WO2012091091A1 - 化合物、核酸、核酸の製造方法および核酸を製造するためのキット - Google Patents
化合物、核酸、核酸の製造方法および核酸を製造するためのキット Download PDFInfo
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- WO2012091091A1 WO2012091091A1 PCT/JP2011/080386 JP2011080386W WO2012091091A1 WO 2012091091 A1 WO2012091091 A1 WO 2012091091A1 JP 2011080386 W JP2011080386 W JP 2011080386W WO 2012091091 A1 WO2012091091 A1 WO 2012091091A1
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- 0 CC(c1c(C)c(*)c(*)c(*)c1*=O)=C Chemical compound CC(c1c(C)c(*)c(*)c(*)c1*=O)=C 0.000 description 2
- CLVUZDJCRWPURC-YRWVKINDSA-N CC(C1/C=C2\Sc(cccc3)c3N2CCCCC(NCCN(CCN)CCNC(CCCCN2c3ccccc3S/C2=C\c2c(cccc3)c3ncc2)=O)=O)C=Nc2c1cccc2 Chemical compound CC(C1/C=C2\Sc(cccc3)c3N2CCCCC(NCCN(CCN)CCNC(CCCCN2c3ccccc3S/C2=C\c2c(cccc3)c3ncc2)=O)=O)C=Nc2c1cccc2 CLVUZDJCRWPURC-YRWVKINDSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
- C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
- C07H19/073—Pyrimidine radicals with 2-deoxyribosyl as the saccharide radical
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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
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
- C09B23/04—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
- C09B23/06—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups three >CH- groups, e.g. carbocyanines
Definitions
- the present invention relates to a compound and a nucleic acid having a dye exhibiting an exciton effect, a method for producing a nucleic acid using this compound, and a kit for producing a nucleic acid.
- Patent Document 1 describes a probe containing a compound having a structure derived from a mononucleoside or a mononucleotide. For example, two fluorescent molecules are bound to this compound. This probe is strained when not bound to DNA, etc., and the fluorescence is extremely weak due to quenching caused by exciton coupling. However, when the probe is hybridized with DNA or RNA, the structure strain is eliminated. Immobilized and exhibits strong fluorescence.
- Patent Document 1 As a method for producing a nucleic acid probe, as described in Patent Document 1, a method of synthesizing by a phosphoramidite method using a nucleoside phosphoramidite body is known.
- Patent Document 1 as a method for producing a nucleic acid probe for detecting a nucleic acid using the exciton effect, first, a nucleoside phosphoramidite body containing no fluorescent molecule is synthesized, and a DNA oligomer is synthesized using this nucleoside phosphoramidite body. A method of introducing a fluorescent molecule after creation is described.
- this method requires a two-step synthesis process, that is, a DNA oligomer synthesis process and a synthesis process for introducing fluorescent molecules, and further requires a purification process for each synthesis process. Further, when the introduction efficiency of the fluorescent molecule is not 100%, an incomplete nucleic acid probe in which only one fluorescent molecule is introduced may be created.
- the fluorescent molecule is introduced after the DNA oligomer is synthesized, it is difficult to introduce different types of dyes (fluorescent molecules) at different positions of the DNA oligomer.
- the present invention has been made in view of the above-described problems of the prior art, and an object thereof is to provide a compound capable of easily producing a nucleic acid having a dye exhibiting an exciton effect, as well as a nucleic acid and a nucleic acid. And a kit for producing a nucleic acid.
- the present inventors paid attention to a method of synthesizing oligonucleic acid using a phosphoramidite compound having a dye in order to facilitate the production of a nucleic acid having a dye exhibiting an exciton effect.
- a molecule having a charge (positive charge) considered to be related to the exertion of the exciton effect is generally used as a dye exhibiting the exciton effect.
- the present inventors tried to synthesize nucleic acids using molecules having such conventional dyes, but there were cases where the synthesis efficiency was not sufficient.
- the present inventors have tried to synthesize nucleic acids using a phosphoramidite compound having a dye using a dye having no charge, and found that the synthesis can be performed with sufficient synthesis efficiency. That is, the present inventors have found that this phosphoramidite compound can be used for nucleic acid synthesis as a nucleoside substrate. Moreover, it discovered that the nucleic acid manufactured using this phosphoramidite compound can exhibit an exciton effect.
- the compound according to the present invention is represented by the following formula (1), (2) or (3).
- G is a phosphoramidite group or a hydroxyl group represented by the following formula (33),
- B is an atomic group having a base skeleton
- E is a deoxyribose skeleton or an atomic group having a ribose skeleton or a derivative thereof
- Z 11 and Z 12 are atomic groups having fluorescence independently and exhibiting an exciton effect, and are atomic groups having no charge, and Z 11 and Z 12 are the same.
- X is a hydrogen atom, a protective group for a hydroxyl group that can be deprotected by an acid, a phosphate group, a diphosphate group, or a triphosphate group;
- R 41 is a protecting group for a phosphate group;
- R 42 and R 43 are each independently an alkyl group or an aryl group,
- R 1 and R 2 are each independently a linker having a main chain, and atoms constituting the main chain may have a substituent,
- R 3 may be a linker having a main chain or may not be present, and an atom constituting the main chain may have a substituent,
- D may be CR 11 , N, P, P ⁇ O, B (boron atom) or SiR 11 , or may not be present, and
- R 11 may be a hydrogen atom, an alkyl group or any substitution It can be a group, If R 3 is not present and D is present, D may be directly bonded to B; if R 3
- the compound according to the present invention is not particularly limited to the more specific examples shown below, but in the above formulas (1) and (3), the atomic group represented by the following formula (1a) is represented by the following formula: (4) or (5) is an atomic group, and in the above formula (2), the atomic group represented by the following formula (2a) is an atom represented by the following formula (6) or (7) A group is preferred.
- A is a hydrogen atom, a hydroxyl group, an alkyl group, or an electron withdrawing group
- M and J are each independently CH 2 , NH, O or S.
- the main chain of R 1 , R 2 and R 3 is independently composed of two or more atoms, although not particularly limited to the more specific examples shown below. Preferably it is.
- the compound according to the present invention is not particularly limited to the more specific examples shown below, but is preferably represented by the following formula (8), (9), (10) or (11).
- l, m and n are each independently a positive integer; R 4 may be a single bond, a double bond or a triple bond, or may not be present.
- the compounds according to the present invention in particular, but not limited to the specific examples from below, l, m and n are each 2, R 4 is preferably a double bond.
- Z 11 and Z 12 are each independently represented by the following formula (12), (28) or (29). It is preferable that it is an atomic group.
