WO2013175656A1 - Nouveau dérivé d'acide nucléique et conjugué dudit dérivé d'acide nucléique et d'un composé amine - Google Patents

Nouveau dérivé d'acide nucléique et conjugué dudit dérivé d'acide nucléique et d'un composé amine Download PDF

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WO2013175656A1
WO2013175656A1 PCT/JP2012/079458 JP2012079458W WO2013175656A1 WO 2013175656 A1 WO2013175656 A1 WO 2013175656A1 JP 2012079458 W JP2012079458 W JP 2012079458W WO 2013175656 A1 WO2013175656 A1 WO 2013175656A1
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compound
group
hydrogen atom
substituent
nucleic acid
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PCT/JP2012/079458
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Japanese (ja)
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真理子 麻生
洋 末宗
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国立大学法人九州大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/18—Acyclic radicals, substituted by carbocyclic rings

Definitions

  • the present invention relates to a nucleic acid derivative that forms a stable conjugate with an amine compound having a primary amino group.
  • the present invention also relates to an amine compound having a primary amino group, particularly a peptide or protein having a primary amino group, or a method of forming a conjugate with a nucleic acid using the nucleic acid derivative, and a conjugate thereof.
  • the conjugate targeted by the present invention means a nucleic acid derivative covalently bonded to an amine compound (covalently bonded nucleic acid derivative and amine compound).
  • nucleic acid medicines are expected as new medicines used for treating diseases by controlling the functions of genes and proteins related to the diseases.
  • a method of conjugating a nucleic acid molecule for example, an aptamer that controls the function of a gene or protein in the body with a molecule having functions such as cell transferability and nuclear transferability has been studied.
  • McGen registered trademark: Pfizer Inc.
  • Pfizer Inc. a therapeutic drug for age-related macular degeneration currently on the market, specifically binds to VEGF 165 , which is highly proinflammatory and deeply involved in pathological angiogenesis in the eye.
  • This is a conjugate of a nucleic acid molecule and PEG, which is obtained by binding polyethylene glycol (PEG) to a synthetic oligonucleotide (aptamer) that inhibits its function.
  • PEG has the effect of increasing the retention in the eyeball of nucleic acid molecules that are aptamers.
  • a conjugate in which a functional molecule having a tissue retention property or other cell migration property or nuclear migration property as described above is bound to a nucleic acid molecule having a function such as an aptamer or the like It is expected to exert a high targeting effect on target cells and tissues by imparting transferability, nuclear transferability, and the like.
  • conjugate formation consists of two steps, a step of introducing a reactive functional group such as thiol or amine into a nucleic acid molecule or a functional molecule, and a conjugate formation step utilizing functional group selective reaction.
  • a functional molecule amine compound
  • a functional molecule having an amino group such as a peptide or protein having a lysine residue is reacted with a nucleic acid molecule having a carbonyl group introduced under reducing conditions.
  • a method of forming a carbon-amine bond is a method of forming a carbon-amine bond.
  • Non-patent Document 1 when a nucleic acid molecule having an abasic site is used as the nucleic acid molecule having a carbonyl group, there is a problem that the abasic site is gradually decomposed and a stable conjugate cannot be obtained (Non-patent Document 1). .
  • the present invention provides a method for more stably producing a conjugate of a nucleic acid molecule and an amine compound (functional molecule) having a function such as tissue retention, cell migration or nuclear migration.
  • an object of the present invention is to provide a method for stably and efficiently producing a conjugate of a nucleic acid molecule and the functional molecule, and a method for producing a conjugate that can be performed in vivo.
  • an object of the present invention is to provide a novel nucleic acid derivative useful for the production of such a conjugate.
  • the inventors of the present invention have made extensive studies in order to solve the above-mentioned problems.
  • a nucleic acid molecule the compound represented by the following formula (I) is used, whereby a peptide, protein, nucleic acid, or the like having a primary amino group is used. It was found that a conjugate can be stably formed with the amine compound.
  • compounds (I) a compound in which two halogen atoms are directly bonded to the C2 ′ position, and a compound represented by the general formula (Ib) can be efficiently conjugated with the amine compound under non-reducing conditions. Since it can be formed, it was confirmed that it is useful as a material for forming a conjugate in vivo.
  • the present invention has been completed based on these findings and has the following embodiments.
  • R 1 is a halogen atom, an alkyl group which may have a substituent, or a hydrogen atom
  • R 2 represents a halogen atom, an alkyl group which may have a substituent, or a hydrogen atom
  • A is a nucleobase sequence-containing oligomer or polymer having a backbone formed from nucleotides or analogs thereof, or a hydrogen atom
  • B means a nucleobase sequence-containing oligomer or polymer having a backbone formed from nucleotides or analogs thereof, or a hydrogen atom.
  • R 1 and R 2 are mutually the same or different halogen atoms, or the same or different alkyl groups which may have a substituent (in this case) , R 1 and R 2 may be linked to each other to form a cycloalkyl group);
  • R 1 is an alkyl group or a hydrogen atom which may have a substituent
  • R 2 is a hydrogen atom or an alkyl group which may have a substituent.
  • a and B are not hydrogen atoms at the same time.
  • R 1 of the nucleic acid derivative (I) is preferably a halogen atom and an alkyl group which may have a substituent.
  • R 1 is preferably an alkyl group which may have a substituent
  • R 2 is preferably a hydrogen atom.
  • the nucleic acid derivative (I) is any compound selected from the group consisting of the following general formulas (Ia-1), (Ia) and (Ib), according to (I-1): Manufacturing method to: (1) Compound represented by general formula (Ia-1):
  • R 1a-1 and R 2a-1 are the same or different halogen atoms;
  • A is a nucleobase sequence-containing oligomer or polymer having a backbone formed from nucleotides or analogs thereof, or a hydrogen atom;
  • B means a nucleobase sequence-containing oligomer or polymer having a backbone formed from nucleotides or analogs thereof, or a hydrogen atom.
  • a and B are not hydrogen atoms at the same time);
  • R 1a and R 2a are mutually the same or different halogen atoms, or the same or different alkyl groups which may have a substituent.
  • R 1a and R 2a are alkyl groups which may have a substituent, they may be linked to each other to form a cycloalkyl group which may have a substituent.
