WO2006027862A1 - Analogue de nucléoside et analogue d'oligonucléotide contenant celui-ci - Google Patents

Analogue de nucléoside et analogue d'oligonucléotide contenant celui-ci Download PDF

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WO2006027862A1
WO2006027862A1 PCT/JP2005/003405 JP2005003405W WO2006027862A1 WO 2006027862 A1 WO2006027862 A1 WO 2006027862A1 JP 2005003405 W JP2005003405 W JP 2005003405W WO 2006027862 A1 WO2006027862 A1 WO 2006027862A1
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group
represented
formula
following formula
nucleoside
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PCT/JP2005/003405
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Japanese (ja)
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Yukio Kitade
Yoshihito Ueno
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Gifu University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
    • C07D473/32—Nitrogen atom
    • C07D473/34—Nitrogen atom attached in position 6, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/52—Two oxygen atoms
    • C07D239/54—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
    • C07D473/18—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 one oxygen and one nitrogen atom, e.g. guanine

Definitions

  • the present invention relates to a nucleoside analog and an oligonucleotide analog containing the same.
  • the anti-sense method involves adding chemically synthesized oligonucleotide analogues (for example, single-stranded DNA consisting of 15-20 base pairs) to cells, and then adding target mRNA (mRNA) and DNA such as ZmRNA double-stranded nucleic acid.
  • mRNA target mRNA
  • mRNA target mRNA
  • ZmRNA double-stranded nucleic acid target mRNA
  • the expression of the target gene is suppressed in a base sequence-specific manner, and the translation process into mRNA protein is inhibited.
  • the antisense method if the base sequence of the pathogenic virus or gene is known, it is possible to theoretically design an antisense molecule and synthesize it. It is expected as one of the effective treatment methods for diseases caused by various viruses and genetic diseases.
  • RNAi RNA interference
  • RNAi refers to a phenomenon in which RNA derived from a cell chromosome having the same base sequence is degraded and cleaved by introducing double-stranded RNA into a cell.
  • the mechanism of RNAi is currently considered as follows.
  • long double-stranded RNA is called a 21-base long double-stranded RNA (siRN A (short interfering RNA)) that has a three-one UU-type dangling end structure by an enzyme (called Dicer). ).
  • RNA-induced silencing complex RISC
  • RNAi-based methods have concentrations around 1Z100 compared to antisense methods. Equivalent effects have been obtained using the RNA. Therefore, a method using RNAi is also expected to be one of effective treatment methods for diseases caused by various viruses and genetic diseases that have been considered difficult to cure.
  • oligonucleotide analogs In methods using oligonucleotide analogs, such as antisense methods and RNAi methods, it is necessary to make oligonucleotide analogs once introduced into cells stable, but there are nucleic acids inside and outside the cells. There was a problem that hydrolyzing enzymes (nucleases) existed, and the oligonucleotide analogues introduced, especially oligonucleotide analogues having a structure very similar to natural oligonucleotides, were easily degraded.
  • oligonucleotide analogs have been developed for the purpose of improving nuclease resistance.
  • phosphorothioate-type one oxygen atom of a phosphodiester bond is replaced with a sulfur atom
  • oligonucleotide analogue or methylphosphonate type (a phosphodiester bond is replaced with a methylphosphonate bond) )
  • methylphosphonate type a phosphodiester bond is replaced with a methylphosphonate bond
  • oligonucleotide analogues have an asymmetric center at the phosphate atom in their phosphorothioates and methylphosphonates, so that the structure of the oligonucleotide analogues is very different from that of natural oligonucleotides. For this reason, it has been known that such oligonucleotide analogues have a reduced ability to form double strands with natural oligonucleotides and the stability of the formed duplexes (for example, non-patents). Reference 1).
  • oligonucleotide analogues have been developed for the purpose of improving the ability to form a double strand with a natural oligonucleotide and improving the stability of the formed double strand.
  • examples of such oligonucleotide analogues include oligonucleotide analogues in which the 2 ′ hydroxyl group of ribose on the nucleoside of a natural oligonucleotide is replaced with an alkoxy group (eg, methoxy group) or a halogen atom (eg, fluorine atom) (eg, Non-Patent Document 2), and oligonucleotide analogues in which a methylene group is inserted into a 5-membered ring of ribose to expand the ring to a 6-membered ring (for example, see Non-Patent Document 3) are known.
  • oligonucleotide analogues have been confirmed to have improved duplex-forming ability with natural oligonucleotides and improved stability of the formed duplex.
  • these oligonucleotide analogues are structurally similar to natural oligonucleotides, and thus improved nuclease resistance is not realized. It wasn't.
  • Non-patent document 1 Jin yan Tang, Jamal Terns amani and ⁇ udhir Agrawal, ⁇ Self-stable antisense oligonucleotide phosphorotnioates: properties and anti-HIV activity ", Nucleic Acids Research, 1993, Vol. 21, ⁇ ⁇ 2729.
  • Non-Special Terms 2 Andrew M. Kawasaki, Martin D. Casper, Susan M. Freier, Maria A. Lesnik, Maryann C. Zounes, Lendell L. Cummins, Carolyn Gonzalez and P. Dan Cook, "Uniformly modified 2 and deoxy -2-fluoro phophorothioate oligonucleotides as nuclease— resistant antisense compounds with highly affinity and specificity for RNA targets, Journal of Medicinal Chemistry, 1993, 36, ⁇ 831.
  • Non-Special Terms 3 Chris Hendrix, Helmut Rosemyer, Bart De Bouvere, Arthur Van Aerschot, Frank Seek and Piet Herdewijin, ,, 5 Anhydrohexitol oligonucleotides: hybridization and strand displacement with
  • the present invention provides a nucleoside analogue that makes it possible to produce an oligonucleotide analogue having excellent properties of nuclease resistance, ability to form a double strand, and stability of the formed double strand, and its nucleoside analogue.
  • the purpose is to provide oligonucleotide analogues containing the body.
  • the present invention is a nucleoside analog represented by the following formula (I) or a salt thereof. [0010] [Chemical 15]
  • R 1 is a group represented by the following formula (1), a group in which the functional group is protected by a protecting group in the group of the following formula (1), and the following formula (2)
  • the functional group is protected by a protecting group, the group represented by the following formula (3), and the group represented by the following formula (3).
  • the group whose power is protected by a protecting group is selected as a group force
  • R 2 is H or a protecting group
  • R 3 is H or an activated phosphate group for solid phase synthesis.
  • the present invention has been completed based on the successful production of a nucleoside analog having a chemical structure that has not existed before.
  • the present invention provides an oligonucleotide analog that is excellent in the three characteristics of nuclease resistance, ability to form a double strand, and stability of the formed double strand by using this nucleoside analog.
  • oligonucleotide refers to, for example, a polymer of nucleoside subunits, and the number of subunits is not particularly limited, but is, for example, 3 to 100. Among them, when the oligonucleotide is DNA, the number of subunits is If 3 to 100 are strong and 3 to 30 is the preferred RNA, 3 to 50 is preferred and 3 to 30 is more preferred.
  • the “oligonucleotide analog” in the present invention is not particularly limited, except that the nucleoside is replaced with the nucleoside analog of the present invention.
