JPWO2013150902A1 - Method for producing 5'-terminal phosphorylated oligodeoxynucleotide by cleaving oligodeoxynucleotide containing non-natural base - Google Patents

Abstract

By introducing deoxynucleotides containing non-natural bases into the DNA strand, the resulting DNA strand is cleaved by chemical reaction at low cost and with simple experimental operations, and the resulting end is phosphorylated. To do. The present invention comprises contacting the nucleophile with the oligodeoxynucleotide represented by formula I to form the 3′-position of deoxynucleoside B containing an unnatural base contained in the oligodeoxynucleotide represented by formula I. The phosphodiester bond between the 5 ′ end of C is cleaved by elimination of the oligodeoxynucleotide moiety phosphorylated at the 5 ′ end of C from the 3 ′ position of B, and the 5 ′ end is phosphorylated. The present invention relates to a method for producing an oligodeoxynucleotide, which comprises a 5′-terminal phosphorylated oligodeoxynucleotide forming step for obtaining the resulting oligodeoxynucleotide C.

Description

  The present invention relates to a method for producing a 5'-terminal phosphorylated oligodeoxynucleotide by cleaving an oligodeoxynucleotide containing a non-natural base.

  In research in the field of molecular biology, techniques for replicating or amplifying, cleaving, and ligating deoxyribonucleic acid (hereinafter also referred to as “DNA”) are used. Among them, a method using a restriction enzyme is generally used as a technique for cleaving DNA, and a method using a DNA ligase (DNA ligase) is generally used as a technique for ligating DNA. For example, a DNA fragment having a protruding end is prepared using a type II restriction enzyme (Non-Patent Documents 1 and 2), and another DNA fragment having a protruding end complementary to the protruding end is prepared. DNA fragments can be ligated using a DNA ligation enzyme.

  However, the above method has several problems. First, overhanging ends made using type II restriction enzymes have short overhangs. Second, the base sequence of the protruding portion at the protruding end is a palindromic sequence. Thirdly, overhanging ends can only be created with restriction enzyme recognition sequences. Among these problems, the first and second problems cause a decrease in the efficiency of ligation between DNA fragments by the DNA ligation enzyme, and the third problem is a new problem formed as a result of ligating DNA fragments. It becomes a big restriction of the base sequence in DNA.

  In order to solve such problems, there is a technique in which deoxy or ribonucleotide containing a non-natural base is introduced into a DNA strand in advance and the DNA strand is cleaved at the site of deoxy or ribonucleotide containing a non-natural base. It has been developed. Patent Documents 1 and 2 and Non-Patent Documents 3 to 5 were amplified after the DNA was amplified by polymerase chain reaction (PCR) or the like using a DNA primer into which uridine (ribonucleotide) was introduced as an unnatural base. A method for cleaving DNA at the position of uridine (ribonucleotide) is described. In Non-Patent Documents 6 to 12, after amplifying DNA by PCR or the like using a DNA primer into which 2'-deoxyuridine is introduced as a non-natural base, the amplified DNA is located at the position of 2'-deoxyuridine. Describes how to cut. The method described in the above document uses an enzyme such as RNase or uracil-removing enzyme for cleaving DNA. Therefore, it is disadvantageous in terms of cost. In general, an enzymatic cleavage reaction must satisfy special reaction conditions relating to pH and / or metal ion concentration.

  Non-Patent Document 13 describes a method for cleaving DNA via a DNA base elimination reaction known as depletion and the resulting basic site. It is known that depurination is easily caused under acidic conditions, but very difficult to occur under basic conditions (Non-patent Document 14). Non-Patent Document 15 describes a deoxynucleotide containing a non-natural base that causes a reaction similar to that of depuration under basic conditions in which the depletion is unlikely to occur with a natural base. However, when the deoxynucleoside described in the document is used, a complicated operation of oxygen bubbling is required for DNA cleavage. In addition, a non-natural base that is stable under conditions of DNA synthesis by an automatic DNA synthesizer and / or DNA replication conditions such as PCR and functions as a template in a DNA replication reaction in the same manner as natural bases. No deoxynucleotides have been reported.

  When an oligodeoxynucleotide (hereinafter also referred to as “DNA oligomer” or “oligo DNA”) is artificially synthesized, an automatic DNA synthesizer is used. A DNA oligomer synthesized using an automatic DNA synthesizer is usually not phosphorylated at the 5 'end. For this reason, when the synthetic DNA oligomer is linked to other DNA (DNA derivative) using a DNA ligation enzyme, it is necessary to phosphorylate the 5 'end in advance.

  Methods for phosphorylating the 5 ′ and / or 3 ′ end of DNA include a method of treating a DNA fragment with a polynucleotide kinase (in the case of 5 ′ end phosphorylation), and a method for terminal phosphorylation of synthetic DNA using phosphorylated amidite (Patent Document 3 and Non-Patent Documents 16 and 17). Among them, in the method using phosphorylated amidite, the phosphorylated amidite has a coupling efficiency not 100%, and thus phosphorylated DNA and non-phosphorylated DNA are generated. Here, in the case of phosphorylation at the 5 'end, there is a problem that it is difficult to separate phosphorylated DNA and unphosphorylated DNA by reverse phase HPLC. As a method to improve the separation between phosphorylated DNA and non-phosphorylated DNA in reverse phase HPLC, DNA oligomers are purified in a form in which the terminal phosphate portion of DNA is protected with a protecting group such as DMTr group, Thereafter, there is a method of deprotecting the DMTr group by acid treatment. However, in this method, since an acid is used for deprotection, there is a problem that a depreciation reaction occurs and DNA is destroyed (Non-patent Document 18).

Japanese Patent Laid-Open No. 10-66576 US Pat. No. 7393635 U.S. Patent No. 5959090 US Patent 7794936 US Pat. No. 5,432,272 US Pat. No. 5,564,985 International Publication No. 2007/058326 Pamphlet International Publication No. 2009/101810 Pamphlet U.S. Patent No. 6864049

How restriction enzymes became the workhorses of molecular biology.Roberts, RJ., PNAS, 2005, 102, 17, p. 5905-5908 Structure and function of type II restriction endonucleases. Pingoud, A. and Jeltsch, A., Nucleic Acids Res., 2001, 29, 18, p. 3705-3727 Cleavage of a DNA-RNA-DNA / DNA chimeric substrate containing a single ribonucleotide at the DNA-RNA junction with prokaryotic RNases HII., Haruki, M., Tsunaka, Y., Morikawa, M. and Kanaya, S., FEBS Letters , 2002, 531, p. 204-208 Universal restriction site-free cloning method using chimeric primers. Chen, G. J., Qiu, N. and Page, M. G. P., Biotechniques, 2002, 32, 3, p. 518-520 Seamless gene engineering using RNA- and DNA-overhang cloning. Coljee, V. W., Mrrray, H. L., Donahue, W. F. and Jarrell, K. A., Nature Biotechnology, 2000, 18, p. 789-791 PHUSER (Primer Help for USER): a novel tool for USER fusion primer design.Olson, LR, Hansen, NB, Bonde, MT, Genee, HJ, Holm, DK, Carlsen, S., Hansen, BG, Patil, KR, Mortensen, UH and Wernersson, R. Nucleic Acids Res. 2011, 39, p. W61-67. A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. Norholm, M. H. BMC Biotechnol. 2010, 10, p. 21. An efficient method to assemble linear DNA templates for in vitro screening and selection systems.Stein, V. and Hollfelder, F. Nucleic Acids Res. 2009, 37, 18, p.e122. USER friendly DNA engineering and cloning method by uracil excision.Bitinaite, J., Rubino, M., Varma, KH, Schildkraut, I., Vaisvila, R. and Vaisskunaite, R. Nucleic Acids Res. 2007, 35, 6, p. 1992-2002. USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products.Geu-Flores, F., Nour-Eldin, HH, Nielsen, MT and Halkier, BA Nucleic Acids Res. 2007, 35, 7, p e55. Total synthesis of long DNA sequences: Synthesis of a contiguous 32-kb polyketide synthase gene cluster.Kodumal, SJ, Patel, KG, Reid, R., Menzella, HG, Welch, M. and Santi, DV PNAS 2004, 101, 44, p. 15573-15578. Generation of cohesive ends on PCR products by UDG-mediated excision of dU, and application for cloning into restriction digest-linearized vectors.Smith, C., Day, PJR and Walker, MR Genome Res. 1993, 2, p. 328- 332 Chemistry of thermal degradation of abasic sites in DNA.Mechanistic investigation on thermal DNA strand cleavage of alkylated DNA.Sugiyama, H., Fujiwara, T., Ur, A., Tahiro, T., Yamamoto, K., Kawanishi, S. And Saito, I., Chem. Res. Toxicol., 1994, 7, p. 673-683 Rate of depurination of native deoxyribonucleic acid.Lidahl, T. and Nyberg, B. Biochemistry, 1972, 11, 19, p. 3610-3618 Chemical restriction: strand cleavage by ammonia treatment at 8-oxoguanine yields biologically active DNA.Meyer, C., Meyer, D., Bickle, T. A. and Giese, B. ChemBioChem, 2003, 4, p. 610-614 A new approach for chemical phosphorylation of oligonucleotides at the 5'-terminus. Guzaev, A., Salo, H., Azhayev, A. and Lonnberg, H., Tetrahedron, 1995, 51, 34, p. 9375-9384 A chemical 5’-phosphorylation of oligodeoxyribonucleotides that can be monitored by trityl cation release. Horn, T. and Urdea, M. S. Tetrahedron Letters, 1986, 27, 39, p. 4705-4708 Rate of depurination of native deoxyribonucleic acid.Lidahl, T. and Nyberg, B., Biochemistry, 1972, 11, 19, p. 3610-3618 Efficient Sonogashira coupling of unprotected halonucleosides in aqueous solvents using water-soluble palladium catalysts. Cho, J. H., Prickett, C. D. and Shaughnessy, K. H. Eur. J. Org. Chem. 2010, p. 3678-3683 Design and studies of novel 5-substituted alkynyl-pyrimidine nucleosides as potent inhibitors of mycobacteria.Rai, D., Johar, M., Manning, T., Agrawal, B., Kunimoto, DY and Kumar, RJ Med. Chem. 2005 Years, 48, 22, p. 7012-7017 Palladium-catalized synthesis of uridines on polystyrene-based solid support.Aucagne, V., Berteina-Raboin, S., Guenot, P. and Agrofoglio, L. A. J. Comb. Chem. 2004, 6, 5, p. 717-723 Palladium-copper catalyzed coupling of terminal alkynes with iodouridines. Yu, J., Zhang, W., Zhang, L., Lu, J. and Zhu, J. Zhongguo aowu Huaxue Zazhi 2001, 11, 6, p. 349- 352 Synthesis, biological activities, and conformation study of 5-alkynyl-2'-deoxyuridines.Vincent, P., Beaucourt, JP, Pichat, L., Balzarini, J. and De Clercq, E. Nucleosides & Nucleotides 1985, 4, 4, p. 429-445 Electron transfer of plurimodified DNA SAMs. Rospigliosi, A., Ehlich, R., Hoerber, H., Middelberg, A. and Moggridge, G. Langmuir 2007, 23, 15, p. 8264-8271 Oligodeoxynucleotides incorporating structurally simple 5-alkynyl-2’-deoxyuridines fluorometrically respond to hybridization. Hudson, R. H. E. and Ghorbani-Choghmarani, A. Org. Biomol. Chem. 2007, 5, p. 1845-1848 Zinc-catalyzed cycloisomerizations.Synthesis of substituted furans and furopyrimidine nucleosides.Sniady, A., Durham, A., Morreale, MS, Marcinek, A., Szafert, S., Lis, T., Brzezinska, KR, Iwasaki, T. , Osshima, T., Mashima, K. and Dembinski, RJ Org. Chem. 2008, 73, p. 5881-5889 Synthesis and antiviral activity of novel acyclic nucleosides in the 5-alkynyl- and 6-alkylfuro [2,3-d] pyrimidine series. Amblard, F., Aucagne, V., Guenot, P., Schinazi, RF and Agrofoglio, LA Bioorg. Med. Chem. 2005, 13, p. 1239-1248 NMR conformational analysis of p-tolyl furanopyrimidine 2'-deoxyribonucleoside and crystal structure of its 3 ', 5'-di-O-acetyl derivative. Esho, N., Desaulniers, J., Davies, B., Chui, HM-P ., Rao, MS, Chow, SS, Szafert, S. and Dembinski, R. Bioorg. Med. Chem. 2005, 13, p. 1231-1238 Highly potent and selective inhibition of varicella-zoster virus by bicyclic furopyridmidine nucleosides bearing an aryl side chain.McGuigan, C., Barucki, H., Blewett, S., Carangio, A., Erichsen, JT, Andrei, G., Snoeck , R., Clercq, ED and Balzarini, JJ Med. Chem. 2000, 43, p. 4993-4997 CyDNA: Synthesis and replication of highly Cy-dye substituted DNA by an evolved polymerase.Ramsay, N., Jemth, A., Brown, A., Crampton, N., Dear, P. and Holliger, PJ Am. Chem. Soc 2010, 132, p. 5096-5104 Cross-coupling reactions of nucleoside triphosphates followed by polymerase incorporation. Construction and application of base-functionalized nucleic acids. Hocek, M. and Fojta, M. Org. Biomol. Chem. 2008, 6, p. 2233-2241 An efficient method for the construction of functionalized DNA bearing amino acid groups through cross-coupling reactions of nucleoside triphosphates followed by primer extension or PCR.Capek, P., Cahove, H., Pohl, R., Hocek, M., Cloeckner, C. and Marx, A. Chem. Eur. J. 2007, 13, p. 6196-6203 Systematic characterization of 2'-deoxynucleoside-5'-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA.Kuwahara, M., Nagashima, J., Hasegawa, M., Tamura, T ., Kitagata, R., Hanawa, K., Hososhima, S., Kasamatsu, T., Ozaki, H. and Sawai, H. Nucleic Acids Res. 2006, 34, 19, p. 5383-5394 Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker and rigidity.Lee, SE, Sidorov, A., Gourlain, T., Mignet, N., Thorpe, SJ, Brazier, JA, Dickman, MJ, Hornby, DP , Grasby, JA and Williams, DM Nucleic Acids Res. 2001, 29, 7, p. 1565-1573 Synthesis of metallointercalator-DNA conjugates on a solid support. Holmlin, R. E., Dandliker, P. J. and Barton, J. K., Bioconjug Chem. 1999, 10, 6, p. 1122-30 Nucleic acid related compounds. 40. Synthesis and biological activities of 5-alkynyluracil nucleosides. De Clercq E., Descamps J., Balzarini J., Giziewicz J., Barr PJ and Robins MJJ Med. Chem. 1983, 26, p. 661-666 Structural requirements of olefinic 5-substituted deoxyuridines for antiherpes activity.Goodchild J., Porter R. A., Raper R. H., Sim I. S., Upton R. M., Viney J. and Wadsworth H. J. J. Med. Chem. 1983, 26, p. 1252-1257 Synthesis and cyclic incorporation of a novel, bicyclic pyrimidine nucleoside: a thymidine mimic.Loakes, D., Brown, DM, Salisbury, SA, McDougall, MG, Neagu, C., Nampalli, S. and Kumar, S. Tetrahedron Lett. 2003, 44, p. 3387-3389 Synthesis and some biochemical properties of a novel 5,6,7,8-tetrahydropyrimido [4,5-c] pyridazine nucleoside. Loakes, D., Brown, DM, Salisbury, SA, McDougall, MG, Neagu, C., Nampalli , S. and Kumar, S. Helvetica Chimica Acta 2003, 86, p. 1193-1204 Pyrrolo-dC and pyrrolo-C: fluorescent analogs of cytidine and 20-deoxycytidine for the study of oligonucleotides.Berry, DA, Jung, KY., Wise, DS, Sercel, AD, Pearson, WH, Mackie, H., Randolph, JB and Somers, RL Tetrahedron Lett. 2004, 45, p. 2457-2461 Design and studies of novel 5-substituted alkynylpyrimidine nucleosides as potent inhibitors of mycobacteria.Rai D., Johar M., Manning T., Agrawal B., Kunimoto DY and Kumar RJ Med. Chem. 2005, 48, p. 7012- 7017 Oligodeoxynucleotides containing C-5 propyne analogs of 2'-deoxyuridine and 2'-deoxycytidine. Froehler, B. C., Wadwani, S., Terhorst, T. J. and Gerrard, S. R. Tetrahedron Lett. 1992, 33, p. 5307-5310 Antisense gene inhibition by oligonucleotides containing C-5 propyne pyrimidines.Wagner, R.W., Matteucci, M.D., Lewis, J. G., Gutierrez, A. J., Molds, C. and Froehler, B. C. Science 1993, 260, 5113, p. 1510-1513 Antisense gene inhibition by C-5-substituted deoxyuridine-containing oligodeoxynucleotides.Gutierrez, AJ, Matteucci, MD, Grant, D., Matsumura, S., Wagner, RW and Froehler, BC Biochemistry. 1997, 36, p. 743- 748 Long-range cooperativity in molecular recognition of RNA by oligodeoxynucleotides with multiple C5- (1-propynyl) pyrimidines. Barnes, T. W. 3rd and Turner, D. H. J. Am. Chem. Soc. 2001, 123, p. 4107-4118 Propynyl groups in duplex DNA: stability of base pairs comprising 7-substituted 8-aza- 7-deazapurines or 5-substituted pyrimidines. He, J. and Seela, F .. Nucleic Acids Res. 2002, 30, p. 5485- 5496 8-Aza-7-deazapurine-pyrimidine base pairs: the contribution of 2- and 7-substituents to the stability of duplex DNA.He, J. and Seela, F. Tetrahedron 2002, 58, p. 4535-4542 DNA triplex formation with 5-dimethylaminopropargyl deoxyuridine. Rusling, D. A., Peng, G., Srinivasan, N., Fox, K. R. and Brown, T. Nucleic Acids Res. 2009, 37, p. 1288-1296 http://www.glenresearch.com/ProductFiles/10-1054.html Nucleic acid related coumpounds. 40. Synthesis and biological activities of 5-alkynyluracil nucleosides. De Clercq, E., Descamps, J., Balzarini, J., Giziewicz, J., Barr, PJ, and Robins, MJJ Med. Chem. 1983, 26, 5, p. 661-6 Synthesis and antiviral activity of the carbocyclic analogues of 5-ethyl-2'-deoxyuridine and of 5-ethynyl-2'-deoxyuridine. Shealy, YF, O'Dell, CA, Arnett, G., and Shannon, WMJ Med. Chem. 1986, 29, 1, p. 79-84 5-Alkynyl-2'-deoxyuridines: chromatography-free synthesis and cytotoxicity evaluation against human breast cancer cells.Meneni, S., Ott, I., Sergeant, C. D ,, Sniady, A., Gust, R., and Dembinski, R. Bioorg Med Chem. 2007, 15, 8, p. 3082-8 C5-Modified nucleosides exhibiting anticancer activity. Lee, YS, Park, SM, Kim, HM, Park, SK, Lee, K., Lee, CW, and Kim, BH Bioorg. Med. Chem. Lett. 2009, 19, 16, p. 4688-91 A chemical method for fast and sensitive detection of DNA synthesis in vivo.Salic, A. and Mitchison, T.J.Proc. Natl. Acad. Sci. USA 2008, 105, 7, p. 2415-20 A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU) .Limsirichaikul, S., Niimi, A., Fawcett, H., Lehmann, A., Yamashita, S. and Ogi , T. Nucleic Acids Res. 2009, 37, 4, e31 Chick embryo proliferation studies using EdU labeling. Warren, M., Puskarczyk, K. and Chapman, S. C. Dev. Dyn. 2009, 238, 4, p. 944-9 Evaluation of 5-ethynyl-2'-deoxyuridinestaining as a sensitive and reliable method for studying cell proliferation in the adult nervous system.Zeng, C., Pan, F., Jones, LA, Lim, MM, Griffin, EA, Sheline, YI, Mintun, MA, Holtzman, DM, and Mach, RH Brain Res. 2010, 1319, p. 21-32 A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine.Kotogany, E., Dudits, D., Horvath, GV and Ayaydin, F. Plant Methods 2010, 6, 1 , p. 5 Fluorogenic "click" reaction for labeling and detection of DNA in proliferating cells., Li, K., Lee, L. A., Lu, X. and Wang, Q., Biotechniques. 2010, 49, 1, p. 525-7 Visualization of mitochondrial DNA replication in individualcells by EdU signalamplification.Haines, K. M., Feldman, E. L., Lentz, S. I. J. Vis. Exp. 2010, 45, pii, 2147 Monitoring DNA replication in fission yeast by incorporation of 5-ethynyl-2'-deoxyuridine. Hua, H. and Kearsey, S. E. Nucleic Acids Res. 2011, 39, 9, e60 Replication Banding Patterns in Human Chromosomes DetectedUsing 5-ethynyl-2'-deoxyuridine Incorporation.Hoshi, O. and Ushiki, T. Acta Histochem Cytochem. 2011, 44, 5, p. 233-7 Super-resolution fluorescence imaging of chromosomal DNA. Zessin, P. J., Finan, K. and Heilemann, M. J. Struct. Biol. 2012, 177, 2, p. 344-8 Imaging of EdU, an alkyne-tagged cell proliferation probe, by Raman microscopy.Yamakoshi, H., Dodo, K., Okada, M., Ando, J., Palonpon, A., Fujita, K., Kawata, S. And Sodeoka, MJ Am. Chem. Soc. 2011, 133, 16, p. 6102-5 5-Alkynyl-2'-deoxyuridines: chromatography-free synthesis and cytotoxicityevaluation against human breast cancercells.Meneni, S., Ott, I., Sergeant, CD, Sniady, A., Gust, R. and Dembinski, R. Bioorg Med Chem. 2007, 15, 8, p. 3082-8 Synthesis and dosage incorporation of modified deoxyuridine triphosphates. Borsenberger, V., Kukwikila, M. and Howorka, S. Org. Biomol. Chem. 2009, 7, 18, p. 3826-35 Cleavage of functionalized DNA containing 5-modifiedpyrimidines by type II restriction endonucleases. Macickova-Cahova, H., Pohl, R., and Hocek, M. Chembiochem. 2011, 12, 3, p. 431-8 DNA duplexes stabilized by modified monomer residues: synthesis and stability.Graham, D., Parkinson, J. A. and Brown, T. J. Chem. Soc. Perkin Trans. 1 1998, p. 1131-1138 Site-directed spin-labeling of DNA by the azide-alkyne 'click' reaction: nanometer distance measurements on 7-deaza-2'-deoxyadenosine and 2'-deoxyuridine nitroxide conjugates spatially separated or linked to a 'dA-dT' base pair Ding, P., Wunnicke, D., Steinhoff, HJ and Seela, F. Chem. Eur. J. 2010, 16, 48, p. 14385-96 Highly sequence specific RNA terminal labeling by DNA photoligation.Yoshimura, Y., Noguchi, Y. and Fujimoto, K. Organic Biomol. Chem. 2007, 5, p. 139-142 Effective synthesis of photosensitive oligonucleotides. Ogino, M., Taya, Y. and Fujimoto, K. Nucleic Acids Symp. Ser. 2008, 52, p. 395-396 Photochemical site-specific mutation of 5-methylcytosine to thymine.Matsumura, T., Ogino, M., Nagayoshi, K. and Fujimoto, K. Chem. Lett. 2008, 37, p. 94-95 The synthesis and antiviral properties of (E) -5- (2-bromovinyl) -2'-deoxyuridine-related compounds. Ashwell, M., Jones, AS, Sayers, JR, Walker, RT, Sakuma, T. and De Clercq , E. Tetrahedron 1987, 43, p. 4601-4608 Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Langer, P. R., Waldrop, A. A. and Ward, D. C. Proc. Natl. Acad. Sci. U S A. 1981, 78, p 6633-6637 The application of a modified nucleotide in aptamer selection: novel thrombin aptamers containing 5- (1-pentynyl) -2'-deoxyuridine. Latham, JA, Johnson, R. and Toole, JJ Nucleic Acids Res. 1994, 22, p 2817 -2822. Expanding the potential of DNA for binding and catalysis: highly functionalized dUTP derivatives that are substrates for thermostable DNA polymerases.Sakthivel, K. and Barbas, III, C. F. Angew. Chem. Int. Ed. 1998, 37, p 2872-2875. Systematic characterization of 2'-deoxynucleoside- 5'-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on experimental production of modified DNA.Kuwahara, M., Nagashima, J., Hasegawa, M., Tamura, T ., Kitagata, R., Hanawa, K., Hososhima, S., Kasamatsu, T., Ozaki, H., and Sawai, H. Nucleic Acids Res. 2006, 34, p. 5383-5394

  The methods described in Patent Documents 1 and 2 and Non-Patent Documents 3 to 12 known as methods for cleaving DNA use an enzyme such as RNase or uracil-removing enzyme for cleaving DNA as described above. Therefore, it is disadvantageous in terms of cost. In general, an enzymatic cleavage reaction must satisfy special reaction conditions relating to pH and / or metal ion concentration. The method described in Non-Patent Document 15 is advantageous in terms of cost and reaction efficiency because DNA is cleaved by a chemical reaction without using an enzyme. However, deoxynucleotides containing non-natural bases described in the literature are under conditions for DNA synthesis by an automatic DNA synthesizer compared to deoxynucleotides containing corresponding natural bases (A, T, G or C). And / or low stability under DNA replication conditions such as PCR, and low fidelity when used in in vivo and in vitro reactions such as DNA replication reactions. In addition, when the deoxynucleoside described in the document is used, a complicated operation of oxygen bubbling is required for DNA cleavage.

  The methods described in Patent Document 3 and Non-Patent Documents 16 and 17 known as methods for phosphorylating the 5 ′ and / or 3 ′ end of DNA have a coupling efficiency of phosphorylated amidite not 100% as described above. In addition, there is a problem that it is difficult to separate phosphorylated DNA from non-phosphorylated DNA.

  In order to cleave the DNA strand at the desired site and phosphorylate the cleaved end, the DNA is cleaved into two fragments by a chemical reaction, and the 5 ′ end of the resulting DNA fragment becomes the phosphate end. The method of introducing a deoxynucleotide containing a non-natural base into a DNA strand as a cleavage site is advantageous in that it is low-cost, has few restrictions on reaction conditions, and is easy to carry out experimental operations. . Therefore, the present invention introduces deoxynucleotides containing unnatural bases into the DNA strand, thereby cleaving the DNA strand with a low-cost and simple experimental operation by a chemical reaction, and the resulting end is phosphorylated. It is an object of the present invention to provide an oligodeoxynucleotide.

  As a result of various studies on means for solving the above-mentioned problems, the present inventors have used the above object by using a deoxynucleotide or deoxynucleoside containing a non-natural base having a moiety removable by a nucleophile. The present invention has been completed.

  That is, the gist of the present invention is as follows.