  • A represents a nucleobase sequence-containing oligomer or polymer having a backbone formed from nucleotides or analogs thereof, or a hydrogen atom
  • B means a nucleobase sequence-containing oligomer or polymer having a backbone formed from nucleotides or analogs thereof, or a hydrogen atom.
  • a and B are not hydrogen atoms at the same time.
  • R 1b is an alkyl group which may have a substituent
  • R 2b is a hydrogen atom or an alkyl group which may have a substituent.
  • A represents a nucleobase sequence-containing oligomer or polymer having a backbone formed from nucleotides or analogs thereof, or a hydrogen atom
  • B means a nucleobase sequence-containing oligomer or polymer having a backbone formed from nucleotides or analogs thereof, or a hydrogen atom.
  • a and B are not hydrogen atoms at the same time. ).
  • R 2b of the nucleic acid derivative (Ib) is preferably a hydrogen atom.
  • nucleic acid derivative (I) is any compound selected from the group consisting of (1) to (3): (1) In the general formula (Ia-1), R 1a-1 and R 2a-1 are the same or different halogen atoms; A compound in which A and B are nucleobase sequence-containing oligomers or polymers having the same or different skeleton formed from nucleotides; (2) In the general formula (Ia), R 1a and R 2a are the same or different and have an alkyl group having a substituent; A compound in which A and B are nucleobase sequence-containing oligomers or polymers having the same or different skeleton formed from nucleotides; (3) In the general formula (Ib), R 2a is an alkyl group which may have a substituent, and R 2b is a hydrogen atom; A compound in which A and B are nucleobase sequence-containing oligomers or polymers having the same or different skeleton formed from nucleotides.
  • R 1a-1 and R 2a-1 are the same or different halogen atoms;
  • A is a nucleobase sequence-containing oligomer or polymer having a backbone formed from nucleotides or analogs thereof, or a hydrogen atom;
  • B means a nucleobase sequence-containing oligomer or polymer having a backbone formed from nucleotides or analogs thereof, or a hydrogen atom.
  • a and B are not hydrogen atoms at the same time).
  • a nucleic acid derivative (Ia-1) represented by the formula (Ib):
  • R 1b is an alkyl group which may have a substituent
  • R 2b is a hydrogen atom or an alkyl group which may have a substituent.
  • A represents a nucleobase sequence-containing oligomer or polymer having a backbone formed from nucleotides or analogs thereof, or a hydrogen atom
  • B means a nucleobase sequence-containing oligomer or polymer having a backbone formed from nucleotides or analogs thereof, or a hydrogen atom.
  • a and B are not hydrogen atoms at the same time.
  • nucleic acid derivative (Ia-1) is a compound shown in the following (1): (1) In the general formula (Ia-1), R 1a-1 and R 2a-1 are the same or different halogen atoms; A compound in which A and B are nucleobase sequence-containing oligomers or polymers having the same or different skeleton formed from nucleotides.
  • (I-6) The production method according to any one of (I-1) to (I-5), wherein the amine compound (II) is a peptide, protein, or nucleic acid having a primary amino group.
  • the amine compound (II) is any one selected from the group consisting of cell migration property, nuclear migration property, target affinity, retention property, nuclease resistance (resistance to degradation enzyme), and labeling property
  • X is a direct bond; R 1 and R 2 are the same or different halogen atoms, or the same or different, optionally substituted alkyl.
  • a group (R 1 and R 2 may be linked to each other to form a cycloalkyl group), (I-1), (I-6) or (I-7) Manufacturing method.
  • a and B are the same or different and are nucleobase sequence-containing oligomers or polymers having a skeleton formed from nucleotides or analogs thereof, as described in (I-8) Production method.
  • X is an oxygen atom
  • R 1 is an optionally substituted alkyl group
  • R 2 is a hydrogen atom (I-1), (I-6) or The production method according to any one of (I-7).
  • a and B are the same or different and are nucleobase sequence-containing oligomers or polymers having a skeleton formed from nucleotides or analogs thereof, as described in (I-10) Production method.
  • the oligomers or polymers represented by A and B are the same or different and are oligomers or polymers containing ribonucleotides or deoxyribonucleotides (I-1), (I-6) Or the production method according to any one of (I-7) to (I-11).
  • the alkyl group which may have a substituent represented by R 1 and R 2 is an alkyl group having 1 to 4 carbon atoms which may or may not have a substituent. (I-1) , (I-6) or (I-7) to (I-12).
  • the substituent of the alkyl group which may have a substituent represented by R 1 and R 2 is selected from a hydroxyl group, an alkoxy group, an amino group, an amide group (carbamoyl group), and an alkylcarbamoyl group
  • Z is a site excluding the primary amino group of amino compound (II);
  • X is a direct bond or an oxygen atom;
  • R 1 is a halogen atom, an alkyl group which may have a substituent, or a hydrogen atom;
  • R 2 represents a halogen atom, an alkyl group which may have a substituent, or a hydrogen atom;
  • A is a nucleobase sequence-containing oligomer or polymer having a backbone formed from nucleotides or analogs thereof, or a hydrogen atom;
  • B means a nucleobase sequence-containing oligomer or polymer having a backbone formed from nucleotides or analogs thereof, or a hydrogen atom.
  • R 1 and R 2 are mutually the same or different halogen atoms, or the same or different alkyl groups which may have a substituent (in this case) , R 1 and R 2 may be linked to each other to form a cycloalkyl group);
  • R 1 is an alkyl group or a hydrogen atom which may have a substituent
  • R 2 is a hydrogen atom or an alkyl group which may have a substituent.
  • a and B are not simultaneously hydrogen atoms.
  • Z is a site excluding the primary amino group of amino compound (II);
  • X is a direct bond or an oxygen atom;
  • R 1 is a halogen atom, an alkyl group which may have a substituent, or a hydrogen atom;
  • R 2 represents a halogen atom, an alkyl group which may have a substituent, or a hydrogen atom;
  • A is a nucleobase sequence-containing oligomer or polymer having a backbone formed from nucleotides or analogs thereof, or a hydrogen atom;
  • B means a nucleobase sequence-containing oligomer or polymer having a backbone formed from nucleotides or analogs thereof, or a hydrogen atom.