  • nucleoside other than the nucleoside analog of the present invention in the oligonucleotide may be a nucleoside analog that is known to those skilled in the art and in which the sugar and base moieties are substituted or converted.
  • the protective group for R 2 a conventionally known primary alcohol protective group can be used.
  • protecting groups include 4,4′-dimethoxytrityl (DMTr), tert-butyldimethylsilyl (TBDMS), 4 monomethoxytrityl (MM Tr), (9-phenol) xanthene 9-yl [pixyl] (pixyl)] Equivalent power.
  • the protecting group for protecting the functional group can be selected from known protecting group forces in nucleic acid chemistry.
  • benzoyl (Bz), isobutyryl (iBu), phenoxyacetyl (Pac), allyloxycarbol (AOC), N, N-dimethylaminomethylene and the like can be used as such a protecting group.
  • R 1 is a group in which the functional group of the group represented by the formula (1), the group represented by the formula (2) and the group represented by the formula (3) is protected with a protecting group. Is, for example, the following formula (3-1)
  • Te R 3 Nitsu ⁇ as the solid-phase synthesis activating phosphate group, can be used a phosphate group of a conventional publicly known Te Contact ⁇ to solid phase synthesis, for example, phosphoramidite, Examples thereof include phosphate groups that form phosphonates, thiophosphites, and the like. Examples of activated phosphate groups for solid phase synthesis that form phosphoramidites include the following formula (10) [0022] [Chemical 18]
  • examples of the salt include a salt with an inorganic base, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, and the like.
  • examples of salts with inorganic bases include, for example, alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; and aluminum salts and ammonium salts.
  • Examples of salts with organic bases include, for example, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, ⁇ , ⁇ '-dibenzylethylenediamine, and the like. Of the salt.
  • salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like.
  • salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, succinic acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, ⁇ -toluenesulfone And salts with acids.
  • the salt is preferably a pharmacologically acceptable salt.
  • R 1 is a group represented by the following formula (1), or a group represented by the following formula (1), wherein the functional group is A group protected by a protecting group, a group represented by the following formula (2), a group represented by the following formula (2), a group whose functional group is protected by a protecting group, a group represented by the following formula (3)
  • the functional group is any group selected as a group force in which the functional group is protected with a protecting group
  • R 2 is ⁇ , 4, 4′-dimethoxytrityl ( DMTr), tert-butyldimethylsilyl (TBDMS), 4 monomethoxy tri chill (MMTr) or (9 - Hue - Le) xanthene 9 is Iru [pixyl (pixyl)], R 3 is H or the following formula (10) [0026] [Chemical 19]
  • nucleoside analog represented by the formula (I) of the present invention or a salt thereof is represented by the following formula (1), wherein R 1 is a group represented by the following formula (1): A group represented by the following formula (3), and a group force represented by the following formula (3-1): any group selected, R 2 is H Or 4,4, -dimethoxytrityl (DMTr)
  • R 3 is more preferably H or a group represented by the following formula (10).
  • the nucleoside analog represented by the formula (I) or a salt thereof of the present invention includes a nucleoside analog represented by the following formula ( ⁇ -1), a nucleoside represented by the following formula ( ⁇ -2) An analog, a nucleoside analog represented by the following formula ( ⁇ -3), R 1 is a group represented by the formula (2), R 2 is DMTr, and R 3 is the formula (10).
  • the nucleoside analog represented by the above formula (I) which is a group represented by (10) or a salt thereof is more preferable.
  • the nucleoside analog represented by the following formula ( ⁇ -1) is a group represented by the above formula (1) wherein R 1 is a group represented by the formula (1), R 2 is H, and R 3 is H. It is a nucleoside analog represented by I).
  • the nucleoside analog represented by the following formula ( ⁇ -2) is a group represented by the formula (I) wherein R 1 is a group represented by the formula (2), R 2 is H, and R 3 is H.
  • the nucleoside analog represented by the following formula ( ⁇ -3) is a group represented by the formula ( 1 ) wherein R 1 is a group represented by the formula (3), R 2 is H, and R 3 is H.
  • Nucleo represented by I) Cid analogue is a group represented by the above formula (1) wherein R 1 is a group represented by the formula (1), R 2 is H, and R 3 is H. It is a nucleoside analog represented by I).
  • nucleoside analog of the present invention or a salt thereof is not limited to the production of the oligonucleotide of the present invention, and can be applied to other uses.
  • the oligonucleotide analogue of the present invention is an oligonucleotide analogue in which one or more nucleosides constituting the oligonucleotide are each replaced with a nucleoside analogue, wherein the nucleoside analogue force is represented by the formula ( ⁇ — A nucleoside analog represented by 1), a nucleoside analog represented by the above formula ( ⁇ —2), a nucleoside analog represented by the above formula ( ⁇ —3), and the following formula ( ⁇ —4) And a nucleoside analog represented by the following formula ( ⁇ -5): a group force that is selected from the group nucleoside analogs.
  • the oligonucleotide analogue is a nucleoside analogue represented by the formula ( ⁇ -4) or a nucleoside analogue represented by the formula ( ⁇ -5), and the nucleoside constituting the oligonucleotide When only one of the above is replaced with the nucleoside analog, the replacement position is at the 3 terminus of the oligonucleotide.
  • all of the nucleosides constituting the oligonucleotide are nucleoside analogues represented by the above formula ( ⁇ -1) and nucleosides represented by the above formula ( ⁇ -2).
  • nucleoside analog represented by the above formula ( ⁇ -3), a nucleoside analog represented by the above formula ( ⁇ -4), and a nucleoside analog represented by the above formula ( ⁇ ⁇ -5) Group power Preferably each is replaced by a selected nucleoside analog.
  • the nucleoside analog to be replaced with thymidine is the nucleoside analog represented by the formula ( ⁇ -1). Preferred.
  • nucleoside analog to be replaced when the nucleoside to be replaced is a uridine, the nucleoside analog to be replaced with the uridine is a nucleoside analog represented by the formula ( ⁇ -2). Is preferred.
  • the nucleoside analog to be replaced with cytidine is the nucleoside analog represented by the formula ( ⁇ -3). Preferred.
  • the nucleoside analog to be replaced with adenosine is the nucleoside analog represented by the formula (I 1-4). Is preferred.
  • the nucleoside analog to be replaced with guanosine is the nucleoside analog represented by the formula (I 1-5) Is preferred.
  • the oligonucleotide analogue of the present invention may be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or the like.
  • Oligonucleotide analog force When double-stranded, one or both of the double-stranded oligonucleotides has a nucleoside analog represented by the above formula ( ⁇ -1) wherein the 3, terminal nucleoside of the single-stranded oligonucleotide is A nucleoside analogue represented by the formula ( ⁇ —2), a nucleoside analogue represented by the formula ( ⁇ —3), a nucleoside analogue represented by the formula ( ⁇ —4), and the formula ( ⁇ ⁇ — Group power consisting of nucleoside analogs represented by 5) is selected, and is preferably replaced by any nucleoside analog.
