC profile of the uridylate dimer.
  • Figures 1A and 1C also show the reversed-phase HPLC profiles of 2, -O-cyanoethylated uridylate dimer and uridine, respectively.
  • CE 2'-cyanoethyl
  • TOM 2'-triylopyl bilyl silyl
  • TDMS 2, -t-butyldimethylsilyl
  • the DNA / RNA synthesizer 392 from Applied Bio System Inc. was used as an automatic nucleic acid synthesizer.
  • 1 (2-Cyanethyl N, N-diisopropyl phosphoramidite) solution of anhydrous acetonitrile with 0.1M Installed on the above automatic synthesizer and purchased from Glen Reserch In.
  • the target RNA strand was extended to a CPG solid phase carrier to which a lysine residue was bound.
  • an activation reagent for phosphoramidite As an activation reagent for phosphoramidite, a 0.25 M 5 benzylthio 1H-tetrazole in water-free nitronitrile solution was used, except that the condensation time was changed to 10 minutes. Synthesis was performed according to (tritylon). After completion of the synthesis, the solid support was immersed in a large excess of concentrated ammonium acetate (10: 1, wZw) and left for 1 hour. By passing the solution through a C18-cartridge column to remove by-products, the 2% trifluoroacetic acid aqueous solution was used to remove the cage dimethoxytrityl group, and the target product was eluted with distilled water containing acetonitrile.
  • DNA / RNA synthesizer 392 is used as an automated nucleic acid synthesizer. I used it. 2, 1 O cyanoethinole 5, -0- (4,4, -dimethoxytritinore) uridine 3, 1 (2 cyanoethyl N, N-diisopropyl phosphoramidite), 4-N-acetyl-2, 1 O cyano etinore 5, 1 O— (4,4, -dimethoxytritinole) cytidine 3, 1 (2 cyanoethyl N, N diisopropyl phosphoramidite) 2, 2, 1 O cyanoethyl 1,5, -0- (4,4′-dimethoxytrityl) 6— N— (N, N dimethylaminomethylene) adenosine 3, 1 (2 cyanoethinole N, N diisopropinorephosphoramidite), 2, 1 O— cyanoethyl-5, -
  • Benzylthio-1H-tetrazole anhydrous acetononitrile solution was used as the phosphoramidite activating reagent, and the condensation time was changed to 10 minutes. Synthesis was performed according to the RNA synthesis protocol (tritylone). After the synthesis, the solid support was immersed in a large excess of concentrated ammonium acetate (10: 1, wZw) and left for 2 hours. The solution was passed through a C18-cartridge column to remove by-products, and then the 2% trifluoroacetic acid aqueous solution was used to remove the dimethoxytrityl group, and the target product was eluted with distilled water containing acetonitrile.
  • the resulting solution was lyophilized to dryness, and 1M tetraptylammonium-fluoride Ztetrahydrofuran-propylamine (20: 1, vZv) was added to the mixture at 630 i u L and left at room temperature for 12 hours. The cyanoethyl group was removed. To the solution was added 0.1 M aqueous ammonium acetate solution and concentrated under reduced pressure. Dissolve the residue in distilled water and pass it through an OASIA MCX cartridge column and then through an H LB cartridge column.
  • DNA / RNA synthesizer 392 is used as an automated nucleic acid synthesizer. I used it. 2, 1 O Shianoethinole 5, -0- (4,4, -dimethoxytritinore) uridine 3, 1 (2-cyanethinole N, N-diisopropyl phosphoramidite) uridine residue purchased from Glen Reserch Inc. The target RNA strand was extended to the CPG solid phase carrier to which was bound.
  • RNA synthesis protocol tritylon set in advance in the above automatic synthesizer, except that it was extended to 30 seconds. After the synthesis, the solid support was immersed in a large excess of concentrated ammonia / ammonium acetate (10: 1, w / w) and left for 1 hour.
  • the 2% trifluoroacetic acid aqueous solution was used to remove the cage dimethoxytrityl group, and the target product was eluted with distilled water containing acetonitrile.
  • the resulting solution was lyophilized to dryness, and 3 mL of 1M tetraptylammonium-fluoride Ztetrahydrofuran propylamine (20: 1, ⁇ ⁇ ) was added to it and allowed to stand at room temperature for 12 hours. The group was removed.
  • To the solution was added 0.1 M aqueous ammonium acetate solution, and the mixture was concentrated under reduced pressure. Dissolve the residue in distilled water and pass it through an OASIA MCX cartridge column and then through an HLB cartridge column. The concentration gradient was loaded at a rate of 1% for 1 minute). The isolation yield was 15%.