  • R 1 and R 2 are mutually the same or different halogen atoms, or the same or different alkyl groups which may have a substituent (in this case) , R 1 and R 2 may be linked to each other to form a cycloalkyl group);
  • R 1 is an alkyl group or a hydrogen atom which may have a substituent
  • R 2 is a hydrogen atom or an alkyl group which may have a substituent.
  • a and B are not simultaneously hydrogen atoms. ).
  • R 1a-1 or R 2a-1 are the same or different halogen atoms, and A and B are the same or different, and a nucleobase sequence having a skeleton formed from nucleotides or analogs thereof
  • the halogen atoms represented by R 1a-1 and R 2a-1 are the same or different and are fluorine atoms or chlorine atoms, and are described in (II-1) Nucleic acid derivatives.
  • the oligomers or polymers represented by A and B are the same or different and are oligomers or polymers containing ribonucleotides or deoxyribonucleotides (II-1) or (II -2) The nucleic acid derivative described in
  • the reducing agent In the presence, an efficient and stable conjugate can be formed with the amine compound (II).
  • the reaction proceeds in one step in an aqueous solution near neutrality. Since the reaction can be performed in an organic solvent in which a reducing agent can be used, the substrate can be used in a wide range and can be applied not only to DNA but also to conjugate formation with RNA that has been difficult to synthesize.
  • the compound represented by the formula (Ia) (wherein R 1a and R 2a are the same or different halogen atoms), or the compound represented by the formula (Ib) is an amine compound (II). It is possible to react efficiently not only under reducing conditions but also under non-reducing conditions to stably produce a conjugate of the nucleic acid derivative (Ia or Ib) and the amine compound (II).
  • a lysine residue originally present in the protein or peptide is used in the conjugate formation of the amine compound (II) and the nucleic acid derivative (I) or (Ia).
  • An amino residue having a primary amino group can be used.
  • Example 2 the electrophoresis image of the reaction liquid which reacted the complementary strand oligonucleotide (amine compound; compound 2) which has a difluoro body (nucleic acid derivative) and an alkylamino group under non-reducing conditions of pH 7 or 8 is shown. . Formation of two conjugates (compounds 6 and 7) was observed.
  • Example 2 the chromatogram which used the reaction liquid which reacted the complementary strand oligonucleotide (amine compound; compound 2) which has a difluoro body (nucleic acid derivative) and the alkylamino group under non-reducing conditions of pH8 for HPLC was shown. Shown (reaction times: 0h, 12h, 24h).
  • Example 3 a chromatogram obtained by subjecting a reaction solution obtained by reacting a difluoro compound (nucleic acid derivative) and a dansylsulfonamide derivative (amine compound; compound 8) having an alkylamino group to pH 8 under non-reducing conditions at pH 8 was subjected to HPLC. Shown (reaction times: 0h, 12h, 24h). As the reaction time elapses, the nucleic acid derivative and the amine compound decreased, and an increased amount of conjugate (compound 9) was observed.
  • the present invention relates to a method for producing a conjugate of nucleic acid derivative (I) and amine compound (II) represented by the following general formula.
  • R 1 and R 2 R 1 means a halogen atom, an alkyl group which may have a substituent, or a hydrogen atom.
  • a halogen atom or an alkyl group which may have a substituent is preferable.
  • R 2 represents a halogen atom, an alkyl group which may have a substituent, or a hydrogen atom.
  • R 1 and R 2 may be the same or different halogen atoms at the same time, or may be the same or different alkyl groups that may have a substituent at the same time. Incidentally, in the case of the latter, if the alkyl group of R 1 and R 2 has a substituent group, to the substituent of R 1 and R 2 may be the same or may be different.
  • R 1 and R 2 are alkyl groups that may have a substituent at the same time, they may be linked to each other to form a cycloalkyl group.
  • alkyl group which may have a substituent includes an alkyl group having a substituent and an alkyl group having no substituent.
  • alkyl group a linear or branched alkyl group usually having 1 to 4 carbon atoms, preferably 1 to 3 carbon atoms, more preferably 1 to 2 carbon atoms can be mentioned.
  • alkyl groups include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, s-butyl group, t-butyl group and the like.
  • they are a methyl group, an ethyl group, and a propyl group, More preferably, they are a methyl group and an ethyl group. Particularly preferred is a methyl group.
  • substituent of the alkyl group examples include, for example, a hydroxyl group, an alkoxy group, an amino group, an amide group (with the limit that the final compound (nucleic acid derivative (I), (Ia)) is acceptable as a compound, that is, can be produced as a compound) And a functional group such as an alkylcarbamoyl group.
  • substituents listed above examples include a group in which one or more hydrogen atoms of the alkyl group are substituted with at least one functional group selected from the group of substituents listed above.
  • examples of the “alkoxy group” include a hydroxyl group in which a hydrogen atom is substituted with an alkyl group having 1 to 4 carbon atoms, preferably 1 to 3 carbon atoms, more preferably 1 to 2 carbon atoms.
  • alkoxy groups include methoxy group, ethoxy group, 1-propoxy group, 2-propoxy group, 1-butoxy group, 2-butoxy group, 2-methyl-1-propoxy group, 2-methyl-2-propoxy group Etc. are included.
  • a methoxy group, an ethoxy group, a 1-propoxy group, and a 2-propoxy group are preferable, a methoxy group or an ethoxy group is more preferable, and a methoxy group is further preferable.
  • examples of the “alkyl group” in the “alkylcarbamoyl group” include a linear or branched alkyl group usually having 1 to 4 carbon atoms.
  • An alkyl group having 1 to 3 carbon atoms, more preferably 1 to 2 carbon atoms is preferable.
  • a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, and the like are included.
  • the “alkyl group having a hydroxyl group” specifically includes a hydroxymethyl group, a 1-hydroxyethyl group, a 2-hydroxyethyl group, a 1-hydroxypropyl group, and a 2-hydroxypropyl group. Examples thereof include a group and a 3-hydroxypropyl group. Preferred are a hydroxymethyl group, a 2-hydroxyethyl group, and a 3-hydroxypropyl group, more preferred are a 2-hydroxyethyl group and a 3-hydroxypropyl group, and particularly preferred is a 2-hydroxyethyl group.