  • the 3-terminal nucleoside of both single-stranded oligonucleotides of the double-stranded oligonucleotide is a nucleoside analogue represented by the above formula ( ⁇ -1), a nucleoside analogue represented by the above formula ( ⁇ -2) Body, nucleoside analogue represented by the formula ( ⁇ -3), nucleoside analogue represented by the formula ( ⁇ -4) and nucleoside analogue represented by the formula ( ⁇ -5) If selected, replaced by any nucleoside analog, both replaced nucleoside analogs may be the same or different.
  • the dangling end sequence at the 3 'end of at least one strand of the double-stranded oligonucleotide strand is represented by the above formula ( ⁇ -1).
  • Analogues and nucleoside analogues represented by the above formula ( ⁇ -5) Group power as a selected group, preferably a sequence comprising one or more nucleoside analogues, preferably a nucleoside represented by the above formula ( ⁇ -4)
  • a substituted dangling end sequence for example, a sequence having two nucleoside analogs represented by the formula ( ⁇ -2) can be used.
  • the oligonucleotide analog When the oligonucleotide analog is double-stranded, it is an oligonucleotide analog that causes RNAi (RNA interference), and has the same base as a part of mRNA that encodes the oligonucleotide analog force endonuclease etc. Oligonucleotide analogs having the sequence are preferred. Such oligonucleotide analogues are useful for RNAi research and the like. Examples of the endonuclease include RNaseL.
  • the oligonucleotide analogue When the oligonucleotide analogue is double-stranded, it is the analogue oligonucleotide-force siRNA (short interfering RNA), and the first part of the mRNA is 75 bases or more upstream from the start codon.
  • siRNA short interfering RNA
  • Such a sense strand can be obtained, for example, as follows.
  • an exonuclease mRNA sequence for example, is obtained using known genes such as NCBI (National and Enter for Biotechnology Information) and EMBL-EBI (European Molecular Biology Laboratory-European Bioinformatics Institute).
  • NCBI National and Enter for Biotechnology Information
  • EMBL-EBI European Molecular Biology Laboratory-European Bioinformatics Institute
  • the 19-base sequence following the AA sequence a total of 21 base sequences, is specific to the target gene again using a known gene database. It is also confirmed that this 19-base sequence has a GC content of around 50%.
  • the 19-base sequence obtained by confirming these two is the sense strand as described above. Examples of such a sense strand include SEQ ID NO: 11 described later, which is the 94th to 112th base sequence from the start codon of the human RNaseL gene.
  • the oligonucleotide analogue of the present invention preferably has double-strand forming ability. This is because the oligonucleotide analog of the present invention can be used for antisense, gene detection, etc. if it has the ability to form a double strand with the natural oligonucleotide.
  • the oligonucleotide analogue of the present invention is preferably resistant to nuclease.
  • the ability of the oligonucleotide analogue of the present invention to prevent degradation by a nuclease when incorporated into cells, and as a result, the activity of the oligonucleotide analogue in cells can be maintained. Power is also.
  • the gene expression inhibitor of the present invention includes the oligonucleotide analog of the present invention.
  • a gene expression inhibitor acts as an oligonucleotide analog, for example, siRNA or antisense, and cleaves the target gene mRNA or forms a double strand with the target gene mRNA. Expression can be suppressed.
  • the pharmaceutical composition of the present invention is for treating a disease associated with gene expression, and includes the gene expression inhibitor.
  • a disease associated with gene expression for example, a disease is caused by the expression of a protein
  • this pharmaceutical composition can be used to suppress the gene expression and treat the disease associated with the gene expression.
  • the test kit of the present invention is a kit that comprises the oligonucleotide analog of the present invention, and the gene is tested by hybridizing the oligonucleotide analog with a gene in a specimen. Examples of such a kit include a DNA chip and a DNA microarray.
  • kits include, in addition to the oligonucleotide analogues of the present invention, plates, fibers, biochips, etc., on which the wells and oligonucleotide analogues are immobilized. It is done.
  • a kit may contain, for example, a drug, a coloring reagent that develops color upon reaction, a detection reagent that facilitates detection, in addition to the oligonucleotide analog and the like.
  • the DNA chip generally, a solution containing the oligonucleotide analog of the present invention using a known gene sequence is spot-fixed on a glass substrate, or the oligonucleotide of the present invention on a glass substrate. Some are fixed by synthesizing analogs.
  • the DNA chip for example, applies a gene in a specimen to an analysis unit on which the oligonucleotide analog is immobilized, and performs hybridization between the gene and the oligonucleotide analog on the substrate, for example, a fluorescent dye or the like. By detecting by this, the presence or absence of expression of the target gene can be detected. According to such a DNA chip, for example, even a small amount of sample can be effectively analyzed, and various DNA probes can be immobilized on one substrate. A multi-item analysis can be performed.
  • the gene expression suppression method of the present invention is a method of suppressing gene expression using an oligonucleotide analog.
  • the ability of oligonucleotide analogues acts as siRNA or antisense, cleaves the target gene mRNA or forms a double strand with the target gene mRNA, and as a result suppresses gene expression. be able to.
  • nucleoside force at the third and terminal ends of the oligonucleotide the nucleoside analog represented by the above formula ( ⁇ -1), the nucleoside analog represented by the above formula ( ⁇ -2), and the above formula ( ⁇ - A nucleoside analog represented by 3), a nucleoside analog represented by the formula ( ⁇ -4), and
  • An example of a method for producing an oligonucleotide analogue in which a selected nucleoside analogue is replaced by a selected nucleoside analogue will be described.
  • the production method includes, for example, removal of R 5 from the solid-phase synthesis unity compound represented by the following formula ( ⁇ ) and solid-phase synthesis unity-combination represented by the following formula (IV).
  • the method includes the step of extending a nucleotide to the free hydroxyl group of the solid phase synthesis unity compound represented by the formula (IV) and then cutting out the solid phase carrier force to obtain the oligonucleotide analog.
  • R 4 is a group represented by the following formula (1), a group of the following formula (1), the functional group of which is protected with a protecting group, and the following formula (2)
  • the functional group is a group protected by a protecting group, the group represented by the following formula (3), and the group represented by the following formula (3):
  • the group represented by the following formula (4) the group represented by the following formula (4)
  • the functional group is protected with a protecting group, and the following formula (5)
  • the functional group is selected from the group consisting of basic groups protected by a protecting group,
  • R 5 is a protecting group
  • A is a group represented by the formula (CH) 1, wherein n is 1
  • n is an integer of 6 and
  • M is a solid phase carrier.
  • nucleoside forces other than the 3, terminal of the oligonucleotide a nucleoside analogue represented by the formula ( ⁇ -1), a nucleoside analogue represented by the formula ( ⁇ -2), Group power selection consisting of a nucleoside analogue represented by the formula ( ⁇ —3), a nucleoside analogue represented by the above formula ( ⁇ —4), and a nucleoside analogue represented by the above formula ( ⁇ —5)
  • a method for producing an oligonucleotide analog that is replaced with V or any of the nucleoside analogs will be described.
  • the manufacturing method includes:
  • a unitary compound for solid phase synthesis represented by the following formula (V) is bound to a nucleotide extended on a solid support,
  • R 4 is a group represented by the following formula (1), a group of the following formula (1), the functional group of which is protected with a protecting group, and the following formula (2)
  • the functional group is a group protected by a protecting group, the group represented by the following formula (3), and the group represented by the following formula (3):
  • the group represented by the following formula (4) the group represented by the following formula (4)
  • the functional group is protected with a protecting group, and the following formula (5)
  • the functional group is selected from the group consisting of basic groups protected by a protecting group,
  • R 6 is a protecting group
  • R 7 is an activated phosphate group for solid phase synthesis.