- X 1 and X 2 are each independently O, S, Se or Te; n is 0 or a positive integer; R 21 to R 30 , R 32 to R 34 , and R 51 to R 60 are each independently a hydrogen atom, a halogen, a lower alkyl group, a lower alkoxy group, a nitro group, or an amino group, and R 28 and R 29 may be bonded to each other, and R 58 and R 59, may be bonded to each other, R 31 is a bond that binds to R 1 or R 2 in the above formula (1), (2), or (3), or NH in the above formula (8), (9), (10), or (11). Group, When n is an integer of 2 or more, R 33 may be the same or different, and R 34 may be the same or different. ).
- R 31 is a polymethylene having 2 or more carbon atoms.
- R 41 is a cyanoethyl group
- R 42 and R 43 are each an isopropyl group.
- the method for producing a nucleic acid according to the present invention comprises a condensation reaction of an oligonucleic acid with any of the compounds described above, wherein G is a phosphoramidite group represented by the above formula (33).
- the nucleic acid according to the present invention is characterized by including an atomic group represented by the following formula (25), (26) or (27) as a nucleotide moiety.
- B is an atomic group having a base skeleton
- E is a deoxyribose skeleton or an atomic group having a ribose skeleton or a derivative thereof
- Z 11 and Z 12 are atomic groups having fluorescence independently and exhibiting an exciton effect, and are atomic groups having no charge, and Z 11 and Z 12 are the same.
- R 1 and R 2 are each independently a linker having a main chain, and atoms constituting the main chain may have a substituent
- R 3 may be a linker having a main chain or may not be present
- an atom constituting the main chain may have a substituent
- D may be CR 11 , N, P, P ⁇ O, B (boron atom) or SiR 11 , or may not be present
- R 11 may be a hydrogen atom, an alkyl group or any substitution It can be a group, If R 3 is not present and D is present, D may be directly bonded to B; if R 3 is present and D is not present , R 1 and R 2 may be directly bonded to R 3, if R 3 and D is not present, R 1 and R 2 may be bonded directly to B, T is phosphoric acid crosslinking, and one or more oxygen atoms may be substituted with sulfur atoms during the phosphoric acid crosslinking. ).
- nucleic acid according to the present invention is not particularly limited, but it is preferably produced by the above-described method for producing nucleic acid.
- the kit for producing the nucleic acid according to the present invention includes any one of the compounds described above.
- a nucleic acid probe having a dye exhibiting an exciton effect can be easily produced.
- phosphoramidite compounds and “compounds” include tautomers and stereoisomers thereof, and salts thereof.
- the compound according to the present invention is a phosphoramidite compound (hereinafter also referred to as “dye-containing phosphoramidite compound”) containing a dye exhibiting an exciton effect (fluorescent dye) or a synthetic intermediate thereof.
- the compound according to the present invention is represented by the following formula (1), (2) or (3).
- B is an atomic group having a base skeleton.
- the atomic group having a base skeleton include an atomic group having a natural nucleobase (adenine, guanine, cytosine, thymine, or uracil) skeleton or an artificial nucleobase skeleton.
- B is preferably a pyrimidine base having a skeleton represented by the following formula (14), a purine base having a skeleton represented by the following formula (15), or a derivative thereof.
- the N atom at the 1-position has a covalent bond bonded to E in the above formulas (1) to (3), and any one of the atoms other than the N atom at the 1-position One may be linked (covalently bonded) to a linker (ie, R 1 , R 2 , R 3 or D) that leads to the dye (Z 11 or Z 12 ).
- a linker ie, R 1 , R 2 , R 3 or D
- At least one carbon atom among the atoms constituting the 6-membered ring may be substituted with an N, S or O atom, and at least one nitrogen atom is a C, S or O atom.
- the N, C, S or O atom may have a charge, a hydrogen atom or a substituent.
- the N atom at the 9th position has a covalent bond bonded to E in the above formulas (1) to (3), and is any atom other than the 9th N atom.
- One may be linked (covalently bonded) to a linker (ie, R 1 , R 2 , R 3 or D) that leads to the dye (Z 11 or Z 12 ).
- a linker ie, R 1 , R 2 , R 3 or D
- At least one of the atoms constituting the 5-membered ring and the 6-membered ring may be substituted with an N, S or O atom.
- at least one nitrogen atom may be substituted with a C, S or O atom, and the N, C, S or O atom may have a charge, a hydrogen atom or a substituent.
- E is a deoxyribose skeleton or an atomic group having a ribose skeleton or a derivative thereof.
- E is preferably an atomic group having a main chain structure of DNA, modified DNA, RNA, or modified RNA, because synthesis is easy.
- the atomic group represented by the following formula (1a) is preferably an atomic group represented by the following formula (4) or (5).
- A is a hydrogen atom, a hydroxyl group, an alkyl group, an alkoxy group or an electron withdrawing group.
- a methoxy group etc. are mentioned as an alkyl group or an alkoxy group.
- Examples of the electron withdrawing group include halogen.
- M and J are each independently CH 2 , NH, O or S.
- the atomic group represented by the following formula (2a) is preferably an atomic group represented by the following formula (6) or (7).
- A represents a hydrogen atom, a hydroxyl group, an alkyl group, an alkoxy group or an electron withdrawing group.
- a methoxy group etc. are mentioned as an alkyl group or an alkoxy group.
- Examples of the electron withdrawing group include halogen.
- M and J are each independently CH 2 , NH, O or S.
- Z 11 and Z 12 are each independently an atomic group having fluorescence (hereinafter also referred to as “dye”) and exhibiting an exciton effect.
- Z 11 and Z 12 are atomic groups having no charge.
- Z 11 and Z 12 may be the same or different.
- exciton effect (exciton coupling)” (also referred to as exciton coupling effect) means that Z 11 and Z 12 are assembled in parallel to each other, or Z 11 or Z 12 and other atomic groups Are gathered in parallel to form an H-aggregate. “Showing an exciton effect” means that fluorescence is emitted when no exciton effect occurs, and quenching occurs when an exciton effect occurs.
- the exciton effect is such that the excited state of the atomic group represented by Z 11 and Z 12 is split into two energy levels by forming a D-association by forming an H-aggregate, and after being excited to an upper energy level, the lower energy level It is considered that this is caused by the internal conversion of the light and the light emission is thermally inhibited.
- these descriptions do not limit the present invention.
- “having no charge” means having no charge (positive charge or negative charge) in at least an aprotic solvent.
- the dipole moment in the atomic group represented by Z 11 and Z 12 is small.
- the atomic groups represented by Z 11 and Z 12 may be those having no charge in the aprotic solvent, and may be polarized and charged in the protic solvent.
- the aprotic solvent refers to, for example, acetonitrile, N, N-dimethylformamide, and the like
- the protic solvent refers to water or the like.
- Z 11 and Z 12 may be, for example, each independently an atomic group represented by the following formula (12).
- X 1 is O, S, Se or Te, and preferably O or S.
- n is 0 or a positive integer. This n is preferably 5 or less, and in one example, is an integer of 0 to 2, preferably 0 or 1.