(1) Nucleophiles and formula I:
(A) _n -BC
[Where:
A is an oligodeoxynucleotide,
B is a deoxynucleoside containing a non-natural base having a moiety E removable by a nucleophile;
C is an oligodeoxynucleotide,
n is 0 or 1,
When n is 1, the 3 ′ end of A and the 5 ′ position of B are linked by a phosphodiester bond,
The 3 ′ position of B and the 5 ′ end of C are linked by a phosphodiester bond,
The phosphodiester bond between B and C is such that the oligodeoxynucleotide part phosphorylated at the 5 'end of C from the 3' position of B with E elimination induced by nucleophile attack. Disconnected by detachment]
By contacting the oligodeoxynucleotide represented by the following formula, the phosphodiester bond between B and C is released from the 3 'position of B by phosphorylation of the 5' end of C. An oligodeoxynucleotide which has been cleaved to phosphorylate the 5 ′ end of C, and a compound of formula II:
(A) _n -B '
[Where:
A and n have the same meaning as defined in Formula I,
B ′ is the residue part of B that is formed with the elimination of E by nucleophile attack]
A method for producing an oligodeoxynucleotide, comprising a 5′-terminal phosphorylated oligodeoxynucleotide forming step, which obtains an oligodeoxynucleotide represented by the formula:

(2) Formula II:
(A) _n -B '
[Where:
A and B ′ have the same meaning as in the above (1),
n is 1]
By reacting with oligodeoxynucleotide represented by Formula IIa:
A-PO ₃ H ₂
The method according to (1), further comprising a 3′-terminal phosphorylated oligodeoxynucleotide forming step of obtaining an oligodeoxynucleotide phosphorylated at the 3 ′ end represented by:

(3) The deoxynucleoside B containing the unnatural base is:

[Where:
R is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, Substituted or unsubstituted heteroarylalkyl or carbonyl;
R ′ is hydrogen or substituted or unsubstituted alkyl, alkenyl or alkynyl;
r is an integer from 0 to 4;
R ″ is halogen or substituted or unsubstituted alkyl, alkenyl or alkynyl;
R ′ ″ is —COZ, —C≡CZ or —CN;
Z is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, Or substituted or unsubstituted heteroarylalkyl]
The method according to (1) or (2), wherein the method is selected from deoxynucleosides represented by:

  (4) Before the 5′-terminal phosphorylated oligodeoxynucleotide forming step, a deoxynucleoside B containing a non-natural base having a moiety E removable by a nucleophile or a derivative thereof, and a natural base Any of the above (1) to (3), further comprising an oligodeoxynucleotide synthesis step of synthesizing an oligodeoxynucleotide represented by formula I using a substrate containing one or more deoxynucleosides or derivatives thereof the method of.

(5) Formula I:
(A) _n -BC
[Where:
A is an oligodeoxynucleotide,
B is a deoxynucleoside containing a non-natural base having a moiety E removable by a nucleophile;
C is an oligodeoxynucleotide,
n is 0 or 1,
When n is 1, the 3 ′ end of A and the 5 ′ position of B are linked by a phosphodiester bond,
The 3 ′ position of B and the 5 ′ end of C are linked by a phosphodiester bond,
The phosphodiester bond between B and C is such that the oligodeoxynucleotide part phosphorylated at the 5 'end of C from the 3' position of B with E elimination induced by nucleophile attack. Disconnected by detachment]
A DNA fragment amplification step of amplifying a linear double-stranded DNA fragment using the oligodeoxynucleotide represented by
By contacting the nucleophile with the amplified linear double-stranded DNA fragment, the phosphodiester bond between B and C becomes an oligodeoxynucleotide moiety phosphorylated at the 5 'end of C. It is cleaved by detaching from the 3 ′ position of B, the 5 ′ end of C contained in the linear double-stranded DNA fragment is phosphorylated, and at least the primer part represented by Formula I Forming a linear double-stranded DNA fragment having a 3 ′ protruding end containing a sequence complementary to a part thereof; and a linear double-stranded DNA fragment having the 3 ′ protruding end; And a ligation step of ligating another double-stranded DNA fragment;
A method for ligating double-stranded DNA.

(6) A kit for use in the method for producing an oligodeoxynucleotide according to any one of (1) to (4) or the method for ligating double-stranded DNA according to (5), wherein the formula I:
(A) _n -BC
[Where:
A is an oligodeoxynucleotide,
B is a deoxynucleoside containing a non-natural base having a moiety E removable by a nucleophile;
C is an oligodeoxynucleotide,
n is 0 or 1,
When n is 1, the 3 ′ end of A and the 5 ′ position of B are linked by a phosphodiester bond,
The 3 ′ position of B and the 5 ′ end of C are linked by a phosphodiester bond,
The phosphodiester bond between B and C is such that the oligodeoxynucleotide part phosphorylated at the 5 'end of C from the 3' position of B with E elimination induced by nucleophile attack. Disconnected by detachment]
Or at least a deoxynucleoside B containing a non-natural base or a derivative thereof having a moiety E removable by a nucleophile.

(7) The following formula:

[Where:
R is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, Substituted or unsubstituted heteroarylalkyl or carbonyl;
R ′ is methyl;
r is an integer from 0 to 4;
R ″ is halogen or substituted or unsubstituted alkyl, alkenyl or alkynyl;
R ′ ″ is —COZ, —C≡CZ or —CN;
Z is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, Or substituted or unsubstituted heteroarylalkyl]
A deoxynucleoside comprising an unnatural base represented by

  According to the present invention, it is possible to cleave a DNA strand by a chemical reaction at a low cost and with a simple experimental operation, and to provide an oligodeoxynucleotide whose resulting end is phosphorylated.

  This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2012-084249, which is the basis of the priority of the present application.

It is process drawing which shows one Embodiment of the manufacturing method of the oligodeoxynucleotide of this invention. In the synthesis example 1-4, it is a figure which shows the reverse phase HPLC chromatogram of the synthetic | combination DNA oligomer (5'-TTTTCTTPTCCTTTT-3 '). A: synthesized DNA oligomer; B: degradation product obtained by enzymatic degradation of the synthesized DNA oligomer. In the synthesis example 1-4, it is a figure which shows the reverse phase HPLC chromatogram of the synthetic | combination DNA oligomer (5'-TTTTTTPTTTTTT-3 '). In the synthesis example 1-4, it is a figure which shows the reverse phase HPLC chromatogram of the synthetic | combination DNA oligomer (5'-TTTTTAPATTTTT-3 '). In the synthesis example 1-4, it is a figure which shows the reverse phase HPLC chromatogram of the synthetic | combination DNA oligomer (5'-TTTTTCPCTTTTT-3 '). In the synthesis example 1-4, it is a figure which shows the reverse phase HPLC chromatogram of the synthetic | combination DNA oligomer (5'-TTTTTGPGTTTTT-3 '). In the synthesis example 1-4, it is a figure which shows the reverse phase HPLC chromatogram of the synthetic | combination DNA oligomer (5'-CGCAATPTAACGC-3 '). In the usage example 1-3, it is a figure which shows the reverse phase HPLC chromatogram before and behind reaction of the synthetic | combination DNA oligomer. A: DNA oligomer before reaction; B: Reaction mixture after reaction with methylamine; C: Degradation product obtained by enzymatic degradation of product 1; D: Degradation product obtained by enzymatic degradation of product 2. In the usage example 1-4, it is a figure which shows the HPLC chromatogram of each reaction mixture. A: Reaction mixture after reaction with methylamine; B: Reaction mixture after reaction with ethylenediamine; C: Reaction mixture after reaction with 2-aminoethanol. In the usage example 1-5, it is a figure which shows the reverse phase HPLC chromatogram before and behind reaction of each synthetic | combination DNA oligomer (5'-TTTTCTTPTCCTTTT-3 '). A: DNA oligomer before reaction; B: reaction mixture after reaction with ethylenediamine. In the usage example 1-5, it is a figure which shows the reverse phase HPLC chromatogram before and behind reaction of each synthetic | combination DNA oligomer (5'-TTTTTTPTTTTTT-3 '). A: DNA oligomer before reaction; B: reaction mixture after reaction with ethylenediamine. In the usage example 1-5, it is a figure which shows the reverse phase HPLC chromatogram before and behind reaction of each synthetic | combination DNA oligomer (5'-TTTTTAPATTTTT-3 '). A: DNA oligomer before reaction; B: reaction mixture after reaction with ethylenediamine. In the usage example 1-5, it is a figure which shows the reverse phase HPLC chromatogram before and behind reaction of each synthetic | combination DNA oligomer (5'-TTTTTCPCTTTTT-3 '). A: DNA oligomer before reaction; B: reaction mixture after reaction with ethylenediamine. In the usage example 1-5, it is a figure which shows the reverse phase HPLC chromatogram before and behind reaction of each synthetic | combination DNA oligomer (5'-TTTTTGPGTTTTT-3 '). A: DNA oligomer before reaction; B: reaction mixture after reaction with ethylenediamine. In the usage example 1-5, it is a figure which shows the reverse phase HPLC chromatogram before and behind reaction of each synthetic | combination DNA oligomer (5'-CGCAATPTAACGC-3 '). A: DNA oligomer before reaction; B: reaction mixture after reaction with ethylenediamine. In the usage example 1-6, it is a figure which shows the reverse phase HPLC chromatogram before and behind reaction of the synthetic | combination DNA oligomer. A: DNA oligomer before reaction; B: Reaction mixture after reaction with 2-aminoethanol; C: Enlarged view of the HPLC chromatogram of the reaction mixture after reaction. In the usage example 1-7, it is a figure which shows the reverse phase HPLC chromatogram of the DNA oligomer after cutting out and deprotecting. In the usage example 1-8, it is a figure which shows the reverse phase HPLC chromatogram of the DNA oligomer after cutting-out and a deprotection. In the usage example 1-8, it is a figure which shows the reverse phase HPLC chromatogram of the DNA oligomer after reaction by ethylenediamine. In the usage example 1-9, it is a figure which shows the reverse phase HPLC chromatogram of the DNA oligomer after cutting out and deprotecting. In the usage example 1-9, it is a figure which shows the reverse phase HPLC chromatogram of the DNA oligomer after 120 degreeC processing in a 100 mM phosphate buffer. In the usage example 1-10, it is a figure which shows the reverse phase HPLC chromatogram of the DNA oligomer after cutting and deprotection. In the usage example 1-10, it is a figure which shows the reverse phase HPLC chromatogram of the DNA oligomer after reaction by ethylenediamine. In the usage example 1-10, it is a figure which shows the reverse phase HPLC chromatogram of the DNA oligomer after 120 degreeC processing in a 100 mM phosphate buffer. In the usage example 1-11, it is a figure which shows the reverse phase HPLC chromatogram of the DNA oligomer by which the 5'-terminal after cut-out and deprotection was DMTr protected. In the usage example 1-11, it is a figure which shows the reverse phase HPLC chromatogram of the DNA oligomer by which the 5'-terminal after the reaction by ethylenediamine was phosphorylated. In the usage example 1-12, it is a figure which shows the reverse phase HPLC chromatogram of TT1 and TT2 after cutting and deprotection. A: Reverse phase HPLC chromatogram of TT1; B: Reverse phase HPLC chromatogram of TT2. In the usage example 1-12, it is a figure which shows the base sequence of each DNA oligomer, and the analysis result by polyacrylamide gel electrophoresis. A: base sequence of each DNA oligomer; B: analysis result by polyacrylamide gel electrophoresis. In the synthesis example 2, it is a figure which shows the reverse phase HPLC chromatogram of the synthetic | combination DNA oligomer (5'-TTTTCTT (Pr) TCCTTTT-3 '). In the synthesis example 2, it is a figure which shows the reverse phase HPLC chromatogram of the synthetic | combination DNA oligomer (5'-TTTTTT (Pr) TTTTTT-3 '). In the usage example 2-1, it is a figure which shows the reverse phase HPLC chromatogram before and behind reaction of the synthetic | combination synthetic | combination DNA oligomer (5'-TTTTCTT (Pr) TCCTTTT-3 '). A: DNA oligomer before reaction; B: reaction mixture after reaction at 70 ° C. for 3 hours; C: reaction mixture after reaction at 70 ° C. for 16 hours. In the usage example 2-1, it is a figure which shows the MALDI TOF-MS spectrum of the product 1 and another by-product. A: Product 1; B: Small peak with retention time of 9.9 minutes; C: Peak with retention time of 10.2 minutes. In the usage example 2-1, it is a figure which shows the reverse phase HPLC chromatogram before and behind reaction of the synthetic | combination DNA oligomer (5'-TTTTTT (Pr) TTTTTT-3 ') synthesize | combined. A: DNA oligomer before reaction; B: reaction mixture after reaction at 70 ° C. for 3 hours; C: reaction mixture after reaction at 70 ° C. for 16 hours. In the usage example 2-1, it is a figure which shows the MALDI TOF-MS spectrum of the product 1 and another by-product. A: Product 1; B: Peak with retention time of 10.3 minutes. In the synthesis example 3, it is a figure which shows the reverse phase HPLC chromatogram of the synthetic | combination DNA oligomer (5'-TTTTCTT (Pa) TCCTTTT-3 '). In the synthesis example 3, it is a figure which shows the reverse phase HPLC chromatogram of the synthetic | combination DNA oligomer (5'-TTTTTT (Pa) TTTTTT-3 '). In the usage example 3-1, it is a figure which shows the reverse phase HPLC chromatogram before and behind reaction of the synthetic | combination DNA oligomer (5'-TTTTCTT (Pa) TCCTTTT-3 '). A: DNA oligomer before reaction; B: reaction mixture after reaction at 70 ° C. for 10 minutes; C: reaction mixture after reaction at 70 ° C. for 30 minutes. In the usage example 3-1, it is a figure which shows the reverse phase HPLC chromatogram before and behind reaction of the synthetic | combination DNA oligomer (5'-TTTTTT (Pa) TTTTTT-3 '). A: DNA oligomer before reaction; B: reaction mixture after reaction at 70 ° C. for 10 minutes; C: reaction mixture after reaction at 70 ° C. for 30 minutes. In the usage example 3-2, it is a figure which shows the reverse phase HPLC chromatogram after reaction of the synthetic | combination DNA oligomer (5'-TTTTTT (Pa) TTTTTT-3 '). A: Reaction mixture after reaction with methylamine; B: Reaction mixture after reaction with ethylenediamine. In the usage example 3-3, it is a figure which shows the reverse phase HPLC chromatogram before and behind cleavage reaction of each synthetic | combination DNA oligomer (5'-TTTTTT (Pa) TTTTTT-3 '). A: DNA oligomer before reaction; B: reaction mixture after cleavage reaction. In the synthesis example 4-3, it is a figure which shows the reverse phase HPLC chromatogram of the synthetic | combination DNA oligomer. A: 5'-TTTTCTT (CvU) TCCTTTT-3 '; B: 5'-TTTTTT (CvU) TTTTTT-3'. In the usage example 4-1, it is a figure which shows the reverse phase HPLC chromatogram before and behind reaction of each synthetic | combination DNA oligomer (5'-TTTTCTT (CvU) TCCTTTT-3 '). A: DNA oligomer before reaction; B: reaction mixture after reaction at 70 ° C. for 3 hours; C: reaction mixture after reaction at 70 ° C. for 8 hours. In the usage example 4-1, it is a figure which shows the MALDI TOF-MS spectrum of the product 1 and another by-product. A: Product 1; B: Peak with retention time of 10.3 minutes. In the usage example 4-1, it is a figure which shows the reverse phase HPLC chromatogram before and behind reaction of each synthetic | combination DNA oligomer (5'-TTTTTT (CvU) TTTTTT-3 '). A: DNA oligomer before reaction; B: reaction mixture after reaction at 70 ° C. for 3 hours; C: reaction mixture after reaction at 70 ° C. for 8 hours. In the usage example 4-1, it is a figure which shows the MALDI TOF-MS spectrum of the product 1 and another by-product. A: Product 1; B: Peak with a retention time of 10.5 minutes. In the synthesis example 5-4, it is a figure which shows the reverse phase HPLC chromatogram of the synthetic | combination DNA oligomer. A: 5'-TTTTCTT (PC) TCCTTTT-3 '; B: 5'-TTTTTT (PC) TTTTTT-3'. In the usage example 5-1, it is a figure which shows the reverse phase HPLC chromatogram before and behind reaction of each synthetic | combination DNA oligomer (5'-TTTTCTT (PC) TCCTTTT-3 '). A: DNA oligomer before reaction; B: reaction mixture after reaction at 70 ° C. for 3 hours; C: reaction mixture after reaction at 70 ° C. for 16 hours. In the usage example 5-1, it is a figure which shows the MALDI TOF-MS spectrum of the product 1 and another by-product. A: Product 1; B: Peak with retention time 10.2 minutes; C: Peak with retention time 10.6 minutes. In the usage example 5-1, it is a figure which shows the reverse phase HPLC chromatogram before and behind reaction of each synthetic | combination DNA oligomer (5'-TTTTTT (PC) TTTTTT-3 '). A: DNA oligomer before reaction; B: reaction mixture after reaction at 70 ° C. for 3 hours; C: reaction mixture after reaction at 70 ° C. for 16 hours. In the usage example 5-1, it is a figure which shows the MALDI TOF-MS spectrum of the product 1 and another by-product. A: product 1; B: peak at retention time 10.4 minutes; C: peak at retention time 10.9 minutes. In Usage Example 6-1, the target DNA was amplified by polymerase chain reaction using a DNA oligomer containing an unnatural base as a primer, and the amplification product was analyzed by 10% non-denaturing polyacrylamide gel electrophoresis. . Lane 1: 100 base pair gene marker; Lane 2: Gene 1 amplification product (X = P); Lane 3: Gene 2 amplification product (X = Pa); Lane 4: Gene 2 amplification product (X = P) Lane 5: Gene 2 amplification product (X = Pa). In Use Example 6-2, the ligation reaction product of the 5′-terminal phosphorylated DNA fragment obtained by cleaving a DNA amplification product containing an unnatural base by the cleavage reaction of the present invention was subjected to 10% non-denaturing polyacrylamide gel electrophoresis. It is a figure which shows the result of having analyzed. Lane 1: 100 base pair gene marker; Lane 2: Amplification product of gene 1 (X = P) was cleaved by the cleavage reaction of the present invention, and obtained 5′-terminal phosphorylated DNA fragment; Lane 3: Gene 2 (X = P) amplification product of the present invention was cleaved by the cleavage reaction of the present invention, and the resulting 5′-end phosphorylated DNA fragment; Lane 4: DNA amplification products of genes 1 and 2 (X = P) were cleaved by the present invention. The ligation product obtained by cleaving and reacting the obtained 5 ′ terminal phosphorylated DNA fragments of genes 1 and 2 with ligase; Lane 5: DNA amplification products of genes 1 and 2 (X = Pa) are cleaved according to the present invention Ligation product obtained by reacting the 5′-terminal phosphorylated DNA fragments of genes 1 and 2 with ligase obtained by cleaving with lane 6; Lane 6: gene 3 (the same nucleotide sequence as the ligation product obtained by linking genes 1 and 2) Amplification product of a gene having 299 base pairs). In the synthesis example 7-4, it is a figure which shows the reverse phase HPLC chromatogram of the synthetic | combination DNA oligomer (5'-TTTTTTQTTTTTT-3 '). In the synthesis example 7-4, it is a figure which shows the reverse phase HPLC chromatogram of the synthetic | combination DNA oligomer (5'-TTTTCTTQTCCTTTT-3 '). In the usage example 7-1, it is a figure which shows the reverse phase HPLC chromatogram after reaction of the synthetic | combination DNA oligomer (5'-TTTTTTQTTTTTT-3 '). In the usage example 7-1, it is a figure which shows the reverse phase HPLC chromatogram after reaction of the synthetic | combination DNA oligomer (5'-TTTTCTTQTCCTTTT-3 '). In the usage example 7-2, it is a figure which shows the reverse phase HPLC chromatogram after reaction of the synthetic | combination DNA oligomer (5'-TTTTTTQTTTTTT-3 '). A: Reaction mixture after reaction with methylamine; B: Reaction mixture after reaction with ethylenediamine. In the usage example 7-2, it is a figure which shows the reverse phase HPLC chromatogram after reaction of the synthetic | combination DNA oligomer (5'-TTTTCTTQTCCTTTT-3 '). A: Reaction mixture after reaction with methylamine; B: Reaction mixture after reaction with ethylenediamine. In the synthesis example 8-1, it is a figure which shows the reverse phase HPLC chromatogram of the synthetic | combination DNA oligomer (5'-TTTTTT (HP) TTTTTT-3 '). In the synthesis example 8-1, it is a figure which shows the reverse phase HPLC chromatogram of the synthetic | combination DNA oligomer (5'-TTTTCTT (HP) TCCTTTT-3 '). In the usage example 8-1, it is a figure which shows the reverse phase HPLC chromatogram after reaction of the synthetic | combination DNA oligomer (5'-TTTTTT (HP) TTTTTT-3 '). In the usage example 8-1, it is a figure which shows the reverse phase HPLC chromatogram after reaction of the synthetic | combination DNA oligomer (5'-TTTTCTT (HP) TCCTTTT-3 '). In the usage example 8-2, it is a figure which shows the reverse phase HPLC chromatogram after reaction of the synthetic | combination DNA oligomer (5'-TTTTTT (HP) TTTTTT-3 '). A: Reaction mixture after reaction with methylamine; B: Reaction mixture after reaction with ethylenediamine. In the usage example 8-2, it is a figure which shows the reverse phase HPLC chromatogram after reaction of the synthetic | combination DNA oligomer (5'-TTTTCTT (HP) TCCTTTT-3 '). A: Reaction mixture after reaction with methylamine; B: Reaction mixture after reaction with ethylenediamine. In the synthesis example 9-5, it is a figure which shows the reverse phase HPLC chromatogram of the synthetic | combination DNA oligomer (5'-TTTTTT (fPG) TTTTTT-3 '). In the synthesis example 9-5, it is a figure which shows the reverse phase HPLC chromatogram of the synthetic | combination DNA oligomer (5'-TTTTCTT (fPG) TCCTTTT-3 '). In the usage example 9-1, it is a figure which shows the reverse phase HPLC chromatogram after reaction of the synthetic | combination DNA oligomer (5'-TTTTTT (fPG) TTTTTT-3 '). In the usage example 9-1, it is a figure which shows the reverse phase HPLC chromatogram after reaction of the synthetic | combination DNA oligomer (5'-TTTTCTT (fPG) TCCTTTT-3 '). In the synthesis example 10-5, it is a figure which shows the reverse phase HPLC chromatogram of the synthetic | combination DNA oligomer (5'-TTTTTT (fPA) TTTTTT-3 '). In the synthesis example 10-5, it is a figure which shows the reverse phase HPLC chromatogram of the synthetic | combination DNA oligomer (5'-TTTTCTT (fPA) TCCTTTT-3 '). In the usage example 10-1, it is a figure which shows the reverse phase HPLC chromatogram after reaction of the synthetic | combination DNA oligomer (5'-TTTTTT (fPA) TTTTTT-3 '). In the usage example 10-1, it is a figure which shows the reverse phase HPLC chromatogram after reaction of the synthetic | combination DNA oligomer (5'-TTTTCTT (fPA) TCCTTTT-3 '). In Use Example 11-1, target DNA was amplified by polymerase chain reaction using a DNA oligomer containing 5-ethynyl-2′-deoxyuridine (Pa) as a primer, and the amplified product was analyzed by 1% agarose gel electrophoresis. It is a figure which shows a result. Lane 1: DNA marker; Lane 2: Amplification product when LL1 and LL3 are used as a primer set; Lane 3: Amplification product when LL2 and LL4 are used as a primer set; Lane 4: LL5 and LL8 as a primer set Amplification product when used; Lane 5: Amplification product when LL6 and LL7 are used as a primer set. In Use Example 11-2, a ligation product of a 5′-terminal phosphorylated DNA fragment obtained by cleaving a DNA amplification product containing 5-ethynyl-2′-deoxyuridine (Pa) by the cleavage reaction of the present invention, It is a figure which shows the result analyzed by 1% agarose gel electrophoresis. Lane 1: DNA marker; Lanes 2 and 3: Ligation reaction product when ligation reaction is performed at 10 temperature cycles; Lanes 4 and 5: Ligation reaction product when ligation reaction is performed at 20 temperature cycles; Lanes 6 and 7: Ligation product when ligation reaction was performed with 30 temperature cycles. Lanes 2, 4, and 6: Ligation product when LL1 and LL3 are used as a primer set, and when LL6 and LL7 are used as a primer set Lanes 3, 5 and 7: Ligation reaction when the amplification product DNA when LL2 and LL4 are used as a primer set and the amplification product DNA when LL6 and LL7 are used as a primer set product. In Use Example 11-2, the 5′-terminal phosphorylated DNA fragment obtained by cleaving the DNA amplification product containing 5-ethynyl-2′-deoxyuridine (Pa) by the cleavage reaction of the present invention was subjected to the step before the ligation reaction. It is a figure which shows the result of having analyzed DNA and DNA after a ligation reaction by 1% agarose gel electrophoresis. Lane 1: DNA marker; Lanes 2 and 3: Cleavage product of DNA cleavage reaction after 9 hours of methylamine treatment; Lanes 4 and 5: DNA cleavage reaction after 24 hours of methylamine treatment Lane 6 and 7: DNA cleavage treatment after 48 hours of methylamine treatment; Lane 8 and 9: DNA cleavage after 48 hours of methylamine treatment Ligation reaction product when ligation reaction is performed with 30 temperature cycles after reaction. Lanes 2, 4, 6, and 8: Cleavage of the amplification product DNA when LL1 and LL3 are used as a primer set and the amplification product DNA when LL6 and LL7 are used as a primer set Reaction product or ligation product when cleaved and ligated; Lanes 3, 5, 7, and 9: DNA of amplification product when LL2 and LL4 are used as a primer set, and LL6 and LL7 are used as a primer set A cleavage reaction product in the case of a cleavage reaction with the amplification product DNA or a ligation reaction product in a cleavage reaction and a ligation reaction. In Use Example 11-3, a ligation reaction product of a 5 ′ terminal phosphorylated DNA fragment obtained by cleaving a DNA amplification product containing 5-ethynyl-2′-deoxyuridine (Pa) by the cleavage reaction of the present invention, It is a figure which shows the result analyzed by 1% agarose gel electrophoresis. Lane 1: DNA marker; Lanes 2, 3, 6, and 7: Amplification product DNA when LL1 and LL4 are used as a primer set and amplification product DNA when LL5 and LL8 are used as a primer set Ligation reaction product when reacted: Lanes 4, 5, 8, and 9: DNA of amplification product when LL2 and LL3 are used as a primer set, and DNA of amplification product when LL6 and LL7 are used as a primer set Ligation reaction product when ligation reaction is performed. Lanes 2 to 5: Ligation reaction product when DNA is ligated using T4 DNA ligase; Lanes 6 to 9: Ligation reaction product when DNA is ligated using Taq DNA ligase. Lanes 2, 4, 6, and 8: Ligation reaction product when DNA is ligated using a mixed solution of 2 μL of the amplified product DNA; Lanes 3, 5, 7, and 9: 4 μL of the amplified product DNA Ligation reaction product when DNA is ligated using a mixed solution of

  Hereinafter, preferred embodiments of the present invention will be described in detail.

<1. Definition>
As used herein, “oligodeoxynucleotide” means deoxyribonucleic acid (DNA), single-stranded or double-stranded oligodeoxynucleotide or polydeoxynucleotide, or a derivative thereof. The oligodeoxynucleotide (DNA oligomer or oligo DNA) may be natural or synthetic and may be a polymerase chain reaction (PCR) product. Suitable oligodeoxynucleotide derivatives include, but are not limited to, for example, oligodeoxynucleotides in which the phosphodiester bond in the oligodeoxynucleotide is converted to a phosphorothioate bond, an N3′-P5 ′ phosphoramidite bond, or a peptide bond Deoxyribose in the derivative, oligodeoxynucleotide is morpholine, 2'-O-propylribose, 2'-O-methylribose, 2'-fluoro-2'-deoxyribose, treofuranose (TNA) or 2'-position Examples include oligodeoxynucleotide derivatives converted to ribose (locked nucleic acid (LNA) or cross-linked nucleic acid (BNA)) obtained by cross-linking O and C at the 4 'position with methylene, and peptide nucleic acids (PNA). it can.

  The oligodeoxynucleotide 5 'end and / or 3' end may be phosphorylated or not phosphorylated unless otherwise specified. Alternatively, in the synthesis of a DNA oligomer using an automatic DNA synthesizer, the 5′-position hydroxyl group is a protecting group as in the case of synthesis under the condition that the 5′-position hydroxyl group protecting group of the last deoxynucleotide is not deprotected. It may be in a protected form. In either case, the oligodeoxynucleotide shall be included.