Abstract

L’invention concerne un processus de déblocage des groupes 2'-hydroxyle des ribonucléosides qui est caractérisé par la réaction d’un dérivé du ribonucléoside représenté par la formule générale (I) où le groupe 2'-hydroxyle est bloqué : [Formule chimique 1] (I) [où X représente l’hydrogène ou similaire; Y est l’hydrogène ou similaire; et B est un résidu de base d’un acide nucléique] avec un composé contenant de la fluorine ou un composé élémentaire pour convertir le dérivé en un ribonucléoside représenté par la formule générale (II) ou une formule similaire : [Formule chimique 2] (II) [où X, Y et B sont chacun tels que défini ci-dessus].
PCT/JP2006/304399 2005-03-09 2006-03-07 Procede de deblocage des groupes 2'-hydroxyle des ribonucleosides WO2006095739A1 (fr)

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JP2005064883A JP4797156B2 (ja) 2005-03-09 2005-03-09 リボヌクレオシドの２’水酸基の脱保護方法
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007099896A1 (fr) * 2006-02-27 2007-09-07 Nippon Shinyaku Co., Ltd. procede pour detacher un groupe protecteur d'un acide nucleique
JP2013533274A (ja) * 2010-07-27 2013-08-22 蘇州瑞博生物技術有限公司 ヌクレオチド及び／又はオリゴヌクレオチド並びにその合成方法
CN103906758A (zh) * 2011-08-25 2014-07-02 株式会社博纳克 配糖体化合物、硫醚的制造方法、醚、醚的制造方法、配糖体化合物的制造方法及核酸的制造方法

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007099896A1 (fr) * 2006-02-27 2007-09-07 Nippon Shinyaku Co., Ltd. procede pour detacher un groupe protecteur d'un acide nucleique
US8158775B2 (en) 2006-02-27 2012-04-17 Nippon Shinyaku Co., Ltd. Method for detaching protecting group on nucleic acid
JP5187189B2 (ja) * 2006-02-27 2013-04-24 日本新薬株式会社 核酸保護基の脱離方法
JP2013533274A (ja) * 2010-07-27 2013-08-22 蘇州瑞博生物技術有限公司 ヌクレオチド及び／又はオリゴヌクレオチド並びにその合成方法
US9567364B2 (en) 2010-07-27 2017-02-14 Suzhou Ribo Life Sciene Co., Ltd. Nucleotide and/or oligonucleotide and preparation process thereof
CN103906758A (zh) * 2011-08-25 2014-07-02 株式会社博纳克 配糖体化合物、硫醚的制造方法、醚、醚的制造方法、配糖体化合物的制造方法及核酸的制造方法
US9481702B2 (en) 2011-08-25 2016-11-01 Bonac Corporation Glycoside compound, method for producing thioether, ether, method for producing ether, method for producing glycoside compound, method for producing nucleic acid
CN103906758B (zh) * 2011-08-25 2017-05-10 株式会社博纳克 配糖体化合物、硫醚的制造方法、醚、醚的制造方法、配糖体化合物的制造方法及核酸的制造方法
US9988415B2 (en) 2011-08-25 2018-06-05 Bonac Corporation Glycoside compound, method for producing thioether, ether, method for producing ether, method for producing glycoside compound, method for producing nucleic acid

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