- R 21 to R 30 and R 32 to R 34 are each independently a hydrogen atom, a halogen, a lower alkyl group, a lower alkoxy group, a nitro group or an amino group.
- the lower alkyl group may be, for example, a linear or branched alkyl group having 1 to 6 carbon atoms.
- the lower alkoxy group may be, for example, a linear or branched alkoxy group having 1 to 6 carbon atoms.
- R 28 and R 29, may be bonded to each other. That is, R 28 and R 29 may be bonded to each other to form a cyclic structure, and examples of the cyclic structure include an aryl group. Moreover, this cyclic structure or aryl group may have a substituent.
- R 31 is R 1 or R 2 in the above formula (1), (2) or (3), or the following formula (8), (9), (10) or (11) It is a linking group that binds to NH.
- R 31 preferably has a main chain having 2 or more atoms.
- the upper limit of the number of atoms constituting the main chain is not particularly limited, but is preferably 100 or less, more preferably 50 or less, still more preferably 30 or less, and particularly preferably 10 or less.
- R 31 is preferably a polymethylene carbonyl group having 2 or more carbon atoms.
- n is an integer of 2 or more
- the plurality of R 33 may be the same or different
- the plurality of R 34 may be the same or different. It may be.
- Z 11 and Z 12 include dyes represented by the following formulas (17) to (20).
- R 31 may be the same as those exemplified in the above formula (12).
- Z 11 and Z 12 may be, for example, each independently an atomic group represented by the following formula (28) or (29).
- R 21 to R 34 , X 1 and n may be the same as those exemplified in the above formula (12).
- X 2 is O, S, Se or Te, and preferably O or S.
- X 1 and X 2 may be the same or different.
- R 51 to R 60 are each independently a hydrogen atom, a halogen, a lower alkyl group, a lower alkoxy group, a nitro group or an amino group.
- the lower alkyl group may be, for example, a linear or branched alkyl group having 1 to 6 carbon atoms.
- the lower alkoxy group may be, for example, a linear or branched alkoxy group having 1 to 6 carbon atoms.
- R 58 and R 59 may be bonded to each other. That is, R 58 and R 59 may be bonded to each other to form a cyclic structure, and examples of the cyclic structure include an aryl group. Moreover, this cyclic structure or aryl group may have a substituent.
- Z 11 and Z 12 may each independently be an atomic group represented by any one of the above formulas (12), (28), or (29).
- X is a hydrogen atom, a hydroxyl protecting group which can be deprotected by an acid, a phosphate group, a diphosphate group, or a triphosphate group.
- a hydroxyl-protecting group that can be deprotected by an acid include a dimethoxytrityl group (DMTr group).
- G represents a phosphoramidite group or a hydroxyl group represented by the following formula (33).
- R 41 is a protecting group of a phosphate group, for example, cyanoalkyl group. Of these, a cyanoethyl group is preferable.
- R 42 and R 43 are each independently an alkyl group or an aryl group.
- the alkyl group include an alkyl group having 1 to 6 carbon atoms, and an isopropyl group is particularly preferable.
- the aryl group include an aryl group having 6 to 10 carbon atoms, and among them, a phenyl group is preferable.
- R 1 and R 2 are each independently a linker having a main chain.
- the atoms constituting the main chain of R 1 and R 2 may have a substituent.
- R 3 may be a linker having a main chain, but may not be present. When R 3 has a main chain, the atoms constituting the main chain may have a substituent.
- the number of atoms constituting the main chain of R 1 and R 2 , and the main chain of R 3 when R 3 has a main chain is independently a positive integer. is there.
- the number of main chain atoms of R 1 , R 2 and R 3 is preferably independently an integer of 2 or more.
- the upper limit of the number of main chain atoms is not particularly limited, but is preferably 100 or less, more preferably 30 or less, and further preferably 10 or less.
- Backbone having R 1, R 2 and R 3 are, C, N, O, S, may comprise at least one of P and Si, also a single bond, double bond, triple bond, an amide bond , At least one of an ester bond, a disulfide bond, an imino group, an ether bond, a thioether bond and a thioester bond.
- R 1 , R 2 and R 3 may be the same as or different from each other.
- D may be CR 11 , N, P, P ⁇ O, B (boron atom) or SiR 11 , and R 11 is a hydrogen atom, It may be an alkyl group or an arbitrary substituent. D may not be present.
- D when R 3 is not present and D is present, D may be directly bonded to B. Further, when R 3 is present and D is not present, R 1 and R 2 may be directly bonded to R 3 . Further, when R 3 and D are not present, R 1 and R 2 may be directly bonded to B.
- T is phosphoric acid crosslinking.
- one or more oxygen atoms may be substituted with sulfur atoms.
- the compound according to the present invention is preferably a compound represented by the following formula (8), (9), (10) or (11).
- l, m and n are each independently a positive integer. It is preferable that l, m and n are each independently an integer of 2 or more. Moreover, although the upper limit of l, m, and n is not specifically limited, Preferably it is 100 or less, More preferably, it is 30 or less, More preferably, it is 10 or less. Here, the upper limit of l, m and n may be 5 or less or 4 or less.
- R 4 may be a single bond, a double bond or a triple bond, or may not exist. Of these, a double bond is preferable.
- the compound according to the present invention is preferably, for example, a compound represented by the following formula (16).
- X, G, Z 11 and Z 12 may be the same as those exemplified in the above formulas (1) to (3).
- A the same as those exemplified in the above formulas (4) to (7) can be used.
- the compound according to the present invention in which G is a hydroxyl group, can be used as a synthetic intermediate for synthesizing a phosphoramidite body.
- the compound according to the present invention wherein G is a phosphoramidite group represented by the above formula (33) (hereinafter also referred to as “phosphoramidite compound according to the present invention”), is a phosphoramidite method.
- the nucleic acid synthesized using the phosphoramidite compound according to the present invention can be suitably used as, for example, a primer, a nucleic acid probe for detecting a nucleic acid, a labeling substance, and the like. That is, the compound according to the present invention can be used as a nucleic acid labeling reagent (nucleic acid labeling reagent).
- the phosphoramidite compound according to the present invention can be produced by various routes. Hereinafter, examples of several routes for producing the phosphoramidite compound according to the present invention will be described, but the present invention is not particularly limited thereto.
- the phosphoramidite compound according to the present invention adds a linker composed of R 1 , R 2 , D, R 3 and the like to a DMTr nucleoside, then adds dyes Z 11 and Z 12 , and then This may be produced by amidite formation (see formulas (1) to (3)). Further, after adding Z 11 and Z 12 to which a linker comprising R 1 , R 2 , D, R 3 and the like has been added in advance to the DMTr form of nucleoside, this may be amidated (formulas (1) to ( See 3)).