  As used herein, “protecting group” means a group that binds to a terminal hydroxyl group to substantially suppress or prevent unwanted side reactions, and includes, but is not limited to, for example, a trityl group, a mono-substituted group, or Mention may be made of disubstituted trityl groups, o-nitrobenzyl groups and levulinyl groups. Dimethoxytrityl (DMTr) is preferred. The protecting group is removed by performing a predetermined deprotection treatment. When the protecting group is a trityl group or a mono-substituted or di-substituted trityl group, examples of the deprotecting agent used for the deprotection treatment include acids such as trichloroacetic acid and trifluoroacetic acid. When the protecting group is a levulinyl group, examples of the deprotecting agent used for performing the deprotecting treatment include hydrazine. When the protecting group is an o-nitrobenzyl group, examples of the deprotection treatment include UV light irradiation treatment.

  The oligodeoxynucleotide may be in a form in which the hydroxyl group at the 5 'end and / or the 3' end and / or the base is labeled with a probe molecule. In either case, the oligodeoxynucleotide shall be included. As used herein, “probe (label) molecule” refers to a molecule conventionally used in the art for labeling oligodeoxynucleotides, such as biotin, polyethylene glycol (PEG), fluorescent dye, dicoxygenin, protein (eg, And a linker molecule for providing a labeling molecule). Fluorescent dyes selected from the group consisting of fluorescein derivatives and cyanine dyes are preferred. The probe molecule may be in its native form, with other functional groups such as active esters (eg succinimidyl esters), azide groups, amino groups, thiol groups, maleimide groups or alkynyl groups. It may be in a form in which a group capable of forming a covalent bond is introduced. Any form shall be included in the probe molecule.

As used herein, “alkyl” means a straight or branched chain aliphatic hydrocarbon group containing the specified number of carbon atoms. For example, “1-20 alkyl” and “C ₁ -C ₂₀ alkyl” mean a straight or branched hydrocarbon chain containing at least 1 and at most 20 carbon atoms. Suitable alkyls include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl and n -Octyl and the like can be mentioned.

  In the present specification, “alkenyl” means a group in which one or more C—C single bonds of the alkyl are substituted with double bonds. Suitable alkenyls include, but are not limited to, vinyl, 1-propenyl, allyl, 1-methylethenyl (isopropenyl), 1-butenyl, 2-butenyl, 3-butenyl, 1-methyl-2-propenyl, 2 Examples include -methyl-2-propenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 1-pentenyl, 1-hexenyl, n-heptenyl and 1-octenyl.

  In the present specification, “alkynyl” means a group in which one or more C—C single bonds of the alkyl are substituted with triple bonds. Suitable alkynyls include but are not limited to ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 1-pentynyl, 1-hexynyl 1-heptynyl, 1-octynyl and the like.

As used herein, “cycloalkyl” means an alicyclic alkyl containing the specified number of carbon atoms. For example, "(ring) cycloalkyl Number 3-20" and "C ₃ -C ₂₀ cycloalkyl" refers to at least three and at most containing 20 carbon atoms, a cyclic hydrocarbon group To do. Suitable cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

  In the present specification, “cycloalkenyl” means a group in which one or more C—C single bonds of the cycloalkyl are substituted with double bonds. Suitable cycloalkenyl includes, but is not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, and the like.

  In the present specification, “cycloalkynyl” means a group in which one or more C—C single bonds of the cycloalkyl are substituted with triple bonds. Suitable cycloalkynyl includes, but is not limited to, cyclobutynyl, cyclopentynyl, cyclohexynyl, and the like.

In the present specification, “heterocyclyl” means that one or more carbon atoms of the cycloalkyl, cycloalkenyl or cycloalkynyl are each independently a nitrogen atom (N), a sulfur atom (S) and an oxygen atom (O). It means a group substituted by a selected hetero atom. For example, “(cyclic) 3-20 membered heterocyclyl” and “C ₃ -C ₂₀ heterocyclyl” include one or more cyclic hydrocarbon groups containing at least 3 and at most 20 carbon atoms. It means a group in which carbon atoms are independently substituted with the above heteroatoms. In this case, substitution with N or S includes substitution with N-oxide or S oxide or dioxide, respectively. Suitable heterocyclyls include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, and piperazinyl. it can.

In the present specification, “aryl” means an aromatic group having 5 to 20 carbon atoms. For example, “aryl of (ring) 5-20” and “C ₅ -C ₂₀ aryl” mean an aromatic group containing at least 5 and at most 20 carbon atoms. Suitable aryls include, but are not limited to, phenyl, naphthyl, anthryl (anthracenyl), and the like.

  In the present specification, “arylalkyl” means a group in which one of hydrogen atoms of the alkyl is substituted with the aryl. Suitable arylalkyls include, but are not limited to, benzyl, 1-phenethyl, 2-phenethyl, and the like.

  In the present specification, “arylalkenyl” means a group in which one of the hydrogen atoms of the alkenyl is substituted with the aryl. Suitable arylalkenyls include, but are not limited to, styryl and the like.

As used herein, “heteroaryl” refers to a heteroatom in which one or more carbon atoms of the aryl are each independently selected from a nitrogen atom (N), a sulfur atom (S), and an oxygen atom (O). Means a substituted group. For example, and "(ring) heteroaryl Number 5-20""C ₅ -C ₂₀ heteroaryl", one or more carbons of the aromatic group containing at least five and at most 20 carbon atoms It means a group in which atoms are each independently substituted with the above heteroatoms. In this case, substitution with N or S includes substitution with N-oxide or S oxide or dioxide, respectively. Suitable heteroaryl include, but are not limited to, furanyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, isothiazolyl, pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, quinolinyl, Examples thereof include isoquinolinyl and indolyl.

  In the present specification, “heteroarylalkyl” means a group in which one of the hydrogen atoms of the alkyl is substituted with the heteroaryl.

  In the present specification, “carbonyl” means a monovalent group having a carbonyl group. Suitable carbonyls include, but are not limited to, formyl, acetyl, benzoyl, carboxy, methoxycarbonyl, halocarbonyl, carbamoyl (aminocarbonyl), and the like.

  Each of the groups described above is independently selected from the group consisting of an oxygen atom (O), a nitrogen atom (N), a sulfur atom (S), a silicon atom (Si), and a phosphorus atom (P). The above heteroatoms may be included. It is preferably an oxygen atom, a nitrogen atom or a sulfur atom. In this case, one or more carbon atoms constituting the above group are each independently substituted with these heteroatoms. Thus, the groups described above are intended to include not only the form itself, but also the form in which some of the carbon atoms are replaced with heteroatoms.

The groups described above are each independently unsubstituted or one or more halogen, hydroxyl group, substituted or unsubstituted amino group, nitro group, cyano group, mercapto group, carbonyl group, amide group or substituted or also contain hetero atoms unsubstituted monovalent hydrocarbon group (e.g., a substituted or unsubstituted C ₁ -C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, C ₂ -C ₂₀ alkynyl, C ₁ -C ₂₀ alkoxyl, C ₃ -C ₂₀ cycloalkyl, C ₃ -C ₂₀ cycloalkyloxy, C ₃ -C ₂₀ cycloalkenyl, C ₃ -C ₂₀ cycloalkenyloxy, C ₄ -C ₂₀ cycloalkynyl, C ₄ ~ C ₂₀ cycloalkylene alkynyloxy, C ₃ -C ₂₀ heterocyclyl, C ₃ -C ₂₀ heterocyclyloxy, C ₅ -C ₂₀ aryl, C ₅ -C ₂₀ aryloxy, C ₆ -C ₂₀ arylalkyl, C ₆ -C ₂₀ aryl alkenyl, C ₅ ~ C ₂₀ heteroaryl, a C ₅ -C ₂₀ heteroaryloxy or C ₆ -C ₂₀ heteroarylalkyl, a part of carbon atoms are replaced by may) be replaced with hetero atoms in these groups You can also. In this case, each of the above groups may independently be further substituted with one or more of the above groups.

  In the present specification, “halogen” or “halo” means fluorine, chlorine, bromine or iodine.

<2. Oligodeoxynucleotide>
[2-1. Oligodeoxynucleotide]
The inventors of the present invention have studied means for performing DNA strand cleavage and phosphorylation of a cleaved end by chemical reaction at low cost and simple experimental operation. As a result, deoxynucleosides containing unnatural bases described below are obtained. When the introduced DNA oligomer is reacted with a nucleophile such as a primary amine, the DNA oligomer is cleaved at a deoxynucleoside site containing a non-natural base to form an oligodeoxynucleotide phosphorylated at the 5 'end. I found out that Since the reaction proceeds by reacting the DNA oligomer in the presence of a nucleophile (under basic conditions), the DNA degradation reaction that proceeds under acidic conditions such as depreciation reaction is substantially suppressed or It becomes possible to prevent. In addition, deoxynucleosides containing unnatural bases are stable under the conditions of DNA synthesis using an automated DNA synthesizer. Chemical synthesis can be achieved with high yield. In addition, since the non-natural base exhibits substantially the same or substantially the same complementarity as the natural base under DNA replication conditions such as PCR, DNA containing the non-natural base can be used in vivo and in vivo. In the in vitro reaction, it is possible to exhibit a function as a template that is substantially the same or substantially the same as DNA comprising a natural base.

In the process of the invention, the formula I:
(A) _n -BC
The oligodeoxynucleotide represented by these is used.

Where in formula I
A is an oligodeoxynucleotide,
B is a deoxynucleoside containing a non-natural base having a moiety E removable by a nucleophile;
C is an oligodeoxynucleotide,
n is 0 or 1,
When n is 1, the 3 ′ end of A and the 5 ′ position of B are linked by a phosphodiester bond,
The 3 ′ position of B and the 5 ′ end of C are linked by a phosphodiester bond,
The phosphodiester bond between B and C is such that the oligodeoxynucleotide part phosphorylated at the 5 'end of C from the 3' position of B with E elimination induced by nucleophile attack. It is cut by detaching.

  In formula I, oligodeoxynucleotides A and C are preferably oligodeoxynucleotides having the characteristics described above. The number of bases of oligodeoxynucleotides A and C is not particularly limited, but in the case of oligodeoxynucleotide A, it is usually in the range of 0 to 100,000, preferably in the range of 0 to 20,000, and in the range of 0 to 100,000. It is more preferable. In the case of oligodeoxynucleotide C, it is usually in the range of 1 to 100,000, preferably in the range of 1 to 20,000, and more preferably in the range of 1 to 10,000.

  The deoxynucleoside B containing a non-natural base having a moiety E removable by a nucleophile is preferably a deoxynucleoside containing a non-natural base having the characteristics described below.

  In Formula I, when n is 0, the hydroxyl group at the 5 ′ end of deoxynucleoside B containing an unnatural base may be in an unprotected form, and is preferably a trityl group or a mono- or di-substituted trityl group, preferably May be in a form protected with a protecting group as described above, such as a DMTr group. Any form shall be encompassed by the oligonucleotide represented by Formula I.

  By using the oligodeoxynucleotide represented by the formula I having the above characteristics, the DNA strand is cleaved by a chemical reaction at a low cost and with a simple experimental operation, and the resulting end is phosphorylated at the end. Can be obtained.

[2-2. Deoxynucleosides containing unnatural bases]
Deoxynucleoside B containing an unnatural base used in the method of the present invention has a moiety that can be eliminated by a nucleophile. Specifically, it has the following characteristics.

  (I) It exists stably under DNA synthesis conditions.

  (Ii) It exists stably under DNA replication conditions such as in vivo and / or in vitro reactions (eg, PCR reactions).

  (Iii) having a moiety E that can be eliminated by reacting in the presence of a nucleophile (under basic conditions).

  (Iv) It has high fidelity under DNA replication conditions such as in vivo and / or in vitro reaction (for example, PCR reaction) and exhibits substantially the same or substantially the same complementarity as a natural base.

  (V) When forming a phosphodiester bond with a phosphate group at the 5 ′ end of another oligodeoxynucleotide C in the hydroxyl group at the 3 ′ position, between the 3 ′ position of B and the 5 ′ end of C The phosphodiester bond is cleaved by elimination of the oligodeoxynucleotide moiety phosphorylated at the 5 'end of C from the 3' position of B upon E elimination induced by nucleophile attack. Is done. This forms another oligonucleotide C phosphorylated at the 5 'end.

  In the present specification, “DNA synthesis conditions” means general synthesis conditions such as phosphoramidite method applied when DNA synthesis is performed by an automatic DNA synthesizer usually used in the art. . Examples of the automatic DNA synthesizer include, but are not limited to, ABI 392 DNA / RNA synthesizer (Applied Biosystems) and NTS H-6 DNA / RNA synthesizer (Nippon Techno Service). . DNA synthesis conditions are based on a combination of deoxynucleoside B containing a non-natural base and one or more deoxynucleosides containing a natural base used as a substrate when synthesizing an oligodeoxynucleotide represented by Formula I. For example, it can be set as appropriate by referring to the instruction manual attached to the DNA automatic synthesizer.

  Feature (i) “Stablely present under DNA synthesis conditions” specifically means that the following features are satisfied.

    (I-1) It does not substantially react with iodine in the presence of iodine used for oxidation of the phosphite part of the coupling reaction product.

    (I-2) It should not substantially react with an acid in the presence of an acid such as trichloroacetic acid (acidic conditions) used for deprotection of the DMTr group.

    (I-3) It should not substantially react with an alkali in the presence of an alkali such as aqueous ammonia used for excision and deprotection of the synthesized DNA (alkaline conditions).

  Moreover, in this specification, "DNA replication conditions" means general reaction conditions applied when replicating DNA by a PCR reaction usually used in the art. DNA replication conditions are based on the base sequence of the oligodeoxynucleotide used as a template when replicating the oligodeoxynucleotide represented by Formula I, for example, Kramer MF and Coen DM, “Enzymatic amplification of DNA by PCR: "standard procedures and optimization.", Curr Protoc Cytom. 2006 Aug; Appendix 3: Appendix 3K.

  Characteristic (ii) “is stably present under DNA replication conditions such as in vivo and / or in vitro reaction (eg, PCR reaction)” specifically means that the following characteristics are satisfied.

    (Ii-1) In a buffer solution usually used in PCR (for example, a tris buffer containing a salt such as magnesium chloride or potassium chloride and having a pH of around 8 at room temperature (optionally containing a surfactant or BSA)), Heating at 95 ° C in the PCR cycle does not substantially decompose.

  More specifically, deoxynucleoside B containing an unnatural base preferably has the following characteristics (vi) and (vii).

  (Vi) The unnatural base has a structure of a purine or pyrimidine base substituted with a substituted or unsubstituted alkenyl or alkynyl.

  (Vii) By reacting in the presence of a nucleophile (under basic conditions), the carbon atom at the 1 'position of deoxyribose is attacked by the nucleophile, and the glycosidic bond is cleaved to form a glycosidic bond. When the flow of the electron pair further attacks the carbon atom of alkenyl or alkynyl, the moiety E derived from the non-natural base is eliminated while causing an intramolecular cyclization reaction at the non-natural base moiety.

  Alternatively, deoxynucleoside B containing an unnatural base preferably has the following characteristics (vi ′) and (vii ′).

  (Vi ') The unnatural base has a structure of a water adduct or a nucleophile adduct of a purine or pyrimidine base substituted with a substituted or unsubstituted alkenyl or alkynyl, or a tautomer thereof. .

  (Vii ') By reacting in the presence of a nucleophile (under basic conditions), water or a nucleophile is added to a purine or pyrimidine type base substituted with a substituted or unsubstituted alkenyl or alkynyl, Form water adducts or nucleophile adducts of deoxynucleosides or form their tautomers. And, the deoxyribose 1'-position carbon atom of the deoxynucleoside water adduct or nucleophile adduct, or their tautomers is attacked by the nucleophile, and the glycosidic bond is cleaved, Part E derived from a non-natural base is eliminated.

  Alternatively, deoxynucleoside B containing an unnatural base preferably has the following characteristics (vi ″) and (vii ″).

  (Vi ″) A non-natural base has a structure of a purine base substituted with a substituted or unsubstituted aryl having carbonyl, substituted or unsubstituted alkynyl or cyano.

  (Vii '') By reacting in the presence of a nucleophile (under basic conditions), the 1'-position carbon atom of deoxyribose is attacked by the nucleophile and the glycosidic bond is cleaved. When the electron pair stream that formed further attacks the carbon atom of carbonyl, substituted or unsubstituted alkynyl or cyano, the moiety E derived from the unnatural base is eliminated.

  In the present specification, “deoxynucleoside B containing a non-natural base” means not only compound B itself but also a water adduct or a nucleophile adduct of the compound or a tautomer thereof. .

For example, deoxynucleoside B containing an unnatural base has the following formulas (vi) and (vii):

[Wherein, R and R ′ represent the same groups as defined below. ]
In the case of a compound represented by the formula, it is considered that the following reaction proceeds when an oligodeoxynucleotide containing B is reacted in the presence of a nucleophile (under basic conditions). In addition, the effect of this invention is not limited to the following reaction mechanism.

  In the above reaction formula, R and R 'are the same groups as defined below, DNA is an oligodeoxynucleotide, and Nu is a nucleophile.

For example, deoxynucleoside B containing an unnatural base has the following characteristics (vi ′) and (vii ′):

[Wherein R represents the same group as defined below. ]
In the case of a compound represented by the formula, it is considered that the following reaction involving tautomerization of the compound proceeds by reacting an oligodeoxynucleotide containing B in the presence of a nucleophile (under basic conditions). It is done. In addition, the effect of this invention is not limited to the following reaction mechanism.

  In the above reaction formula, R means the same group as defined below, DNA means an oligodeoxynucleotide, and Nu means a nucleophile.

For example, deoxynucleoside B containing an unnatural base has the following characteristics (vi ′) and (vii ′):

[Wherein R represents the same group as defined below. ]
In the case of a compound represented by the formula, by reacting an oligodeoxynucleotide containing B in the presence of a nucleophile (under basic conditions), the water addition reaction or nucleophile addition reaction of the compound can proceed. There is sex.

It is considered that the following reaction proceeds by further reacting an oligodeoxynucleotide containing a water adduct or nucleophile adduct of B produced by the reaction in the presence of a nucleophile (basic conditions). In addition, the effect of this invention is not limited to the following reaction mechanism.

In the above reaction formula, R means the same group as defined below, DNA means an oligodeoxynucleotide, Nu means a nucleophile, and R ^Nu means a group generated upon addition of a nucleophile. .

For example, deoxynucleoside B containing an unnatural base has the following characteristics (vi ″) and (vii ″):

[Wherein r, R ″, R ′ ″ and Z mean the same groups as defined below. ]
In the case of a compound represented by the formula, it is considered that the following reaction proceeds when an oligodeoxynucleotide containing B is reacted in the presence of a nucleophile (under basic conditions). In the following reaction formula, the case where R ′ ″ is a formyl group (—COZ, Z = H) is exemplified, but it is considered that the same reaction proceeds even when R ′ ″ is another group. . In addition, the effect of this invention is not limited to the following reaction mechanism.

  In the above reaction formula, r and R ″ represent the same groups as defined below, DNA represents an oligodeoxynucleotide, and Nu represents a nucleophile.

  By using deoxynucleoside B containing an unnatural base having the above-mentioned characteristics in the method of the present invention, a DNA strand can be reacted at a low cost and with a simple experimental operation by reacting in the presence of a nucleophile (under basic conditions). As a result, it is possible to obtain an oligodeoxynucleotide whose resulting end is phosphorylated. Further, since the cleavage of DNA by the method of the present invention is accompanied by the elimination of E from the deoxynucleoside containing a non-natural base induced by the attack of a nucleophile, the single-base deoxynucleoside B It becomes possible to introduce a cleavage site at an arbitrary position of the DNA strand by insertion or substitution.

  When preparing an oligodeoxynucleotide represented by Formula I using an automatic DNA synthesizer, the hydroxyl group at the 5 ′ end of deoxynucleoside B containing a non-natural base prepared as a monomer unit is a trityl group or mono-substituted. Alternatively, it is preferably in a form protected with a protecting group as described above, such as a disubstituted trityl group, preferably a DMTr group. In addition, in the oligodeoxynucleotide represented by formula I, when n is 0, the hydroxyl group at the 5 ′ end of deoxynucleoside B containing an unnatural base is unprotected, or is a trityl group, monosubstituted or disubstituted It is preferably in a form protected with a protecting group as described above, such as a trityl group, preferably a DMTr group.

  In the present specification, “deoxynucleotide B containing an unnatural base” means a form in which the hydroxyl group at the 5 ′ end of deoxynucleoside B containing an unnatural base is phosphorylated.

Deoxynucleoside B containing unnatural bases is:

Is preferably a deoxynucleoside represented by the formula: B-1, B-2, B-3, B-5, B-6, B-7, B-21 or B-22 It is more preferable that Of the above compounds, formulas B-3, B-7, B-21 and 22 wherein R is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl , Substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl or carbonyl; R ′ is methyl; Is an integer of 4; R ″ is halogen or substituted or unsubstituted alkyl, alkenyl, or alkynyl; R ′ ″ is —COZ, —C≡CZ, or —CN; Z is hydrogen Substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted aryl Le, a substituted or unsubstituted heteroaryl, or substituted or a compound represented by unsubstituted heteroaryl alkyl) is a novel compound discovered by the present inventors.

In formulas B-1 to B-7, B-11 to B-14, B-21 and B-22, R is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted A heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl or carbonyl. Specifically, R, hydrogen, a substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₃ -C ₂₀ heterocyclyl, substituted or unsubstituted substituted C ₅ -C ₂₀ aryl, substituted or unsubstituted C ₆ -C ₂₀ arylalkyl, substituted or unsubstituted C ₅ -C ₂₀ heteroaryl, substituted or unsubstituted C ₅ -C ₂₀ heteroarylalkyl or carbonyl It is preferable that Particularly suitable R can include hydrogen, methyl, ethyl, phenyl, hydroxymethyl, aminomethyl, mercaptomethyl, halomethyl, formyl, carboxyl, carbamoyl and acetyl. Particular preference is given to hydrogen, methyl, phenyl or carbamoyl.

In formulas B-1 to B-7, B-11 to B-14, B-21 and B-22, when R ′ is present, R ′ is hydrogen or substituted or unsubstituted alkyl, alkenyl or alkynyl. It is. Specifically, R ′ is preferably a hydrogen atom or a substituted or unsubstituted C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl or C ₂ -C ₁₀ alkynyl, particularly methyl. preferable.

In formulas B-21 and B-22, r is an integer from 0 to 4 and R ″ is halogen or substituted or unsubstituted alkyl, alkenyl or alkynyl. Specifically, r is an integer from 0 to 4, R '' is halogen, or substituted or unsubstituted C ₁ -C ₁₀ alkyl, with C ₂ -C ₁₀ alkenyl or C ₂ -C ₁₀ alkynyl Preferably, r is particularly preferably 0.

  In formulas B-21 and B-22, R ″ ″ is —COZ, —C≡C—Z or —CN. Specifically, R ″ ″ is preferably —COZ.

In formulas B-21 and B-22, Z is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted aryl Alkyl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heteroarylalkyl. Specifically, Z is hydrogen, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₃ -C ₂₀ cycloalkyl, substituted or unsubstituted C ₃ -C ₂₀ heterocyclyl, substituted or unsubstituted C ₅ -C ₂₀ aryl substituted, substituted or unsubstituted C ₆ -C ₂₀ arylalkyl, substituted or unsubstituted C ₅ -C ₂₀ heteroaryl, or substituted or unsubstituted of C ₅ -C ₂₀ heteroarylalkyl Preferably there is. Particularly suitable R may include hydrogen, methyl, ethyl, phenyl, hydroxymethyl, aminomethyl, mercaptomethyl or halomethyl. Hydrogen, methyl or phenyl is preferable, and hydrogen is more preferable.

  In the above groups, some of the carbon atoms may be replaced with one or more, preferably 1 to 5, more preferably 1 to 3 heteroatoms. The hetero atom is preferably a hetero atom selected from the group consisting of an oxygen atom, a nitrogen atom, a sulfur atom, a silicon atom and a phosphorus atom.

When the above group is substituted, the group is one or more halogen, hydroxyl group, substituted or unsubstituted amino group, nitro group, cyano group, mercapto group, carbonyl group, amide group, or hetero atom. the comprise also be substituted or unsubstituted optionally monovalent hydrocarbon group (e.g., a substituted or unsubstituted C ₁ -C ₂₀ alkyl, C ₁ -C ₂₀ alkoxyl, C ₃ -C ₂₀ cycloalkyl, C ₃ -C ₂₀ cycloalkyloxy, C ₃ -C ₂₀ heterocyclyl, C ₃ -C ₂₀ heterocyclyloxy, C ₅ -C ₂₀ aryl, C ₅ -C ₂₀ aryloxy, C ₆ -C ₂₀ arylalkyl, C ₅ -C ₂₀ heteroaryl , C ₅ -C ₂₀ heteroaryloxy or C ₆ -C ₂₀ heteroarylalkyl, in which some of the carbon atoms may be replaced by heteroatoms). That's right.

  Deoxynucleoside B containing a non-natural base includes not only the compound itself but also a salt thereof. The counter ion of the salt of deoxynucleoside B containing a non-natural base includes, but is not limited to, for example, a cation such as sodium ion, potassium ion, calcium ion or magnesium ion, or chloride ion, bromide ion, Formate, acetate, maleate, fumarate, benzoate, ascorbate, pamoate, succinate, bismethylenesalicylate, methanesulfonate, ethanedisulfonate, propionate, tartaric acid Ion, salicylate ion, citrate ion, gluconate ion, aspartate ion, stearate ion, palmitate ion, itaconic acid ion, glycolate ion, p-aminobenzoate ion, glutamate ion, benzene Phosphonate ion, cyclohexylsulfamate ion, methanesulfonate ion, ethanesulfonate ion, isethionate ion, benzenesulfonate ion, p-toluenesulfonate ion, naphthalenesulfonate ion, phosphate ion, nitrate ion, sulfate ion, Anions such as carbonate ions, bicarbonate ions or perchlorate ions are preferred.

  The deoxynucleoside B containing an unnatural base includes not only the compound itself but also a solvate thereof. Solvents that can form solvates with deoxynucleoside B containing an unnatural base include, but are not limited to, for example, methanol, ethanol, 2-propanol (isopropyl alcohol), dimethyl sulfoxide (DMSO), acetic acid, Organic solvents such as ethanolamine or ethyl acetate, or water are preferred.

  In addition, deoxynucleoside B containing a non-natural base includes not only the compound itself but also a protected form thereof. In the present specification, “protected form” means a form in which a protecting group is introduced into one or more functional groups.

  By using deoxynucleoside B containing an unnatural base having the above-mentioned characteristics in the method of the present invention, a DNA strand can be reacted at a low cost and with a simple experimental operation by reacting in the presence of a nucleophile (under basic conditions). As a result, it is possible to obtain an oligodeoxynucleotide whose resulting end is phosphorylated.

<3. Method for producing oligodeoxynucleotide>
FIG. 1 is a process diagram showing one embodiment of a method for producing an oligodeoxynucleotide of the present invention. Hereinafter, a preferred embodiment of the method of the present invention will be described in detail with reference to FIG.

[3-1. 5′-terminal phosphorylated oligonucleotide formation process]
The method of the present invention comprises a nucleophile and a compound of formula I:
(A) _n -BC
And an oligodeoxynucleotide phosphorylated at the 5 ′ end of C, by contacting with an oligodeoxynucleotide represented by formula II:
(A) _n -B '
A 5′-terminal phosphorylated oligonucleotide formation step for obtaining an oligodeoxynucleotide represented by (Step S2). In this step, the phosphodiester bond between B and C is cleaved by removing the oligodeoxynucleotide moiety phosphorylated at the 5 ′ end of C from the 3 ′ position of B. 'To obtain oligodeoxynucleotides phosphorylated at the ends.

  In formulas I and II, A, B, C and n represent the same meaning as defined above.

  In Formula II, B 'is the residue part of B that is formed upon elimination of E by nucleophilic attack.

  In Formula II, A and B 'are linked by a phosphodiester bond. More specifically, B 'is linked to the 3' end of A by a phosphodiester bond at a position derived from the 5 'position of B. In the present specification, “the 5′-position of B ′” means a position derived from the 5′-position of B in B ′.