  • examples of the “alkyl group having an alkoxy group” include a C1-4-alkoxy-C1-4-alkyl group. Specific examples include a methoxymethyl group, a 1-methoxyethyl group, 2 -Methoxyethyl group, 1-methoxypropyl group, 2-methoxypropyl group, 3-methoxypropyl group, ethoxymethyl group, 1-ethoxyethyl group, 2-ethoxyethyl group, 1-ethoxypropyl group, 2-ethoxypropyl group And 3-ethoxypropyl group.
  • alkyl group having an amino group is preferably an alkyl group having a primary amino group.
  • Specific examples include amino-C1-4-alkyl groups as described above, and specific examples include aminomethyl group, 1-aminoethyl group, 2-aminoethyl group, 1-aminopropyl group. , 2-aminopropyl group, 3-aminopropyl group and the like.
  • Preferred are an aminomethyl group, a 2-aminoethyl group, and a 3-aminopropyl group, more preferred are a 2-aminoethyl group and a 3-aminopropyl group, and particularly preferred is a 2-aminoethyl group.
  • alkyl group having an amide group examples include an amide-C1-4-alkyl group as described above, and specific examples include an amide methyl group and a 1-amidoethyl group. 2-amidoethyl group, 1-amidopropyl group, 2-amidopropyl group, 3-amidopropyl group and the like. Preferred are amidomethyl group, 2-amidoethyl group, and 3-amidopropyl group, more preferred are amidomethyl group and 2-amidoethyl group, and particularly preferred is amidomethyl group.
  • alkyl group having an alkylcarbamoyl group examples include a C1-4-alkylcarbamoyl-C1-4-alkyl group as described above, and specific examples thereof include a methylcarbamoylmethyl group and a 1-methylcarbamoyl group.
  • Ethyl group 2-methylcarbamoylethyl group, 1-methylcarbamoylpropyl group, 2-methylcarbamoylpropyl group, 3-methylcarbamoylpropyl group, ethylcarbamoylmethyl group, 1-ethylcarbamoylethyl group, 2-ethylcarbamoylethyl group, Examples include 1-ethylcarbamoylpropyl group, 2-ethylcarbamoylpropyl group, 3-ethylcarbamoylpropyl group, and the like.
  • methylcarbamoylmethyl group 2-methylcarbamoylethyl group, and 3-methylcarbamoylpropyl group
  • more preferred are methylcarbamoylmethyl group and 2-methylcarbamoylethyl group, and particularly preferred is methylcarbamoylmethyl group. is there.
  • halogen atom is preferably a fluorine atom and a chlorine atom, and more preferably a fluorine atom.
  • cycloalkyl group is preferably a cycloalkyl group having 1 to 6 carbon atoms such as cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexyl group and the like. More preferably, it is a cyclopropyl group.
  • R 1 and R 2 may be the same or different halogen atoms, or the same or different alkyl groups that may have a substituent. preferable. More preferably, they are the same or different halogen atoms.
  • a and B A and B are each a nucleobase sequence-containing oligomer or polymer having a backbone formed from nucleotides or analogs thereof, or a hydrogen atom. However, A and B do not become hydrogen atoms at the same time.
  • the nucleotide or its analog may be derived from nature or may be artificially synthesized.
  • the nucleotide includes ribonucleotide or deoxyribonucleotide.
  • Preferred oligomers or polymers are RNA consisting of ribonucleotides and DNA consisting of deoxyribonucleotides.
  • RNA is classified into mRNA, tRNA, rRNA, ncRNA, siRNA, dsRNA, shRNA, and the like based on the structure and its action, but is not particularly limited to these types.
  • DNA is also classified into cDNA, single-stranded DNA (including sense and anti-sense DNA strands), double-stranded DNA, etc., depending on its structure and action, but is not limited to these types. Absent.
  • LNA LockedleNucleic Acid
  • N- instead of sugar PNA (Peptide Nucleic Acid) in which 2-aminoethyl) glycine is bonded via an amide bond to the main chain, and a purine ring or pyrimidine ring corresponding to a nucleobase is bonded to the main chain via a methylene group and a carbonyl group
  • PNA Peptide Nucleic Acid
  • 2-aminoethyl Peptide Nucleic Acid
  • 2-aminoethyl 2-aminoethyl
  • a purine ring or pyrimidine ring corresponding to a nucleobase is bonded to the main chain via a methylene group and a carbonyl group
  • Other examples include ribonucleotides in which an optional substituent such as a hydroxyl group, an alkyl group, an amide group, or an alkylcarbamoyl group is bonded to the oxygen atom at the 2-position
  • the nucleobase sequence-containing oligomer or polymer targeted by the present invention may be a chimera or hybrid of the above nucleotides and analogs thereof.
  • the chimera include DNA / RNA chimera molecules, DNA / PNA chimera molecules, Examples thereof include DNA / LNA chimeric molecules and PNA / LNA chimeric molecules.
  • the hybrid include a double-stranded DNA / RNA hybrid molecule, a double-stranded DNA / PNA hybrid molecule, a double-stranded DNA / LNA hybrid molecule, and a double-stranded PNA / LNA hybrid molecule.
  • oligomer means a nucleic acid in which the above nucleotides are usually formed in a continuous sequence of about 2 to 10
  • polymer means a nucleic acid in which a larger number of nucleotides are formed in a continuous manner.
  • a and B may be the same type of oligomer or polymer as each other, or may be different types of oligomers or polymers from each other.
  • the bonding mode between A and B and the oxygen atom of the compound represented by formula (I) varies depending on the types of A and B, but when A and B are phosphate-containing nucleotides or analogs thereof, Usually, a phosphoric acid diester bond can be mentioned.
  • X means a direct bond or an oxygen atom.
  • nucleic acid derivative (I) targeted by the present invention is preferably a compound represented by the following formula (Ia).
  • a and B are each a nucleobase sequence-containing oligomer or polymer having a skeleton formed from nucleotides or analogs thereof, or a hydrogen atom. However, A and B do not become hydrogen atoms at the same time. Details thereof are as described above in the section of “R 1 and R 2” , and the above description can be incorporated herein as it is.
  • R 1a and R 2a are the same or different halogen atoms at the same time, or the same or different, optionally substituted alkyl groups.