- the phosphoramidite compound according to the present invention can be produced by synthesizing a synthetic intermediate represented by the following formula (21), (22) or (23) and then amidating it. .
- Z 11 and Z 12 are dyes having no charge in an aprotic solvent
- Z 12 have no charge in the aprotic solvent. Therefore, it is easy to amidite these synthetic intermediates in an aprotic solvent, and the phosphoramidite compound according to the present invention can be easily produced.
- the type of aprotic solvent used as a solvent in the amidation is not particularly limited, and examples thereof include acetonitrile and N, N-dimethylformamide.
- nucleic acid in the present specification means a polynucleotide, and includes DNA, RNA, and the like.
- the nucleic acid includes an oligonucleic acid.
- the nucleic acid may be single-stranded or double-stranded.
- oligonucleic acid means an oligonucleotide and may be composed of one or more nucleotides.
- the length of the nucleic acid according to the present invention is not particularly limited, but is preferably 10 bp to 10 kb, for example, and more preferably 10 bp to 1 kb.
- the nucleic acid according to the present invention may be an oligonucleic acid, and the length thereof is not particularly limited, but is preferably, for example, 10 bp to 100 bp, more preferably 10 bp to 50 bp, more preferably 10 bp. More preferably, it is 30 bp or less.
- the nucleic acid according to the present invention contains an atomic group represented by the following formula (25), (26) or (27) as a nucleotide moiety.
- the nucleic acid according to the present invention only needs to contain at least one atomic group represented by the above formula (25) or (26) as a nucleotide moiety, but may contain two or more. Thereby, the nucleic acid according to the present invention includes one nucleotide portion having two dyes or two nucleotide portions having one dye in succession.
- the nucleic acid according to the present invention preferably contains the atomic group represented by the above formula (27) as a nucleotide moiety together with another atomic group having a structure exhibiting an exciton effect.
- This other atomic group may be an atomic group having any one of those exemplified as Z 11 and Z 12, for example.
- the nucleic acid contains at least two atomic groups represented by the above formula (27) as nucleotide portions. These two atomic groups may be arranged in succession in the nucleic acid, or may be arranged across several nucleotides that do not contain other dyes. It is preferably arranged so as to exhibit the exciton effect.
- the nucleic acid according to the present invention is preferably produced using the phosphoramidite compound according to the present invention.
- the nucleic acid produced using the phosphoramidite compound according to the present invention contains a portion other than the phosphoramidite group in the phosphoramidite compound (hereinafter also referred to as “dye-containing nucleoside”) as a constituent element.
- the nucleic acid according to the present invention contains a dye exhibiting an exciton effect, and can be suitably used as a probe (nucleic acid probe) for detecting the nucleic acid to be detected using this exciton effect.
- the nucleic acid according to the present invention may constitute a part of such a probe.
- the nucleic acid according to the present invention can also be suitably used as a labeling substance that utilizes the fluorescence emission of the dye contained therein.
- a labeling substance used for tissue staining or the like may be used.
- the nucleic acid according to the present invention can also be suitably used as a primer, for example, a primer for PCR.
- the nucleic acid according to the present invention may be a primer, a probe, a labeling substance, or the like for use in research, clinical practice, diagnosis, in vitro gene detection, in vivo gene detection and the like.
- the compound represented by the above formula (1) or (2) having two dyes At least one molecule may be used for nucleic acid synthesis.
- the nucleic acid contains one molecule of a nucleoside having two dyes or two molecules of a nucleoside having one dye in succession.
- the compound represented by the above formula (3) having one dye is used for nucleic acid synthesis together with other compounds having an atomic group exhibiting an exciton effect. It is preferable.
- the other compound may be a compound having any one of those exemplified as Z 11 and Z 12, for example.
- the nucleic acid may be synthesized using at least two molecules of the compound having one dye described above.
- this nucleic acid probe contains at least two dyes exhibiting an exciton effect. When the nucleic acid probe is in a single-stranded state, these dyes associate with each other, thereby causing an exciton binding effect and quenching. On the other hand, when the nucleic acid probe is in a double-stranded state bound to the nucleic acid to be detected, these dyes intercalate or groove bind to the double-stranded nucleic acid to eliminate exciton coupling and to emit fluorescence. happenss. By detecting this fluorescence emission, the nucleic acid can be detected.
- the nucleic acid according to the present invention may have at least two regions containing a nucleoside (pigment-containing nucleoside) derived from the phosphoramidite compound according to the present invention. These regions in one nucleic acid are configured so that the dye contained in the dye-containing nucleoside contained in one region does not exhibit an exciton effect with the dye contained in the dye-containing nucleoside contained in another region. Is preferred. Moreover, it is preferable that the pigment
- the nucleic acid according to the present invention has a plurality of different dyes, when used as a nucleic acid probe, by simultaneously observing the light emission and quenching of each dye, which region of the nucleic acid probe is to be detected. It can be detected whether it is bound to a nucleic acid. That is, when only a part of the nucleic acid probe forms, for example, a double strand with the nucleic acid to be detected, it is possible to detect which sequence in the nucleic acid probe forms a double strand, etc. Become.
- a dye-containing nucleoside having a red dye and a dye-containing nucleoside having a blue dye may be introduced into separate regions of the nucleic acid probe.
- the region into which the dye-containing nucleoside having a blue dye is introduced is hybridized with the nucleic acid to be detected, but the red dye It can be seen that the region into which the dye-containing nucleoside having ⁇ is introduced is not hybridized with the nucleic acid to be detected.
- the nucleic acid probe which can detect various nucleic acid structures can be manufactured.
- the nucleic acid probe can be used for investigating the translocation of genes related to cancer, the splicing position of RNA, and the like, and can be suitably used for diagnosis of various diseases, elucidation of causes, and the like.
- the nucleic acid according to the present invention is used as a probe for detecting hybridization with a complementary strand, it is preferably used in an environment having a pH of 10.5 or less from the viewpoint of obtaining stronger fluorescence intensity.
- the following environment is more preferable, and an environment having a pH of 8 or less is more preferable. Note that background noise from a probe that is not hybridized under any conditions is sufficiently low.
- the method for producing a nucleic acid according to the present invention includes a dye-containing nucleoside addition step (addition step) in which the phosphoramidite compound according to the present invention and an oligonucleic acid are subjected to a condensation reaction.
- a dye-containing nucleoside addition step in which the phosphoramidite compound according to the present invention and an oligonucleic acid are subjected to a condensation reaction.
- a nucleoside having an atomic group having fluorescence and exhibiting an exciton effect can be added to the oligonucleic acid.
- the dye-containing nucleoside addition step is a step of subjecting the phosphoramidite compound according to the present invention, that is, the dye-containing phosphoramidite compound, to a condensation reaction with the 5'-hydroxyl of the nucleotide in the oligonucleic acid by the phosphoramidite method. Thereby, the dye-containing nucleoside is added to the oligonucleic acid, and the oligonucleic acid containing the dye-containing nucleoside is synthesized.