In formula I, when n is 0, a nucleophile and formula I ′:
BC
The phosphodiester bond between B and C is removed from the 3 'position of B by contacting the phosphodiester bond between B and C by contacting with the oligodeoxynucleotide represented by Resulting in oligodeoxynucleotides phosphorylated at the 5 ′ end of the C. In Formula I, when n is 1, the phosphodiester bond between B and C is brought into contact with the oligodeoxynucleotide represented by Formula I by contacting the nucleophile with the 5 ′ end of C. An oligodeoxynucleotide that is cleaved by removing the phosphorylated oligodeoxynucleotide moiety from the 3 ′ position of B and phosphorylated at the 5 ′ end of C and an oligodeoxynucleotide represented by Formula II And produce. Therefore, after synthesizing the oligodeoxynucleotide represented by formula I ′ using an automatic DNA synthesizer, this step can be performed to produce oligodeoxynucleotide C phosphorylated at the 5 ′ end. It becomes possible.

  In this step, enzymatic treatment is not required, and the oligodeoxynucleotide represented by Formula I or Formula I ′ is cleaved and phosphorylated at the 5 ′ end by a chemical reaction (nucleophilic reaction by a nucleophile). Therefore, it is possible to produce oligodeoxynucleotides phosphorylated at the 5 'end with a low-cost and simple experimental operation as compared with the prior art method using an enzyme such as polynucleotide kinase.

For example, when deoxynucleoside B containing an unnatural base is a compound represented by formula B-1, the reaction in this step is considered to proceed as follows. In addition, the effect of this invention is not limited to the following principles.

In the above reaction formula, A, C, and E have the same meaning as in formula I defined above, and R ^Nu is a non-natural type obtained by adding one molecule of a nucleophile (Nu) to a degraded nucleotide. This means that the base has been removed.

  In this step, deoxynucleoside B-1 containing unnatural base is reacted with an oligodeoxynucleotide represented by formula I containing deoxynucleoside B-1 containing unnatural base in the presence of a nucleophile. Part E is considered to be eliminated (feature (iii)). More specifically, the 1'-position carbon atom of deoxyribose was attacked by the nucleophile Nu, the glycosidic bond was cleaved, and the flow of electron pairs that had formed the glycosidic bond replaced the alkynyl carbon atom. By further attack, it is considered that the portion E derived from the non-natural base is eliminated while causing an intramolecular cyclization reaction at the non-natural base portion (feature (vii)). In the above formula, the reaction mechanism is shown when the carbon atom at the 1 'position is attacked by a nucleophile, but the same reaction occurs when the carbon atom at the 4' position is attacked by a nucleophile. It is thought to progress. Here, deoxynucleoside B-1 containing an unnatural base corresponding to thymine having characteristics (vi) and (vii) has been described as an example. The above reaction mechanism applies not only to non-natural bases corresponding to pyrimidine-type bases such as thymine and cytosine, but also to deoxynucleoside B containing non-natural bases corresponding to purine-type bases such as adenine and guanine. Is done. In addition, the reaction mechanism includes not only deoxynucleoside B containing unnatural bases having characteristics (vi) and (vii), but also characteristics (vi ′) and (vii ′) or characteristics (vi ″) and ( The same applies to deoxynucleoside B containing non-natural bases having vii ″) in consideration of the above explanation regarding deoxynucleoside B containing these non-natural bases.

This reaction process is considered to be similar to the decomposition reaction of the basic site generated by the depreciation reaction shown below (Non-patent Document 13).

  The depurination reaction is a reaction in which adenine and / or guanine, which are purine bases contained in DNA, are eliminated from the nucleoside. Here, since adenine and guanine are easily released by protonation, the depurination reaction hardly occurs under basic conditions, and easily occurs under acidic conditions. In addition, the pyrimidine bases cytosine and thymine are less likely to desorb than purine bases, and the pyrimidine base is not likely to be desorbed from DNA not only under basic and neutral conditions but also under acidic conditions.

Moreover, as shown below, the cyclization reaction in which hydrogen at the 3-position of the base moiety is eliminated is known (Non-Patent Documents 26 to 29). However, as described above, a reaction in which DNA is cleaved by a mechanism considered to be eliminated from a nucleoside at the same time that an unnatural base contained in deoxynucleoside B is cyclized has not been reported so far. Is a new reaction found.

  DNAs containing unnatural bases of thymidine derivatives that act as thymidine in DNA replication reactions using DNA polymerase are known (Patent Documents 4 and 5 and Non-Patent Documents 30 to 34). However, as in this step, when reacted under basic conditions (in the presence of a nucleophile), the natural bases contained in the DNA are not damaged and are specific only at the sites of deoxynucleotides containing unnatural bases. A reaction in which DNA is cleaved and the 5 ′ end resulting from cleavage is phosphorylated has not been reported so far, and is a novel reaction found by the present inventors.

  The oligodeoxynucleotide represented by the formula I used in this step may be prepared by purchasing a pre-synthesized one or by performing the oligodeoxynucleotide synthesis step described below. May be. In either case, it is included in the embodiment of this step.

  The nucleophilic agent used in this step attacks the deoxynucleoside B site containing the unnatural base contained in the oligodeoxynucleotide represented by Formula I, and removes the detachable moiety E from B. And an oligodeoxynucleotide moiety phosphorylated at the 5 ′ end of C along with the elimination of E, can be cleaved by elimination from the 3 ′ position of B. Although a nucleophile is not limited, For example, a primary amine can be mentioned. A primary amine selected from the group consisting of methylamine, ethylenediamine, 2-aminoethanol, ethylamine, propylamine, 2-aminoethanethiol, 1,3-diaminopropane and tris (2-aminoethyl) amine is preferred. . By using the above nucleophile, the phosphodiester bond between B and C contained in the oligodeoxynucleotide represented by formula I is converted to the oligodeoxynucleotide moiety phosphorylated at the 5 ′ end of C. It is possible to obtain oligodeoxynucleotide C in which the 5 ′ end is phosphorylated by cleaving it from the 3 ′ position.

  This step is usually carried out by bringing a nucleophile and an oligodeoxynucleotide represented by formula I into contact under predetermined reaction conditions. The reaction conditions may be set as appropriate based on the combination of the nucleophile used in this step and deoxynucleoside B containing an unnatural base. Specifically, the reaction temperature is preferably in the range of 0 to 90 ° C, and more preferably in the range of 4 to 70 ° C. When the reaction conditions are heating conditions, the reaction temperature is preferably in the range of 60 to 90 ° C, more preferably in the range of 70 to 80 ° C. When the reaction temperature is 90 ° C. or lower, particularly in the range of 4 to 70 ° C., the lower the temperature, the more alkali denaturation of the long-chain oligonucleotide can be suppressed. The reaction time is preferably a time in the range of 0.1 to 168 hours, more preferably a time in the range of 0.1 to 48 hours, further preferably a time in the range of 0.1 to 24 hours, A time in the range of 12 hours is particularly preferred. By carrying out this step under the above reaction conditions, the reaction between the nucleophile and the oligodeoxynucleotide represented by formula I can be promoted.

  This step is usually performed by bringing a nucleophile and an oligodeoxynucleotide represented by formula I into contact in a medium. The medium may be appropriately set based on the combination of the nucleophile used in this step and deoxynucleoside B containing an unnatural base. Specifically, water is preferable. By carrying out this step in the above medium, it is possible to suppress undesirable side reactions and promote the reaction between the nucleophile and the oligodeoxynucleotide represented by formula I.

  This step may be carried out using a deprotected oligodeoxynucleotide represented by Formula I, for example, a 5 ′ end is a trityl group or a mono- or di-substituted trityl group, preferably a DMTr group. Alternatively, the oligodeoxynucleotide represented by the formula I protected with the protecting group described above may be used. In the latter case, the oligodeoxynucleotide represented by Formula II obtained in this step is in a form in which the 5 'end is protected. In this case, if desired, deprotection may be performed by performing the deprotection treatment described above in the 3′-terminal phosphorylated oligodeoxynucleotide forming step described below.

  The oligodeoxynucleotide C phosphorylated at the 5 ′ end obtained in this step and optionally the oligodeoxynucleotide represented by the formula II are separated from each other, the raw material and the auxiliary by a purification means usually used in the art. By being separated from the product, it can be purified and isolated. Therefore, this step may further include a purification and isolation step. Examples of purification means include, but are not limited to, medium pressure liquid chromatography (MPLC), high performance liquid chromatography (HPLC), vacuum concentration (evaporation), lyophilization, recrystallization, agarose gel electrophoresis, poly Mention may be made of acrylamide gel electrophoresis, reverse phase or treatment with a simple cartridge filled with an ion exchange carrier and ethanol precipitation, and combinations thereof. Alternatively, the purification and isolation step is not performed in this step, and in the 3′-terminal phosphorylated oligodeoxynucleotide forming step described below, oligodeoxynucleotide C having a phosphorylated 5 ′ end and the 3 ′ end are phosphorylated. The purification and isolation of the oligodeoxynucleotides may be performed together. By performing the above-described purification and isolation step, it is possible to produce oligodeoxynucleotide C having a highly purified 5 'end phosphorylated.

[3-2. 3'-terminal phosphorylated oligodeoxynucleotide forming step]
The method of the present invention optionally comprises Formula II:
(A) _n -B '
Is reacted with an oligodeoxynucleotide of formula IIa:
A-PO ₃ H ₂
A 3′-terminal phosphorylated oligodeoxynucleotide forming step of obtaining an oligodeoxynucleotide phosphorylated at the 3 ′ end represented by the formula (3) may be further included (step S3). This step cleaves the phosphodiester bond between B ′ and A by removing the oligodeoxynucleotide moiety phosphorylated at the 3 ′ end of A from B 3 'To obtain oligodeoxynucleotides phosphorylated at the ends.

In Formulas II and IIa, A and B ′ have the same meaning as described above,
n is 1.

  In this step, the 3 'end of A is phosphorylated by cleaving the phosphodiester bond between B' and A contained in the oligodeoxynucleotide represented by Formula II by a chemical reaction (elimination reaction). Resulting in oligodeoxynucleotides. Therefore, it is possible to produce an oligodeoxynucleotide phosphorylated at the 3 'end with a low-cost and simple experimental operation, compared to the terminal phosphorylation method of synthetic DNA using phosphorylated amidite.

When deoxynucleoside B containing an unnatural base is a compound represented by formula B-1, the reaction in this step is considered to proceed as follows. In addition, the effect of this invention is not limited to the following principles.

In the above reaction formula, A, C, and E have the same meaning as in formula I defined above, and R ^Nu is a non-natural type obtained by adding one molecule of a nucleophile (Nu) to a degraded nucleotide. This means that the base has been removed.

  The oligodeoxynucleotide A- (B-1 ′) represented by the formula II formed along with the elimination of E due to the attack of the nucleophile in the previous step is subjected to heat treatment in this step to obtain B-1 The phosphodiester bond between 'and A is cleaved by elimination of the oligodeoxynucleotide moiety phosphorylated at the 3' end of A from B-1 'and phosphorylated at the 3' end of A Resulting in oligodeoxynucleotides.

  This step is usually performed by reacting the oligodeoxynucleotide represented by formula II under predetermined reaction conditions. The reaction conditions may be set as appropriate based on deoxynucleosides B and B 'formed by cleavage of the unnatural base used. Specifically, the reaction temperature is preferably in the range of 0 to 120 ° C, and more preferably in the range of 4 to 70 ° C. When the reaction condition is a heating condition, the reaction temperature is preferably in the range of 90 to 120 ° C, more preferably in the range of 110 to 120 ° C. The reaction time is preferably a time in the range of 0.1 to 168 hours, more preferably a time in the range of 0.1 to 48 hours, further preferably a time in the range of 0.1 to 24 hours, A time in the range of 1 hour is particularly preferred. Further, the pH of the reaction system is usually neutral conditions, and specifically, it is preferably a value in the range of 6 to 8, more preferably a value in the range of 6.8 to 7.2. By carrying out this step under the above conditions, the oligodeoxynucleotide part phosphorylated at the 3 'end of A is eliminated from B' and the phosphodiester bond between B 'and A is cleaved. Is possible.

  This step is usually performed by reacting an oligodeoxynucleotide represented by formula II in a medium. The medium may be appropriately set based on the oligodeoxynucleotide represented by formula II used in this step. Specifically, water is preferable. The medium may also contain one or more buffer components in order to maintain the pH of the reaction system in the preferred pH range described above. Examples of the buffer component include, but are not limited to, phosphate and cacodylate, and combinations thereof. A phosphate is preferred. The concentration of the buffer component is preferably in the range of 10 to 1000 mM, more preferably in the range of 10 to 100 mM. By carrying out this step in the above medium, an undesirable side reaction is suppressed, and the oligodeoxynucleotide moiety phosphorylated at the 3 ′ end of A is eliminated from B ′, and B ′ and A It becomes possible to cleave the phosphodiester bond between.

  The oligodeoxynucleotide phosphorylated at the 3 ′ end represented by Formula IIa obtained in this step is purified by being separated from the raw materials and by-products by purification means usually used in the art. And can be isolated. Therefore, this step may further include a purification and isolation step. Examples of purification means include, but are not limited to, medium pressure liquid chromatography (MPLC), high performance liquid chromatography (HPLC), vacuum concentration (evaporation), lyophilization and recrystallization, agarose gel electrophoresis, poly Mention may be made of acrylamide gel electrophoresis, reverse phase or treatment with a simple cartridge filled with an ion exchange carrier and ethanol precipitation, and combinations thereof. By performing the above-described purification and isolation step, it is possible to produce a highly pure oligodeoxynucleotide having a phosphorylated 3 ′ end represented by Formula IIa.

  This step may be performed using a deprotected oligodeoxynucleotide represented by Formula II, for example, a 5 ′ end is a trityl group or a mono- or di-substituted trityl group, preferably a DMTr group. Alternatively, the oligodeoxynucleotide represented by the formula II protected with the protecting group described above may be used. In the latter case, the oligodeoxynucleotide phosphorylated at the 3 'end represented by Formula IIa obtained in this step is in a form in which the 5' end is protected. In this case, if desired, the deprotection treatment described above may be performed to obtain a deprotected oligodeoxynucleotide having a phosphorylated 3 ′ end represented by Formula IIa. It becomes possible.

[3-3. Oligodeoxynucleotide synthesis process]
You may implement the method of this invention by purchasing the oligodeoxynucleotide represented by Formula I previously synthesized. However, after synthesizing the oligodeoxynucleotide represented by Formula I, a 5′-terminal phosphorylated oligodeoxynucleotide forming step may be performed in situ or sequentially. Therefore, in the method of the present invention, deoxynucleoside B containing a non-natural base or a portion thereof having a moiety E removable by a nucleophile, optionally before the 5′-terminal phosphorylated oligodeoxynucleotide forming step. Using a substrate comprising a derivative and one or more deoxynucleosides or derivatives thereof comprising a natural base, the formula I:
(A) _n -BC
An oligodeoxynucleotide synthesis step for synthesizing the oligodeoxynucleotide represented by the formula (1) may be further included (step S1). The purpose of this step is to obtain an oligodeoxynucleotide represented by the formula I, which is a raw material for the 5′-terminal phosphorylated oligodeoxynucleotide forming step.

In the formula I, A, B and C have the same meaning as above,
n is 0 or 1.

  In this step, as the deoxynucleoside B containing a non-natural base having a moiety E that can be eliminated by a nucleophile, the deoxynucleoside B containing a non-natural base described above can be used. In addition, one or more deoxynucleosides containing a natural base are adenine, thymine, guanine and other than deoxynucleotide B containing an unnatural base among the deoxynucleotides contained in the oligodeoxynucleotide represented by Formula I. It means the one corresponding to a deoxynucleotide containing a natural base selected from the group consisting of cytosine, and a deoxynucleoside containing a natural base having a purity usually used in the art can be used.

  A derivative of deoxynucleoside B containing a non-natural base and a derivative of deoxynucleoside containing a natural base having a moiety E that can be eliminated by a nucleophile is not limited, for example, at the 3 ′ position. Mention may be made of derivatives of the deoxynucleoside having a phosphoramidite group. By using the above derivative, it is possible to obtain the oligodeoxynucleotide represented by the formula I by an automatic DNA synthesizer usually used in the art.

  The substrate used in this step is a deoxynucleoside B containing a non-natural base having a moiety E that can be eliminated by a nucleophile described above or a derivative thereof, and one or more deoxy containing a natural base. A nucleoside or a derivative thereof. The substrate may optionally include other components such as deoxynucleosides or derivatives thereof containing other unnatural bases, or labeling molecules such as fluorescent dyes, biotin or dichogigenin.

  This step is carried out by contacting the substrate with a medium such as acetonitrile or N, N-dimethylformamide and an activator such as tetrazole, a tetrazole derivative or 4,5-dicyanoimidazole, if desired. can do. In this case, this step may be performed under the DNA synthesis conditions described above.

  By carrying out this step under the above conditions, the oligodeoxynucleotide represented by formula I can be obtained in high yield.

<4. Method of linking double-stranded DNA>
According to the method of the present invention, a DNA strand can be cleaved by a low-cost and simple experimental operation, and the resulting oligodeoxynucleotide whose end is phosphorylated can be obtained. Therefore, the present invention also provides formula I:
(A) _n -BC
[Wherein A, B, C and n represent the same meaning as defined above,
When n is 1, the 3 ′ end of A and the 5 ′ position of B are linked by a phosphodiester bond,
The 3 ′ position of B and the 5 ′ end of C are linked by a phosphodiester bond,
The phosphodiester bond between B and C is such that the oligodeoxynucleotide part phosphorylated at the 5 'end of C from the 3' position of B with E elimination induced by nucleophile attack. Disconnected by detachment]
A DNA fragment amplification step of amplifying a linear double-stranded DNA fragment using the oligodeoxynucleotide represented by
By contacting the nucleophile with the amplified linear double-stranded DNA fragment, the phosphodiester bond between B and C becomes an oligodeoxynucleotide moiety phosphorylated at the 5 'end of C. It is cleaved by detaching from the 3 ′ position of B, the 5 ′ end of C contained in the linear double-stranded DNA fragment is phosphorylated, and at least the primer part represented by Formula I Forming a linear double-stranded DNA fragment having a 3 ′ protruding end containing a sequence complementary to a part thereof; and a linear double-stranded DNA fragment having the 3 ′ protruding end; And a ligation step of ligating another double-stranded DNA fragment;
And a method for ligating double-stranded DNA.

  In the DNA fragment amplification step, means for amplifying a linear double-stranded DNA fragment is not limited, and examples thereof include a polymerase chain reaction (PCR). In this case, this step may be performed under the DNA replication conditions described above. In the oligodeoxynucleotide represented by the formula I, the oligodeoxynucleotide C may have a base sequence complementary to at least a part of the template DNA that is a target of the polymerase chain reaction.

  As the nucleophile used in the 3 'protruding end forming step and the specific reaction conditions of this step, it is preferable to apply the conditions described above with respect to the 5' end phosphorylated oligodeoxynucleotide forming step.

  Compared with a method using a type II restriction enzyme, the method of the present invention can cleave DNA without considering the presence or absence of a restriction enzyme recognition sequence in the DNA fragment to be cleaved, and the length of the protruding portion at the protruding end. And it has the advantage that the arrangement can be set arbitrarily. Therefore, a linear double-stranded DNA fragment having a 3 ′ overhang produced by the method of the present invention is a DNA fragment produced using a type II restriction enzyme during ligation using a DNA ligation enzyme. Compared to the above, it is possible to exhibit high connection efficiency.

<5. Kit for use in the production method of oligodeoxynucleotide>
By the method of the present invention, oligodeoxynucleotide C phosphorylated at the 5 ′ end and / or oligodeoxynucleotide phosphorylated at the 3 ′ end represented by Formula IIa can be obtained. Therefore, the present invention also relates to a kit for use in the above-described method for producing the oligodeoxynucleotide of the present invention or the method for ligating double-stranded DNA.

The kit has the formula I:
(A) _n -BC
Or a deoxynucleoside B containing a non-natural base or a derivative thereof having a moiety E removable by a nucleophile.

In the formula I, A, B and C have the same meaning as above,
n is 0 or 1.

  The kit further comprises a natural base selected from the group consisting of adenine, thymine, guanine and cytosine in addition to the oligodeoxynucleotide represented by formula I or deoxynucleoside B containing a non-natural base or a derivative thereof. It may contain one or more additional elements selected from the group consisting of one or more deoxynucleosides or derivatives thereof, nucleophiles, media, catalysts, DNA-ligating enzymes (ligases) and instructions for use. Here, the nucleophile, the medium, the catalyst, and the DNA ligating enzyme can be appropriately selected from those that can be used in the method of the present invention described above. By including the above-described elements, the method for producing an oligodeoxynucleotide of the present invention can be carried out using the kit.

  The kit of the present invention does not necessarily need to be provided in an integrated form, and may be provided in a form in which the elements are packaged separately. Any form shall be included in the kit.

  By using the kit having the above characteristics, the oligodeoxynucleotide production method of the present invention is carried out, and the oligodeoxynucleotide C and / or formula IIa phosphorylated at the 5 ′ end by a low-cost and simple experimental operation It is possible to obtain an oligodeoxynucleotide phosphorylated at the 3 ′ end represented by

  As described above in detail, according to the present invention, a DNA strand is cleaved by a chemical reaction at a low cost and with a simple experimental operation, and further, the terminal phosphorylation of a synthetic DNA using an enzymatic method such as polynucleotide kinase or a phosphorylated amidite. It becomes possible to perform terminal phosphorylation with higher yield compared to the oxidation method.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

<1. Organic synthesis and DNA synthesis reagents and equipment>
Reagents for organic synthesis and / or DNA synthesis were purchased from Sigma-Aldrich, Wako Pure Chemicals, Tokyo Kasei, Nacalai Tesque or Glen Research. The solvent used for the synthesis reaction was anhydrous. The synthesis reaction was performed under a nitrogen atmosphere. Wako Gel C-200 (Wako Pure Chemical Industries) was used as the silica gel used for purification of the synthesized compound. An HPLC system manufactured by Gilson was used for DNA purification. The reverse phase column used was Chemco Bond 5-ODS-H (10 × 150 mm) manufactured by Chemco. The NMR spectrum was measured using JNM-ECS400 manufactured by JEOL. The chemical shift of ¹ H-NMR was described in ppm based on the chemical shift (2.49 ppm) of the residual proton in DMSO-d6. The HRMS spectrum was measured using SYNAPT G2 MS manufactured by Waters. The MALDI TOF-MS spectrum was measured using AXIMA Assurance manufactured by Shimadzu Corporation.

<2. Synthesis Example 1: DNA oligomer containing 5-phenylethynyl-2′-deoxyuridine>
[Synthesis Example 1-1: Synthesis of 5-phenylethynyl-2′-deoxyuridine]
3.54 g (10.0 mmol) 5-iodo-2'-deoxyuridine (molecular weight 354.10), 1.16 g (2.00 mmol) Pd (PPh ₃ ) ₄ (molecular weight 1155.56) and 190 mg (1.00 mmol) CuI (molecular weight 190.45) ) With a stir bar, 20 mL of DMF, 2.78 mL (20.0 mmol) Et ₃ N (molecular weight 101.19, specific gravity 0.73 g / mL) and 2.52 mL (23.0 mmol) ethynylbenzene (molecular weight). 102.13, specific gravity 0.93 g / mL) was added, and the mixture was stirred for 1 hour while heating at 80 ° C. in a nitrogen atmosphere. The solvent was distilled off under reduced pressure, and the target substance was purified with a silica gel column (developing solvent was 2 to 10% MeOH / AcOEt). The solvent of the desired fraction was distilled off under reduced pressure, dissolved in a small amount of methanol, about 200 mL of ethyl acetate was added, the solution was concentrated under reduced pressure, and the resulting white precipitate was filtered and dried with a vacuum pump to obtain 2.02 g. The target substance (molecular weight 328.32) (6.15 mmol, yield 61.5%) was obtained as a white powder.

The ¹ H NMR spectrum of the obtained material was measured, Non-Patent Document 19 and (Cho, JH et al., Eur. J. Org. Chem. 2010 years, p. 3678-3683) ¹ H NMR spectral data according to Almost matched.

¹ H NMR (400 MHz, DMSO-d6) δ11.67 (s, 1H), 8.37 (s, 1H), 7.47-7.38 (m, 5H), 6.12 (t, J = 6.6 Hz, 1H), 5.25 ( d, J = 4.1 Hz, 1H), 5.15 (t, J = 4.8 Hz, 1H), 4.26-4.24 (m, 1H), 3.81-3.79 (m, 1H), 3.64-3.58 (m, 2H), 2.18 -2.14 (m, 2H).
[Synthesis Example 1-2: Synthesis of 5-phenylethynyl-5′-O-dimethoxytrityl-2′-deoxyuridine]
656 mg (2.00 mmol) 5-phenylethynyl-2′-deoxyuridine (molecular weight 328.32) and 813 mg (4.00 mmol) DMTrCl (molecular weight 338.83) were placed in an eggplant flask, and 10 mL of pyridine was added thereto, Stir at room temperature for 1 hour. About 2 mL of methanol was added to stop the reaction, and the solvent was distilled off under reduced pressure. The target substance was purified by a silica gel column (developing solvent was 2-5% MeOH, 2% Et ₃ N / CH ₂ Cl ₂ ). The solvent of the objective fraction was distilled off under reduced pressure and dried with a vacuum pump. Dissolve in a small amount of methylene chloride, add about 400 mL of hexane, filter the resulting white precipitate, and dry with a vacuum pump to obtain 1.20 g (1.64 mmol, 82.0% yield) of the target substance (molecular weight 731.88 (one triethylamine molecule) Was obtained as a white powder.

When the ¹ H NMR spectrum of the obtained substance was measured, it was found that non-patent document 24 (Rospigliosi, A. et al., Langmuir 2007, 23, 15, p. 8264-8271, except that one molecule of triethylamine remained. ¹ H NMR spectrum data described in ( ¹ ).

¹ H NMR (400 MHz, DMSO-d6) δ11.75 (s, 1H), 8.06 (s, 1H), 7.41-7.39 (m, 2H), 7.33-7.26 (m, 9H), 7.16-7.10 (m , 3H), 6.84-6.80 (m, 4H), 6.14 (t, J = 6.9 Hz, 1H), 5.40 (d, J = 4.1 Hz, 1H), 4.34-4.28 (m, 1H), 3.98-3.95 ( m, 1H), 3.644 (s, 3H), 3.640 (s, 3H), 3.24-3.14 (m, 2H), 3.04-3.03 (m, 6H), 2.32-2.21 (m, 2H), 1.19 (t, J = 7.3 Hz, 9H).
[Synthesis Example 1-3: Synthesis of 5-phenylethynyl-5′-O-dimethoxytrityl-2′-deoxyuridine-3′-N, N-diisopropyl (cyanoethyl) phosphoramidite]
366 mg (0.500 mmol) of 5-phenylethynyl-5′-O-dimethoxytrityl-2′-deoxyuridine (molecular weight 731.88 (including one molecule of triethylamine)) was placed in an eggplant flask, 5 mL of CH ₃ CN and 261 μL (1.50 mmol) N, N-diisopropylethylamine (molecular weight 129.25, specific gravity 0.742 g / mL) was added, stirred, and 223 μL (1.00 mmol) 2-cyanoethyl N, N, N ', N'-tetraisopropylphospho Loamidite (molecular weight 236.68, specific gravity 1.061 g / mL) was added, and the mixture was stirred at room temperature for 30 minutes. 25 mL of AcOEt and 25 mL of saturated saline were added and separated, and the organic layer was further washed twice with saturated saline. The organic layer was dried over anhydrous magnesium sulfate and filtered through a filter, and the solvent of the filtrate was distilled off under reduced pressure. After azeotroping with acetonitrile three times, 10 mL of acetonitrile was added. The solution was passed through a 0.45 μm filter and a small amount of molecular sieve was added. Attached to an automatic DNA synthesizer and used for DNA synthesis

[Synthesis Example 1-4: Synthesis of DNA oligomer containing 5-phenylethynyl-2′-deoxyuridine]
A synthetic strategy and parameters involved in the synthesis of oligodeoxyribonucleotides according to the phosphoramidite method. Beaucage, SL and Caruthers, MH Current Protocols in Nucleic Acid Chemistry UNIT 3.3). Cutting out from the carrier was performed with 28% aqueous ammonia. Deprotection was performed by cutting out the DNA oligomer and then allowing to stand at room temperature for 16 hours. After deprotection, the ammonia was distilled off under reduced pressure using a centrifugal concentrator, and the DNA-containing solution was passed through a 0.45 μm filter. -50% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, detected at 254 nm), ammonium formate and the solvent were distilled off by centrifugal concentrator and lyophilization. The obtained DNA oligomer was identified by MALDI TOF-MS measurement. The synthesized DNA oligomer was made into an aqueous solution by adding about 500 μL of distilled water.

