WO2012077800A1 - Nouvel analogue de nucléoside et son utilisation - Google Patents

Nouvel analogue de nucléoside et son utilisation Download PDF

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Publication number
WO2012077800A1
WO2012077800A1 PCT/JP2011/078604 JP2011078604W WO2012077800A1 WO 2012077800 A1 WO2012077800 A1 WO 2012077800A1 JP 2011078604 W JP2011078604 W JP 2011078604W WO 2012077800 A1 WO2012077800 A1 WO 2012077800A1
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group
oxo
oligodeoxynucleotide
adap
target dna
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PCT/JP2011/078604
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Japanese (ja)
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茂貴 佐々木
陽祐 谷口
亮太 河口
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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
    • 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
    • C07H19/16—Purine radicals
    • C07H19/173—Purine radicals with 2-deoxyribosyl as the saccharide radical
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to a novel compound and use thereof.
  • the novel compound of the present invention is useful as a fluorescent probe capable of selectively recognizing 8-oxo-dG in a DNA strand.
  • 8-oxo-deoxyguanosine (8-oxo-dG) is one of the DNA damage bodies generated from deoxyguanosine (dG) by reactive oxygen species in the body, and it is suggested to be related to aging, neurodegenerative diseases, and oxidative stress Yes.
  • dG deoxyguanosine
  • oxidative stress Yes it is known that the structural change enables base pairing not only with cytosine but also with adenosine, thereby inducing transversion mutation.
  • detection methods using electrochemical methods, mass spectrometry, antibodies, etc. are currently being developed.
  • the inventors of the present invention selectively detect 8-oxoG-clamp, which was designed based on the known compound G-clamp, which has high binding ability to guanine, with 8-oxo-dG, and detect it by the change in fluorescence intensity due to the binding. It was shown that it is possible (Non-Patent Document 1). In addition, 8-oxoG-clamp derivatives and their 8-oxo-dG recognition ability (Non-patent Document 2), and 8-oxo-dG and dG in DNA can be partially distinguished by incorporation into oligonucleotides. The possibility was also shown (Non-Patent Document 3).
  • the 8-oxoG-clamp that we have already developed is capable of distinguishing 8-oxo-dGG and dG effectively in organic solvents because the molecular recognition ability is mainly carried out by hydrogen bonding, but it is still not recognized in water. Not achieved. At present, only antibodies can discriminate 8-oxo-dG in water.
  • the present inventors have created a molecule that can effectively distinguish 8-oxo-dG and dG even when present in DNA.
  • the present invention provides the following: [1] Adenosine-1,3-diazaphenoxazine (Adap) nucleoside analog-containing oligodeoxynucleotide represented by the following formula
  • R 1 is an oligodeoxynucleotide group 1 (ODN group 1); R 2 is an oligodeoxynucleotide group 2 (ODN group 2). ).
  • [2] The oligodeoxynucleotide according to [1] for detection of 8-oxodeoxyguanosine (8-oxo-dG).
  • [3] The oligodeoxynucleotide according to [1], which is labeled
  • Consists of a base sequence complementary to a part of the base sequence containing 8-oxo-dG, but contains an Adapt nucleoside analog at a position complementary to 8-oxo-dG, thereby targeting oligodeoxynucleotides A method for detecting a target DNA containing 8-oxo-dG that selectively hybridizes to DNA.
  • [5] Labeled with one of a fluorescent substance or a quencher substance and composed of the base sequence A and labeled with the first oligodeoxynucleotide according to [1] and the other of the fluorescent substance or the quencher substance and complementary to the base sequence A
  • a double-stranded oligodeoxynucleotide probe comprising at least a second oligodeoxynucleotide comprising a natural base sequence B but containing a natural nucleotide at a position complementary to an Adap nucleoside analog.
  • [6] The probe according to [5], wherein the 5 ′ end of the first oligodeoxynucleotide is labeled with a fluorescent substance, and the 3 ′ end of the second oligodeoxynucleotide is labeled with a quencher substance. [7] including a step of providing the probe according to [5] or [6] to a target DNA containing 8-oxo-dG, wherein the first oligodeoxynucleotide is at least 8-oxo-dG of the target DNA.
  • R 1 is H, a protecting group for a 3′-hydroxyl group, or —P (OR 3 ) R 4 , where R 3 is a protecting group for a phosphate group, and R 4 is a nitrogen atom.
  • R 2 is a hydrogen atom or a protecting group for a 5′-hydroxyl group.
  • R 1 is —P (OR 3 ) R 4 , wherein R 3 is a methyl group, a 2-cyanoethyl group, or a 2-trimethylsilylethyl group, and R 4 is a diisopropylamino group.