- the dye-containing phosphoramidite compound and the oligonucleic acid are preferably reacted at room temperature for 2 to 20 minutes. About other reaction conditions, the normal conditions used in the phosphoramidite method can be used conveniently.
- the method for producing a nucleic acid according to the present invention includes a step of obtaining a phosphoramidite compound by amidating a compound according to the present invention, wherein G is a hydroxyl group, prior to the dye-containing nucleoside addition step. May be.
- the aforementioned dye-containing nucleoside addition step may be incorporated into a known nucleic acid production method.
- the known nucleic acid production methods include, for example, a nucleoside addition step of adding a normal nucleoside substrate to an oligonucleic acid by a phosphoramidite method, and a deprotection step of deprotecting the oligonucleic acid synthesized by the nucleoside addition step.
- a set including a purification step for purifying the oligonucleic acid deprotected by the deprotection step is repeated a predetermined number of times.
- the method for producing a nucleic acid according to the present invention may be such that in this known nucleic acid production method, the nucleoside addition step in at least one set is replaced with the above-described dye-containing nucleoside addition step.
- the present invention may be a known nucleic acid production method in which each of the nucleoside addition steps in a plurality of sets is replaced with a dye-containing nucleoside addition step.
- a nucleic acid containing a plurality of dye-containing nucleosides derived from the compound according to the present invention can be produced.
- the plurality of sets may be a set that is continuously performed or a set that is performed with one or more other sets interposed therebetween.
- a nucleic acid having at least two regions containing a dye-containing nucleoside derived from the compound according to the present invention can be produced.
- each of the dye-containing nucleoside addition steps may use phosphoramidite compounds having different dyes.
- a plurality of different dyes can be introduced into one nucleic acid, that is, a nucleic acid having a plurality of different dyes can be produced.
- These dye-containing nucleoside addition step, nucleoside addition step, deprotection step, and purification step can be performed by known methods, conditions, apparatuses, etc., for example, using a known automatic nucleic acid synthesizer. Therefore, a known nucleic acid synthesizer or the like can be used in the method for producing a nucleic acid according to the present invention.
- the production method according to the present invention can be used to produce a nucleic acid containing a dye-containing nucleoside, that is, a nucleic acid according to the present invention.
- the present invention does not require a two-step synthesis process of synthesis of a DNA oligomer and synthesis for introducing a dye, a nucleic acid having a dye exhibiting an exciton effect can be easily produced. Furthermore, since a nucleoside into which a dye has been introduced in advance is used for DNA synthesis, it is possible to avoid the possibility of synthesizing an incomplete nucleic acid into which a desired number of dyes have not been introduced.
- the kit for producing the nucleic acid according to the present invention includes the above-described compound according to the present invention. Thereby, a nucleic acid can be easily manufactured by performing the nucleic acid manufacturing method described above using the phosphoramidite compound according to the present invention, that is, the dye-containing phosphoramidite compound.
- the kit according to the present invention may contain a plurality of types of dye-containing phosphoramidite compounds each containing different types of dyes. Moreover, various nucleosides which do not contain a pigment may be further included. These nucleosides are preferably contained in an embodiment that can be suitably used for nucleic acid synthesis, for example, as a phosphoramidite.
- the kit according to the present invention includes an apparatus for synthesizing nucleic acid, a reagent used for nucleic acid synthesis, a detection reagent for detecting nucleic acid using the produced nucleic acid, and a kit instruction manual (manufacturing nucleic acid using the kit). And the like) may be further included.
- Example 1 Synthesis of fluorescent dye
- the NHS active form of a fluorescent dye (fluorescent atomic group) represented by the following formula (24) was synthesized by the following reaction formulas (A) to (D). The specific method used is shown below.
- the obtained powder was washed with 500 mM distilled water, the precipitate was filtered, further washed with distilled water, and then dried under reduced pressure. This powder was similarly washed with 500 mL of acetonitrile and dried under reduced pressure to obtain 8.31 g (15.7 mmol, 30%) of a red powder as a target compound.
- the measurement data of the target compound is shown below.
- the resulting red powder was mixed with 300 mL of methanol and mixed well. The precipitate was then filtered and dried under reduced pressure. Thereafter, 300 mL of dichloromethane was added to the obtained red powder, mixed well, and the supernatant was discarded by decantation. Thereafter, 300 mL of acetone was added, and the resulting precipitate was filtered and dried under reduced pressure to obtain 2.24 g (5.95 mmol, 51%) of a red powder as the target compound.
- the measurement data of the target compound is shown below.
- the solvent was evaporated by evaporation and purified using a silica gel column (5% MeOH, 1% Et 3 N / CH 2 Cl 2 ). The solvent was evaporated by evaporation, dissolved in a small amount of acetone, and then diethyl ether was added. The produced orange powder was filtered, washed with ether and dried to obtain 83 mg (FW 14445.75, yield 18%) as a target compound.
- the measurement data of the target compound is shown below.
- DMF N, N-dimethylformamide
- Example 3 DNA probe
- a DNA probe (nucleic acid) containing a nucleoside having a fluorescent dye was synthesized. A specific method will be described below.
- FIG. 1 is a diagram showing a mass spectrum of a DNA probe in one example of the present invention.
- the calculated molecular weight of the DNA probe 5′-d (TTTTTTNTTTTT) -3 ′ is a calculated value of 4793.7, and the actually measured value is 4793.2. Met.
- a DNA probe containing a fluorescent dye could be easily synthesized by using a nucleoside phosphoramidite having a fluorescent dye. That is, when a DNA probe is synthesized, a set composed of a nucleoside addition step, a deprotection step and a purification step is repeated in order to introduce a nucleoside, one of which is used with the compound according to the present invention. It was shown that a DNA probe containing a fluorescent dye can be easily produced by carrying out the process.
- the compound according to the present invention already contains a fluorescent dye, and it is not necessary to introduce a fluorescent dye separately after DNA synthesis. Therefore, according to the present invention, it was shown that a DNA probe can be produced easily and with low labor.
- Example 4 UV absorption spectrum measurement and fluorescence spectrum measurement using a DNA probe
- UV absorption spectrum measurement and fluorescence spectrum measurement were performed.
- Shimadzu UV-2550 (trade name) spectrophotometer manufactured by Shimadzu Corporation was used for UV absorption spectrum measurement
- RF-5300PC trade name fluorescence spectrophotometer was used for fluorescence spectrum measurement.
- the UV absorption spectrum measurement and the fluorescence spectrum measurement a sample having a DNA strand concentration of 0.4 ⁇ M, a phosphate buffer of 50 mM (pH 7.0), and NaCl of 100 mM was used.