In the following, 5-phenylethynyl-2′-deoxyuridine:

Is indicated by “P”.

  The reverse phase HPLC chromatogram of the synthesized DNA oligomer is shown in FIGS.

  The retention time in the reverse phase HPLC of the synthesized DNA oligomer is as follows.

10.9 minutes for 5'-TTTTCTTPTCCTTTT-3 '(SEQ ID NO: 1)
11.0 minutes for 5'-TTTTTTPTTTTTT-3 '(SEQ ID NO: 2)
10.9 minutes for 5'-TTTTTAPATTTTT-3 '(SEQ ID NO: 3)
10.8 minutes for 5'-TTTTTCPCTTTTT-3 '(SEQ ID NO: 4)
10.6 minutes for 5'-TTTTTGPGTTTTT-3 '(SEQ ID NO: 5)
10.0 minutes for 5'-CGCAATPTAACGC-3 '(SEQ ID NO: 6)
The MALDI TOF-MS data of the synthesized DNA oligomer is as follows.

For 5′-TTTTCTTPTCCTTTT-3 ′, the molecular formula C ₁₅₄ H ₁₉₅ N ₃₃ O ₁₀₀ P ₁₄ , [MH] ⁻ calculated value 4541.0, measured value 4541.9 (SEQ ID NO: 1)
5'-TTTTTTPTTTTTT-3 'molecular formula _{C 137 H 172 N 26 O 89} P 12 about, [MH] ^- Calculated 3977.6, Found 3978.0 (SEQ ID NO: 2)
For 5′-TTTTTAPATTTTT-3 ′, the molecular formula C ₁₃₇ H ₁₇₀ N ₃₂ O ₈₅ P ₁₂ , [MH] ⁻ calculated value 3995.7, measured value 3996.3 (SEQ ID NO: 3)
For 5′-TTTTTCPCTTTTT-3 ′, the molecular formula C ₁₃₅ H ₁₇₀ N ₂₈ O ₈₇ P ₁₂ , calculated value of [MH] ⁻ 3947.6, measured value 3948.4 (SEQ ID NO: 4)
For 5′-TTTTTGPGTTTTT-3 ′, the molecular formula C ₁₃₇ H ₁₇₀ N ₃₂ O ₈₇ P ₁₂ , calculated for [MH] ⁻ 4027.7, measured value 4028.2 (SEQ ID NO: 5)
For 5′-CGCAATPTAACGC-3 ′, the molecular formula C ₁₃₃ H ₁₆₂ N ₄₈ O ₇₅ P ₁₂ , [MH] ⁻ calculated value 4003.7, measured value 4004.7 (SEQ ID NO: 6)
<3. Use example 1: DNA oligomer containing 5-phenylethynyl-2′-deoxyuridine>
[Use Example 1-1: Determination of DNA oligomer concentration]
Determine the concentration of DNA. For 5'-TTTTCTTPTCCTTTT-3 ', 10μL of DNA aqueous solution contains 10μL of enzymatic degradation solution (calf intestinal alkaline phosphatase (100 U / mL) and P1 nuclease (100 U / mL) And then decomposed to deoxynucleoside by standing at room temperature for 12 hours by reverse-phase HPLC (3-20% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, detected at 254 nm) Went by. From this result, the molar absorption constant at 260 nm of 5′-TTTTCTTPTCCTTTT-3 ′ was calculated to be 119,000. This value was very close to the calculated value of 119,400 for the molar absorption constant at 260 nm of 5'-TTTTCTTTTCCTTTT-3 '(Predicting ultraviolet spectrum of single and double stranded deoxyribonucleic acids. Tataurov, AV, You , Y. and Owczarzy, R. Biophys. Chem. 2008, 133, 66-70), the molar extinction coefficient at 260 nm of a DNA oligomer containing one P is the molar extinction of DNA calculated by substituting P for T. It was suggested that the value is close to the coefficient. Therefore, in this example, the DNA oligomer was diluted and the absorbance at 260 nm was measured, and the molar extinction coefficient at 260 nm calculated by substituting P for T (Predicting ultraviolet spectrum of single and double stranded deoxyribonucleic acids) Tataurov, AV, You, Y. and Owczarzy, R. Biophys. Chem. 2008, 133, 66-70), the concentration of the original DNA oligomer was determined.

  The molar extinction constant ε of each DNA oligomer at 260 nm when P is replaced with T is as follows.

For 5'-TTTTCTTPTCCTTTT-3 'ε = 119,400
For 5'-TTTTTTPTTTTTT-3 ', ε = 105,900
Ε = 117,700 for 5'-TTTTTAPATTTTT-3 '
For 5'-TTTTTCPCTTTTT-3 'ε = 104,100
For 5'-TTTTTGPGTTTTT-3 ', ε = 111,000
For 5'-CGCAATPTAACGC-3 ', ε = 122,700
[Use Example 1-2: Cleavage reaction of DNA oligomer containing 5-phenylethynyl-2′-deoxyuridine]
In a general method, when an equal volume of primary amine is added to an aqueous DNA solution and heated to 70 ° C. in a sealed state, the DNA oligomer is cleaved. Here, when the primary amine used is different, the rate of the cleavage reaction and the one DNA fragment produced by the cleavage are different. Of the two DNA fragments generated by cleavage, the DNA fragment generated by the primary amine used is different because the amine used is added to the newly generated DNA fragment at the 3′-end. This DNA fragment may be further degraded to the 3′-phosphate end, in which case the same DNA fragment is generated regardless of the primary amine used. Of the two DNA fragments generated by cleavage, a DNA fragment that newly generates a 5′-phosphate terminus, the same DNA fragment is generated regardless of the primary amine used when the source DNA oligomer is the same. Specific reaction of the primary amine is shown below.

[Use Example 1-3: Cleavage reaction of DNA oligomer containing 5-phenylethynyl-2′-deoxyuridine with methylamine and identification of product]
30 μL of 5′-TTTTCTTPTCCTTTT-3 ′ aqueous solution (strand concentration 500 μM) and 30 μL of methylamine (40% aqueous solution) were added to the screw tube, and heated in a water bath at 70 ° C. for 12 hours. After the reaction, methylamine was distilled off under reduced pressure using a centrifugal concentrator and analyzed by reverse phase HPLC to fractionate the product (0-50% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, at 254 nm. detection). The molecular weight of the fractionated product was measured by MALDI TOF-MS. Further, the ratio of deoxynucleoside obtained by enzymatic decomposition was determined for a part of the product. Regarding pTCCTTTT, it was confirmed that the retention time in the reverse phase HPLC of the product treated with alkaline phosphatase coincided with TCCTTTT synthesized separately, and the molecular weight by MALDI TOF-MS measurement also coincided. When desalting of the DNA oligomer is insufficient and ammonium formate is contained in the DNA oligomer, a part of the product 2 may be further decomposed to become a 3′-phosphate end.

  FIG. 8 shows reverse phase HPLC chromatograms of the synthesized DNA oligomer before and after the reaction.

The MALDI TOF-MS data for products 1 and 2 are as follows. In the following, “R ^Nu ” means a P-derived moiety that is generated in association with the cleavage reaction.

Product 1: pTCCTTTT molecular formula _{C 68 H 91 N 16 O 48} P 7 FW 2117.3 [MH] - 2116.3 Found 2116.4
Product 2: TTTTCTTpR ^Nu molecular formula _{C 75 H 103 N 16 O 51} P 7 FW 2261.5 [MH] - 2260.5 Found 2262.2
The ratio of deoxynucleosides generated by decomposing DNA oligomers is as follows.

Product 1: dC: dT = 2: 5
Product 2: dC: dT = 1: 6
Degradation reaction of DNA oligomer by methylamine is shown below. In the following reaction formula, the product on the left corresponds to the product 2, and the product on the right corresponds to the product 1.

In the above reaction formula, A and C have the same meaning as in formula I defined above. Based on the molecular weight of the product, R ^Nu is expected to be formed by adding one molecule of nucleophile to the degraded nucleotide and removing the unnatural base.

[Use Example 1-4: Comparison of cleavage reaction rate of DNA oligomer containing 5-phenylethynyl-2′-deoxyuridine with methylamine, ethylenediamine or 2-aminoethanol]
30 μL of 5′-TTTTCTTPTCCTTTT-3 ′ aqueous solution (strand concentration 500 μM) and 30 μL of primary amine (methylamine, ethylenediamine or 2-aminoethanol) were added to the screw tube and heated in a water bath at 70 ° C. for 1 hour. The reaction mixture was neutralized with acetic acid and analyzed by reverse phase HPLC (0-50% CH ₃ CN / 50 mM aqueous ammonium formate, 20 min, detected at 254 nm). The HPLC chromatogram of each reaction mixture is shown in FIG.

  Using 5'-TCCTTTT-3 'molar extinction coefficient at 260 nm ε = 55,500, 5'-TTTTCTTTTCCTTTT-3' molar extinction coefficient at 260 nm ε = 119,400, based on the peak area on the HPLC chromatogram As a result, the disappearance of the raw material in the reaction mixture obtained using methylamine was 24%, the disappearance of the raw material in the reaction mixture obtained using ethylenediamine was 76%, and in the reaction mixture obtained using 2-aminoethanol. The disappearance of raw material was calculated as 31%. From these results, it was suggested that in the cleavage reaction of DNA oligomer using primary amine of methylamine, ethylenediamine or 2-aminoethanol, the one having the fastest reaction rate is the cleavage reaction using ethylenediamine.

[Use Example 1-5: Cleavage reaction of DNA oligomers having various sequences including 5-phenylethynyl-2′-deoxyuridine with ethylenediamine and identification of products]
30 μL of DNA oligomer aqueous solution (strand concentration 500 μM) and 30 μL of ethylenediamine (anhydrous) were added to the screw tube, and heated in a water bath at 70 ° C. for 3 hours. The reaction mixture was neutralized with acetic acid and analyzed by reverse phase HPLC to fractionate the product (0-50% CH ₃ CN / 50 mM aqueous ammonium formate, 20 min, detected at 254 nm). The molecular weight of the fractionated product was measured by MALDI TOF-MS.

  The reverse phase HPLC chromatogram before and behind reaction of each synthetic | combination DNA oligomer is shown to FIGS.

The MALDI TOF-MS data of products 1 and 2 in each DNA oligomer are as follows. In the following, “R ^Nu ” means a P-derived moiety that is generated in association with the cleavage reaction.

5'-TTTTCTTPTCCTTTT-3 'product for 1: pTCCTTTT molecular formula _{C 68 H 91 N 16 O 48} P 7 FW 2117.3 [MH] - 2116.3 Found 2116.4
Product 2: TTTTCTTpR ^Nu molecular formula _{C 76 H 106 N 17 O 51} P 7 FW 2290.6 [MH] - 2289.5 Found 2290.4
5'-TTTTTTPTTTTTT-3 'product for 1: pTTTTTT molecular formula _{C 60 H 80 N 12 O 43} P 6 FW 1843.2 [MH] - 1842.2 Found 1842.3
Product 2: TTTTTTpR ^Nu molecular formula _{C 67 H 94 N 14 O 45} P 6 FW 2001.4 [MH] - 2000.4 Found 2000.3
5'-TTTTTAPATTTTT-3 'product for 1: pATTTTT molecular formula _{C 60 H 79 N 15 O 41} P 6 FW 1852.2 [MH] - 1851.2 Found 1851.0
Product 2: TTTTTApR ^Nu molecular formula _{C 67 H 93 N 17 O 43} P 6 FW 2010.4 [MH] - 2009.4 Found 2009.2
5'-TTTTTCPCTTTTT-3 'product for 1: pCTTTTT molecular formula _{C 59 H 79 N 13 O 42} P 6 FW 1828.2 [MH] - 1827.2 Found 1826.9
Product 2: TTTTTCpR ^Nu molecular formula _{C 66 H 93 N 15 O 44} P 6 FW 1986.4 [MH] - 1985.4 Found 1985.5
5'-TTTTTGPGTTTTT-3 'product for 1: pGTTTTT molecular formula _{C 60 H 79 N 15 O 42} P 6 FW 1868.2 [MH] - 1867.2 Found 1867.2
Product 2: TTTTTGpR ^Nu molecular formula _{C 67 H 93 N 17 O 44} P 6 FW 2026.4 [MH] - 2025.4 Found 2025.2
5'-CGCAATPTAACGC-3 'product for 1: pTAACGC molecular formula _{C 58 H 75 N 23 O 36} P 6 FW 1856.2 [MH] - 1855.2 Found 1855.2
Product 2: CGCAATpR ^Nu molecular formula _{C 65 H 89 N 25 O 38} P 6 FW 2014.4 [MH] - 2013.4 Found 2013.2
Degradation reaction of DNA oligomer by ethylenediamine is shown below. In the following reaction formula, the product on the left corresponds to the product 2, and the product on the right corresponds to the product 1.

In the above reaction formula, A and C have the same meaning as in formula I defined above. Based on the molecular weight of the product, R ^Nu is expected to be formed by adding one molecule of nucleophile to the degraded nucleotide and removing the unnatural base.

[Use Example 1-6: Cleavage reaction of DNA oligomer containing 5-phenylethynyl-2′-deoxyuridine with 2-aminoethanol and identification of the product]
30 μL of 5′-TTTTCTTPTCCTTTT-3 ′ aqueous solution (strand concentration: 500 μM) and 30 μL of 2-aminoethanol (anhydrous) were added to the screw tube and heated in a water bath at 70 ° C. for 12 hours. The reaction mixture was neutralized with acetic acid and analyzed by reverse phase HPLC to fractionate the product (0-50% CH ₃ CN / 50 mM aqueous ammonium formate, 20 min, detected at 254 nm). The molecular weight of the fractionated product was measured by MALDI TOF-MS.

  FIG. 16 shows reverse phase HPLC chromatograms of the synthesized DNA oligomer before and after the reaction.

MALDI TOF-MS data for products 1, 2 and 3 are as follows: In the following, “R ^Nu ” means a P-derived moiety that is generated in association with the cleavage reaction.

Product 1: pTCCTTTT molecular formula _{C 68 H 91 N 16 O 48} P 7 FW 2117.3 [MH] - 2116.3 Found 2116.2
Product 2: TTTTCTTp molecular formula _{C 69 H 92 N 15 O 49} P 7 FW2132.4 [MH] - 2131.3 Found 2131.1
Product 3: TTTTCTTpR ^Nu molecular formula _{C 76 H 105 N 16 O 52} P 7 FW 2291.5 [MH] - 2290.5 Found 2290.5
The degradation reaction of DNA oligomer with 2-aminoethanol is shown below. In the case of 2-aminoethanol, even if ammonium formate is not contained in the reaction mixture, a part of the product on the 5′-terminal side generated by cleavage is further decomposed to form a 3′-phosphate terminal. In the following reaction formula, the product on the left corresponds to the product 3, and the product on the right corresponds to the product 1. Product 2 corresponds to a compound in which the sugar-derived residue is eliminated from product 3 to form a 3′-phosphate end.

In the above reaction formula, A and C have the same meaning as in formula I defined above. Based on the molecular weight of the product, R ^Nu is expected to be formed by adding one molecule of nucleophile to the degraded nucleotide and removing the unnatural base.

[Use Example 1-7: 5′-Phosphorylation Using DNA Oligomers Containing 5-Phenylethynyl-2′-Deoxyuridine (1)]
The deprotection of the DNA oligomer and the DNA cleavage reaction at the position of the thymidine derivative were performed simultaneously.

A DNA oligomer having a 5′-PTTATTGTT-3 ′ sequence was synthesized by the same method as described above using an automatic DNA synthesizer. Cutting out from the carrier was performed with a 1: 1 mixed solution of 28% aqueous ammonia and 40% aqueous methylamine. The DNA oligomer solution thus cut out was put in a screw tube and deprotected by heating in a water bath at 70 ° C. for 16 hours. Ammonia and methylamine were removed under reduced pressure using a centrifugal concentrator and passed through a 0.45 μm filter. Thereafter, the fraction containing the main peak was separated by reverse phase HPLC (0-50% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, detected at 254 nm), and formic acid was collected using a centrifugal concentrator and a freeze dryer. Ammonium and solvent were distilled off. By MALDI TOF-MS measurement, it was confirmed that the obtained DNA oligomer coincided with the molecular weight of 5′-TTATTGTT-3 ′ phosphorylated at the 5′-end. 10 μL of the obtained aqueous solution of DNA oligomer was mixed with 10 μL of alkaline phosphatase (calf intestine-derived alkaline phosphatase (100 U / mL)) and heated at 40 ° C. for 2 hours to remove the terminal phosphate portion. When the product and 5′-TTATTGTT-3 ′ were mixed and analyzed by reverse phase HPLC, it was confirmed that both compounds showed the same retention time. Further, it was confirmed by MALDI TOF-MS that this product had the same molecular weight as 5′-TTATTGTT-3 ′.

  FIG. 17 shows a reverse phase HPLC chromatogram of the DNA oligomer after excision and deprotection.

Main peak: 5'-pTTATTGTT-3 'molecular formula _{C 80 H 104 N 22 O 54} P 8 FW 2485.6 [MH] - 2484.6 Found 2484.7
The reaction scheme is shown below.

[Use Example 1-8: 5′-Phosphorylation (2) using DNA oligomer containing 5-phenylethynyl-2′-deoxyuridine]
The excision and deprotection of the DNA oligomer and the DNA cleavage reaction at the position of the thymidine derivative were performed separately.

A DNA oligomer having a 5′-PTTATTGTT-3 ′ sequence was synthesized by the same method as described above using an automatic DNA synthesizer. Cleavage from the carrier was performed with 28% aqueous ammonia, and deprotection was performed by standing at room temperature (about 25 ° C.) for 16 hours. Ammonia was distilled off under reduced pressure using a centrifugal concentrator and passed through a 0.45 μm filter. Thereafter, the fraction containing the main peak was separated by reverse phase HPLC (0-50% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, detected at 254 nm), and formic acid was collected using a centrifugal concentrator and a freeze dryer. Ammonium and solvent were distilled off. 30 μL of distilled water and 30 μL of ethylenediamine were added to the resulting lyophilized product and heated in a water bath at 70 ° C. for 3 hours. After neutralizing the reaction mixture with acetic acid, the DNA oligomer was purified by the same method as in Use Example 1-7. The obtained DNA oligomer was identified by MALDI TOF-MS. 10 μL of the obtained aqueous solution of DNA oligomer was mixed with 10 μL of alkaline phosphatase (calf intestine-derived alkaline phosphatase (100 U / mL)) and heated at 40 ° C. for 2 hours to remove the terminal phosphate portion. When the product and 5′-TTATTGTT-3 ′ were mixed and analyzed by reverse phase HPLC, it was confirmed that both compounds showed the same retention time. Further, it was confirmed by MALDI TOF-MS that this product had the same molecular weight as 5′-TTATTGTT-3 ′.

  FIG. 18 shows a reverse phase HPLC chromatogram of the DNA oligomer after excision and deprotection, and FIG. 19 shows a reverse phase HPLC chromatogram of the DNA oligomer after the reaction with ethylenediamine.

Excised and DNA oligomers main peak after deprotection: 5'-PTTATTGTT-3 'molecular formula _{C 97 H 118 N 24 O 58} P 8 FW 2795.9 [MH] - 2794.9 Found 2795.1
DNA oligomers main peak after reaction with ethylene diamine: 5'-PTTATTGTT-3 'molecular formula _{C 97 H 118 N 24 O 58} P 8 FW 2795.9 [MH] - 2794.9 Found 2795.1
The reaction scheme is shown below.

[Use Example 1-9: 3′-Phosphorylation (1) using a DNA oligomer containing 5-phenylethynyl-2′-deoxyuridine]
The deprotection of the DNA oligomer and the DNA cleavage reaction at the position of the thymidine derivative were performed simultaneously.

Using an automatic DNA synthesizer, a DNA oligomer having the sequence 5′-TTATTGTTPT-3 ′ was synthesized by the same method as described above. Cutting out from the carrier was performed with a 1: 1 mixed solution of 28% aqueous ammonia and 40% aqueous methylamine. The DNA oligomer solution thus cut out was put in a screw tube and deprotected by heating in a water bath at 70 ° C. for 16 hours. Ammonia and methylamine were removed under reduced pressure using a centrifugal concentrator and passed through a 0.45 μm filter. Then, fractions containing a peak with a retention time of around 10 minutes were collected by reverse-phase HPLC (0-50% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, detected at 254 nm), centrifugal concentrator and lyophilized. The ammonium formate and the solvent were distilled off using a machine. On the preparative HPLC chromatogram, the small peak close to the main peak was a 3′-phosphate terminal as a result of elimination of the sugar-derived portion from the main peak product. To the obtained DNA oligomer, 100 μM phosphate buffer (500 μL, pH 7) was added, transferred to a screw tube, and allowed to stand in a drier set at 120 ° C. for 1 hour. Fractions containing the main peak were separated by reverse phase HPLC (0-50% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, detected at 254 nm), and ammonium formate and lyophilizer were used. The solvent was distilled off. On the preparative HPLC chromatogram, the peak eluted shortly after the main peak was one in which the 3′-terminal phosphate was eliminated. By MALDI TOF-MS measurement, it was confirmed that the obtained DNA oligomer coincided with the molecular weight of 5′-TTATTGTT-3 ′ phosphorylated at the 3′-terminus. 10 μL of the obtained aqueous solution of DNA oligomer was mixed with 10 μL of alkaline phosphatase (calf intestine-derived alkaline phosphatase (100 U / mL)) and heated at 40 ° C. for 2 hours to remove the terminal phosphate portion. When the product and 5′-TTATTGTT-3 ′ were mixed and analyzed by reverse phase HPLC, it was confirmed that both compounds showed the same retention time. Further, it was confirmed by MALDI TOF-MS that this product had the same molecular weight as 5′-TTATTGTT-3 ′.

  FIG. 20 shows a reverse phase HPLC chromatogram of the DNA oligomer after excision and deprotection, and FIG. 21 shows a reverse phase HPLC chromatogram of the DNA oligomer after treatment at 120 ° C. in 100 mM phosphate buffer.

Excised and DNA oligomers main peak after deprotection: 5'-TTATTGTTpR ^Nu -3 'molecular formula _{C 86 H 115 N 23 O 56} P 8 FW 2614.7 [MH] - 2613.7 Found 2613.4
DNA oligomers main peak of 120 ° C. after treatment in phosphoric acid buffer: 5'-TTATTGTTp-3 'molecular formula _{C 80 H 104 N 22 O 54} P 8 FW 2485.6 [MH] - 2484.6 Found 2484.6
The reaction scheme is shown below.

Based on the molecular weight of the product, R ^Nu is expected to be formed by adding one molecule of nucleophile to the degraded nucleotide and removing the unnatural base.

[Use Example 1-10: 3′-Phosphorylation (2) using a DNA oligomer containing 5-phenylethynyl-2′-deoxyuridine]
The excision and deprotection of the DNA oligomer and the DNA cleavage reaction at the position of the thymidine derivative were performed separately.

Using an automatic DNA synthesizer, a DNA oligomer having the sequence 5′-TTATTGTTPT-3 ′ was synthesized by the same method as described above. Cutting out from the carrier was performed with 28% aqueous ammonia, and deprotection was performed by standing at room temperature (about 25 ° C.) for 16 hours. Ammonia was distilled off under reduced pressure using a centrifugal concentrator and passed through a 0.45 μm filter. Thereafter, the fraction containing the main peak was separated by reverse phase HPLC (0-50% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, detected at 254 nm), and formic acid was collected using a centrifugal concentrator and a freeze dryer. Ammonium and solvent were distilled off. 30 μL of distilled water and 30 μL of ethylenediamine were added to the resulting lyophilized product and heated in a water bath at 70 ° C. for 3 hours. The reaction mixture was neutralized with acetic acid and then passed through a 0.45 μm filter. After that, the peak with a retention time of around 10 minutes was purified by reverse phase HPLC (0-50% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, detected at 254 nm), using a centrifugal concentrator and a freeze dryer. Ammonium formate and the solvent were distilled off. After adding 500 μL of 100 mM phosphate buffer (pH 7) to the resulting lyophilized product, it was transferred to a screw tube and allowed to stand in a dryer set at 120 ° C. for 1 hour. Fractions containing the main peak were separated by reverse phase HPLC (0-50% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, detected at 254 nm), and ammonium formate and lyophilizer were used. The solvent was distilled off. The obtained DNA oligomer was identified by MALDI TOF-MS. 10 μL of the obtained aqueous solution of DNA oligomer was mixed with 10 μL of alkaline phosphatase (calf intestine-derived alkaline phosphatase (100 U / mL)) and heated at 40 ° C. for 2 hours to remove the terminal phosphate portion. When the product and 5′-TTATTGTT-3 ′ were mixed and analyzed by reverse phase HPLC, it was confirmed that both compounds showed the same retention time. Further, it was confirmed by MALDI TOF-MS that this product had the same molecular weight as 5′-TTATTGTT-3 ′.

  Fig. 22 shows the reverse-phase HPLC chromatogram of the DNA oligomer after excision and deprotection, and Fig. 23 shows the reverse-phase HPLC chromatogram of the DNA oligomer after reaction with ethylenediamine. DNA after treatment at 120 ° C in 100 mM phosphate buffer. The reverse phase HPLC chromatogram of the oligomer is shown in FIG. 24, respectively.

Excised and DNA oligomers main peak after deprotection: 5'-TTATTGTTPT-3 'molecular formula _{C 107 H 131 N 26 O 65} P 9 FW 3100.1 [MH] - 3099.1 Found 3099.4
DNA oligomers main peak after reaction with ethylene diamine: 5'-TTATTGTTpR ^Nu -3 'molecular formula _{C 87 H 118 N 24 O 56} P 8 FW 2643.8 [MH] - 2642.8 Found 2642.9
DNA oligomers main peak of 120 ° C. after treatment in phosphoric acid buffer: 5'-TTATTGTTp-3 'molecular formula _{C 80 H 104 N 22 O 54} P 8 FW 2485.6 [MH] - 2484.6 Found 2484.7
The reaction scheme is shown below.

Based on the molecular weight of the product, R ^Nu is expected to be formed by adding one molecule of nucleophile to the degraded nucleotide and removing the unnatural base.

[Use Example 1-11: DMTr ON synthesis using a DNA oligomer containing 5-phenylethynyl-2′-deoxyuridine]
In DNA oligomer synthesis using an automatic DNA synthesizer, synthesis was carried out under conditions that do not deprotect the DMTr group at the 5′-end of the last deoxynucleoside (hereinafter also referred to as “DMTr ON”).