  • the unit compound for solid phase synthesis represented by the formula (V) may be produced in-house with reference to known literature, or represented by the following formula ( ⁇ -7). It may be made in-house with reference to a method for producing a nucleoside analogue and a method for producing a nucleoside analogue represented by the formula ( ⁇ -8).
  • R 4 is a group represented by the formula (2)
  • R 6 is R 11
  • the R 7 force is 12 in the formula (V).
  • R 4 is a group in which the functional group of the group represented by the formula (3) is protected with a protecting group, and R 6 is R 11 Corresponding to the nucleoside analogue of formula (V) wherein R 7 is R 12 .
  • nucleoside analog represented by the above formula ( ⁇ -1), represented by the above formula ( ⁇ -2) Nucleoside analogues represented by the formula ( ⁇ -3), nucleoside analogues represented by the formula (II 4), and nucleosides represented by the formula ( ⁇ -5)
  • ⁇ -1 nucleoside analog represented by the above formula ( ⁇ -2)
  • Nucleoside analogues represented by the formula ( ⁇ -3) Nucleoside analogues represented by the formula (II 4), and nucleosides represented by the formula ( ⁇ -5)
  • An example of a method for producing an oligonucleotide analogue that is selected from the group consisting of analogues and is replaced by a shift will be described.
  • the production method includes, for example, (a) a solid-phase synthesis unity compound represented by the following formula (III) by removing R 5 and a solid-phase synthesis represented by the following formula (IV) Get unity compound,
  • a solid phase synthesis unit compound represented by the following formula (V) is bound to a nucleotide extended on a solid phase carrier,
  • step (e) repeating the step (c) by the number of substitutions with the nucleoside analog at a position other than the 3 ′ end of the oligonucleotide;
  • R 4 is a group represented by the following formula (1) and a group represented by the following formula (1), respectively.
  • the functional group is protected by a protecting group, the group represented by the following formula (4), and the group represented by the following formula (4):
  • the functional group is selected from a basic group protected by a protecting group. It ’s the base of either,
  • R 5 and R 6 are independently of each other a protecting group
  • A is a group represented by the formula (CH) 1, wherein n is an integer of 1 to 6,
  • M is a solid support
  • R 7 is an activated phosphate group for solid phase synthesis.
  • the solid phase carrier is not limited as long as it is a solid phase carrier suitable for synthesizing DNA, RNA, and the like on the solid phase carrier.
  • CPG control pore glass
  • HCP Highly Cross- linked polystyrene etc.
  • a conventionally known primary alcohol protecting group can be used as the protecting group.
  • protecting groups include 4,4′-dimethoxytrityl (DMTr), tert-butyldimethylsilyl (TBDMS), 4 monomethoxytrityl (MMTr), (9-phenol) xanthene 9-yl [ Pixyl] and the like.
  • a protecting group that is protected by a functional group is selected from those known in nucleic acid chemistry.
  • benzoyl (Bz), isoptyryl (iBu), phenoxycetyl (Pac), aryloxycarbol (AOC), N, N-dimethylaminomethylene and the like can be used as such protecting groups.
  • R 4 a group a group a functional group of groups represented by the formula (1) is protected with a protecting group, the functional group of the group represented by the formula (2) is protected by a protecting group, A group in which the functional group of the group represented by formula (3) is protected with a protecting group, a group in which the functional group of the group represented by formula (4) is protected with a protecting group, and a group represented by (5)
  • Examples of the group in which the functional group is protected with a protecting group include, for example, the following formula (3-1)
  • R 13 described later a conventionally known primary amino protecting group can be used.
  • a protecting group for example, acetyl (Ac), benzoyl (Bz), phenoxy acetyl (Pac) and the like can be used.
  • the lower alkyl group for R 8 and R 15 described later is a linear or branched alkyl group, for example, containing 16 to 16 carbon atoms.
  • Examples of the lower alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl 2-ethylbutyl, isobutyl, tert-butyl, pentyl, n-hexyl and the like.
  • the aryl group for R 15 described later is an aromatic hydrocarbon residue, for example, one containing 6-30 carbon atoms.
  • Examples of the aryl group include a monocyclic aryl group such as phenyl, and a condensed polycyclic aryl group such as naphthyl, indenyl, fluoroenyl and the like.
  • R 15 to be described later may be substituted with lower alkyl !, but examples of the aryl group include an aryl group substituted with 115 of the lower alkyl.
  • a specific example thereof is, for example, triisopropyl file.
  • the halogen atom is a fluorine atom, a chlorine atom, a bromine atom, an iodine atom or the like.
  • the activated phosphate group for solid-phase synthesis a conventionally known phosphate group can be used in solid-phase synthesis.
  • Forms phosphoramidites As the activated phosphate group for solid phase synthesis, for example, the following formula (10)
  • nucleoside is sequentially added according to the sequence of the oligonucleotide analog, using a conventionally known technique in the field of oligonucleotide synthesis. It can be done by coupling.
  • the nucleoside As the nucleoside, coupling reagent, deprotection reagent, washing reagent, etc., those usually used for nucleic acid solid phase synthesis are used.
  • the obtained oligonucleotide analog on the solid phase carrier can also cut out the solid phase carrier force to obtain a crude oligonucleotide analog.
  • Deprotection of the oligonucleotide side chains may be performed before, simultaneously with and after Z or after the excision of the solid support, if necessary.
  • the reagent used for excision can be appropriately selected from conventionally known reagents according to the structure of the solid phase carrier and the linker (the portion connecting the solid phase carrier and the oligonucleotide analog).
  • This crude oligonucleotide analog may be purified by HPLC or the like, if necessary.
  • a single-stranded oligonucleotide analog is first prepared.
  • a single-stranded natural oligonucleotide having a sequence complementary to the oligonucleotide analog is separately produced according to a conventionally known method.
  • the resulting single-stranded oligonucleotide analog is then dissolved in an annealing buffer (eg, a buffer containing lOOmM KOAc in water, 2 mM MgOAc, and 30 mM HEPES- KOH (pH 7.4)).
  • an annealing buffer eg, a buffer containing lOOmM KOAc in water, 2 mM MgOAc, and 30 mM HEPES- KOH (pH 7.4)
  • a solution in which a single-stranded natural oligonucleotide is dissolved in an annealing buffer solution for example, mixed, treated at 95 ° C for 5 minutes, and then gradually cooled to 25 ° C.
  • Double stranded oligonucleotide analogues can be obtained.
  • This double-stranded oligonucleotide analog can be extracted with phenol Z It can be isolated and purified by further ethanol precipitation or the like.
  • the solid-phase synthesis unit compound ( ⁇ ⁇ ⁇ ⁇ ) used in the production method can be produced, for example, by the following method, which will be described with reference to Scheme 1.
  • a benzene derivative represented by the formula (VI) and an anhydride represented by the formula (VII) are optionally mixed with a base (eg, pyridine, triethylamine, etc.), a catalyst (eg, 4-dimethylaminopyridine (
  • a benzene derivative represented by the formula (VIII) can be obtained by condensation in the presence of DMAP) and the like.