- the complementary strand 5′-d (AAAAAAAAAAAA) -3 ′ of the DNA probe is 0 ⁇ M (that is, only the DNA probe; the result is indicated by a in the figure) or 0.4 ⁇ M (the result in the figure is the result). Is indicated by b).
- 488 nm (1.5 nm width) light was used as excitation light.
- FIG. 2 is a diagram showing a UV absorption spectrum of a DNA probe in one example of the present invention
- FIG. 3 is a diagram showing a fluorescence spectrum of a DNA probe in one example of the present invention.
- the DNA probe emits fluorescence by binding to complementary strand DNA. Therefore, it was shown that the DNA probe of this example has a clear fluorescence emission switching function.
- Example 5 A DNA probe 5′-d (TACCANCCCAT) -3 ′ (N is a nucleoside having a fluorescent dye) was synthesized by the same method and conditions as in Example 3, and then purified. Next, using this DNA probe, UV absorption spectrum measurement and fluorescence spectrum measurement were performed under the same method and conditions as in Example 4.
- FIG. 4 is a diagram showing a UV absorption spectrum of a DNA probe in another example of the present invention
- FIG. 5 is a diagram showing a fluorescence spectrum of a DNA probe in another example of the present invention. 4 and 5, the result when the complementary strand of the DNA probe is not added to the sample is indicated by c, and the result when the complementary strand is added is indicated by d.
- the DNA probe emits fluorescence by binding to complementary strand DNA. Therefore, it was shown that the DNA probe of this example has a clear fluorescence emission switching function.
- Example 6 Synthesis of DMTr body of nucleoside having fluorescent dye
- a nucleoside DMTr body having a fluorescent dye represented by the following formula (32) was synthesized by the following reaction formulas (H) to (J). Specifically, a nucleoside NHTr active DMTr body and a fluorescent dye were respectively synthesized, and then a nucleoside DMTr body having a fluorescent dye was synthesized. The specific method used is shown below.
- nucleoside NHS active DMTr body represented by the following formula (30) was synthesized.
- the target compound (593 mg, FW863.15, yield 28%) was obtained as an orange powder.
- the measurement data of the target compound is shown below.
- the solvent was removed by evaporation and separated on a silica gel column (7-15% MeOH, 1% Et 3 N / CH 2 Cl 2 ). Dissolved in a small amount of methanol, diethyl ether was added, and the precipitate was filtered and dried. The powder was washed with water, filtered and dried.
- the target compound (256 mg, FW 14445.75, yield 25%) was obtained as an orange powder.
- the measurement data of the target compound is shown below.
- the DMTr nucleoside having a fluorescent dye synthesized by this method can be converted to a dye-containing phosphoramidite compound by amidation using the method described in 2-3 of Example 2.
- the present invention can easily produce a nucleic acid having a dye exhibiting an exciton effect, it can be suitably used for production of a primer, a probe capable of effectively detecting a detection target, a labeling substance, and the like. . Therefore, the present invention can be suitably used for nucleic acids for a wide range of uses such as research, clinical, diagnostic, in vitro gene detection, and in vivo gene detection, and the production thereof.
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Abstract
Description
Gは、下記式(33)で表されるホスホロアミダイト基または水酸基であり、
Eは、デオキシリボース骨格もしくはリボース骨格を有する原子団またはその誘導体であり、
Z11およびZ12は、それぞれ独立して蛍光性を有し、かつエキシトン効果を示す原子団であって、電荷を有さない原子団であり、Z11とZ12とは、同一であっても異なっていてもよく、
Xは、水素原子、酸により脱保護が可能な水酸基の保護基、リン酸基、二リン酸基、または三リン酸基であり、
R41はリン酸基の保護基であり、
R42およびR43は、それぞれ独立してアルキル基またはアリール基であり、
R1およびR2は、それぞれ独立して主鎖を有するリンカーであり、当該主鎖を構成する原子が置換基を有していてもよく、
R3は、主鎖を有するリンカーであってもよく、または存在していなくてもよく、当該主鎖を構成する原子は置換基を有していてもよく、
Dは、CR11、N、P、P=O、B(ホウ素原子)もしくはSiR11であってもよく、または存在していなくてもよく、R11は、水素原子、アルキル基もしくは任意の置換基であってもよく、