A DNA oligomer having a 5′-PTTATTGTT-3 ′ sequence was synthesized by the same method as described above using an automatic DNA synthesizer. However, the DMTr group at the 5′-end of the last deoxynucleoside was not deprotected and was synthesized under the conditions of “DMTr ON”. Cleavage from the carrier was performed with 28% aqueous ammonia, and deprotection was performed by standing at room temperature (about 25 ° C.) for 16 hours. Ammonia was distilled off under reduced pressure using a centrifugal concentrator and passed through a 0.45 μm filter. Thereafter, the fraction containing the main peak was separated by reverse phase HPLC (0-50% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, detected at 254 nm), and formic acid was collected using a centrifugal concentrator and a freeze dryer. Ammonium and solvent were distilled off. 100 μL of distilled water and 100 μL of ethylenediamine were added to the resulting lyophilized product and heated in a water bath at 70 ° C. for 8 hours. The reaction mixture was diluted to 1 mL with water and was neutralized with acetic acid, it was collected fractions containing the main peak by reverse phase _{HPLC (0-50% CH 3 CN /} 50 mM ammonium formate solution, 20 minutes, 254 (detected at nm), ammonium formate and the solvent were distilled off using a centrifugal concentrator and a freeze dryer. It was confirmed by MALDI TOF-MS that the obtained DNA oligomer had the same molecular weight as 5′-TTATTGTT-3 ′ phosphorylated at the 5′-end.

  Figure 25 shows the reverse-phase HPLC chromatogram of the DNA oligomer with DMTr protection at the 5'-end after excision and deprotection. Figure 25 shows the reverse-phase HPLC chromatogram of the DNA oligomer phosphorylated at the 5'-end after reaction with ethylenediamine. Are shown in FIG.

Cut and deprotection after the 5'-terminal DMTr protected DNA oligomer main peak: 5'-DMTr-PTTATTGTT-3 ' molecular formula _{C 118 H 137 N 24 O 60} P 8 FW 3099.3 [MH] - 3098.3 Found 3097.7
DNA oligomers 5'-end of the post-reaction with ethylenediamine was phosphorylated main peak: 5'-pTTATTGTT-3 'molecular formula _{C 80 H 104 N 22 O 54} P 8 FW 2485.6 [MH] - 2484.6 Found 2484.6
The reaction scheme is shown below.

[Use Example 1-12: Ligation reaction between a DNA oligomer prepared from a DNA oligomer containing 5-phenylethynyl-2′-deoxyuridine and phosphorylated at the 5 ′ end and another DNA oligomer]
Using an automatic DNA synthesizer, a DNA oligomer having the following sequence was synthesized in the same manner as described above. However, TS3 was synthesized under the conditions of “DMTr ON” without deprotecting the DMTr group at the 5 ′ end of the last deoxynucleoside. FAM modifications at the 5 'and 3' ends include 5'-fluorescein phosphoramidite (Catalog No. 10-5901) and 3 '-(6-FAM) -CPG (Catalog No. 20-2961) from Glen Research, respectively. It was used. The amino group was introduced into the 5 ′ end of TF3 according to the method described in Non-Patent Document 35.

TS1: 5'-FAM-ATAGTGAGTCGTATTAGGCCCGATCCAGGCCATCGCGCATGCTCAG-3 '(SEQ ID NO: 7)
TS3: 5'-ATAGTGAGTCGTATTAGGCCCGATCCAGGCCATCGCGCATGCTCAG-3 '(SEQ ID NO: 8)
TF3: 5'-NH ₂ -TTAACTGAGCATGCGCGATGGCCTG-3 '(SEQ ID NO: 9)
TT1: 5'-pGATCGGGCCTAATACGACTCACTAT-FAM-3 '(SEQ ID NO: 10)
TT2: 5'-pATCGGGCCTAATACGACTCACTAT-FAM-3 '(SEQ ID NO: 11)
TT1 and TT2, which are DNA oligomers phosphorylated at the 5 ′ end, were prepared by the following method. Using 5-phenylethynyl-2′-deoxyuridine, a DNA oligomer having sequences of 5′-PGATCGGGCCTAATACGACTCACTAT-FAM-3 ′ (SEQ ID NO: 12) and 5′-PATCGGGCCTAATACGACTCACTAT-FAM-3 ′ (SEQ ID NO: 13) The compound was synthesized in the same manner as in the above Use Example 1-7. Cutting out from the carrier was performed with a 1: 1 mixed solution of 28% aqueous ammonia and 40% aqueous methylamine. The DNA oligomer solution thus cut out was put in a screw tube and deprotected by heating in a water bath at 70 ° C. for 16 hours. Ammonia and methylamine were removed under reduced pressure using a centrifugal concentrator and passed through a 0.45 μm filter. Thereafter, the fraction containing the main peak was separated by reverse phase HPLC (0-50% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, detected at 254 nm), and formic acid was collected using a centrifugal concentrator and a freeze dryer. Ammonium and solvent were distilled off. It was confirmed by MALDI TOF-MS measurement that the obtained DNA oligomers were in agreement with the molecular weights of TT1 and TT2, which are DNA oligomers phosphorylated at the 5 ′ end.

  The reverse phase HPLC chromatograms of TT1 and TT2 after excision and deprotection are shown in FIGS. 27A and B, respectively.

TT1:
Main peak: 5'-pGATCGGGCCTAATACGACTCACTAT-FAM-3 ' molecular formula _{C 270 H 332 N 94 O 161} P 26 FW 8275.4 [MH] - 8274.4 Found 8273.7
TT2:
Main peak: 5'-pATCGGGCCTAATACGACTCACTAT-FAM-3 ' molecular formula _{C 260 H 320 N 89 O 155} P 25 FW 7946.2 [MH] - 7945.2 Found 7946.1
The structure of a DNA oligomer (hereinafter also referred to as “5′-FAM”) in which the 5 ′ end is FAM-modified is shown below.

The structure of a DNA oligomer whose 5 ′ end is modified with an amino group (hereinafter also referred to as “5′-NH ₂ ”) is shown below.

The structure of a DNA oligomer having the 3 ′ end modified with FAM (hereinafter also referred to as “FAM-3 ′”) is shown below.

  For the DNA oligomer ligation reaction, 15 μL of Ligation high (TOYOBO catalog number LGK-101) is mixed with 15 μL of a mixed solution of 40 μM TF3 and TS3 and 33 μM TT1 (TT2 for comparison) This was done by shaking at 16 ° C. for 1 to 3 hours. As a control, a sample in which TT1 was changed to TT2 was reacted in the same manner as described above. The obtained reaction mixture was analyzed by 15% polyacrylamide gel electrophoresis containing 7 M urea to confirm the ligation reaction. FIG. 28 shows the base sequence of each DNA oligomer and the analysis results by polyacrylamide gel electrophoresis.

  As shown in FIG. 28B, when the polyacrylamide gel is irradiated with ultraviolet rays, the fluorescent dye FAM emits fluorescence, so that the FAM-modified DNA oligomer is detected as a white band on the gel. The first lane band from the left is TS1 which is a DNA oligomer consisting of 46 bases, and the second and sixth lane bands from the left are the DNA oligomers consisting of 25 bases and 24 bases, TT1 and TT2, respectively. , Respectively. The third to fifth lanes from the left are the DNA oligomers obtained by ligation reaction of TT1 and TF3 for 1, 2 or 3 hours using TS3 as a template on the gel of FAM-modified TT1-derived DNA oligomers. Indicates the position. Since the position of the main band detected in these lanes is close to the position of TS1 consisting of 46 bases, TT1 and TF3 were connected in the ligation reaction mixture, and a DNA oligomer consisting of 50 bases was formed. It is suggested.

  The first lane from the right shows the position on the gel of the DNA oligomer in the control group obtained by ligating TT2 and TF3 for 3 hours using TS3 as a template. Since the position of the main band detected in this lane is close to the position of TT2, which is a DNA oligomer before ligation consisting of 24 bases, TT2 annealed with the template TS3 but was not ligated with TF3. Is suggested. TT2 is a DNA oligomer consisting of a base sequence with one base deleted from the 5 'end of TT1, so when TT2 and TF3 are annealed using TS3 as a template, a 1 base gap is generated between TT2 and TF3. (Figure 28A). For this reason, it is considered that the ligation reaction did not proceed in the control sample.

<4. Synthesis Example 2: DNA oligomer containing 5- (1-propynyl) -2′-deoxyuridine>
Phosphoramidite (non-patent documents 36 to 41), which is a monomer unit of deoxynucleoside (5- (1-propynyl) -2'-deoxyuridine) containing an unnatural base, was purchased from Glen Research (non-patent document). Reference 49). Using an automatic DNA synthesizer, a DNA oligomer containing 5- (1-propynyl) -2'-deoxyuridine was synthesized by the same method as described above (Non-patent Documents 42 to 49). Cleavage from the carrier was performed with 28% aqueous ammonia, and deprotection was performed by standing at room temperature (about 25 ° C.) for 16 hours. Ammonia was distilled off under reduced pressure using a centrifugal concentrator and passed through a 0.45 μm filter. Thereafter, the fraction containing the main peak was separated by reverse phase HPLC (0-50% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, detected at 254 nm), and formic acid was collected using a centrifugal concentrator and a freeze dryer. Ammonium and solvent were distilled off. The obtained DNA oligomer was identified by MALDI TOF-MS measurement.

In the following, 5- (1-propynyl) -2′-deoxyuridine:

Is indicated by “Pr”.

  The reverse phase HPLC chromatogram of the synthesized DNA oligomer is shown in FIGS.

  The retention time in the reverse phase HPLC of the synthesized DNA oligomer is as follows.

10.5 minutes for 5'-TTTTCTT (Pr) TCCTTTT-3 '(SEQ ID NO: 14)
10.7 minutes for 5'-TTTTTT (Pr) TTTTTT-3 '(SEQ ID NO: 15)
The MALDI TOF-MS data of the synthesized DNA oligomer is as follows.

For 5′-TTTTCTT (Pr) TCCTTTT-3 ′, the molecular formula C ₁₄₉ H ₁₉₃ N ₃₃ O ₁₀₀ P ₁₄ , [MH] ⁻ calculated value 4478.8, measured value 4479.4 (SEQ ID NO: 14)
For 5′-TTTTTT (Pr) TTTTTT-3 ′, the molecular formula C ₁₃₂ H ₁₇₀ N ₂₆ O ₈₉ P ₁₂ , [MH] ⁻ calculated value 3915.6, measured value 3915.8 (SEQ ID NO: 15)
<5. Use Example 2: DNA oligomer containing 5- (1-propynyl) -2′-deoxyuridine>
[Use Example 2-1: Cleavage reaction of DNA oligomer containing 5- (1-propynyl) -2′-deoxyuridine]
30 μL of DNA oligomer aqueous solution (strand concentration 500 μM) and 30 μL of ethylenediamine (anhydrous) were added to the screw tube, and heated in a water bath at 70 ° C. for 3 hours or 16 hours. The reaction mixture was neutralized with acetic acid and analyzed by reverse phase HPLC to fractionate the product (0-50% CH ₃ CN / 50 mM aqueous ammonium formate, 20 min, detected at 254 nm). The molecular weight of the fractionated product was measured by MALDI TOF-MS (sample product after reaction for 16 hours).

  Reverse phase HPLC chromatograms of the synthesized DNA oligomers before and after the reaction are shown in FIGS. 31 and 33, and MALDI TOF-MS spectra of the product 1 and other by-products are shown in FIGS. 32 and 34, respectively.

  The MALDI TOF-MS data of product 1 in each DNA oligomer is as follows.

5'-TTTTCTT (Pr) TCCTTTT- 3 ' product for 1: pTCCTTTT molecular formula _{C 68 H 91 N 16 O 48} P 7 FW 2117.3 [MH] - 2116.3 Found 2116.4
5'-TTTTTT (Pr) TTTTTT- 3 ' product for 1: pTTTTTT molecular formula _{C 60 H 80 N 12 O 43} P 6 FW 1843.2 [MH] - 1842.2 Found 1841.9
Degradation reaction of DNA oligomer by ethylenediamine is shown below. In the following reaction formula, the product on the right side corresponds to product 1. From the MALDI TOF-MS spectrum, the product on the left side shows that due to the longer reaction time, the elimination of the sugar residue or the reaction with an increase in molecular weight occurred to form another by-product. is expected.

In the above reaction formula, A and C have the same meaning as in formula I defined above. Based on the molecular weight of the product, R ^Nu is expected to be formed by adding one molecule of nucleophile to the degraded nucleotide and removing the unnatural base.

<6. Synthesis Example 3: DNA oligomer containing 5-ethynyl-2′-deoxyuridine>
5-Ethynyl-2′-deoxyuridine, which is a deoxynucleoside containing an unnatural base, is a known compound having antiviral activity and anticancer activity (Non-patent Documents 50 to 53). The deoxynucleoside is also used for DNA labeling in cells (Non-Patent Documents 54 to 63). When the deoxynucleoside is added to the cell culture medium, the deoxynucleoside is mistaken for thymidine and incorporated into the DNA of the cell. Since this deoxynucleoside has an ethynyl group, it can be easily coupled with another labeled molecule (for example, a fluorescent dye) having an azide group by click chemistry. By the above mechanism, the deoxynucleoside incorporated into the DNA of the cell can be detected with a label such as a fluorescent dye. Alternatively, by using Raman spectroscopy, the deoxynucleoside can be detected without performing the labeling reaction as described above. Therefore, the deoxynucleoside can be monitored even in a living cell (Non-patent Document 64).

  Many methods have so far been reported for the synthesis of 5-ethynyl-2'-deoxyuridine (for example, non-patent documents 65 to 67). In addition, a method of synthesizing a DNA oligomer containing 5-ethynyl-2'-deoxyuridine using an automatic DNA synthesizer has already been reported (Non-patent Documents 68 and 69). However, there is no example in which the deoxynucleoside is applied to DNA strand breaks and phosphorylated phosphorylated ends.

The phosphoramidite, which is a monomer unit of 5-ethynyl-2′-deoxyuridine, was synthesized according to the methods described in Non-Patent Documents 66 and 68. Using an automatic DNA synthesizer, a DNA oligomer containing 5-ethynyl-2′-deoxyuridine was synthesized by the same method as in Synthesis Example 1-4 (Non-patent Document 69). Cleavage from the carrier and deprotection were performed by standing in 50 mM K ₂ CO ₃ / MeOH at room temperature (about 25 ° C.) for 16 hours. If the DNA oligomer contains adenine and / or guanine, 500 μL of water is added to the DNA oligomer, and then methanol is distilled off under reduced pressure using a centrifugal concentrator. However, it is possible to completely deprotect the base moiety by leaving it to stand for 24 hours. Methanol was distilled off under reduced pressure using a centrifugal concentrator, 1 mL of distilled water was added, and then passed through a 0.45 μm filter. Thereafter, the fraction containing the main peak was separated by reverse phase HPLC (0-50% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, detected at 254 nm), and formic acid was collected using a centrifugal concentrator and a freeze dryer. Ammonium and solvent were distilled off. The obtained DNA oligomer was identified by MALDI TOF-MS measurement.

In the following, 5-ethynyl-2′-deoxyuridine:

Is indicated by “Pa”.

  The reverse phase HPLC chromatogram of the synthesized DNA oligomer is shown in FIGS.

  The retention time in the reverse phase HPLC of the synthesized DNA oligomer is as follows.

10.4 minutes for 5'-TTTTCTT (Pa) TCCTTTT-3 '(SEQ ID NO: 16)
10.7 minutes for 5'-TTTTTT (Pa) TTTTTT-3 '(SEQ ID NO: 17)
The MALDI TOF-MS data of the synthesized DNA oligomer is as follows.

For 5′-TTTTCTT (Pa) TCCTTTT-3 ′, the molecular formula C ₁₄₈ H ₁₉₁ N ₃₃ O ₁₀₀ P ₁₄ , [MH] ⁻ calculated value 4464.9, measured value 4465.3 (SEQ ID NO: 16)
5'-TTTTTT (Pa) molecular formula for _{TTTTTT-3 'C 131 H 168} N 26 O 89 P 12, [MH] - Calculated 3901.5, Found 3901.8 (SEQ ID NO: 17)
<7. Use Example 3: DNA oligomer containing 5-ethynyl-2′-deoxyuridine>
[Use Example 3-1: Cleavage reaction of DNA oligomer containing 5-ethynyl-2'-deoxyuridine]
30 μL of DNA oligomer aqueous solution (strand concentration 500 μM) and 30 μL of methylamine (40% aqueous solution) were added to the screw tube, and heated in a water bath at 70 ° C. for 10 minutes or 30 minutes. Methylamine was distilled off from the reaction mixture under reduced pressure using a centrifugal concentrator, the reaction mixture was analyzed by reverse phase HPLC, and the product was separated (0-50% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 Min, detected at 254 nm). The molecular weight of the fractionated product was measured by MALDI TOF-MS (sample product after reaction for 30 minutes).

  The reverse phase HPLC chromatogram before and after the reaction of each synthesized DNA oligomer is shown in FIGS. 37 and 38, respectively.

  The MALDI TOF-MS data of product 1 in each DNA oligomer is as follows.

5'-TTTTCTT (Pa) TCCTTTT- 3 ' product for 1: pTCCTTTT molecular formula _{C 68 H 91 N 16 O 48} P 7 FW 2117.3 [MH] - 2116.3 Found 2116.2
Product 2: TTTTCTTR ^Nu molecular formula _{C 75 H 103 N 16 O 51} P 7 FW 2261.5 [MH] - 2260.5 Found 2260.6
5'-TTTTTT (Pa) TTTTTT- 3 ' product for 1: pTTTTTT molecular formula _{C 60 H 80 N 12 O 43} P 6 FW 1843.2 [MH] - 1842.2 Found 1841.8
Product 2: TTTTTTR ^Nu molecular formula _{C 66 H 91 N 13 O 45} P 6 FW 1972.3 [MH] - 1971.3 Found 1971.3
Degradation reaction of DNA oligomer by methylamine is shown below. In the following reaction formula, it was shown from the MALDI TOF-MS spectrum that the product on the right side corresponds to product 1 and the product on the left side corresponds to product 2.

In the above reaction formula, A and C have the same meaning as in formula I defined above. Based on the molecular weight of the product, R ^Nu is expected to be formed by adding one molecule of nucleophile to the degraded nucleotide and removing the unnatural base.

  As described above, the cleavage reaction of the DNA oligomer containing 5-ethynyl-2'-deoxyuridine was almost completed by heating at 70 ° C for 30 minutes. From this result, a DNA oligomer containing 5-ethynyl-2′-deoxyuridine is a DNA oligomer containing 5-phenylethynyl-2′-deoxyuridine (Use Example 1) and 5- (1-propynyl) -2′- It was revealed that the cleavage reaction proceeded very rapidly as compared with a DNA oligomer containing deoxyuridine (Use Example 2).

[Use Example 3-2: Comparison of cleavage reaction rates of DNA oligomers containing 5-ethynyl-2′-deoxyuridine (Pa) with methylamine or ethylenediamine at 25 ° C.]
Add 200 μL of 5′-T ₆ (Pa) T ₆ -3 ′ aqueous solution (strand concentration 100 μM) and 200 μL of methylamine or ethylenediamine (40% aqueous solution) to the screw tube, stir well, and add 40 ° C. at 25 ° C. Let stand for hours. In the case of the reaction with methylamine, after the reaction, the reaction solution was concentrated with a centrifugal concentrator without heating, and methylamine was distilled off from the reaction solution under reduced pressure. In the case of reaction with ethylenediamine, the reaction solution was neutralized with acetic acid. Each resulting reaction mixture was analyzed by reverse phase HPLC (8-20% CH ₃ CN / 50 mM aqueous ammonium formate, 20 min, detected at 254 nm). An HPLC chart of the reaction solution is shown in FIG.

As shown in FIG. 39, in the case of reaction with methylamine, the retention time was around 8.2 minutes and 10.3 minutes (FIG. 39A), and in the case of reaction with ethylenediamine, the retention time was around 8.1 minutes and 10.3 minutes (FIG. 39B), respectively. Was detected. The same MALDI TOF-MS spectrum measurement as in Use Example 3-1 confirmed that these peaks correspond to products derived from cleavage of 5′-T ₆ (Pa) T ₆ -3 ′. From the above results, it was confirmed that the cleavage of the DNA oligomer proceeds even at 25 ° C.

[Use Example 3-3: Cleavage reaction of Pa-containing DNA oligomer in potassium carbonate aqueous solution and product identification]
100 μL of 5′-T ₆ PaT ₆ -3 ′ aqueous solution (strand concentration 400 μM) and 100 μL of potassium carbonate aqueous solution (1 M) were added to the screw tube, and heated in a dryer at 120 ° C. for 4 hours. The reaction solution was neutralized with acetic acid. The resulting reaction mixture was analyzed by reverse phase HPLC to fractionate the product (5-20% CH ₃ CN / 50 mM aqueous ammonium formate, 20 min, detected at 254 nm). An HPLC chart before and after the cleavage reaction of the DNA oligomer is shown in FIG.

  As shown in FIG. 40, products 1 and 2 were produced by the cleavage reaction. The fractionated products 1 and 2 were measured for molecular weight by MALDI TOF-MS. The MALDI TOF-MS data for products 1 and 2 are as follows.

Product 1: pTTTTTT molecular formula _{C 60 H 80 N 12 O 43} P 6 FW 1843.2 [MH] - 1842.2 Found 1842.2
HPLC retention time 12.1 min product 2: TTTTTTp molecular formula _{C 60 H 80 N 12 O 43} P 6 FW 1843.2 [MH] - 1842.2 Found 1842.0
HPLC retention time 12.8 minutes Degradation reaction of DNA oligomer with potassium carbonate is shown below. In the following reaction formula, it was shown from the MALDI TOF-MS spectrum that the product on the right side corresponds to product 1 and the product on the left side corresponds to product 2.

In the above reaction formula, A and C have the same meaning as in formula I defined above. Based on the molecular weight of the product, R ^Nu is expected to be formed by adding one molecule of nucleophile to the degraded nucleotide and removing the unnatural base.

  Product 1 was confirmed to have the same retention time by HPLC analysis by overstrike with the degradation product by methylamine. The nucleophilic species of this reaction is considered to be hydroxide ions. In this reaction, it is expected that a reaction similar to the decomposition reaction of DNA having an abasic site shown in Non-Patent Document 13 in water occurred. The reaction of DNA oligomers containing cytosine resulted in more than two product peaks. This result suggests that deamination of cytosine occurs when heated under alkaline conditions.

<8. Synthesis Example 4: DNA oligomer containing 5-[(E) -2-carbamoylvinyl] -2'-deoxyuridine>
[Synthesis Example 4-1: Synthesis of 5′-O-dimethoxytrityl-5-[(E) -2-carbomethoxyvinyl] -2′-deoxyuridine]
767 mg (2.46 mmol) 5-[(E) -2-carbomethoxyvinyl] -2'-deoxyuridine (molecular weight 312.28) and 1.05 g (3.10 mmol) DMTrCl (molecular weight 338.83) synthesized according to Non-Patent Document 73 Were placed in an eggplant flask, 20 mL of pyridine was added thereto, and the mixture was stirred at room temperature for 1 hour. About 2 mL of methanol was added to stop the reaction, and the solvent was distilled off under reduced pressure. The target substance was purified by a silica gel column (developing solvent was 2-5% MeOH, 2% Et ₃ N / CH ₂ Cl ₂ ). The solvent of the objective fraction was distilled off under reduced pressure and dried with a vacuum pump. Dissolve in a small amount of methylene chloride, add hexane, distill off the solvent under reduced pressure, and dry with a vacuum pump to obtain 1.49 g (1.94 mmol, yield 79%) of the target substance (including 1.5 molecules of molecular weight 766.43 triethylamine) Was obtained as a white powder.

¹ H NMR (400 MHz, DMSO-d6) δ11.70 (s, 1H), 8.01 (s, 1H), 7.39-7.36 (m, 2H), 7.29-7.15 (m, 8H), 6.86-6.81 (m , 5H), 6.15 (t, J = 6.4 Hz, 1H), 5.34 (d, J = 5.0Hz, 1H), 4.23-4.20 (m, 1H), 3.93-3.88 (m, 1H), 3.70 (s, 1H), 3.69 (s, 1H), 3.63 (s, 1H), 3.26-3.12 (m, 2H), 3.07-2.98 (m, 9H), 2.36-2.28 (m, 1H), 2.24-2.16 (m, 1H), 1.20-1.15 (m, 13.5 H); HRMS (ESI): Calculated for C ₃₄ H ₃₄ N ₂ O ₉ Na: 637.2157 [M + Na] ⁺ ; Found: 637.2162.
[Synthesis Example 4-2: 5′-O-dimethoxytrityl-5-[(E) -2-carbomethoxyvinyl] -2′-deoxyuridine-3′-N, N-diisopropyl (cyanoethyl) phosphoramidite Composition]
300 mg (0.391 mmol) of 5′-O-dimethoxytrityl-5-[(E) -2-carbomethoxyvinyl] -2′-deoxyuridine (molecular weight 766.43; containing 1.5 molecules of triethylamine) was placed in an eggplant flask, 5 mL of CH ₃ CN and 261 μL (1.50 mmol) of N, N-diisopropylethylamine (molecular weight 129.25, specific gravity 0.742 g / mL) were added, stirred, and further 223 μL (1.00 mmol) of 2-cyanoethyl N, N, N ′, N′-tetraisopropyl phosphoramidite (molecular weight 236.68, specific gravity 1.061 g / mL) was added, and the mixture was stirred at room temperature for 30 minutes. 25 mL of AcOEt and 25 mL of saturated saline were added and separated, and the organic layer was further washed twice with saturated saline. The organic layer was dried over anhydrous magnesium sulfate and filtered through a filter, and the solvent of the filtrate was distilled off under reduced pressure. After azeotroping with acetonitrile three times, 10 mL of acetonitrile was added. The solution was passed through a 0.45 μm filter and a small amount of molecular sieve was added. Attached to an automatic DNA synthesizer and used for DNA synthesis

[Synthesis Example 4-3: Synthesis of DNA oligomer containing 5-[(E) -2-carbamoylvinyl] -2'-deoxyuridine]
Using an automatic DNA synthesizer, a DNA oligomer containing 5-[(E) -2-carbamoylvinyl] -2′-deoxyuridine was synthesized by the same method as described above (Non-patent Documents 70 to 72). Cleavage from the carrier was performed with 28% aqueous ammonia, and deprotection was performed by standing at room temperature (about 25 ° C.) for 16 hours. The carbomethoxyvinyl group in the base moiety is converted to a carbamoylvinyl group under deprotection conditions. Ammonia was distilled off under reduced pressure using a centrifugal concentrator and passed through a 0.45 μm filter. Thereafter, the fraction containing the main peak was separated by reverse phase HPLC (0-50% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, detected at 254 nm), and formic acid was collected using a centrifugal concentrator and a freeze dryer. Ammonium and solvent were distilled off. The obtained DNA oligomer was identified by MALDI TOF-MS measurement.

In the following, 5-[(E) -2-carbamoylvinyl] -2′-deoxyuridine:

Is indicated by “CvU”.

  FIG. 41 shows a reverse phase HPLC chromatogram of the synthesized DNA oligomer.

  The retention time in the reverse phase HPLC of the synthesized DNA oligomer is as follows.

10.5 minutes for 5'-TTTTCTT (CvU) TCCTTTT-3 '(SEQ ID NO: 18)
10.6 minutes for 5'-TTTTTT (CvU) TTTTTT-3 '(SEQ ID NO: 19)
The MALDI TOF-MS data of the synthesized DNA oligomer is as follows.

For 5′-TTTTCTT (CvU) TCCTTTT-3 ′, the molecular formula C ₁₄₉ H ₁₉₄ N ₃₄ O ₁₀₁ P ₁₄ , [MH] ⁻ calculated value 4509.9, actual value 4510.0 (SEQ ID NO: 18)
For 5′-TTTTTT (CvU) TTTTTT-3 ′, the molecular formula C ₁₃₂ H ₁₇₁ N ₂₇ O ₉₀ P ₁₂ , [MH] ⁻ calculated value 3946.6, measured value 3946.6 (SEQ ID NO: 19)
<9. Use Example 4: DNA oligomer containing 5-[(E) -2-carbamoylvinyl] -2'-deoxyuridine>
[Use Example 4-1: Cleavage reaction of DNA oligomer containing 5-[(E) -2-carbamoylvinyl] -2'-deoxyuridine]
30 μL of DNA oligomer aqueous solution (strand concentration 500 μM) and 30 μL of ethylenediamine (anhydrous) were added to the screw tube, and heated in a water bath at 70 ° C. for 3 hours or 16 hours. The reaction mixture was neutralized with acetic acid and analyzed by reverse phase HPLC to fractionate the product (0-50% CH ₃ CN / 50 mM aqueous ammonium formate, 20 min, detected at 254 nm). The molecular weight of the collected product was measured by MALDI TOF-MS (sample product after reaction for 8 hours).