  • TBDMS protected 8-oxo-dG and dG Fluorescence quenching by addition of TBDMS protected 8-oxo-dG and dG to TBDMS protected Adapt.
  • TBDMS-protected 8-oxo-dG and dG in CHCl 3 were dropped into a 1 ⁇ M solution of TBDMS-protected Adap in CHCl 3 , and changes in the fluorescence spectrum were observed.
  • the 13PuY buffer solution was dropped into 13PyX 50 nM buffer solution (100 M NaCl, 10 Msodium phosphate buffer, pH 7.0, 30 ° C).
  • the 13PuY concentration in the solution is 25 nM and 50 nM. Specific quenching by 8-oxo-dG in 13PyY by the fluorescence spectrum of 13PuX incorporating Adap.
  • the 13PyY buffer solution was dropped into 13PuX 50 nM buffer solution (100 M NaCl, 10 Msodium phosphate buffer, pH 7.0, 30 ° C).
  • the 13PuY concentration in the solution is 25 nM and 50 nM.
  • the 80 PuY buffer solution was dropped into 13 PuX 100 nM buffer solution (100 mM NaCl, 10 mM sodium phosphate buffer, pH 7.0, 25 ° C.). 80PuY concentration: 0, 10, 20, 50, 100, 200 nM. Fluorescence spectrum of ODN1-ODN3 probe. Measurements were carried out in 100 mM NaCl, 10 mM sodium phosphate buffer, pH 7.0, 30 ° C., and Cy3 was present at 546 nm.
  • A duplex-containing Cy3-5'-Adap (ODN1) and T-, C-, G- or A- 3'-BHQ probe (ODN3-6), and natural AT pair (ODN2-ODN3)
  • B Double-stranded Adap-3′-BHQ (ODN7) and Cy3-5′-T, -C, -G or -A probes (ODN9-12), and natural AT pair (ODN8-ODN9).
  • Measurements were performed in 100 mM NaCl, 10 mM sodium phosphate buffer, pH 7.0, 30 ° C., with 10 nM probes and 10 nM target ODN13 (gray bar) or ODN14 (black bar), Cy3 is Present at 546 nm and detected at 562 nm. Time course for detecting 8-oxo-dG in long DNA. Measurements were performed in 100 mM NaCl, 10 mM sodium phosphate buffer, pH 7.0, 40 ° C. using an ODN1-ODN3 probe (10 nM) and a target 80 mM ODN (ODN15 or ODN16, 10 nm). Present at 546 nm and detected at 562 nm.
  • the base sequence and the number of bases are not particularly limited unless otherwise specified, and the base sequence may be the base of the target nucleic acid, if necessary. Designed to be complementary to the sequence, the number of bases can be any number capable of hybridizing with the target nucleic acid.
  • nucleic acid refers to DNA or RNA unless otherwise specified. In the present invention, “nucleic acid” is preferably DNA unless otherwise specified.
  • oligodeoxynucleotide group means an oligodeoxynucleotide moiety that is bound to or can be bound to a group adjacent to the 3 ′ end or 5 ′ end unless otherwise specified. Point to.
  • both ODN group 1 and ODN group 2 have a phosphate group at the end, and the phosphate group has It may be an oligodeoxynucleotide moiety capable of binding to an adenosine-1,2-diazaphenoxazine (Adap) nucleoside analog moiety.
  • ODN group 1 and ODN group 2 are each independently preferably 5 to 100 bases long, and more preferably 10 to 30 bases long.
  • ODN group 1 and ODN group 2 can be synthesized from the compound of the present invention, which is a DNA synthesis precursor, by an automatic nucleic acid synthesizer using the phosphoramidite method.
  • R 1 and / or R 2 in the formula (I) are a hydroxyl-protecting group, they can be independently selected from the conventional hydroxyl-protecting groups.
  • TBDMS may be described as TBS) (tert-butyldimethylsilyl) group, DMTr (4,4'-dimethoxytrityl) group, TBDPS (tert-butyldiphenylsilyl) group, TES (triethylsilyl) group A TIPS (triisopropylpropyl) group, a DMES (dimethylethylsilyl) group, a THP (tetrahydropyranyl) group, an EE (ethoxyethyl) group, a MOM (methoxymethyl) group, and a Bn (benzyl) group.
  • the hydroxyl protecting group for R 1 and R 2 is preferably a TBDMS group, and when R 1 is —P (OR 3 ) R 4 , a particularly preferred
  • R 1 in formula (I) may be —P (OR 3 ) R 4 .