R3が存在しておらず、かつDが存在している場合には、DはBに直接結合していてもよく、R3が存在しており、かつDが存在していない場合には、R1およびR2はR3に直接結合していてもよく、R3およびDが存在していない場合には、R1およびR2はBに直接結合していてもよく、
Tは、リン酸架橋であり、当該リン酸架橋中、1つ以上の酸素原子が硫黄原子で置換されていてもよい。)
また、本発明に係る化合物は、特に、以下に示すより具体的な例示に限定されないが、上記式(1)および(3)中、下記式(1a)で表される原子団が、下記式(4)または(5)で表される原子団であり、上記式(2)中、下記式(2a)で表される原子団が、下記式(6)または(7)で表される原子団であることが好ましい。
Aは水素原子、水酸基、アルキル基、または電子吸引基であり、
MおよびJは、それぞれ独立してCH2、NH、OまたはSである。)
また、本発明に係る化合物では、特に、以下に示すより具体的な例示に限定されないが、R1、R2およびR3が有する主鎖は、それぞれ独立して2以上の原子により構成されていることが好ましい。
l、mおよびnは、それぞれ独立して正の整数であり、
R4は、単結合、二重結合もしくは三重結合であるか、または存在していなくてもよい。)
また、本発明に係る化合物では、特に、以下に示すより具体的な例示に限定されないが、l、mおよびnは、それぞれ2であり、R4は、二重結合であることが好ましい。
X1およびX2は、それぞれ独立してO、S、SeまたはTeであり、
nは、0または正の整数であり、
R21~R30、R32~R34、R51~R60は、それぞれ独立して水素原子、ハロゲン、低級アルキル基、低級アルコキシ基、ニトロ基またはアミノ基であり、R28とR29とは、互いに結合されていてもよく、R58とR59とは、互いに結合されていてもよく、
R31は、上記式(1)、(2)もしくは(3)中のR1もしくはR2、または上記式(8)、(9)、(10)もしくは(11)中のNHに結合する連結基であり、
nが2以上の整数である場合には、R33は、それぞれ同一であっても異なっていてもよく、R34は、それぞれ同一であっても異なっていてもよい。)。
Bは、塩基骨格を有する原子団であり、
Eは、デオキシリボース骨格もしくはリボース骨格を有する原子団またはその誘導体であり、
Z11およびZ12は、それぞれ独立して蛍光性を有し、かつエキシトン効果を示す原子団であって、電荷を有さない原子団であり、Z11とZ12とは、同一であっても異なっていてもよく、
R1およびR2は、それぞれ独立して主鎖を有するリンカーであり、当該主鎖を構成する原子が置換基を有していてもよく、
R3は、主鎖を有するリンカーであってもよく、または存在していなくてもよく、当該主鎖を構成する原子は置換基を有していてもよく、
Dは、CR11、N、P、P=O、B(ホウ素原子)もしくはSiR11であってもよく、または存在していなくてもよく、R11は、水素原子、アルキル基もしくは任意の置換基であってもよく、
R3が存在しておらず、かつDが存在している場合には、DはBに直接結合していてもよく、R3が存在しており、かつDが存在していない場合には、R1およびR2はR3に直接結合していてもよく、R3およびDが存在していない場合には、R1およびR2はBに直接結合していてもよく、
Tは、リン酸架橋であり、当該リン酸架橋中、1つ以上の酸素原子が硫黄原子で置換されていてもよい。)。
まず、本発明に係る化合物について説明する。
本発明に係るホスホロアミダイト化合物は、種々の経路にて製造することができる。以下に、本発明に係るホスホロアミダイト化合物を製造するいくつかの経路の例について説明するが、本発明は、特にこれに限定されるものではない。
次に、本発明に係る核酸について説明する。
次に、本発明に係る核酸の製造方法について説明する。
本発明に係る核酸を製造するためのキットは、上述した本発明に係る化合物を含むものである。これにより、本発明に係るホスホロアミダイト化合物、すなわち色素含有ホスホロアミダイト化合物を用いて、上述した核酸の製造方法を行なうことにより、核酸を容易に製造することができる。
下記反応式(A)~(D)により、下記式(24)で表される蛍光色素(蛍光性を有する原子団)のNHS活性体を合成した。用いた具体的な方法を以下に示す。
13C NMR(DMSO-d6):171.4,150.7,147.7,137.3,135.7,130.8,129.8,129.6,121.9,118.9,53.2,33.3。
13C NMR(CD3OD):178.0,176.4,161.1,150.7,145.6,141.2,138.5,134.4,129.4,128.2,126.6,126.0,125.8,125.7,123.7,118.7,113.7,109.5,88.8,53.2,47.3,37.2,35.1,27.7,23.5。
13C NMR(DMSO-d6):174.2,153.5,145.8,144.9,142.6,140.5,130.8,127.4,126.1,124.7,124.24,124.19,123.0,122.8,122.2,111.0,110.6,85.8,44.6,33.2,25.8,21.7。
13C-NMR(DMSO-d6):170.2,169.0,158.3,149.0,140.8,140.0,137.8,132.7,128.1,126.9,125.0,124.2,123.6,123.3,122.8,120.6,112.7,108.4,87.2,45.0,29.8,25.8,25.4,21.5;
HRMS(ESI)calcd for C26H24N3O4S([M+H]+)474.15;found,474,12。
次に、下記反応式(E)~(G)により、蛍光色素を有するヌクレオシドのホスホロアミダイト体(色素含有ホスホロアミダイト化合物)を合成した。用いた具体的な方法を以下に示す。
まず、下記反応式(E)により、ヌクレオシドのDMTr体において、アミノ基の脱保護を行なった。
13C-NMR(CD3OD):169.3,163.7,160.2,151.0,146.1,143.4,137.1,137.0,134.6,131.3,131.2,129.3,128.9,128.0,122.4,114.3,110.8,88.2,88.0,72.7,65.0,55.8,54.5,52.8,41.8,38.5,38.4;
HRMS(ESI)calcd for C39H49N6O8([M+H]+),729.36;found,729.3114。
次に、下記反応式(F)により、蛍光色素を有するヌクレオシドのDMTr体を合成した。
次に、下記反応式(G)により、上記2-2で合成したDMTr体を非プロトン性溶媒中においてアミダイト化し、色素含有ホスホロアミダイト化合物を合成した。
実施例2で合成した色素含有ホスホロアミダイト化合物を用いて、蛍光色素を有するヌクレオシドを含むDNAプローブ(核酸)を合成した。具体的な方法について、以下に説明する。
DNA合成機(Applied Biosystems社の392 DNA/RNA synthesizer(商品名))を用い、固相合成法にしたがってDNAプローブ5’-d(TTTTTTNTTTTTT)-3’(Nは、蛍光色素を有するヌクレオシド)を合成した。蛍光色素を有するヌクレオシド(N)を導入する際の反応時間を15分とし、蛍光色素を有さないヌクレオシド(T)を導入する際の反応時間を2分とした。これ以外の条件については、通常のホスホロアミダイト法において用いる条件を用いた。固相担体からの切り出しは、通常用いられる方法(28%アンモニア水を用いる方法)により行なった。また、脱保護は、25℃、終夜(16時間)にて行なった。
次に、合成したDNAプローブを、逆相HPLC(ギルソン社の装置Gilson Chromatograph, Model 305(商品名))により精製した。カラムには、CHEMCOBOND 5-ODS-Hカラム(商品名、10×150mm)を使用した。溶媒には、5-55%CH3CN/100mMのTEAA buffer 100mM(pH7.0)を用い、40分の勾配にて3mL/分の流速で流した。UV検出器Model 118(商品名)により、260nmのUV吸収をモニターすることによってDNAプローブを検出した。
実施例3によって合成されたDNAプローブを用いて、UV吸収スペクトル測定および蛍光スペクトル測定を行なった。UV吸収スペクトル測定には、株式会社島津製のShimadzu UV-2550(商品名)分光光度計を用い、蛍光スペクトル測定には、RF-5300PC(商品名)蛍光分光光度計を用いた。
実施例3と同様の方法および条件により、DNAプローブ5’-d(TACCAGNCACCAT)-3’(Nは、蛍光色素を有するヌクレオシド)を合成し、その後精製した。次に、このDNAプローブを用いて、実施例4と同様の方法および条件により、UV吸収スペクトル測定および蛍光スペクトル測定を行なった。
下記反応式(H)~(J)により、下記式(32)で表される、蛍光色素を有するヌクレオシドのDMTr体を合成した。具体的には、ヌクレオシドのNHS活性体のDMTr体と、蛍光色素とをそれぞれ合成した後、蛍光色素を有するヌクレオシドのDMTr体を合成した。用いた具体的な方法を以下に示す。