  42 and 44 show reverse phase HPLC chromatograms of the synthesized DNA oligomers before and after the reaction, and FIGS. 43 and 45 show MALDI TOF-MS spectra of the product 1 and other by-products, respectively.

  The MALDI TOF-MS data of product 1 in each DNA oligomer is as follows.

5'-TTTTCTT (CvU) TCCTTTT- 3 ' product for 1: pTCCTTTT molecular formula _{C 68 H 91 N 16 O 48} P 7 FW 2117.3 [MH] - 2116.3 Found 2116.2
5'-TTTTTT (CvU) TTTTTT- 3 ' product for 1: pTTTTTT molecular formula _{C 60 H 80 N 12 O 43} P 6 FW 1843.2 [MH] - 1842.2 Found 1841.9
Degradation reaction of DNA oligomer by ethylenediamine is shown below. In the following reaction formula, the product on the right side corresponds to product 1 which is a small peak on the HPLC chromatogram. When a fraction containing a peak eluted at the same retention time as the raw material DNA oligomer in preparative HPLC after the reaction was measured by MALDI TOF-MS, a peak corresponding to the raw material +60 was detected on the MALDI TOF-MS spectrum. From this result, it was suggested that the fraction after the reaction contains a large amount of a compound obtained by adding one molecule of ethylenediamine to the raw material DNA oligomer. Therefore, it is expected that a reaction other than the degradation reaction of the DNA oligomer occurred at the same time.

In the above reaction formula, A and C have the same meaning as in formula I defined above. Based on the molecular weight of the product, R ^Nu is expected to be formed by adding one molecule of nucleophile to the degraded nucleotide and removing the unnatural base.

<10. Synthesis Example 5: DNA oligomer containing N6- (3-propynyl) -5-methyl-2'-deoxycytidine>
[Synthesis Example 5-1: Synthesis of N6- (3-propynyl) -5-methyl-2′-deoxycytidine]
1.21 g (5.00 mmol) thymidine (molecular weight 242.23) was suspended in 20 mL acetonitrile, and 5.58 mL (40.0 mmol) triethylamine (molecular weight 101.19, specific gravity 0.73 g / mL) and 1.89 mL (20.0 mmol) anhydrous Acetic acid (molecular weight 102.09, specific gravity 1.08 g / mL) was added, and the mixture was stirred at 25 ° C. for 21 hours. The solvent was distilled off under reduced pressure, and 100 mL of ethyl acetate and 100 mL of saturated brine were added for liquid separation. The organic layer was washed twice with 100 mL of saturated brine, and then the organic layer was dried over magnesium sulfate. The organic layer after drying was filtered, and the solvent of the filtrate was distilled off under reduced pressure, followed by drying with a vacuum pump. 3.45 g (50.0 mmol) of 1,2,4-triazole (molecular weight 69.07) was added, 30 mL of acetonitrile was added, and 7.67 mL (55.0 mmol) of triethylamine (molecular weight 101.19, specific gravity 0.73 g / mL) was added. 932 μL (10.0 mmol) of phosphoryl chloride (molecular weight 153.33, specific gravity 1.645 g / mL) was added while stirring the mixture well. Stir at 25 ° C. for 1 hour. The solvent was distilled off under reduced pressure, and 100 mL of ethyl acetate and 100 mL of saturated brine were added for liquid separation. The organic layer was washed twice with 100 mL of saturated brine, and then the organic layer was dried over magnesium sulfate. The organic layer after drying was filtered, and the solvent of the filtrate was distilled off under reduced pressure, followed by drying with a vacuum pump. About 30 mL of 1,4-dioxane was added and dissolved, and then 961 μL (15.0 mmol) of propargylamine (molecular weight 55.08, specific gravity 0.86) was added, and the mixture was stirred at 25 ° C. for 2 hours. The solvent was distilled off under reduced pressure, 30 mL of methanol and 30 mL of 28% aqueous ammonia were added, and the mixture was stirred at 25 ° C. for 24 hours. The solvent was removed under reduced pressure and the desired product was purified on a silica gel column (10-15% MeOH / CH ₂ Cl ₂ ). 873 mg (3.13 mmol, yield 63%) of the target substance (molecular weight 279.29) was obtained as a white powder.

¹ H NMR (400 MHz, DMSO-d6) δ7.65 (d, J = 0.9Hz, 1H), 7.57 (t, J = 5.7Hz, 1H), 5.19 (d, J = 4.6Hz, 1H), 5.01 (t, J = 5.3Hz, 1H), 4.23-4.18 (m, 1H), 4.14-4.01 (m, 2H), 3.76-3.74 (m, 1H), 3.62-3.50 (m, 2H), 3.04 (t , J = 2.5 Hz, 1H), 2.12-2.05 (m, 1H), 1.99-1.92 (m, 1H), 1.84 (s, 3H); HRMS (ESI): calculation for C ₁₃ H ₁₇ N ₃ O ₄ Na Value: 302.1116 [M + Na] ⁺ ; Found: 302.1117.
[Synthesis Example 5-2: Synthesis of 5′-O-dimethoxytrityl-N6- (3-propynyl) -5-methyl-2′-deoxycytidine]
312 mg (1.12 mmol) N6- (3-propynyl) -5-methyl-2'-deoxycytidine (molecular weight 279.29) and 454 mg (1.34 mmol) DMTrCl (molecular weight 338.83) were placed in an eggplant flask. 10 mL of pyridine was added and stirred at room temperature for 1 hour. About 2 mL of methanol was added to stop the reaction, and the solvent was distilled off under reduced pressure. The target substance was purified by a silica gel column (developing solvent was 2-5% MeOH, 2% Et ₃ N / CH ₂ Cl ₂ ). The solvent of the objective fraction was distilled off under reduced pressure and dried with a vacuum pump. Dissolve in a small amount of methylene chloride, add hexane, evaporate the solvent under reduced pressure, and dry with a vacuum pump to obtain 631 mg (0.924 mmol, 83% yield) of the target substance (molecular weight 682.85; including one triethylamine molecule) Was obtained as a white powder.

¹ H NMR (400 MHz, DMSO-d6) δ7.63 (t, J = 5.7Hz, 1H), 7.51 (s, 1H), 7.39-7.35 (m, 2H), 7.32-7.19 (m, 7H), 6.90-6.86 (m, 4H), 6.21 (t, J = 6.6 Hz, 1H), 5.34 (d, J = 4.1Hz, 1H), 4.31-4.25 (m, 1H), 4.14-4.02 (m, 2H) , 3.90-3.86 (m, 1H), 3.72 (s, 6H), 3.22-3.16 (m, 2H), 3.09-3.00 (m, 6H), 2.21-2.06 (m, 2H), 1.50 (s, 3H) , 1.23-1.15 (m, 9H); HRMS (ESI): Calculated for C ₃₄ H ₃₅ N ₃ O ₆ Na: 604.2525 [M + Na] ⁺ ; Found: 604.2424.
[Synthesis Example 5-3: Synthesis of 5′-O-dimethoxytrityl-N6- (3-propynyl) -5-methyl-2′-deoxycytidine-3′-N, N-diisopropyl (cyanoethyl) phosphoramidite]
291 mg (0.426 mmol) of 5′-O-dimethoxytrityl-N6- (3-propynyl) -5-methyl-2′-deoxycytidine (molecular weight 682.85; containing one molecule of triethylamine) was placed in an eggplant flask and 5 mL Of CH ₃ CN and 261 μL (1.50 mmol) of N, N-diisopropylethylamine (molecular weight: 129.25, specific gravity: 0.742 g / mL), stirred, and further 223 μL (1.00 mmol) of 2-cyanoethyl N, N, N ′ , N'-tetraisopropyl phosphoramidite (molecular weight 236.68, specific gravity 1.061 g / mL) was added, and the mixture was stirred at room temperature for 30 minutes. 25 mL of AcOEt and 25 mL of saturated saline were added and separated, and the organic layer was further washed twice with saturated saline. The organic layer was dried over anhydrous magnesium sulfate and filtered through a filter, and the solvent of the filtrate was distilled off under reduced pressure. After azeotroping with acetonitrile three times, 10 mL of acetonitrile was added. The solution was passed through a 0.45 μm filter and a small amount of molecular sieve was added. Attached to an automatic DNA synthesizer and used for DNA synthesis

[Synthesis Example 5-4: Synthesis of DNA oligomer containing N6- (3-propynyl) -5-methyl-2′-deoxycytidine]
Using an automatic DNA synthesizer, a DNA oligomer containing N6- (3-propynyl) -5-methyl-2'-deoxycytidine was synthesized by the same method as described above. Cutting out from the carrier was performed with 28% aqueous ammonia, and deprotection was performed by standing at room temperature (about 25 ° C.) for 16 hours. Ammonia was distilled off under reduced pressure using a centrifugal concentrator and passed through a 0.45 μm filter. Thereafter, the fraction containing the main peak was separated by reverse phase HPLC (0-50% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, detected at 254 nm), and formic acid was collected using a centrifugal concentrator and a freeze dryer. Ammonium and solvent were distilled off. The obtained DNA oligomer was identified by MALDI TOF-MS measurement.

In the following, N6- (3-propynyl) -5-methyl-2′-deoxycytidine:

Is indicated by “PC”.

  FIG. 46 shows a reverse phase HPLC chromatogram of the synthesized DNA oligomer.

  The retention time in the reverse phase HPLC of the synthesized DNA oligomer is as follows.

10.6 minutes for 5'-TTTTCTT (PC) TCCTTTT-3 '(SEQ ID NO: 20)
10.9 minutes for 5'-TTTTTT (PC) TTTTTT-3 '(SEQ ID NO: 21)
The MALDI TOF-MS data of the synthesized DNA oligomer is as follows.

5'-TTTTCTT (PC) TCCTTTT- 3 ' molecular formula _{C 150 H 196 N 34 O 99} P 14 about, [MH] ^- calcd 4492.0, found 4491.9 (SEQ ID NO: 20)
5'-TTTTTT (PC) TTTTTT- 3 molecular formula C ₁₃₃ for _{'H 173 N 27 O 88 P} 12, [MH] - Calculated 3928.6, Found 3928.8 (SEQ ID NO: 21)
<11. Use Example 5: DNA oligomer containing N6- (3-propynyl) -5-methyl-2'-deoxycytidine>
[Use Example 5-1: Cleavage reaction of a DNA oligomer containing N6- (3-propynyl) -5-methyl-2'-deoxycytidine]
30 μL of DNA oligomer aqueous solution (strand concentration 500 μM) and 30 μL of ethylenediamine (anhydrous) were added to the screw tube, and heated in a water bath at 70 ° C. for 3 hours or 16 hours. The reaction mixture was neutralized with acetic acid and analyzed by reverse phase HPLC to fractionate the product (0-50% CH ₃ CN / 50 mM aqueous ammonium formate, 20 min, detected at 254 nm). The molecular weight of the fractionated product was measured by MALDI TOF-MS (sample product after reaction for 16 hours).

  47 and 49 show the reverse phase HPLC chromatograms of the synthesized DNA oligomers before and after the reaction, and FIGS. 48 and 50 show the MALDI TOF-MS spectra of the product 1 and other by-products, respectively.

  The MALDI TOF-MS data of product 1 in each DNA oligomer is as follows.

5'-TTTTCTT (PC) TCCTTTT- 3 ' product for 1: pTCCTTTT molecular formula _{C 68 H 91 N 16 O 48} P 7 FW 2117.3 [MH] - 2116.3 Found 2116.5
5'-TTTTTT (PC) TTTTTT- 3 ' product for 1: pTTTTTT molecular formula _{C 60 H 80 N 12 O 43} P 6 FW 1843.2 [MH] - 1842.2 Found 1841.8
Degradation reaction of DNA oligomer by ethylenediamine is shown below. In the following reaction formula, the product on the right side corresponds to product 1 which is a small peak on the HPLC chromatogram. The fraction containing a peak eluted at the same retention time as the raw material DNA oligomer in preparative HPLC after the reaction was measured by MALDI TOF-MS, and it was suggested that the raw material DNA oligomer was contained in a large amount. Therefore, it was suggested that the degradation reaction of the DNA oligomer was considerably slower than that of the DNA oligomer containing 5-phenylethynyl-2′-deoxyuridine in Use Example 1.

In the above reaction formula, A and C have the same meaning as in formula I defined above. Based on the molecular weight of the product, R ^Nu is expected to be formed by adding one molecule of nucleophile to the degraded nucleotide and removing the unnatural base.

<12. Use Example 6: DNA amplification by polymerase chain reaction using DNA oligomers containing unnatural bases and ligation of amplified products>
[Example of use 6-1: Amplification of DNA by polymerase chain reaction using a DNA oligomer containing an unnatural base]
Using λDNA (catalog number N3011 manufactured by New England BioLabs) as template DNA and KOD FX Neo (catalog number KFX-201 manufactured by TOYOBO) as an enzyme, 5-phenylethynyl-2′-deoxyuridine (P) or 5 Polymerase chain reaction was performed using a DNA oligomer containing -ethynyl-2′-deoxyuridine (Pa) as a primer. By performing the DNA chain cleavage reaction of the present invention on the amplified product after the polymerase chain reaction, the sequence design of the DNA oligomer containing an unnatural base is performed so that a protruding end that can be ligated by ligase is generated, Polymerase chain reaction was performed using two target DNAs as templates.

  The base sequence of the target DNA consists of gene 1 (136 base pairs) corresponding to the 28634-28770th position from the 5 'end and gene 2 (179 base pairs) corresponding to the 28743-28932 position from the 5' end in the lambda DNA. 5′-GTAGCGATCAAGCCATGAATGTAACG-3 ′ (SEQ ID NO: 22) as the forward primer strand of gene 1 and 5′-ACAGGCGAATCGCAAXCACTG-3 ′ (X = 5-phenylethynyl-2′-deoxyuridine (P) or 5 as the reverse primer strand -Ethynyl-2′-deoxyuridine (Pa)) (SEQ ID NO: 23) as a forward primer strand of gene 2 5′-ATTGCGATTCGCCTGXCTCTGC-3 ′ (5-phenylethynyl-2′-deoxyuridine (P) or 5- Ethinyl-2′-deoxyuridine (Pa)) (SEQ ID NO: 24) and 5′-GGTTGACTTAAATCGACCAGTAACAGG-3 ′ (SEQ ID NO: 25) were designed as reverse primer strands.

  Polymerase chain reaction was performed using TaKaRa PCR Thermal Cycler Dice (registered trademark) Gradient (Takara thermal cycler) using a final concentration of 0.3 μM primer, 1 ng λDNA, 0.4 mM dNTPs, 1 U KOD Fx neo, and a buffer attached to the product. -, Catalog number TP600), annealing: 94 ° C., 2 minutes, denaturation: 98 ° C., 10 seconds, elongation: 68 ° C., 10 seconds (denaturation → extension repeated 35 times). The amplified product was subjected to 10% non-denaturing polyacrylamide gel to confirm the amplified product by polymerase chain reaction. The results are shown in FIG.

  In FIG. 51, the gel after electrophoresis is infiltrated into an ethidium bromide solution at room temperature for 30 minutes, thereby performing a double-stranded DNA selective staining operation and detecting fluorescence emitted by ultraviolet irradiation to detect DNA. The strand is detected as a white band. The band in Lane 1 was a TrackIt ™ 100 bp DNA ladder (catalog number 10488-058 manufactured by Invitrogen) in which a band was detected every 100 base pairs as a gene marker. The bands in lanes 2 and 3 are genes when a DNA oligomer containing X = 5-phenylethynyl-2′-deoxyuridine (P) or 5-ethynyl-2′-deoxyuridine (Pa) is used as a primer chain, respectively. 1 shows amplification products, and the bands in lanes 4 and 5 are DNA oligomers containing X = 5-phenylethynyl-2′-deoxyuridine (P) or 5-ethynyl-2′-deoxyuridine (Pa), respectively. The amplification product of gene 2 when using as a primer strand is shown. The amplification of the target product was confirmed from the mobility of 100 and 200 base pairs derived from the marker.

[Use Example 6-2: Cleavage reaction of DNA amplification product containing unnatural base and ligation reaction of the obtained 5′-terminal phosphorylated DNA fragment]
50 μL of the DNA amplification product prepared in Use Example 5-1 and 50 μL of ethylenediamine were added to the PCR tube, and heated by TaKaRa PCR Thermal Cycler Dice (registered trademark) Gradient at 70 ° C. for 3 hours. After the reaction, add 50 μL of distilled water, and then add 150 μL of PCI (phenol: chloroform: isoamyl alcohol = 25: 24: 1) mixed solution, then vigorously mix the solution using a vortex mixer to make two layers even Centrifugation until layer separation. The supernatant was collected, 150 μL of PCI mixed solution was added again, and then the solution was vigorously mixed using a vortex mixer and centrifuged until the two layers were uniformly separated. This operation was repeated a total of 5 times. The supernatant obtained by the above operation was applied to a Bio-Spin 6 column (catalog number 732-6227 manufactured by BIO-RAD), and then purified by ethanol precipitation. The 5′-end phosphorylated DNA fragment was dried using a centrifugal concentrator and redissolved in 5 μL of TE buffer.

  A solution containing 5 μL of the gene 1 and the 2′-phosphorylated DNA fragment of gene 2 was mixed, 1.5 μL of 1 M NaCl and 3.5 μL of distilled water were added, and TaKaRa PCR Thermal Cycler Dice (registered trademark) Gradient After heating at 98 ° C. for 15 minutes, it was gradually cooled to room temperature. 15 μL of Ligation High (catalog number LGK-101, manufactured by TOYOBO) was added and shaken at 16 ° C. for 40 hours. The sample after the ligase reaction was purified by PCI extraction operation and ethanol precipitation, and subjected to 10% non-denaturing polyacrylamide gel to analyze the ligation reaction. The results are shown in FIG.

  The lane 1 band used was a TrackIt ™ 100 bp DNA ladder in which a band was detected every 100 base pairs as a gene marker. The bands of lanes 2 and 3 are obtained by cleaving the amplification product of gene 1 or gene 2 containing X = 5-phenylethynyl-2′-deoxyuridine (P) by the cleavage reaction of the present invention, respectively. Phosphorylated DNA fragments are shown, and the bands in lanes 4 and 5 are genes 1 containing X = 5-phenylethynyl-2′-deoxyuridine (P) or 5-ethynyl-2′-deoxyuridine (Pa), respectively. 2 shows a ligation product obtained by cleaving the DNA amplification products of No. 1 and No. 2 by the cleavage reaction of the present invention and reacting the obtained 5 ′ terminal phosphorylated DNA fragments of Genes 1 and 2 with ligase. The band in lane 6 is a DNA fragment having the nucleotide sequence corresponding to the 28634-28932th position from the 5 ′ end in λDNA, that is, gene 3 (299 nucleotides) having the same nucleotide sequence as the ligation product obtained by linking genes 1 and 2. The amplification product of the pair) is shown. A ligation product obtained by cleaving the DNA amplification products of genes 1 and 2 containing X = P or Pa by the cleavage reaction of the present invention and reacting the resulting 5′-terminal phosphorylated DNA fragment with ligase (lanes 4 and 5) Is consistent with the mobility of gene 3 (lane 6), indicating that the ligation reaction proceeds with high efficiency.

<13. Synthesis Example 7: DNA oligomer containing 2′-deoxy-4-N- {N ′, N′-di (n-butyl) formamidine} -5-phenylethynylcytidine>
[Synthesis Example 7-1: Synthesis of 2′-deoxy-4-N- {N ′, N′-di (n-butyl) formamidine} -5-phenylethynylcytidine]
2′-Deoxy-5-phenylethynylcytidine was synthesized according to Non-Patent Document 41. 708 mg (2.16 mmol) of 2′-deoxy-5-phenylethynylcytidine (molecular weight of 327.33) was dissolved in 10 mL of methanol. To the resulting solution, 2.0 mL (8.6 mmol) of N, N-di (n-butyl) formamidine dimethyl acetal (molecular weight 203.32, density 0.87 g / mL) (Dialkylformamidines: depurination resistant N6-protecting group for deoxyadenosine. , BC and Matteucci, MD Tetrahedron Letters 1983, 11, p. 8031-8036), and stirred at 25 ° C. for 2 hours. The solvent of the reaction solution was distilled off under reduced pressure, and the target substance was purified with a silica gel column (developing solvent: CH ₂ Cl ₂ only to 5 to 10% MeOH / CH ₂ Cl ₂ ). The solvent of the objective fraction was distilled off under reduced pressure to obtain 700 mg (1.50 mmol, yield 69%) of the objective substance (molecular weight 466.57) as a light brown oily substance.

¹ H NMR (400 MHz, DMSO-d6) δ8.64 (s, 1H), 8.44 (s, 1H), 7.43-7.34 (m, 5H), 6.12 (t, J = 6.2 Hz, 1H), 5.23 ( d, J = 4.1 Hz, 1H), 5.13 (t, J = 4.8 Hz, 1H), 4.26-4.21 (m, 1H), 3.84-3.81 (m, 1H), 3.70-3.44 (m, 6H), 2.26 -2.20 (m, 1H), 2.08-2.01 (m, 1H), 1.66-1.52 (m, 4H), 1.32-1.22 (m, 4H), 0.90 (t, J = 7.3 Hz, 3H), 0.82 (t , J = 7.3 Hz, 3H); ¹³ C NMR (100 MHz, DMSO-d6) δ169.7, 157.4, 153.4, 145.3, 130.7, 128.6, 128.1, 123.2, 97.5, 91.6, 87.6, 85.8, 84.5, 69.7, 60.7, 54.9, 51.4, 45.4, 41.0, 30.4, 28.5, 19.6, 19.1, 13.5; HRMS (ESI): Calculated for C ₂₆ H ₃₅ N ₄ O ₄ : 467.2658 [M + H] ⁺ ; Found: 467.2653.
[Synthesis Example 7-2: Synthesis of 4-N- {N ′, N′-di (n-butyl) formamidine} -5-phenylethynyl-5′-O-dimethoxytrityl-2′-deoxycytidine]
700 mg (1.50 mmol) 2'-deoxy-4-N- {N ', N'-di (n-butyl) formamidine} -5-phenylethynyl-cytidine (molecular weight 466.57) in 10 mL pyridine Dissolved. To the obtained solution, 763 mg (2.25 mmol) of 4,4′-dimethoxytrityl chloride (molecular weight: 338.83) was added and stirred at 25 ° C. for 1 hour. About 10 mL of methanol was added to the reaction solution, and the solvent was distilled off under reduced pressure. A small amount of methylene chloride and hexane were added to the resulting residue and mixed. After the mixture was azeotroped three times, the target substance was purified with a silica gel column (developing solvent was 2% Et ₃ N, 0 to _{2 to} 5% MeOH / CH ₂ Cl ₂ ). The solvent of the objective fraction was distilled off under reduced pressure, and a small amount of methylene chloride and hexane were added and azeotroped. 924 mg (1.20 mmol, 80% yield) of the desired product was obtained as a white powder.

¹ H NMR (400 MHz, DMSO-d6) δ8.65 (s, 1H), 8.17 (s, 1H), 7.44-6.81 (m, 18H), 6.14 (t, J = 6.6 Hz, 1H), 5.32 ( d, J = 4.6 Hz, 1H), 4.31-4.26 (m, 1H), 4.02-3.97 (m, 1H), 3.644 (s, 3H), 3.637 (s, 3H), 3.57-3.49 (m, 4H) , 3.23-3.16 (m, 2H), 2.36-2.30 (m, 1H), 2.17-2.10 (m, 1H), 1.66-1.53 (m, 4H), 1.32-1.22 (m, 4H), 0.91 (t, J = 7.3 Hz, 3H), 0.82 (t, J = 7.3 Hz, 3H); ¹³ C NMR (100 MHz, DMSO-d6) δ169.7, 158.03, 158.01, 157.5, 153.4, 144.7, 144.3, 135.6, 135.4 , 130.6, 129.7, 129.6, 128.3, 127.93, 127.86, 127.6, 126.6, 123.0, 113.2, 97.9, 91.7, 86.2, 85.9, 83.9, 70.6, 63.5, 54.9, 54.5, 45.4, 41.2, 30.4, 28.5, 19.6, 19.1 , 13.5; HRMS (ESI): Calculated for C ₄₇ H ₅₃ N ₄ O ₆ : 769.3965 [M + H] ⁺ ; Found: 769.3985.
[Synthesis Example 7-3: 4-N- {N ′, N′-di (n-butyl) formamidine} -5-phenylethynyl-5′-O-dimethoxytrityl-2′-deoxycytidine-3′- Synthesis of N, N-diisopropyl (cyanoethyl) phosphoramidite]
109 mg (0.142 mmol) 4-N- {N ', N'-di (n-butyl) formamidine} -5-phenylethynyl-5'-O-dimethoxytrityl-2'-deoxycytidine (molecular weight 768.94) Was added to an eggplant flask, 5 mL of CH ₃ CN and 157 μL (1.13 mmol) of triethylamine (molecular weight 101.19, specific gravity 0.726 g / mL) were added, stirred, and 126 μL (0.56 mmol) of 2-cyanoethyl N, N, N ′, N′-tetraisopropyl phosphorodiamidite (molecular weight 236.68, specific gravity 1.061 g / mL) was added, and the mixture was stirred at room temperature for 30 minutes. To the obtained mixture, 25 mL of AcOEt and 25 mL of saturated brine were added and separated, and the organic layer was further washed twice with saturated brine. The obtained organic layer was dried over anhydrous magnesium sulfate and filtered through a filter, and the solvent of the filtrate was distilled off under reduced pressure. After azeotroping with acetonitrile three times, 4.7 mL of acetonitrile was added. A small amount of molecular sieve was added to the solution. Attached to an automatic DNA synthesizer and used for DNA synthesis

[Synthesis Example 7-4: Synthesis of DNA oligomer containing 5-phenylethynyl-2′-deoxycytidine (Q)]
The DNA was synthesized by the phosphoramidite method with 1 μmol scale and DMTr ON according to the protocol for DNA synthesis of a normal automatic DNA synthesizer. Cutting out from the carrier was performed with 28% aqueous ammonia, and deprotection was performed by standing at 25 ° C. for 16 hours. After deprotection, the ammonia was distilled off under reduced pressure using a centrifugal concentrator, the solution containing DNA was passed through a 0.45 μm filter, and the main peak was purified by reverse-phase HPLC (0-50% CH ₃ CN / 50 mM formic acid. Ammonium aqueous solution, 20 minutes, detected at 254 nm), ammonium formate and the solvent were distilled off by centrifugal concentrator and lyophilization. To the obtained residue, 100 μL of 80% aqueous acetic acid solution was added, mixed well, and allowed to stand at 25 ° C. for 30 minutes. Thereafter, distilled water was added to the mixture so as to be about 1 mL, passed through a 0.45 μm filter, and the main peak eluted at a retention time of 13 to 14 minutes was purified by reverse phase HPLC (8- 20% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, detected at 254 nm), ammonium formate and the solvent were distilled off by centrifugal concentrator and lyophilization. The obtained DNA oligomer was identified by MALDI TOF-MS measurement. The synthesized DNA oligomer was made into an aqueous solution by adding about 500 μL of distilled water.

In the following, 5-phenylethynyl-2′-deoxycytidine:

Is indicated by “Q”.

  The concentration of DNA was determined by calculating the molar extinction coefficient at 260 nm by approximating Q to C and calculating the concentration from the UV absorption at 260 nm.

  53 and 54 show reverse phase HPLC chromatograms of the synthesized DNA oligomers.

  The retention time in the reverse phase HPLC of the synthesized DNA oligomer is as follows.