  • R 3 is a protecting group for a phosphoric acid group, and the protecting group for the phosphoric acid group can be appropriately selected from the protecting groups of the prior art as long as it is used in the phosphoramidite method. Specific examples are a methyl group, a 2-cyanoethyl group, and a 2-trimethylsilylethyl group.
  • R 4 is a dialkylamino group in which two identical or different alkyl groups having 1 to 6 carbon atoms are bonded to the nitrogen atom.
  • dialkylamino group are a diethylamino group, a diisopropylamino group, and a dimethylamino group.
  • Two alkyl groups may be bonded to each other to form a ring.
  • An example of this is the morpholin-1-yl group.
  • those that are -P (OR 3 ) R 4 can be used as a DNA synthesis precursor.
  • those that are DNA synthesis precursors particularly preferred examples are as follows. belongs to.
  • the advantage of this molecular design is that the 8-oxo-dG to be recognized targets a special conformation that can be taken in DNA. That is, in general, anti-conformation is preferred for dG in DNA, but 8-oxo-dG can have a syn-conformation due to repulsion between the oxygen atom at the 8-position and the phosphate of the backbone. Adap itself is expected to prioritize the anti-conformation, and this conformation was expected to effectively form a multipoint hydrogen bond with the syn-conformed 8-oxo-dG [Chem. 7]. On the other hand, in order for the anti-conformational dG to complex with Adap, it must be converted to an unstable syn-conformation.
  • Adap is expected to have a favorable interaction with 8-oxo-dG in DNA, whereas many disadvantageous factors are expected with dG, and effective discrimination in DNA is achieved. The inventors expected.
  • TBDMS protecting group of 5 was deprotected with TBAF to obtain 6, which was reacted with DMTrCl in pyridine to protect the 5'-hydroxyl group and react with 2-Cyanoethyl-N, N'-diisopropylchlorophosphorodiamidite.
  • a precursor ⁇ -phosphoramidite (7) can be obtained.
  • 7 CH 3 CN solution was incorporated into ODN (oliogodeoxynucleotide) using a solid-phase DNA synthesizer. The ODN synthesized in this way was used for evaluation of 8-oxo-dG recognition ability in DNA (see Examples).
  • the oligodeoxynucleotide (Adap-containing ODN) represented by the formula (I) is: It has the following functions. (1) A double strand and a complementary strand having 8-oxo-dG are formed at a position corresponding to Adapt and stabilized (increase in melting temperature Tm). (2) It can distinguish dG and 8-oxo-dG in DNA. (3) Quench the fluorescent label during double strand formation.
  • the Adap-containing ODN of the present invention can be used for detection of 8-oxo-dG in nucleic acids.
  • Adap-containing ODN recognizes 8-oxodG by multipoint hydrogen bonding and can be used as an artificial nucleic acid that can be detected by specific fluorescence quenching (Y. Taniguchi, R. Kawaguchi, S. Sasaki, J. Am. Chem. Soc. 2011, 133, 7272-7275), and can also be used to enhance fluorescence when a target sequence is detected (Y. Taniguchi, Y. Koga, K. Fukabori , R. Kawaguchi, S. Sasaki, Bioorg. Med. Chem. Lett. In press, Available online 4 November 2011).
  • the Adap-containing ODN (sometimes referred to as “first deoxyoligonucleotide”) labeled with one of the fluorescent substance or the quencher substance, and the base labeled with the other fluorescent substance or the quencher substance. It consists of a base sequence B complementary to sequence A, but can also be used as a double-stranded probe (duplex prove) comprising at least a second oligodeoxynucleotide containing a natural nucleotide at a position complementary to an Adapt nucleoside analog. .
  • a diagram illustrating this concept is shown below.
  • fluorescent substance refers to a substance that can emit fluorescence unless otherwise specified.
  • quencher substance refers to a substance that is an excitation energy absorber and has a function of suppressing the fluorescence of a nearby fluorescent substance, unless otherwise specified.
  • a fluorescent substance and a quencher substance are used in combination.
  • This combination is not particularly limited as long as the desired effect can be exhibited, but can be selected with reference to a combination of a fluorescent substance and a quencher substance used in real-time PCR or the like.