  • the benzene derivative represented by the formula (VI) may be made in-house with reference to known literature.
  • the benzene derivative represented by the formula (VI) is produced by a method for producing a nucleoside analogue represented by the formula ( ⁇ —7) and a method for producing a nucleoside analogue represented by the formula ( ⁇ —8). You can also make your own by referring to ⁇ .
  • a benzene derivative represented by the above formula (VIII) and a solid phase carrier having an amino group represented by the formula (IX) are combined with a coupling reagent (for example, WSC (1-ethylol). 3- (3-dimethylaminopropyl) carpositimide'hydrochloride))) in the presence of the compound, unitary compound ( ⁇ I) can be obtained.
  • a coupling reagent for example, WSC (1-ethylol). 3- (3-dimethylaminopropyl) carpositimide'hydrochloride)
  • R 4 represents a group represented by the following formula (1), a group in which the functional group is protected with a protecting group in the group represented by the following formula (1)
  • the functional group is protected by a protecting group, the group represented by the following formula (3), and the group of the following formula (3).
  • a group represented by the following formula (5) wherein the functional group is selected from the group consisting of basic groups protected by a protecting group:
  • R 5 is a protecting group
  • A is a group represented by the formula (CH) 1, wherein n is an integer of 1 to 6,
  • M is a solid phase carrier.
  • R 4 is a group represented by the following formula (1), a group in which the functional group is protected by a protecting group in the group of the following formula (1), and a group represented by the following formula (2).
  • the functional group is a group protected by a protecting group, the group represented by the following formula (3), and the group represented by the following formula (3).
  • the functional group is selected from the group consisting of basic groups protected by a protecting group!
  • R is a protecting group
  • A is a group represented by the formula (CH) 1, wherein n is an integer of 1 to 6,
  • M is a compound that is a solid phase carrier.
  • R 4 is a group represented by the following formula (1), a group represented by the following formula (2), and a group represented by the following formula (3), respectively.
  • R 5 is DMTr
  • a compound in which M is a solid phase carrier is preferred.
  • nucleoside analogues represented by the following formulas ( ⁇ -2), (II-6) and ( ⁇ -7) will be described with reference to Scheme 2.
  • an ⁇ , ⁇ -unsaturated ketone derivative represented by the formula (X) is reacted with AgOCN, and then an aryl derivative represented by the formula (XI) is condensed to form the formula ( ⁇ ) -Derived derivatives can be obtained.
  • the arrin derivative represented by the formula (XII) is treated with an alkali (for example, sodium hydroxide, etc.) to give a nucleoside analog represented by the formula ( ⁇ —2) or a salt thereof.
  • an alkali for example, sodium hydroxide, etc.
  • the ⁇ , j8-unsaturated ketone derivative of the formula (X) and the arine derivative of the formula (XI) can be obtained commercially, or can be made in-house using known literature.
  • nucleoside analog represented by the formula ( ⁇ -2) and a formula ( ⁇ ) can be obtained by optionally condensing the compound with a compound in the presence of a base (eg, pyridine, triethylamine, etc.).
  • a base eg, pyridine, triethylamine, etc.
  • nucleoside analog represented by the above formula ( ⁇ -6) and the compound represented by the formula (XIV) are optionally added in the presence of a base (for example, diisopropylethyleneamine).
  • a base for example, diisopropylethyleneamine.
  • the compounds represented by the formulas ( ⁇ ) and (XIV) may be obtained commercially or manufactured in-house using known literature.
  • R 8 represents a lower alkyl group
  • R 9 , R 1Q and R 11 are independently of each other a protecting group
  • R 12 is an activated phosphate group for solid phase synthesis
  • X 1 , X 2 and X 3 are halogen atoms.
  • a compound represented by the formula (XVI) can be obtained by condensing the compound represented by the formula (XV).
  • the compound represented by the formula (XVI) and the compound represented by the formula (XVII) are optionally combined with a base (for example, pyridine, triethylamine, etc.), a catalyst (for example, 4-dimethylaminopyridine (D MAP)). And the like, and then reacted with NH 4 OH to obtain a compound represented by the formula (XVIII)
  • the compound represented by the formula (XVIII) and the compound represented by the formula (XIX) are optionally condensed in the presence of a base (for example, pyridine, triethylamine, etc.), and then R " After deprotection, it is condensed with a compound represented by the formula (XIV), optionally in the presence of a base (for example, diisopropylethylamine, etc.), and represented by the formula ( ⁇ -8) Nucleoside analogues can be obtained. Deprotection of R "can be carried out under conditions depending on the type of group R".
  • TBAF tetraptyl ammonium fluoride
  • the compounds represented by the formulas (XV), (XVII), (XIX) and (XIV) may be obtained commercially V, and are publicly known. You can make a homemade article using literature!
  • R U , R 13 and R 14 are each independently a protecting group
  • R 12 is an activated phosphate group for solid phase synthesis
  • R 15 is optionally substituted with a lower alkyl group or a lower alkyl group, and is an aryl group,
  • X 3 , X 4 , X 5 and X 6 are each independently a halogen atom.
  • the nucleoside analogue represented by the formula ( ⁇ -1), the nucleoside analogue represented by the formula ( ⁇ -2), and the nucleoside analogue represented by the formula ( ⁇ -3) The body can be produced in the same manner as in the production method.
  • the nucleoside analog represented by the formula ( ⁇ -3) can be obtained by removing R 11 and R ′′ of the compound represented by the formula (XVIII). Depending on the structure of R 11 and R 14 , it can be carried out using conventionally known conditions.
  • a protecting group may be introduced into each functional group, the protecting group may be deprotected, or the protecting group may be changed.
  • the selection of the protecting group, the introduction of the protecting group and the removal of the protecting group can be carried out according to methods known in the art, for example, “Protective Groups in Organic Synthesis”, T Greene et al., Published by John Wiley & Sons, Inc.
  • Phosphorylation reagent 2-Chanoethyl N, N-Diisopropyl black phosphoramidite Ar: Anolegon
  • DIPEA N, N-Diisopropylethylamine
  • mRNA messenger ribonucleic acid
  • TBDMSC1 tert-butyldimethylsilylchloride (tert-butyldimethylsilylchloride)
  • TPSC1 2, 4, 6-Triisopropylbenzenesulfurolide
  • the activity of the compound on CPG was calculated as follows. [0114] Place 6 mg of dried CPG on a glass filter and add HCIO and ethanol.
  • weight is the weight of the measured CPG.
  • dimethyl 5-aminoisophthalate (2.00 g, 9.58 mmol) was charged with anhydrous THF (48 ml), and further lithium lithium boron (1.13 g, 51.80 mmol, 5. 4 eq) was added.
  • the reaction mixture was stirred for 20 hours and then neutralized by adding a few drops of acetic acid to the reaction mixture in a water bath. After stirring for several minutes, a large amount of crystals precipitated from the reaction mixture were collected by filtration.
  • reaction mixture was further calo-free with benzoinolecide, (0.04 ml, 0.35 mmol, 0.2 eq).