  • R 1a and R 2a may be the same halogen atom or alkyl group (including both those having a substituent and those not having a substituent), but are not necessarily the same alkyl group (substituent). It is not necessary to be a halogen atom or both having and not having).
  • R 1a and R 2a are alkyl groups which may have a substituent at the same time, R 1a and R 2a are linked to each other to form an optionally substituted cycloalkyl group. It may be.
  • the alkyl group, the cycloalkyl group, the substituent, and the halogen atom are as described above in the section of “R 1 and R 2” , and the above description can be used as it is.
  • the alkyl group is preferably a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, and an isobutyl group; more preferably a methyl group, an ethyl group, a propyl group; still more preferably a methyl group and an ethyl group; It is a group.
  • the alkyl group may have no substituent, but when it has a substituent, preferably a hydroxyl group, an alkoxy group, an amino group, an amide group (carbamoyl group), and an alkylcarbamoyl group can be exemplified.
  • the halogen atom is preferably a fluorine atom and a chlorine atom; more preferably a fluorine atom.
  • the cycloalkyl group is preferably a cyclopropyl group.
  • the nucleic acid derivative (I) targeted by the present invention is preferably a compound represented by the following formula (Ib).
  • a and B are each a nucleobase sequence-containing oligomer or polymer having a skeleton formed from nucleotides or analogs thereof, or a hydrogen atom. However, A and B do not become hydrogen atoms at the same time. Details thereof are as described above in the section of “R 1 and R 2” , and the above description can be incorporated herein as it is.
  • R 1b is an alkyl group which may have a substituent
  • R 2b is a hydrogen atom or an alkyl group which may have a substituent.
  • R 1b is an alkyl group which may have a substituent
  • R 2b is a hydrogen atom or an alkyl group which may have a substituent.
  • it is a hydrogen atom.
  • the alkyl group and the substituent are as described above in the section of “R 1 and R 2” , and the above description can be incorporated herein as it is.
  • the alkyl group is preferably a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, and an isobutyl group; more preferably a methyl group, an ethyl group, a propyl group; still more preferably a methyl group and an ethyl group; It is a group.
  • the alkyl group may have no substituent, but when it has a substituent, preferably a hydroxyl group, an alkoxy group, an amino group, an amide group (carbamoyl group), and an alkylcarbamoyl group can be exemplified.
  • nucleic acid derivatives (I) A and B are deoxyribonucleotide oligomers, X is a direct bond, and R 1 and R 2 are fluorine atoms. Details of the method for producing the nucleic acid derivative (I) will be described later. This is described in Production Example 1 (see Formula A). Details of the method for producing nucleic acid derivative (I) (corresponding to nucleic acid derivative (Ia) at the same time) in which A and B are deoxyribonucleotide oligomers, X is a direct bond, and R 1 and R 2 are methyl groups It describes in the manufacture example 2 mentioned later (refer Formula B).
  • a and B are deoxyribonucleotide oligomers
  • X is an oxygen atom
  • R 1 is a methyl group
  • R 2 is a hydrogen atom
  • the nucleic acid derivative (I) (corresponding to the nucleic acid derivative (Ib) at the same time) Details of the production method will be described in Production Example 3 described later (see Formula C).
  • X is directly produced using 2,2-dichloro-2-deoxy-3,5-bis-O- [benzoyl] -D-erythro-pentose represented by the following formula.
  • the nucleic acid derivative (I) in which the bond, R 1 and R 2 are all chlorine atoms can be produced.
  • X is a direct bond
  • any of R 1 and R 2 A nucleic acid derivative (I) in which one is a fluorine atom and the other is a chlorine atom can be produced.
  • nucleic acid derivative (I) wherein X is a direct bond, and R 1 and R 2 are the same or different alkyl groups other than a methyl group. (Corresponding to the nucleic acid derivative (Ia) at the same time); or X is a direct bond, R 1 and R 2 are the same or different, a substituent (for example, hydroxyl group, alkoxy group, amino group, amide group (carbamoyl group) And at least one selected from the group consisting of alkylcarbamoyl groups) can be produced nucleic acid derivative (I) which is an alkyl group (corresponding to nucleic acid derivative (Ia) at the same time).
  • nucleic acid derivative (I) (corresponding to the nucleic acid derivative (Ib) at the same time); or X is an oxygen atom, R 1 is a substituent (for example, a hydroxyl group, an alkoxy group, an amino group, an amide group (carbamoyl group), and an alkylcarbamoyl group)
  • R 1 is a substituent (for example, a hydroxyl group, an alkoxy group, an amino group, an amide group (carbamoyl group), and an alkylcarbamoyl group)
  • a nucleic acid derivative (I) having an alkyl group having at least one substituent selected from the group consisting of a group and R 2 is a hydrogen atom (corresponding to the nucleic acid derivative (Ib) at the same time) can be produced.
  • R 1a is a production example (a) of a compound in which the alkyl group has a hydroxyl group
  • R 1a is a production example (b) of a compound in which the alkyl group has an alkoxy group
  • R 1a is A production example (c) of a compound which is an alkyl group having an alkylcarbamoyl group is shown below.
  • a 2′-O-alkoxyethyl compound (Compound B) can be obtained by alkylating the 2′-O-hydroxyethyl compound.
  • a 2′-O-alkamoylmethyl compound (compound C) can be obtained by reacting a 2′-O-alkoxycarbonylmethyl compound with ammonia, a primary amine, or a secondary amine. it can.
  • the amine compound (II) targeted by the present invention may be a compound having a primary amino group, preferably a peptide having a primary amino group, a protein having a primary amino group, a nucleic acid having a primary amino group, etc. be able to. More preferred are peptides having a primary amino group and nucleic acids having a primary amino group, and particularly preferred are nucleic acids having a primary amino group.
  • Preferred examples of the peptide having a primary amino group include a peptide having a lysine having a primary amine in the side chain as a constituent amino acid residue, and a peptide having an artificially introduced lysine or primary amino group.
  • a protein having a primary amino group is preferably a protein having a lysine having a primary amine in the side chain as a constituent amino acid residue, or a protein having an artificially introduced lysine or primary amino group.
  • the nucleic acid having a primary amino group is preferably a nucleic acid into which lysine or a primary amino group has been artificially introduced.