  • Examples include FAM and IBFQ, FAM and TAMRA, TET and IBFQ, HEX and IBFQ, Cy5 and IBFQ, FAM and BHQ1 or 2, TET and BHQ1 or 2, HEX and BHQ1 or 2, MAX2NHS ester and IBFQ , MAX NHS ester and BHQ1 or 2, Cy3 and IBRQ, Cy3 and BHQ2, TYE563 and IBRQ, TYE563 and BHQ2, TEX615 and IBRQ, TEX615 and BHQ2, Cy5 and IBRQ, Cy5 and BHQ2, TYE665 and IBRQ, TYE665 and BHQ2, FAM And TAMRA NHS ester, JOE NHS ester and IBFQ, JOE NHS ester and BHQ1 or 2, TAMRA NHS ester and IBRQ, TAMRA NHS ester and BHQ2, ROX NHS ester and IBRQ, ROX NHS ester and BHQ2, Texas Red-X NHS ester and IBRQ, Texas
  • the present invention can be used to detect oxidative mutations of guanine in nucleic acids, and more specifically to detect 8-oxo-dG in DNA. Such detection is useful for the study of diseases or conditions associated with oxidative mutations of guanine in DNA.
  • Diseases or conditions associated with oxidative mutations in DNA, particularly guanine mutations in DNA include aging, neurodegenerative diseases, and brain aging.
  • the DNA to be detected can be DNA in a living cell or DNA contained in a sample derived from a living body.
  • the “detection method” refers to a method for evaluating the presence and / or abundance of the target 8-oxo-dG unless otherwise specified.
  • reaction solution was diluted with AcOEt, washed successively with saturated aqueous NaHCO 3 solution and saturated brine, dried over Na 2 SO 4 , and the solvent was evaporated under reduced pressure.
  • Adap was incorporated into the X part of the following sequence.
  • the 13-base ODN synthesized the sequence 13PyX sandwiched between pyrimidine bases and 13PuX sandwiched between purine bases.
  • CG-X a self-complementary sequence incorporated at the terminal portion, was synthesized as a sequence for examining the stacking effect.
  • the molecular recognition function was evaluated by dropping a TBDMS-protected 8-oxo-dG and dG CHCl 3 solution into a 1 ⁇ M solution of TBDMS-protected Adap in CHCl 3 and observing changes in the fluorescence spectrum.
  • FIG. 1 shows changes in the fluorescence spectrum by titration of TBDMS-protected 8-oxo-dG and dG against TBDMS-protected Adap (5).
  • the 8-oxo-dG body had higher quenching efficiency than the dG body, but no effective distinction was observed under this condition.
  • the melting temperature was measured using the self-complementary sequence CG-X.
  • Thermodynamic parameters were determined from plots of melting temperature at each concentration. The melting temperature was expressed as a calculated value at 0.1 mM ODN.
  • Adap at the terminal part also showed an effect of increasing the melting temperature, but this value was not significantly different from the effect of adenosine, and there was almost no stacking effect due to the phenoxazine part of Adap. It has been shown.
  • the 13PuY buffer solution was dropped into 13PyX 50 nM buffer solution (100 mM NaCl, 10 mM sodium phosphate buffer, pH 7.0, 30 ° C.).
  • the 13PuY concentration in the solution was 25 nM and 50 nM.
  • the 80 PuY buffer solution was dropped into 13 PuX 100 nM buffer solution (100 mM NaCl, 10 mM sodium phosphate buffer, pH 7.0, 25 ° C.). The concentration of 80 PuY was 0, 10, 20, 50, 100, 200 nM.
  • ODN1 labeled with Cy3 at the 5 ′ end and ODN7 labeled with BHQ2 at the 3 ′ end were synthesized.
  • ODN2 and ODN8 having adenosine at the same position as Adap of ODN1 and ODN7 were synthesized as control probes.
  • BHQ2-labeled ODN3-6 and Cy3-labeled ODN9-12 were synthesized to determine the appropriate complementary pair. What was synthesize
  • combined in the present Example is as follows.
  • Tm value of the duplex containing the Adap and 8oxo-dG pair is higher than that of the corresponding natural dA-T base pair (Tm, ° C; 47.2 vs 41.9), but the Adap and thymine pair is naturally It had a Tm value similar to that of dA-T base pair (Tm, ° C; 39.6 vs 41.9).
  • the pair with the double-stranded probe ODN1-ODN3 including the pair of Adap and T was determined as a probe suitable for detection of 8-oxo-dG in DNA.
  • the natural double-stranded ODN2 -ODN3 (AT base pair) was tested and fluorescence intensity did not increase with either addition of ODN13 (8-oxo-dG) or ODN14 (dG). .
  • the natural ODN8-ODN9 probe (dA-T) showed similar fluorescence changes with ODN13 and ODN14, indicating that perfectly matched base pairs are not suitable for use in this detection system.