  • TLC ethyl acetate only
  • a small amount of saturated aqueous NaHCO solution was added to the reaction mixture, stirred for several minutes, and distilled.
  • N 6 -benzoyl 9- (3,5-bis-tert-butyldimethylsilyloxymethyl-phenol) -adene (0.215 g, 0.30 mmol) in THF (2.8 ml) ) And dichloromethane (2.8 ml).
  • TBAF (0.15 ml, 0.15 mmol, 0.5 eq) was added to the solution while cooling in an ice bath. Then, after returning to normal temperature, TBAF (0.3 ml, 0.3 mmol, 1. Oeq) was further added to the reaction mixture. After 4 hours, the reaction mixture was extracted with black mouth form. Extracted black mouth form layer into saturated aqueous NaHCO solution
  • N 6 -benzoyl 9 (3-hydroxymethyl-5-tert-butyldimethylsilyloxymethyl-phenol) -adenine (0.38 g, 0.78 mmol) was added to pyridine ( 3. 4ml) [This was dissolved. Add the solution [DMAP (5. Omg, 0.04mmol, 0.05.eq) and 4,4, -dimethoxytrityl chloride (0.53g, 1.56mmol, 2.Oeq) and the reaction mixture in an argon atmosphere. Stirred under for 16 hours.
  • the extracted black-form layer was washed with saturated saline and dried over anhydrous sodium sulfate. Its chloroform solution power The solvent was azeotroped with toluene and distilled under reduced pressure.
  • N 6 Benzoyl 9— (3— (4,4, -Dimethoxytrityloxy) methyl-5—0— [(2 —Cyanoethyl) (N, N-diisopropyl)] Phosphieroxy Methylphenol-denine (N 6 -benzoy ⁇ 9- (3- (4,4,
  • the solvent was distilled off from the obtained organic layer under reduced pressure.
  • the solvent was distilled off from the organic layer under reduced pressure, and the resulting residue was dried overnight.
  • N-(N, N-Dimethylaminomethylene) — 9— (3— (4, 4, Dimethoxytrityloxy) methyl-5 — O— ⁇ (2-Cyanoethyl) (N, N— Diisopropyl) ⁇ phosphieroxymethylphenol) -guanine
  • N 2- (N, N-dimethylaminomethylene)-9- (3- (4,4, — dimethoxytrityloxy) methyl— 5— O— [(2— cyanoethyl) — ( ⁇ , ⁇ — dusopropyl)] — phosphinyloxy methyiphenyl)-guanine): ⁇
  • N 2 — (N, N-dimethylaminomethylene) — 9— (3— (4, 4, -dimethoxytrityloxy) methyl-5-hydroxymethyl-phenol) -guanine ( 0.2 g, 0.31 mmol) was added THF (1.6 ml) under an argon atmosphere.
  • THF 1.6 ml
  • Hunig's base 0.31 ml, 1.86 mmol, 6. Oeq
  • phosphite reagent 0.14 ml, 0.62 mmol, 2. Oeq
  • the filtrate was extracted with ethyl acetate, and the resulting organic layer was washed with saturated brine and dried over anhydrous sodium sulfate.
  • the solvent was distilled off from the organic layer under reduced pressure to obtain the title compound as a white solid.
  • the obtained title compound was a total of 17.697 g (0.17 mol, 68%).
  • N 4 Benzoyl 1— (3—Hydroxymethyl-5— (4,4, -dimethoxytrityloxy) methinorefe-nore) —cytosine (N 4 — benzoy ⁇ 1— (3—hydroxymethy ⁇ 5— (4 ,Four,
  • N 4 Benzoyl 1— (3— (4,4, -Dimethoxytrityloxy) methyl-5—0— ⁇ (2-Cyanethyl) (N, N-diisopropyl) ⁇ phosphioxymethylphenol) -Cytosine (N 4 -benzoy ⁇ 1- (3- (4,4, -Dimethoxytrityloxy) methyl-5—0— ⁇ (2-Cyanethyl) (N, N-diisopropyl) ⁇ phosphioxymethylphenol) -Cytosine (N 4 -benzoy ⁇ 1- (3- (4,4, -Dimethoxytrityloxy) methyl-5—0— ⁇ (2-Cyanethyl) (N, N-diisopropyl) ⁇ phosphioxymethylphenol) -Cytosine (N 4 -benzoy ⁇ 1- (3- (4,4, -Dimethoxytrityloxy) methyl-5—
  • N 6 Benzo Luo 9— (3— (4, 4, Dimethoxytrityloxy) methyl-5-hydroxymethyl roof)
  • Adenine (0.15 g, 0.22 mmol) in pyridine (2.2 ml)
  • DMA P 26.9 mg, 0.22 mmol, 1. Oeq
  • succinic anhydride 73. lmg, 0.73 mmol, 3.3 eq
  • DMAP (28.5 mg, 0.23 mmol, 1. Oeq)
  • succinic anhydride 76.9 mg, 0.777 mmol, 3.3 eq
  • the reaction mixture was separated with black mouth form.
  • the resulting organic layer was washed with saturated aqueous NaHCO solution,
  • the extract was washed with saturated brine and dried over anhydrous sodium sulfate. After the solvent was distilled off from the organic layer under reduced pressure, the obtained residue was vacuum-dried. DMF (4.2 ml) was added to the residue (0.13 g, 0.17 mmol, 4. Oeq) and dissolved, and CPG (250 mg, 222 ⁇ mol / g) was allowed to stand in the solution for 30 minutes. To the reaction mixture was added WSC (45 mg, 0.17 mmol, 4. Oeq) and shaken at room temperature for 6 days. The reaction mixture was filtered under reduced pressure, and the filtered product was washed with pyridine.
  • an oligonucleotide (DNA type) was produced by the phosphoramidite method using an automatic nucleic acid synthesizer.
  • the sequence indicated by A B is N 6 -benzoyl 9- (3— (4,4, -dimethoxytrityloxy) methyl-5-O— [(2-cyanethyl) prepared in Reference Example 1. (N, N-diisopropyl)] phosphioxymethylphenol)
  • Adene was introduced as a nucleoside monomer.
  • Other sequences were introduced using deoxyribose type nucleosides. 1 ⁇ mol CPG resin for solid phase synthesis was used, and each condensation time was 15 minutes.
  • the oligonucleotide of SEQ ID NO: 1 bound to the CPG resin was synthesized by an automatic nucleic acid synthesizer while protected with a benzoyl group, an isobutylyl group, and a cyanoethyl group.
  • the oligonucleotide bound to the CPG resin was reacted in an aqueous ammonia solution (2 mL) at 55 ° C for 12 hours.
  • the reaction mixture was filtered under reduced pressure, and the resulting filtrate was transferred to an Eppendorf tube, and then concentrated under reduced pressure.
  • the resulting concentrates were each dissolved in packing solution (1 XTBE solution in 90% formamide) (100 ⁇ L).
  • the solutions were separated using 20% PAGE (20A, 6 hours) (1 XTBE buffer was used as the running buffer), and the target oligonucleotide band was excised. To the cut out band, 0.1 M EDTA aqueous solution (20 mL) was added, and left to stand.
  • a nucleotide analogue was obtained.