  • Such an amine compound (II) is preferably a functional molecule.
  • Such functions include, but are not limited to, cell transferability (cell permeability), nuclear transferability, target affinity, retention, nuclease resistance (resistance to degrading enzymes), labeling (function that can be a detectable label), etc. Can be mentioned.
  • Non-patent Document 2 Biological Activity of Oligonucleotide-Poly (L-lysine) Conjugates: Mechanism of Cell Uptake, Bioconjugate Chem., 1990, 1, 149-153).
  • a nuclear translocation peptide a peptide derived from SV 40T antigen, nuclear localization signal peptide (PKKKRKV (SEQ ID NO: 1); P is proline, K is lysine, R is arginine, and V is valine
  • Patent document 3 Gene delivery: A single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus, Proc. Natl.
  • Non-patent document 4 Controlled intracellular localization and enhanced antisense effect of oligonucleotides by chemical conjugation, Org. Biomol. Chem., 2005, 3, 3257-3259.”
  • the nuclear export signal (NES) sequence of HIV-1 rev protein and the nuclear localization signal (NLS) sequence of HIV-1 tat protein are described in “Non-patent Document 5: C Dingwell and RA Laskey, Annu. Rev. Cell.
  • Non-Patent Document 6 DS Goldfarb, J. Gariepy, G. Schoolnik, RD Kornberg, Nature, 1986, 322, 641-642.
  • Non-patent Document 7 DD Newmeyer, JM Lucocq, TR Burglin, EM ”. De Robertis, EMBO J., 1986, 5, 501-510.
  • Nucleic acid derivative (I) described above the nucleic acid derivative includes the nucleic acid derivatives (Ia) and (Ib) described above) II
  • Nucleic acid derivative (I) II the nucleic acid derivative includes the nucleic acid derivatives (Ia) and (Ib) described above) II
  • efficiently react under reducing conditions to stably produce a conjugate of the nucleic acid derivative (I) and the amine compound (II) (reductive amination of the nucleic acid derivative (I)).
  • Z—NH 2 means an amine compound (II) having a primary amino group (—NH 2 ).
  • X is a direct bond or an oxygen atom
  • R 1 is a halogen atom or an alkyl group which may have a substituent
  • R 2 may be a halogen atom or a substituent.
  • a and B mean the same or different nucleobase sequence-containing oligomer or polymer having a backbone formed from nucleotides or analogs thereof, or a hydrogen atom. However, A and B are not hydrogen atoms at the same time.
  • R 1 and R 2 are the same or different halogen atoms, or are alkyl groups that may have the same or different substituents (in this case, R 1 1 and R 2 may be linked to each other to form a cycloalkyl group); when X is an oxygen atom, R 1 is an optionally substituted alkyl group, R 2 is hydrogen Is an atom.
  • conjugate 1 a compound in which both A and B are nucleobase sequence-containing oligomers or polymers having a skeleton formed from nucleotides or analogs thereof
  • conjugate 2 A compound in which A is a hydrogen atom and B is a nucleobase sequence-containing oligomer or polymer having a backbone formed from nucleotides or analogs thereof
  • Conjugate 3 A compound in which A is a nucleotide or Included are compounds that are nucleobase sequence-containing oligomers or polymers having a backbone formed from the analog.
  • Conjugate 3 a compound in which both A and B are nucleobase sequence-containing oligomers or polymers having a skeleton formed from nucleotides or analogs thereof
  • the reaction between the nucleic acid derivative (I) and the amine compound (II) under reducing conditions is a method in which both compounds are dissolved in a solvent and reacted in the presence of a reducing agent under conditions of room temperature to 50 ° C. and pH of 5 to 8. Can be mentioned.
  • the temperature condition is preferably 30 to 50 ° C, more preferably 40 to 50 ° C.
  • the pH condition is preferably pH 6 to 8, more preferably pH 7 to 8.
  • the solvent for dissolving the nucleic acid derivative (I) and the amine compound (II) may be any solvent that can dissolve both compounds, but is preferably an aqueous solution. More preferably, it is an aqueous solution containing a buffer having a buffering ability in the above pH range, particularly preferably a physiologically acceptable aqueous solution, and specifically, a phosphate buffer solution of about 1 to 200 mM or about 10 to 50 mM is exemplified. (In the examples described below, a 10 mM phosphate buffer containing 100 mM sodium chloride is used).
  • Examples of the concentration of the nucleic acid derivative (I) in the reaction solution are usually 10 to 100 ⁇ M, preferably 20 to 100 ⁇ M, more preferably 50 to 100 ⁇ M.
  • Examples of the concentration of the amine compound (II) in the reaction solution are usually 10 ⁇ M to 10 mM, preferably 100 ⁇ M to 10 mM, more preferably 1 to 10 mM.
  • sodium cyanoborohydride can be usually used, but a reducing agent equivalent to the above, such as sodium borohydride or dimethylamine borane, may be used.
  • concentration of the reducing agent in the reaction system is usually 1 to 30 mM, preferably 2 to 20 mM, more preferably 5 to 15 mM.
  • the proportion of the nucleic acid derivative (I) and the amine compound (II) in the reaction system can be 1 to 100 parts by mole of the amine compound (II) with respect to 1 part by mole of the nucleic acid derivative (I).
  • the amount is preferably 10 to 100 parts by mole, more preferably 50 to 100 parts by mole, based on 1 part by mole of the nucleic acid derivative (I).
  • a conjugate of the nucleic acid derivative (I) and the amine compound (II) can be stably obtained in a high yield. be able to.
  • nucleic acid derivatives (I) described above the compounds represented by the following formulas (Ia) and (Ib) are not limited to the above-described amine compound (II), and are effective not only under reducing conditions but also under non-reducing conditions. By reacting, a conjugate of the nucleic acid derivative (Ia or Ib) and the amine compound (II) is stably produced (reaction under non-reducing conditions of the nucleic acid derivative).
  • the reaction conditions (temperature, pH, ratio of nucleic acid derivative and amine compound, time, etc.) under non-reducing conditions are the same as the reaction conditions under reducing conditions described above except that no reducing agent is used. be able to. Therefore, the reaction conditions described above can be directly incorporated herein. Also in the production method in this case, in the same manner as in the production method under the above-mentioned reducing conditions, not only one of conjugates 1 to 3, particularly only conjugate 1 is produced, but also the conjugate 1 and conjugate described above. The case where the gate 2 or 3 is simultaneously generated is also included.