  • the double-stranded ODN probe having the Adap-T pair was determined again as a probe suitable for detection of 8-oxo-dG.
  • the fluorescence recovery value of the ODN7 double-stranded probe was slightly lower than that of the ODN1 probe. Hydrophobic fluorescent substances introduced at the 3′- or 5′-end can affect the thermodynamic and / or kinetic stability of the double-stranded probe.
  • the FRET probe containing the Adapt-T mask ODN is a useful tool for detecting fluorescence of 8-oxo-dG in DNA.
  • the present inventors have used an OFF-to-ON type fluorescent probe for detection of 8-oxo-dG in DNA, including non-natural Adap- T base pair 8-oxo-dG. Designed. This pair masks the base recognition of Adap and results in an effective strand exchange reaction in the presence of 8-oxo-dG in the target DNA. As a result, the Adapt-T probe fluoresces efficiently and complete discrimination between 8-oxo-dG and dG in DNA is achieved. This system is useful for the detection of 8-oxo-dG in various nucleic acids.

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Abstract

L'invention concerne une technique de détection de 8-oxo-dG dans l'ADN en utilisant un nouveau composé représenté par la formule (I). Dans la formule (I) : R1 représente un atome d'hydrogène, un groupe protecteur pour le groupe 3'-hydroxyle, un groupe oligodésoxynucléotide 1 (groupe ODN 1), ou -P(OR3)R4 [dans lequel R3 représente un groupe protecteur pour le groupe phosphate; et R4 représente un groupe dialkylamino contenant deux groupes alkyle en C1-6 attachés à un atome d'azote, lesdits groupes alkyle en C1-6 étant identiques ou différents]; et R2 représente un atome d'hydrogène, un groupe protecteur pour le groupe 5'-hydroxyle, ou un groupe oligodésoxynucléotide 2 (groupe ODN 2). Le composé selon la présente invention est utile pour détecter une mutation oxydative dans un acide nucléique.
PCT/JP2011/078604 2010-12-10 2011-12-09 Nouvel analogue de nucléoside et son utilisation WO2012077800A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014069645A1 (fr) * 2012-11-01 2014-05-08 国立大学法人九州大学 Dérivé d'un analogue d'un nucléoside et son utilisation
US10577481B2 (en) 2017-04-19 2020-03-03 Canon Kabushiki Kaisha Polymer

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2009256645A1 (en) * 2008-06-10 2009-12-17 Novartis Ag Pyrazine derivatives as epithelial sodium channel blockers

Non-Patent Citations (6)

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Title
OSAMU NAKAGAWA ET AL.: "8- Oxoguanosine Keiko Probe no Kaihatsu", ABSTRACTS, SYMPOSIUM ON BIOFUNCTIONAL CHEMISTRY, vol. 22, 2007, pages 88 - 89 *
OSAMU NAKAGAWA ET AL.: "Specific Fluorescent Probe for 8-Oxoguanosine", ANGEW. CHEM. INT. ED., vol. 46, no. 24, 2007, pages 4500 - 4503, XP055133776, DOI: doi:10.1002/anie.200700671 *
TAMER NASR ET AL.: "Selective fluorescence quenching of the 8-oxoG- clamp by 8-oxodeoxyguanosine in ODN", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 19, 2009, pages 727 - 730 *
ZHICHUN LI ET AL.: "8-Oxoguanosine Tokuiteki Oto o Shimesu Keiko Probe no Kaihatsu", ABSTRACTS SYMPOSIUM ON PROGRESS IN ORGANIC REACTIONS AND SYNTHESIS, vol. 35, 2009, pages 272 - 273 *
ZHICHUN LI ET AL.: "Keiko Probe "8-oxoG -clamp" no 8-Oxoguanosine Ninshiki Mechanism no Kaimei Oyobi Ninshikino no Kojo o Mezashita Shinki Yudotai no Gosei to Hyoka'", BANYU LIFE SCIENCE FOUNDATION INTERNATIONAL, FUKUOKA SYMPOSIUM, vol. 20, May 2010 (2010-05-01), pages 64 *
ZHICHUN LI ET AL.: "Synthesis of new derivatives of 8-oxoG-Clamp for better understanding the recognition mode and improvement of selective affinity", BIOORGANIC & MEDICINAL CHEMISTRY, June 2010 (2010-06-01), pages 3992 - 3998 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014069645A1 (fr) * 2012-11-01 2014-05-08 国立大学法人九州大学 Dérivé d'un analogue d'un nucléoside et son utilisation
US10577481B2 (en) 2017-04-19 2020-03-03 Canon Kabushiki Kaisha Polymer

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