  • a 0.1 M TEAA buffer was prepared as follows. First, 2N acetic acid (114. 38 mL) and triethylamine (277. 6 mL) were made up to 1 L with water. The solution was prepared by adding acetic acid to adjust the pH to 7.0, and then diluting the solution 20 times.
  • a 0.1 M aqueous EDTA solution was prepared by dissolving EDTA'4Na (l. 80 g) in water (40 mL).
  • 10 XTBE buffer was prepared by dissolving 1 U of Tris (109 g), boric acid (55 g) and EDTA '2Na (7.43 g) in water.
  • nucleotide sequence of SEQ ID NO: 2 instead of SEQ ID NO: 1, the sequence shown in U B, prepared in Example 2 1- (3- (4, 4 Mr. dimethoxytrityl O) methyl -5-0- ⁇ (2-Cyanoethyl) (N, N-diisopropyl) ⁇ Phosphooxymethylphenol) -uracil was introduced as a nucleoside monomer in the same manner as in Example 10 except that the Rigonucleotide analogs were produced.
  • oligonucleotide analog (SEQ ID NO: 1) prepared in Example 10 (1.2 nmol) and the single-stranded oligonucleotide analog (SEQ ID NO: 2) prepared in Example 11 (1.2nmol) mol) was dissolved in annealing buffer (10 mM sodium phosphate (pH 7.0) and 100 mM NaCl). Incubate the solution at 90 ° C for 1 minute, then at 37 ° C for 1 hour to obtain an oligonucleotide analog consisting of SEQ ID NO: 1 and an oligonucleotide analog consisting of SEQ ID NO: 2, as shown in Figure 1.
  • annealing buffer 10 mM sodium phosphate (pH 7.0) and 100 mM NaCl
  • the oligonucleotide analog consisting of SEQ ID NO: 3 (2.4 nmol) prepared in Example 13 was dissolved in annealing buffer (10 mM sodium phosphate (pH 7.0) and 1 M NaCl). Incubate the solution at 90 ° C for 1 minute and then at 37 ° C for 1 hour to obtain an oligonucleotide analog consisting of SEQ ID NO: 3 and an oligonucleotide analog consisting of SEQ ID NO: 3, as shown in Figure 2.
  • a double-stranded oligonucleotide analogue consisting of the body was obtained.
  • Example 8 According to the base sequence of SEQ ID NO: 4 (see FIG. 2) instead of SEQ ID NO: 1, except that the solid-phase synthesis unit compound ( ⁇ -3) produced in Example 8 was used as a starting material. A single-stranded oligonucleotide analog was prepared in the same manner as in Example 10.
  • the oligonucleotide analogue consisting of SEQ ID NO: 3 produced in Example 13
  • the oligonucleotide analogue comprising SEQ ID NO: 4 produced in Example 15 was used, and the sequence as shown in FIG.
  • a double-stranded oligonucleotide analog was obtained consisting of the oligonucleotide analog having the number 4 force and the oligonucleotide analog having the sequence number 4 force.
  • the oligonucleotide consisting of SEQ ID NO: 5 produced in Comparative Example 1 was used, and the oligonucleotide consisting of SEQ ID NO: 5 as shown in FIG. A double-stranded oligonucleotide consisting of a nucleotide and an oligonucleotide consisting of SEQ ID NO: 5 was obtained.
  • a single-stranded oligonucleotide was produced in the same manner as in Example 10 according to the base sequence of SEQ ID NO: 6 (see FIG. 2) instead of SEQ ID NO: 1.
  • the oligonucleotide analogue consisting of SEQ ID NO: 3 produced in Example 13 was used, as shown in FIG. A double-stranded oligonucleotide consisting of a nucleotide and an oligonucleotide consisting of SEQ ID NO: 6 was obtained.
  • the AG value was calculated as follows.
  • Tm is measured by dividing the oligonucleotide concentration into several steps. Based on the following formula (B), a graph of 1 / Tm vs. In Ct / 4 (Ct is the total oligonucleotide concentration) was drawn, and ⁇ ° and AS ° were calculated from the slope and intercept. And AG ° was calculated from the following equation (A).
  • oligonucleotide analogues replaced with nucleoside analogues have an increased Tm value, which improves the double-stranded stability and has an absolute AG of
  • the solid-phase synthesis unit compound ( ⁇ -1) prepared in Example 6 was used as a starting material, and other than the 3 'end
  • the sequence indicated by A B is N 6 -benzoyl 9- (3- (4, 4-dimethoxytrityloxy) methyl-5-O- [(2-cyanoethyl) prepared in Reference Example 1. )-(N, N-diisopropyl)]-phosphioxymethylphenol)
  • SEQ ID NO: 8 SEQ ID NO: 1 for solid phase synthesis Yunitti ⁇ prepared in accordance Example 8 on the nucleotide sequence of (see FIG. 3) ( ⁇ - 3) as a starting material, the sequence shown in U B 1- (3- (4,4 dimethoxytrityloxy) methyl-5-0- ⁇ (2-cyanethyl)-(N, N-diisopropyl) ⁇ phosphioxymethylphenol prepared in Example 2
  • a single-stranded oligonucleotide analog was produced in the same manner as in Example 10 except that it was introduced using uracil as a nucleoside monomer.
  • a single-stranded oligonucleotide was produced in the same manner as in Example 10 according to the base sequence of SEQ ID NO: 10 (see FIG. 3) instead of SEQ ID NO: 1.
  • oligonucleotide analogue consisting of SEQ ID NO: 8 produced in Example 18 and the oligonucleotide analogue (1.2 nmol) consisting of SEQ ID NO: 9 produced in Comparative Example 5 were mixed with a annealing buffer (10 mM). Dissolved in sodium phosphate (pH 7.0) and 1M NaCl. The solution was incubated at 90 ° C for 1 minute, then at 37 ° C for 1 hour, and the oligonucleotide analog consisting of SEQ ID NO: 8 and the oligonucleotide consisting of SEQ ID NO: 9 as shown in FIG. A double-stranded oligonucleotide analogue consisting of
  • oligonucleotide analog consisting of SEQ ID NO: 7 (1.2 nmol) prepared in Example 17 and an oligonucleotide analog consisting of SEQ ID NO: 8 (1.2 nmol) prepared in Example 18 were subjected to annealing buffer (10 mM). Dissolved in sodium phosphate (pH 7.0) and 1 M NaCl. The solution was incubated at 90 ° C for 1 minute and then at 37 ° C for 1 hour to obtain an oligonucleotide analogue consisting of SEQ ID NO: 7 and an oligonucleotide consisting of SEQ ID NO: 8, as shown in Figure 3. A double-stranded oligonucleotide analogue consisting of the nucleotide analogue was obtained.
  • the oligonucleotide consisting of SEQ ID NO: 9 (1.2 nmol) produced in Comparative Example 5 and the oligonucleotide consisting of SEQ ID NO: 10 (1.2 nmol) produced in Comparative Example 6 were annealed (10 mM sodium phosphate). Dissolved in salt (pH 7.0) and 1M NaCl). The solution is incubated at 90 ° C for 1 minute, then at 37 ° C for 1 hour, and consists of the oligonucleotide consisting of SEQ ID NO: 9 and the oligonucleotide consisting of SEQ ID NO: 10, as shown in Fig. 3. A double-stranded oligonucleotide was obtained.