まず、ヌクレオシドのNHS活性体を合成した。
次に、下記式(31)で表される蛍光色素を合成した。
次に、下記式(32)で表される、蛍光色素を有するヌクレオシドのDMTr体を合成した。
Claims (13)
- 下記式(1)、(2)または(3)で表されることを特徴とする、化合物。
Gは、下記式(33)で表されるホスホロアミダイト基または水酸基であり、
Eは、デオキシリボース骨格もしくはリボース骨格を有する原子団またはその誘導体であり、
Z11およびZ12は、それぞれ独立して蛍光性を有し、かつエキシトン効果を示す原子団であって、電荷を有さない原子団であり、Z11とZ12とは、同一であっても異なっていてもよく、
Xは、水素原子、酸により脱保護が可能な水酸基の保護基、リン酸基、二リン酸基、または三リン酸基であり、
R41はリン酸基の保護基であり、
R42およびR43は、それぞれ独立してアルキル基またはアリール基であり、
R1およびR2は、それぞれ独立して主鎖を有するリンカーであり、当該主鎖を構成する原子が置換基を有していてもよく、
R3は、主鎖を有するリンカーであってもよく、または存在していなくてもよく、当該主鎖を構成する原子は置換基を有していてもよく、
Dは、CR11、N、P、P=O、B(ホウ素原子)もしくはSiR11であってもよく、または存在していなくてもよく、R11は、水素原子、アルキル基もしくは任意の置換基であってもよく、
R3が存在しておらず、かつDが存在している場合には、DはBに直接結合していてもよく、R3が存在しており、かつDが存在していない場合には、R1およびR2はR3に直接結合していてもよく、R3およびDが存在していない場合には、R1およびR2はBに直接結合していてもよく、
Tは、リン酸架橋であり、当該リン酸架橋中、1つ以上の酸素原子が硫黄原子で置換されていてもよい。) - R1、R2およびR3が有する主鎖は、それぞれ独立して2以上の原子により構成されていることを特徴とする請求項1または2に記載の化合物。
- l、mおよびnは、それぞれ2であり、R4は、二重結合であることを特徴とする請求項4に記載の化合物。
- Z11およびZ12が、それぞれ独立して下記式(12)、(28)または(29)で表される原子団であることを特徴とする請求項1~5のいずれか1項に記載の化合物。
X1およびX2は、それぞれ独立してO、S、SeまたはTeであり、
nは、0または正の整数であり、
R21~R30、R32~R34、R51~R60は、それぞれ独立して水素原子、ハロゲン、低級アルキル基、低級アルコキシ基、ニトロ基またはアミノ基であり、R28とR29とは、互いに結合されていてもよく、R58とR59とは、互いに結合されていてもよく、
R31は、上記式(1)、(2)もしくは(3)中のR1もしくはR2、または上記式(8)、(9)、(10)もしくは(11)中のNHに結合する連結基であり、
nが2以上の整数である場合には、R33は、それぞれ同一であっても異なっていてもよく、R34は、それぞれ同一であっても異なっていてもよい。) - 上記式(12)、(28)および(29)中、R31は、炭素数2以上のポリメチレンカルボニル基であり、カルボニル基部分において上記式(1)、(2)もしくは(3)中のR1もしくはR2、または上記式(8)、(9)、(10)もしくは(11)中のNHに結合することを特徴とする請求項6に記載の化合物。
- R41がシアノエチル基であり、R42およびR43がそれぞれイソプロピル基であることを特徴とする請求項1~7のいずれか1項に記載の化合物。
- 請求項1~8のいずれか1項に記載の化合物であって、Gが上記式(33)で表されるホスホロアミダイト基である化合物と、オリゴ核酸とを縮合反応させることを特徴とする核酸の製造方法。
- 下記式(25)、(26)または(27)で表される原子団をヌクレオチド部分として含むことを特徴とする核酸。
Bは、塩基骨格を有する原子団であり、
Eは、デオキシリボース骨格もしくはリボース骨格を有する原子団またはその誘導体であり、
Z11およびZ12は、それぞれ独立して蛍光性を有し、かつエキシトン効果を示す原子団であって、電荷を有さない原子団であり、Z11とZ12とは、同一であっても異なっていてもよく、
R1およびR2は、それぞれ独立して主鎖を有するリンカーであり、当該主鎖を構成する原子が置換基を有していてもよく、
R3は、主鎖を有するリンカーであってもよく、または存在していなくてもよく、当該主鎖を構成する原子は置換基を有していてもよく、
Dは、CR11、N、P、P=O、B(ホウ素原子)もしくはSiR11であってもよく、または存在していなくてもよく、R11は、水素原子、アルキル基もしくは任意の置換基であってもよく、
R3が存在しておらず、かつDが存在している場合には、DはBに直接結合していてもよく、R3が存在しており、かつDが存在していない場合には、R1およびR2はR3に直接結合していてもよく、R3およびDが存在していない場合には、R1およびR2はBに直接結合していてもよく、
Tは、リン酸架橋であり、当該リン酸架橋中、1つ以上の酸素原子が硫黄原子で置換されていてもよい。) - 請求項9に記載の核酸の製造方法により製造されたことを特徴とする請求項10に記載の核酸。
- 請求項1~8のいずれか1項に記載の化合物を含むことを特徴とする核酸を製造するためのキット。
- 請求項1~8の何れか1項に記載の化合物であって、式中のGが上記式(33)で表されるホスホロアミダイト基である化合物を製造する方法であって、
請求項1~8の何れか1項に記載の化合物であって、式中のGが水酸基である化合物を、非プロトン性溶媒中でホスホロアミダイト化する工程を含むことを特徴とする製造方法。
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WO2014038561A1 (ja) * | 2012-09-04 | 2014-03-13 | 株式会社ダナフォーム | 化合物、核酸、標識物質および検出方法 |
US11597744B2 (en) | 2017-06-30 | 2023-03-07 | Sirius Therapeutics, Inc. | Chiral phosphoramidite auxiliaries and methods of their use |
US11981703B2 (en) | 2016-08-17 | 2024-05-14 | Sirius Therapeutics, Inc. | Polynucleotide constructs |
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EP2873731B1 (en) * | 2012-07-16 | 2018-08-29 | Kabushiki Kaisha Dnaform | Nucleic acid probe, method for designing nucleic acid probe, and method for detecting target sequence |
CN107502318A (zh) * | 2017-08-08 | 2017-12-22 | 中国石油集团渤海钻探工程有限公司 | 一种高密度盐水完井液用复配缓蚀剂及其制备方法 |
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WO2014034818A1 (ja) * | 2012-08-30 | 2014-03-06 | 株式会社ダナフォーム | 標的核酸の分析方法、キットおよび分析機器 |
JP5598784B2 (ja) * | 2012-08-30 | 2014-10-01 | 株式会社ダナフォーム | 標的核酸の分析方法、キットおよび分析機器 |
JPWO2014034818A1 (ja) * | 2012-08-30 | 2016-08-08 | 株式会社ダナフォーム | 標的核酸の分析方法、キットおよび分析機器 |
US10066264B2 (en) | 2012-08-30 | 2018-09-04 | Kabushiki Kaisha Dnaform | Method for analyzing target nucleic acid, kit, and analyzer |
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JPWO2014038561A1 (ja) * | 2012-09-04 | 2016-08-12 | 株式会社ダナフォーム | 化合物、核酸、標識物質および検出方法 |
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US11597744B2 (en) | 2017-06-30 | 2023-03-07 | Sirius Therapeutics, Inc. | Chiral phosphoramidite auxiliaries and methods of their use |
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