  • the conjugate thus produced can be isolated and purified from the reaction solution using a conventional isolation and purification method such as an extraction method, a dialysis method, column chromatography, or electrophoresis.
  • nucleic acid derivatives (I) described above a nucleic acid derivative represented by the following general formula (Ia-1) is a novel compound.
  • a and B are as described above, and mean the same or different nucleobase sequence-containing oligomer or polymer having a skeleton formed from nucleotides or analogs thereof.
  • R 1a-1 and R 2a-1 are the same or different from each other and are simultaneously halogen atoms.
  • the halogen atom is as described above, preferably a fluorine atom and a chlorine atom, more preferably a fluorine atom.
  • nucleic acid derivative (Ia-1) in which R 1a-1 and R 2a-1 are chlorine atoms can be produced by producing according to the method described in Production Example 1.
  • R 1a-1 and R can be obtained by using 2-chloro-2-deoxy-2-fluoro-3,5-bis-O- [benzoyl] -D-erythro-pentose instead of compound 1-1.
  • a nucleic acid derivative (Ia-1) in which any one of 2a-1 is a fluorine atom and the other is a chlorine atom can be produced.
  • Such a compound (Ia-1) is a kind of the nucleic acid derivative (I) described above, and reacts with an amine compound (II) having a primary amino group under a reducing condition or a non-reducing condition to obtain a nucleic acid derivative (Ia-1). -1) and a conjugate of amine compound (II) can be stably produced.
  • the compound is a nucleobase having a skeleton formed from nucleotides, wherein X is a direct bond, R 1 and R 2 are the same halogen atom, and A and B are different from the compounds represented by the general formula (I) It corresponds to a compound that is a sequence-containing oligomer.
  • R 1a and R 2a are the same halogen atoms, and A and B are different nucleobase sequence-containing oligomers having different skeletons formed from nucleotides. Equivalent to.
  • R 1a-1 and R 2a-1 are the same halogen atoms, and A and B are different from each other, and each has a skeleton formed from nucleotides. It corresponds to the compound which is a contained oligomer.
  • Triethylamine (7.2 ml, 0.052 mol) was added dropwise, and the mixture was stirred for 3 hours while returning from 0 ° C. to room temperature.
  • the solvent was evaporated under reduced pressure, a saturated aqueous sodium hydrogen carbonate solution was added to the residue, and the mixture was extracted with ether. The organic layer was washed with saturated brine, dried, and the solvent was evaporated.
  • the compound is formed of a nucleotide represented by the general formula (I) wherein X is directly bonded; R 1 and R 2 are the same alkyl group having no substituent; and A and B are different from each other. It corresponds to a compound that is a nucleobase sequence-containing oligomer having a skeleton.
  • R 1a and R 2a have the same alkyl group having no substituent; A and B are different from each other, and have a nucleobase sequence having a skeleton formed from nucleotides It corresponds to a compound that is an oligomer.
  • R 1a-1 and R 2a-1 are the same alkyl groups having no substituent; A and B are different skeletons formed from nucleotides, respectively. It corresponds to a compound which is a nucleobase sequence-containing oligomer having
  • the 5-iodo compound (2.34 g, 5.72 mmol) was dissolved in dry DMF (50 ml), DBU (2.35 ml, 2.39 g, 15.73 mmol) was added, and the mixture was reacted at 60 ° C. for 3 hours.
  • Phosphonamidic chloride (0.11 ml, 0.48 mmol, 3equiv) and N-methylimidazole (0.01 ml, 0.013 mmol) were added, and the mixture was stirred at 0 ° C. for 30 minutes and at room temperature for 1 hour.
  • Isopropylethylamine (0.11 ml, 0.64 mmol) and N, N-diisopropylamino cyanoethyl phosphonamidic chloride (0.22 ml, 0.96 mmol) were added and reacted for 2 hours. After confirming disappearance of the raw materials, a cooled 5% aqueous sodium hydrogen carbonate solution was added, extracted with CH 2 Cl 2 and dried over Na 2 SO 4 .
  • the compound is formed of a nucleotide represented by the general formula (I), wherein X is an oxygen atom; R 1 is an alkyl group having no substituent; R 2 is a hydrogen atom; and A and B are different from each other. It corresponds to a compound which is a nucleobase sequence-containing oligomer having a skeleton.
  • Example 1 Conjugate Formation with Amine Compound (Part 1)
  • the amine compound a compound having a primary amino group having a complementary strand oligonucleotide corresponding to the oligonucleotide of the difluoro form, dimethyl form and methoxy form was used.
  • the following formula shows the methoxy compound of the present invention produced in Production Example 3 as a nucleic acid derivative (I) (compound 1 in the following formula) and a complementary-strand oligonucleotide having an alkylamino group as the amine compound (following formula
  • a method for producing a conjugate (compound 3) of a nucleic acid derivative (compound 1) and an amine compound (compound 2) by reacting with compound 2) is shown.
  • an oligodeoxynucleotide (5′-CC) of a methoxy compound (compound 1) corresponding to the substituent A of the nucleic acid derivative (I), and the nucleic acid derivative (I) Primary amino groups having complementary oligodeoxynucleotides (GG-3 ′ and CACCTATTGT-5 ′) corresponding to each of the oligodeoxynucleotides (GTGGATAACA-3 ′) of the methoxy compound (compound 1) corresponding to the substituent B of A nucleic acid having
  • each of the difluoro form, dimethyl form and methoxy form of the present invention and the amine compound (compound 2) represented by the above formula are dissolved in a 10 mM phosphate buffer (pH 7) containing 100 mM sodium chloride. Then, a solution containing each at a concentration of 10 ⁇ M was prepared. An excess amount (1000 equivalents) of sodium cyanoborohydride (NaCNBH 3 ) was added thereto and incubated at 37 ° C. According to the reaction analysis by HPLC, the reaction for 24 hours produced about 40% conjugate in the difluoro form, about 50% conjugate in the dimethyl form, and about 75% conjugate in the methoxy form. It was confirmed.