  • the double-stranded oligonucleotide analog prepared in Example 20 and the comparative example 7 The measured Tm values for the double-stranded oligonucleotides are shown in Table 2 and FIG. 4 below.
  • oligonucleotide analogues that are all replaced with nucleoside analogues have increased Tm values, so that the ability to form double strands and the stability of double strands 1S It was confirmed that it was improved as compared with the natural oligonucleotide.
  • CD spectra were measured for the double-stranded oligonucleotide analogue produced in Example 19 and the double-stranded oligonucleotide produced in Comparative Example 7 (temperature 10 ° C.). The results obtained are shown in FIG.
  • a double-stranded oligonucleotide analog comprising a single-stranded oligonucleotide analog that has been completely replaced with a nucleoside analog and a natural single-stranded oligonucleotide, It was confirmed that the same helical structure as that of the natural double-stranded oligonucleotide was formed.
  • the exonuclease resistance of the single-stranded oligonucleotide analog consisting of SEQ ID NO: 7 obtained in Example 17 and the natural single-stranded oligonucleotide consisting of SEQ ID NO: 9 obtained in Comparative Example 5 was evaluated.
  • snake venom phosphorodiesterase SVP
  • SVP selectively cleaves phosphodiester bonds and cleaves oligonucleotides into 5-monophosphate nucleotides.
  • the results are shown in Table 3 below.
  • Buffer solution (0.1M Tris-HCl, 2 mM MgCl a (pH7.0)) 495 L
  • Buffer solution (0.1M Tris-HCL 2 mM MgCl 2 (pH7.0)) 495 L
  • RNA type an oligonucleotide (RNA type) was produced by the phosphoramidite method using an automatic nucleic acid synthesizer (PerSeptive Biosystems Model 8909).
  • the oligonucleotide bound to the CPG resin was reacted in an aqueous ammonia solution (2 mL) at 55 ° C for 12 hours.
  • the reaction mixture was filtered under reduced pressure, and the resulting filtrate was transferred to an Eppendorf tube, and then concentrated under reduced pressure.
  • the resulting concentrates were each dissolved in packing solution (1 XTBE solution in 90% formamide) (100 ⁇ L).
  • the solutions were separated using 20% PAGE (20A, 6 hours) (1 XTBE buffer was used as the running buffer), and the target oligonucleotide band was excised. To the cut out band, 0.1 M EDTA aqueous solution (20 mL) was added, and left to stand.
  • a nucleotide analogue was obtained.
  • a single-stranded oligonucleotide analog was produced in the same manner as in Example 21 except that the base sequence of SEQ ID NO: 12 was used instead of SEQ ID NO: 11.
  • oligonucleotide analogue consisting of SEQ ID NO: 11 produced in Example 21 and the oligonucleotide analogue (1.2 nmol) consisting of SEQ ID NO: 12 produced in Example 22 were subjected to annealing buffer (10 mM). Dissolved in sodium phosphate (pH 7.0) and 1M NaCl). The solution was incubated at 90 ° C for 1 minute, then at 37 ° C for 1 hour, and consisted of an oligonucleotide analog consisting of SEQ ID NO: 11 and SEQ ID NO: 12, as shown in Figure 6. A double-stranded oligonucleotide analogue consisting of the oligonucleotide analogue was obtained.
  • nucleoside analog of the present invention is useful, for example, as a nucleoside for producing an oligonucleotide for a DNA test kit.
  • FIG. 1 is a diagram showing examples of oligonucleotide analogues.
  • FIG. 2 is a diagram showing another example of an oligonucleotide analogue.
  • FIG. 3 is a diagram showing another example of an oligonucleotide analogue.
  • FIG. 4 is a graph showing Tm values of oligonucleotide analogues.
  • FIG. 5 is a graph showing CD spectra of oligonucleotide analogues.
  • FIG. 6 is a view showing another example of an oligonucleotide analogue.
  • SEQ ID NO: 2 oligonucleotide analogue
  • SEQ ID NO: 3 oligonucleotide analogue

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Abstract

Analogue de nucléoside lequel permet la production d'un analogue d'oligonucléotide excellent en ce qui concerne trois propriétés, c'est-à-dire en termes de résistance à des nucléases, d'aptitude à former un duplex et de stabilité du duplex formé ; et analogue d'oligonucléotide contenant l'analogue de nucléoside. L'analogue de nucléoside est un analogue de nucléoside représenté par la formule (I) suivante ou un sel de celui-ci. L'analogue d'oligonucléotide contient un ou plusieurs éléments sélectionnés dans le groupe constitué de l'analogue de nucléoside représenté par la formule (II-1) suivante, de l'analogue de nucléoside représenté par la formule (II-2) suivante, de l'analogue de nucléoside représenté par la formule (II-3) suivante, de l'analogue de nucléoside représenté par la formule (II-4) suivante et de l'analogue de nucléoside représenté par la formule (II-5) suivante. [Dans la formule (I), R1 est tel que défini dans la description.]
PCT/JP2005/003405 2004-09-07 2005-03-01 Analogue de nucléoside et analogue d'oligonucléotide contenant celui-ci WO2006027862A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007094135A1 (fr) * 2006-02-15 2007-08-23 Gifu University Derive d'oligonucleotide et son utilisation
JP2009136157A (ja) * 2007-12-03 2009-06-25 Gifu Univ オリゴヌクレオチド類似体またはその塩
WO2010032704A1 (fr) * 2008-09-22 2010-03-25 財団法人岐阜県研究開発財団 Préparation pharmaceutique comprenant un dérivé de micro-arn-143
WO2010055789A1 (fr) * 2008-11-14 2010-05-20 独立行政法人科学技術振興機構 Dérivé d'oligonucléotide, agent de marquage et utilisation de l'agent de marquage
CN102666480A (zh) * 2009-12-08 2012-09-12 国立大学法人岐阜大学 芳香族化合物、低聚核苷酸衍生物合成用修饰载体、低聚核苷酸衍生物及低聚核苷酸构建物

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WO2010055789A1 (fr) * 2008-11-14 2010-05-20 独立行政法人科学技術振興機構 Dérivé d'oligonucléotide, agent de marquage et utilisation de l'agent de marquage
JP4815015B2 (ja) * 2008-11-14 2011-11-16 独立行政法人科学技術振興機構 オリゴヌクレオチド誘導体、ラベル化剤及びその利用
JPWO2010055789A1 (ja) * 2008-11-14 2012-04-12 独立行政法人科学技術振興機構 オリゴヌクレオチド誘導体、ラベル化剤及びその利用
US8354515B2 (en) 2008-11-14 2013-01-15 Japan Science And Technology Agency Oligonucleotide derivative, labeling agent and use for labeling agent
CN102666480A (zh) * 2009-12-08 2012-09-12 国立大学法人岐阜大学 芳香族化合物、低聚核苷酸衍生物合成用修饰载体、低聚核苷酸衍生物及低聚核苷酸构建物
CN102666480B (zh) * 2009-12-08 2014-04-09 国立大学法人岐阜大学 芳香族化合物、低聚核苷酸衍生物合成用修饰载体、低聚核苷酸衍生物及低聚核苷酸构建物

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