  • Example 2 Conjugate formation with amine compound (Part 2)
  • the difluoro compound of the present invention produced in Production Example 1 was reacted with an amine compound under non-reducing conditions to produce a conjugate.
  • As the amine compound a complementary strand oligonucleotide (compound 2) having an alkylamino group was used as in Example 1.
  • the difluoro compound of the present invention and the amine compound (compound 2) are dissolved in a 50 mM phosphate buffer solution (pH 7 or pH 8) containing 100 mM sodium chloride, and the difluoro compound of the present invention is dissolved.
  • a solution containing 20 ⁇ M and an amine compound at a concentration of 10 ⁇ M was prepared. This solution was incubated at 37 ° C. for 24 hours.
  • the results obtained by subjecting the obtained reaction solution to HPLC and electrophoresis are shown in FIGS. 1 and 2, respectively.
  • the HPLC conditions are as follows.
  • a double-stranded oligodeoxynucleotide composed of a difluoro derivative (nucleic acid derivative) and compound 2 (amine compound), and a conjugate (compound 6) are each added in 2.5 ⁇ m of 10 ⁇ m phosphate buffer containing 100 ⁇ mM sodium chloride, pH 7 Adjusted to ⁇ M.
  • the change in absorbance at 260 nm was measured when the temperature was changed from 20 ° C. to 90 ° C. at a rate of 0.5 ° C. per minute.
  • FIG. 5 shows a temperature-dependent UV change (arrow a) of a double-stranded oligodeoxynucleotide composed of a difluoro derivative (nucleic acid derivative) and compound 2 (amine compound), and a temperature-dependent UV change (conjugate 6).
  • Arrow b) is shown.
  • double-stranded oligodeoxynucleotides consisting of a nucleic acid derivative and an amine compound (compound 2) dissociate 50% of the double strand at 42 ° C.
  • compound 6 in the double-stranded structure of the conjugate
  • Example 3 Conjugate Formation with Amine Compound (Part 3)
  • the difluoro compound of the present invention produced in Production Example 1 was reacted with an amine compound under non-reducing conditions to produce a conjugate.
  • an amine compound As the amine compound, a dansylamide derivative (compound 8) having an alkylamino group represented by the following formula was used.
  • the difluoro compound of the present invention and the above amine compound (compound 8) are dissolved in a 50 ⁇ mM phosphate buffer solution (pH 8) containing 100 ⁇ mM sodium chloride, and the difluoro compound of the present invention is dissolved in 20 ⁇ M amine.
  • a solution containing the compound at a concentration of 2 mM was prepared. This solution was incubated at 37 ° C. for 24 hours.
  • nucleic acid conjugates can be widely applied both as pharmaceuticals and as molecular biological research tools, and there is a need for synthetic techniques that serve as a basis for their supply. This technology is valuable at the laboratory level and at the company level as a technology for producing nucleic acid conjugates that can be used as the tool.
  • SEQ ID NO: 1 is the amino acid sequence of the nuclear localization signal peptide
  • SEQ ID NO: 2 is the base sequence of the compound (1-11) described in Formula A of Preparation Example 1
  • SEQ ID NO: 3 is described in Formula A of Preparation Example 1.
  • the base sequence of the difluoro compound of the present invention SEQ ID NO: 4 is the base sequence of the compound (2-12) described in Formula B of Preparation Example 2
  • SEQ ID NO: 5 is the dimethyl compound of the present invention described in Formula B of Preparation Example 2
  • SEQ ID NO: 6 is the base sequence of the compound (3-9) described in Formula C of Preparation Example 3
  • SEQ ID NO: 7 is the base sequence of the methoxy compound of the present invention described in Formula C of Preparation Example 3, Each is shown.

Abstract

Procédé de production d'un conjugué d'un dérivé d'acide nucléique (I) et d'un composé amine (II), comportant une étape de mise en réaction du dérivé d'acide nucléique (I) représenté par la formule générale (I) (dans la formule, R1, R2, X, A, et B sont tels que mentionnés dans la Revendication 1) et du composé amine (II) en présence d'un agent réducteur.
PCT/JP2012/079458 2012-05-25 2012-11-14 Nouveau dérivé d'acide nucléique et conjugué dudit dérivé d'acide nucléique et d'un composé amine WO2013175656A1 (fr)

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Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
SATOSHI INOUE ET AL.: "2-i ni Chikanki o Motsu C4'-i Sankagata Datsu Enki Bui Yudotai no Gosei to Amine Bunshi tono Hannosei Kenkyu", ABSTRACTS OF ANNUAL MEETING OF PHARMACEUTICAL SOCIETY OF JAPAN, vol. 130, no. 2, 2010, pages 159 *
SATOSHI INOUE ET AL.: "C4'-i Sankagata RNA Oyobi DNA no Amine Bunshi tono Hannosei Kenkyu", ABSTRACTS OF ANNUAL MEETING OF PHARMACEUTICAL SOCIETY OF JAPAN, vol. 129, 2009, pages 193 *
SATOSHI INOUE ET AL.: "Caged Zenkutai o Mochiite Seisei shita C4' Sankagata DNA Oyobi RNA no Hannosei", THE PHARMACEUTICAL SOCIETY OF JAPAN KYUSHU SHIBU TAIKAI KOEN YOSHISHU, vol. 25, 2008, pages 68 *
SATOSHI INOUE ET AL.: "Hikari Hanno o Mochiita 2-i Chikan C4' Sankagata DNA Yudotai no Seisei to Hannosei Kenkyu", ABSTRACTS SYMPOSIUM ON PROGRESS IN ORGANIC REACTIONS AND SYNTHESES, vol. 36, 2010, pages 312 - 313 *
SHOKO JINNOUCHI ET AL.: "2'-i Chikan C4' Sankagata DNA Yudotai no Gosei to Amine Hannosei Kenkyu", THE PHARMACEUTICAL SOCIETY OF JAPAN KYUSHU SHIBU TAIKAI KOEN YOSHISHU, vol. 28, 2011, pages 157 *
TOMI ERIKO ET AL.: "Photochemical generation and reaction of 2'-substituted analogs of C4'-oxidized abasic lesion", NUCLEIC ACIDS SYMPOSIUM SERIES, vol. 53, no. 1, 2009, pages 185 - 186 *
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