13.6 minutes for 5'-TTTTTTQTTTTTT-3 '(SEQ ID NO: 26)
13.1 minutes for 5'-TTTTCTTQTCCTTTT-3 '(SEQ ID NO: 27)
The MALDI TOF-MS data of the synthesized DNA oligomer is as follows.

For 5′-TTTTTTQTTTTTT-3 ′, the molecular formula C ₁₃₇ H ₁₇₃ N ₂₇ O ₈₈ P ₁₂ , calculated value of [MH] ⁻ 3976.6, measured value 3977.1 (SEQ ID NO: 26)
5'-TTTTCTTQTCCTTTT-3 molecular formula C ₁₅₄ for _{'H 196 N 34 O 99 P} 14, [MH] - Calculated 4540.0, Found 4540.0 (SEQ ID NO: 27)
<14. Use Example 7: DNA oligomer containing 5-phenylethynyl-2′-deoxycytidine>
[Use Example 7-1: Cleavage reaction of DNA oligomer containing 5-phenylethynyl-2′-deoxycytidine with methylamine and identification of the product]
Add 300 μL of 5′-T ₆ QT ₆ -3 ′ or 5′-TTTTCTTQTCCTTTT-3 ′ aqueous solution (strand concentration: 100 μM) and 300 μL of methylamine (40% aqueous solution) to the screw tube. Heated for 12 hours. After the reaction, methylamine was distilled off under reduced pressure with a centrifugal concentrator and analyzed by reverse phase HPLC to fractionate the product (8-20% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, at 254 nm. detection). The molecular weight of the fractionated product was measured by MALDI TOF-MS.

  The reverse phase HPLC chromatogram after reaction of each synthesized DNA oligomer is shown in FIGS.

The MALDI TOF-MS data of products 1 and 2 in each DNA oligomer are as follows. In the following, “R ^Nu ” means a Q-derived moiety that is generated in accordance with the cleavage reaction.

5'-TTTTTTQTTTTTT-3 'product for 1: pTTTTTT molecular formula _{C 60 H 80 N 12 O 43} P 6 FW 1843.2 [MH] - 1842.2 Found 1842.0
HPLC retention time 8.5 min product 2: TTTTTTpR ^Nu molecular formula _{C 66 H 91 N 13 O 45} P 6 FW 1972.3 [MH] - 1971.3 Found 1971.1
HPLC retention time 10.6 minutes
5'-TTTTCTTQTCCTTTT-3 'product for 1: pTCCTTTT molecular formula _{C 68 H 91 N 16 O 48} P 7 FW 2117.3 [MH] - 2116.3 Found 2116.3
HPLC retention time 7.1 min product 2: TTTTCTTpR ^Nu molecular formula _{C 75 H 103 N 16 O 51} P 7 FW 2261.5 [MH] - 2260.5 Found 2260.5
HPLC retention time 9.7 minutes Degradation reaction of DNA oligomer by methylamine is shown below. In the following reaction formula, the product on the left corresponds to the product 2, and the product on the right corresponds to the product 1.

In the above reaction formula, A and C have the same meaning as in formula I defined above. Based on the molecular weight of the product, R ^Nu is expected to be formed by adding one molecule of nucleophile to the degraded nucleotide and removing the unnatural base.

[Use Example 7-2: Comparison of cleavage reaction rate of DNA oligomer containing 5-phenylethynyl-2′-deoxycytidine with methylamine or ethylenediamine]
Add 30 μL of 5′-T ₆ (Q) T ₆ -3 ′ or 5′-TTTTCTT (Q) TCCTTTT-3 ′ aqueous solution (strand concentration 100 μM) and 30 μL of methylamine 40% aqueous solution or ethylenediamine to the screw tube. The mixture was heated in a water bath at 70 ° C. for 2 hours. The reaction mixture was neutralized with acetic acid and analyzed by reverse phase HPLC (8-20% CH ₃ CN / 50 mM aqueous ammonium formate, 20 min, detected at 254 nm). An HPLC chart of the reaction mixture of 5′-T ₆ (Q) T ₆ -3 ′ is shown in FIG. 57, and an HPLC chart of the reaction mixture of 5′-TTTTCTT (Q) TCCTTTT-3 ′ is shown in FIG.

5'-T ₆ QT ₆ -3 was quantified based on the peak area of 'which is the 5'-cleavage product of _{5'-T 6 p-3'} , the 5'-T ₆ QT ₆ -3 ' The amount of cleavage product on the 5′-side was calculated to be 3.6 times higher with methylamine treatment than with ethylenediamine treatment. In addition, when quantified based on the peak area of 5'-TTTTCTTp-3 'which is the 5'-side cleavage product of 5'-TTTTCTTQTCCTTTT-3', 5'-TTTTCTTQTCCTTTT-3 '5'-side cleavage The amount of product was calculated to be 2.6 times higher with methylamine treatment than with ethylenediamine treatment. From this result, it was suggested that in the cleavage reaction of DNA oligomer using methylamine or primary amine of ethylenediamine, the one having the highest reaction rate is the cleavage reaction using methylamine.

Q obtained in Synthesis Example 7 has a plurality of tautomers as shown below. Therefore, in oligodeoxynucleotides containing Q, it is considered that the elimination reaction of E and the cleavage reaction of oligodeoxynucleotide proceed when a tautomer of Q reacts with a nucleophile.

  In the above reaction formula, DNA means oligodeoxynucleotide and Nu means nucleophile.

<15. Synthesis Example 8: DNA oligomer containing 5- (2-oxy-2-phenylethyl) -2′-deoxyuridine (HP)>
[Synthesis Example 8-1: Synthesis of DNA oligomer containing 5- (2-oxy-2-phenylethyl) -2′-deoxyuridine (HP)]
Based on a known reaction at the nucleoside level, in the DNA oligomer containing P obtained in Synthesis Example 1, the base portion of P was cyclized with silver ions (A facile synthesis of fluorophores based on 5-phenylethynyluracils.Hudson, HER And Moszybski, JM Synlett 2006, 18, p. 2997-3000), followed by sodium hydroxide treatment to convert P to 5- (2-oxy-2-phenylethyl) -2'-deoxyuridine (Non-patent document 39).

To 200 μL of the 5′-T ₆ PT ₆ -3 ′ aqueous solution (1 mM strand concentration) obtained in Synthesis Example 1, 200 μL of an aqueous silver nitrate solution (100 mM) was added, mixed well, and allowed to stand at 25 ° C. for 16 hours. To the mixture, 20 μL of NaOH aqueous solution (10N) was added and mixed well. The resulting mixture was allowed to stand at 25 ° C. for 2 hours. Thereafter, water was added to the mixture to make about 1 mL, and 20 μL of acetic acid was added. The obtained solution was passed through a 0.45 μm filter, and purified by reverse phase HPLC (8-20% CH ₃ CN / 50 mM aqueous ammonium formate solution, 20 minutes, detected at 254 nm) and desalted. The product obtained as the main peak was identified by MALDI TOF-MS measurement.

In the following, 5- (2-oxy-2-phenylethyl) -2′-deoxyuridine:

Is indicated by “HP”.

  The concentration of DNA was determined by approximating HP with T and calculating the molar extinction coefficient at 260 nm, and calculating the concentration from UV absorption at 260 nm.

  The reverse phase HPLC chromatogram of the synthesized DNA oligomer is shown in FIGS.

  The retention time in the reverse phase HPLC of the synthesized DNA oligomer is as follows.

12.5 minutes for 5'-TTTTTT (HP) TTTTTT-3 '(SEQ ID NO: 28)
11.4 minutes for 5'-TTTTCTT (HP) TCCTTTT-3 '(SEQ ID NO: 29)
The MALDI TOF-MS data of the synthesized DNA oligomer is as follows.

5'-TTTTTT (HP) molecular formula for _{TTTTTT-3 'C 137 H 174} N 26 O 90 P 12, [MH] - Calculated 3995.6, Found 3995.6 (SEQ ID NO: 28)
For 5′-TTTTCTT (HP) TCCTTTT-3 ′, the molecular formula C ₁₅₄ H ₁₉₇ N ₃₃ O ₁₀₁ P ₁₄ , [MH] ⁻ calculated value 4559.0, measured value 4559.4 (SEQ ID NO: 29)
<16. Use Example 8: DNA oligomer containing 5- (2-oxy-2-phenylethyl) -2'-deoxyuridine>
[Use Example 8-1: Cleavage reaction of DNA oligomer containing 5- (2-oxy-2-phenylethyl) -2'-deoxyuridine with methylamine and identification of the product]
Add 300 μL of 5′-T ₆ (HP) T ₆ -3 ′ or 5′-TTTTCTT (HP) TCCTTTT-3 ′ aqueous solution (strand concentration 100 μM) and 300 μL methylamine (40% aqueous solution) to the screw tube. Heated in a water bath at 70 ° C. for 12 hours. After the reaction, methylamine was distilled off under reduced pressure with a centrifugal concentrator and analyzed by reverse phase HPLC to fractionate the product (8-20% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, at 254 nm. detection). The molecular weight of the fractionated product was measured by MALDI TOF-MS.

  61 and 62 show reverse phase HPLC chromatograms after the reaction of each synthesized DNA oligomer.

The MALDI TOF-MS data of products 1 and 2 in each DNA oligomer are as follows. In the following, “R ^Nu ” means a part derived from HP that is generated in accordance with the cleavage reaction.

5'-TTTTTT (HP) TTTTTT- 3 ' product for 1: pTTTTTT molecular formula _{C 60 H 80 N 12 O 43} P 6 FW 1843.2 [MH] - 1842.2 Found 1841.6
HPLC retention time 8.5 min product 2: TTTTTTpR ^Nu molecular formula _{C 66 H 91 N 13 O 45} P 6 FW1972.3 [MH] - 1971.3 Found 1971.0
HPLC retention time 10.6 minutes
5'-TTTTCTT (HP) TCCTTTT- 3 ' product for 1: pTCCTTTT molecular formula _{C 68 H 91 N 16 O 48} P 7 FW 2117.3 [MH] - 2116.3 Found 2116.2
HPLC retention time 7.3 min product 2: TTTTCTTpR ^Nu molecular formula _{C 75 H 103 N 16 O 51} P 7 FW 2261.5 [MH] - 2260.5 Found 2260.5
HPLC retention time 9.9 minutes Degradation reaction of DNA oligomer by methylamine is shown below. In the following reaction formula, the product on the left corresponds to the product 2, and the product on the right corresponds to the product 1.

In the above reaction formula, A and C have the same meaning as in formula I defined above. Based on the molecular weight of the product, R ^Nu is expected to be formed by adding one molecule of nucleophile to the degraded nucleotide and removing the unnatural base.

[Use Example 8-2: Comparison of cleavage reaction rate of DNA oligomer containing 5- (2-oxy-2-phenylethyl) -2'-deoxyuridine with methylamine or ethylenediamine]
Add 30 μL of 5′-T ₆ (HP) T ₆ -3 ′ or 5′-TTTTCTT (HP) TCCTTTT-3 ′ aqueous solution (strand concentration 100 μM) and 30 μL of methylamine 40% aqueous solution or ethylenediamine to the screw tube. The mixture was heated in a water bath at 70 ° C. for 2 hours. The reaction mixture was neutralized with acetic acid and analyzed by reverse phase HPLC (8-20% CH ₃ CN / 50 mM aqueous ammonium formate, 20 min, detected at 254 nm). An HPLC chart of the reaction mixture of 5′-T ₆ (HP) T ₆ -3 ′ is shown in FIG. 63, and an HPLC chart of the reaction mixture of 5′-TTTTCTT (HP) TCCTTTT-3 ′ is shown in FIG.

Was quantified based on the peak area of the _{5'-T 6 (HP) T} 6 -3 '5'a cleavage product of the side _{5'-T 6 p-3'} , 5'-T 6 (HP) The amount of cleavage product on the 5′-side of T ₆ -3 ′ was calculated to be 2.4 times greater for the methylamine treatment than for the ethylenediamine treatment. In addition, when quantified based on the peak area of 5'-TTTTCTTp-3 'which is a 5'-side cleavage product of 5'-TTTTCTT (HP) TCCTTTT-3', 5'-TTTTCTT (HP) TCCTTTT-3 The amount of cleavage product on the 5'-side of 'was calculated to be 2.1 times higher with methylamine treatment than with ethylenediamine treatment. From this result, it was suggested that in the cleavage reaction of DNA oligomer using methylamine or primary amine of ethylenediamine, the one having the highest reaction rate is the cleavage reaction using methylamine.

The HP obtained in Synthesis Example 8 has a structure in which water is added to the triple bond of P obtained in Synthesis Example 1. P can be heated in the presence of an amine used as a nucleophile to form an amine adduct in which an amine is added to a triple bond, and to form HP via tautomerism and amine elimination. There is sex.

In the above reaction formula, R ^Nu means a group generated with the addition of a nucleophile. The oligodeoxynucleotide is considered to be cleaved by elimination of a portion derived from an unnatural base while causing the following intramolecular cyclization reaction.

Alternatively, an oligodeoxynucleotide containing an amine adduct is considered to be cleaved after causing an intramolecular cyclization reaction as described below.

<17. Synthesis Example 9: DNA oligomer containing 8- (2-formylphenyl) -2′-deoxyguanosine>
[Synthesis Example 9-1: Synthesis of 8- (2-formylphenyl) -2′-deoxyguanosine]

8-Bromo-2'-deoxyguanosine is known in the literature (Cation-Mediated Energy Transfer in G-Quadruplexes Revealed by an Internal Fluorescent Probe. Dumas A. and Luedtke, NWJ Am. Chem. Soc. 2010, 132, p. 18004-18007). 3.46 g (10.0 mmol) 8-bromo-2'-deoxyguanosine (molecular weight 346.14), 3.00 g (20.0 mmol) 2-formylbenzeneboronic acid (molecular weight 149.94) and 224 mg (1.00 mmol) palladium acetate ( (Molecular weight 224.51), 787 mg (3.00 mmol) of triphenylphosphine (molecular weight 262.29) and 2.76 g (20.0 mmol) of potassium carbonate were added to an eggplant flask, and 80 mL of acetonitrile and 40 mL of water were added thereto, For 4 hours. The solvent of the reaction solution was distilled off under reduced pressure, 200 mL of methanol was added and filtered. The residue was washed with methanol. The filtrate was collected, the solvent was distilled off under reduced pressure, and the target substance was purified with a silica gel column (developing solvent was 10 to 20% MeOH / CH ₂ Cl ₂ ). The solvent of the objective fraction was distilled off under reduced pressure, and the precipitate formed with a small amount of methanol and acetonitrile was filtered and dried under reduced pressure. Concentrate the filtrate, filter the precipitate formed with a small amount of methanol and acetonitrile, and repeat the operation of drying under reduced pressure three times in total.2.97 g (8.00 mmol, 80% yield) of the target substance (molecular weight 371.35) Obtained as a pale yellow powder.

¹ H NMR (400 MHz, DMSO-d6) δ10.80 (br, 1H), 9.85 (s, 1H), 7.98 (dd, J = 7.8, 1.4 Hz, 1H), 7.84-7.80 (m, 1H), 7.75-7.67 (m, 2H), 6.47 (br, 2H), 5.92 (dd, J = 8.2, 6.4 Hz, 1H), 5.10 (d, J = 4.1 Hz, 1H), 4.89-4.86 (m, 1H) , 4.21-4.18 (m, 1H), 3.72-3.69 (m, 1H), 3.49-3.38 (m, 2H), 2.98 (ddd, J = 13.2, 8.2, 5.9 Hz, 1H), 4.20 (ddd, J = 13.3, 6.4, 2.3 Hz, 1H); ¹³ C NMR (100 MHz, DMSO-d6) δ191.7, 156.5, 153.3, 151.8, 143.4, 135.5, 133.5, 132.5, 131.4, 130.1, 128.4, 117.5, 87.8, 84.6 , 71.0, 61.9, 36.8; HRMS (ESI): Calculated for C ₁₇ H ₁₇ N ₅ O ₅ Na: 394.1127 [M + Na] ⁺ ; Found: 394.1115.
[Synthesis Example 9-2: Synthesis of 2-N- {N ′, N′-di (n-butyl) formamidine} -8- (2-formylphenyl) -2′-deoxyguanosine]

2.32 g (6.25 mmol) of 8- (2-formylphenyl) -2′-deoxyguanosine (molecular weight 371.35) was dissolved in 20 mL of methanol. 11.7 mL (50.0 mmol) N, N-di (n-butyl) formamide dimethyl acetal (molecular weight 203.32, density 0.87 g / mL) (Dialkylformamidines: depurination resistant N6-protecting group for deoxyadenosine.Froehler, BC And Matteucci, MD Tetrahedron Letters 1983, 11, p. 8031-8036), and stirred at 25 ° C. for 2 hours. The solvent was distilled off under reduced pressure, and the target substance was purified with a silica gel column (developing solvent was 5 to 10% MeOH / CH ₂ Cl ₂ ). The solvent of the objective fraction was distilled off under reduced pressure, and the precipitate formed with a small amount of ethyl acetate and hexane was filtered and dried under reduced pressure. 2.10 g (4.11 mmol, 66% yield) of the target substance (molecular weight 510.59) was obtained as a pale yellow powder.

¹ H NMR (400 MHz, DMSO-d6) δ11.51 (s, 1H), 9.89 (s, 1H), 8.53 (s, 1H), 8.02-8.00 (m, 1H), 7.87-7.83 (m, 1H ), 7.77-7.73 (m, 1H), 7.69-7.67 (m, 1H), 5.92 (dd, J = 7.8, 6.9 Hz, 1H), 5.16 (d, J = 4.2 Hz, 1H), 4.85-4.82 ( m, 1H), 4.33-4.29 (m, 1H), 3.76-3.73 (m, 1H), 3.58-3.35 (m, 6H), 3.14 (ddd, J = 13.7, 7.3, 6.4 Hz, 1H), 2.05 ( ddd, J = 13.3, 6.4, 2.8 Hz, 1H), 1.63-1.54 (m, 4H), 1.35-1.23 (m, 4H), 0.94-0.89 (m, 6H); ¹³ C NMR (100 MHz, DMSO- d6) δ191.7, 157.8, 157.4, 157.3, 150.5, 144.6, 135.5, 133.6, 132.1, 131.3, 130.2, 128.7, 120.5, 87.8, 85.0, 71.0, 62.0, 51.2, 44.9, 37.3, 30.3, 28.5, 19.6, 19.1, 13.7, 13.5; HRMS (ESI): Calculated for C ₂₆ H ₃₄ N ₆ O ₅ Na: 533.2488 [M + Na] ⁺ ; Found: 533.2481.
[Synthesis Example 9-3: 2-N- {N ′, N′-di (n-butyl) formamidine} -8- (2-formylphenyl) -5′-O-dimethoxytrityl-2′-deoxyguanosine Synthesis]

16 mL of 2.10 g (4.11 mmol) 2-N- {N ', N'-di (n-butyl) formamidine} -8- (2-formylphenyl) -2'-deoxyguanosine (molecular weight 510.59) In pyridine. 1.53 g (4.52 mmol) of 4,4′-dimethoxytrityl chloride (molecular weight 338.83) was added to the resulting solution, and the mixture was stirred at 25 ° C. for 1 hour. About 10 mL of methanol was added, and the solvent was distilled off under reduced pressure. A small amount of methylene chloride and hexane were added to the residue and azeotroped three times, and then the target substance was purified with a silica gel column (developing solvent was 2% Et ₃ N, AcOEt: n-hexane = 8: 2 to AcOEt). The solvent of the objective fraction was distilled off under reduced pressure, and a small amount of methylene chloride and hexane were added and azeotroped. 2.20 g (2.71 mmol, 66% yield) of the desired product (molecular weight 812.95) was obtained as a pale yellow powder.

¹ H NMR (400 MHz, DMSO-d6) δ11.52 (s, 1H), 9.88 (s, 1H), 8.38 (s, 1H), 7.98 (d, J = 7.8 Hz, 1H), 7.80-7.79 ( m, 2H), 7.74-7.70 (m, 1H), 7.31-7.29 (m, 2H), 7.20-7.15 (m, 7H), 6.79-6.72 (m, 4H), 6.00 (dd, J = 8.0, 4.8 Hz, 1H), 5.19 (d, J = 5.1 Hz, 1H), 4.54-4.48 (m, 1H), 3.84-3.80 (m, 1H), 3.70 (s, 3H), 3.69 (s, 3H), 3.47 -3.43 (m, 2H), 3.31-3.26 (m, 1H), 3.21-3.13 (m, 3H), 3.07-3.04 (m, 1H), 2.17 (ddd, J = 13.7, 8.0, 5.8 Hz, 1H) , 1.59-1.51 (m, 2H), 1.45-1.36 (m, 2H), 1.33-1.24 (m, 2H), 1.15-1.09 (m, 2H), 0.93-0.89 (m, 3H), 0.79-0.75 ( m, 3H); ¹³ C NMR (100 MHz, DMSO-d6) δ 191.6, 158.0, 157.9, 157.5, 157.3, 156.9, 150.3, 144.9, 144.8, 135.7, 135.6, 133.4, 132.5, 131.2, 130.1, 129.6, 129.4, 128.3, 127.6, 126.5, 120.4, 112.9, 85.4, 85.2, 84.0, 70.6, 63.6, 55.0, 54.9, 51.2, 45.0, 37.6, 30.2, 28.5, 19.6, 19.0, 13.7, 13.5; HRMS (ESI): C Calculated for ₄₇ H ₅₃ N ₆ O ₇ : 813.3976 [M + H] ⁺ ; Found: 813.3976.
[Synthesis Example 9-4: 2-N- {N ′, N′-di (n-butyl) formamidine} -8- (2-formylphenyl) -5′-O-dimethoxytrityl-2′-deoxyguanosine Of 3'-N, N-diisopropyl (cyanoethyl) phosphoramidite]

163 mg (0.200 mmol) 2-N- {N ', N'-di (n-butyl) formamidine} -8- (2-formylphenyl) -5'-O-dimethoxytrityl-2'-deoxyguanosine (Molecular weight 812.95) was added to an eggplant flask, 5 mL of CH ₃ CN and 223 μL (1.600 mmol) of triethylamine (molecular weight 101.19, specific gravity 0.726 g / mL) were added, stirred, and further 178 μL (0.800 mmol) of 2-cyanoethyl. N, N, N ′, N′-tetraisopropyl phosphoramidite (molecular weight 236.68, specific gravity 1.061 g / mL) was added, and the mixture was stirred at room temperature for 30 minutes. 25 mL of AcOEt and 25 mL of saturated saline were added, and the mixture was separated. The organic layer was further washed twice with saturated brine. The obtained organic layer was dried over anhydrous magnesium sulfate and filtered through a filter, and the solvent of the filtrate was distilled off under reduced pressure. After azeotroping with acetonitrile three times, 10 mL of acetonitrile was added and dissolved, and a small amount of molecular sieve was added. Attached to an automatic DNA synthesizer and used for DNA synthesis

[Synthesis Example 9-5: Synthesis of DNA oligomer containing 8- (2-formylphenyl) -2'-deoxyguanosine (fPG)]

The DNA was synthesized by the phosphoramidite method with DMTr ON using a DNA synthesis protocol of a normal automatic DNA synthesizer with 1 μmol scale. Cutting out from the carrier was performed with 28% aqueous ammonia, and deprotection was performed by standing at 25 ° C. for 16 hours. After deprotection, the ammonia was distilled off under reduced pressure using a centrifugal concentrator, the solution containing DNA was passed through a 0.45 μm filter, and the main peak was purified by reverse-phase HPLC (0-50% CH ₃ CN / 50 mM formic acid. Ammonium aqueous solution, 20 minutes, detected at 254 nm), ammonium formate and solvent were distilled off by centrifugal concentrator and lyophilization. To the obtained residue, 100 μL of 80% aqueous acetic acid was added, mixed well, and allowed to stand at 25 ° C. for 30 minutes. Thereafter, distilled water was added to the mixture to make about 1 mL, passed through a 0.45 μm filter, and then the main peak eluted at a retention time of 13 to 14 minutes was purified by reverse phase HPLC (5- 20% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, detected at 254 nm), ammonium formate and the solvent were distilled off by centrifugal concentrator and lyophilization. The obtained DNA oligomer was identified by MALDI TOF-MS measurement. The synthesized DNA oligomer was made into an aqueous solution by adding about 500 μL of distilled water.

In the following, 8- (2-formylphenyl) -2′-deoxyguanosine:

Is indicated by “fPG”.

  For determination of DNA concentration, the molar extinction coefficient at 260 nm was calculated by approximating fPG to C, and the concentration was calculated from UV absorption at 260 nm.

  65 and 66 show reverse phase HPLC chromatograms of the synthesized DNA oligomers.

  The retention time in the reverse phase HPLC of the synthesized DNA oligomer is as follows.

14.6 minutes for 5'-TTTTTT (fPG) TTTTTT-3 '(SEQ ID NO: 30)
14.0 minutes for 5'-TTTTCTT (fPG) TCCTTTT-3 '(SEQ ID NO: 31)
The MALDI TOF-MS data of the synthesized DNA oligomer is as follows.

5'-TTTTTT (fPG) TTTTTT- 3 ' molecular formula _{C 137 H 173 N 29 O 89} P 12 about, [MH] ^- calcd 4020.7, found 4022.0 (SEQ ID NO: 30) of
For 5′-TTTTCTT (fPG) TCCTTTT-3 ′, the molecular formula C ₁₅₄ H ₁₉₆ N ₃₆ O ₁₀₀ P ₁₄ , calculated value of [MH] ⁻ 4584.0, measured value 4584.3 (SEQ ID NO: 31)
<18. Use Example 9: DNA oligomer containing 8- (2-formylphenyl) -2'-deoxyguanosine (fPG)>
[Use Example 9-1: Cleavage reaction of methyl oligomer containing 8- (2-formylphenyl) -2'-deoxyguanosine (fPG) with methylamine and identification of the product]
Add 300 μL of 5′-T ₆ (fPG) T ₆ -3 ′ or 5′-TTTTCTT (fPG) TCCTTTT-3 ′ aqueous solution (strand concentration 100 μM) and 300 μL methylamine (40% aqueous solution) to the screw tube. Heated in a water bath at 70 ° C. for 6 hours. After the reaction, methylamine was distilled off under reduced pressure with a centrifugal concentrator and analyzed by reverse phase HPLC to fractionate the product (5-20% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, at 254 nm. detection). The molecular weight of the fractionated product was measured by MALDI TOF-MS.

  67 and 68 show reverse phase HPLC chromatograms after the reaction of each synthesized DNA oligomer.

The MALDI TOF-MS data of products 1 and 2 in each DNA oligomer are as follows. In the following, “R ^Nu ” means a part derived from fPG that is generated along with the cleavage reaction.

5'-TTTTTT (fPG) TTTTTT- 3 ' product for 1: pTTTTTT molecular formula _{C 60 H 80 N 12 O 43} P 6 FW 1843.2 [MH] - 1842.2 Found 1842.2
HPLC retention time 12.0 min product 2: TTTTTTpR ^Nu molecular formula _{C 66 H 91 N 13 O 45} P 6 FW 1972.3 [MH] - 1971.3 Found 1971.0
HPLC retention time 13.9 minutes
5'-TTTTCTT (fPG) TCCTTTT- 3 ' product for 1: pTCCTTTT molecular formula _{C 68 H 91 N 16 O 48} P 7 FW 2117.3 [MH] - 2116.3 Found 2116.2
HPLC retention time 11.1 min product 2: TTTTCTTpR ^Nu molecular formula _{C 75 H 103 N 16 O 51} P 7 FW 2261.5 [MH] - 2260.5 Found 2260.5
HPLC retention time 13.3 minutes Degradation reaction of DNA oligomer by methylamine is shown below. In the following reaction formula, the product on the left corresponds to the product 2, and the product on the right corresponds to the product 1.

In the above reaction formula, A and C have the same meaning as in formula I defined above. Based on the molecular weight of the product, R ^Nu is expected to be formed by adding one molecule of nucleophile to the degraded nucleotide and removing the unnatural base.

<19. Synthesis Example 10: DNA oligomer containing 8- (2-formylphenyl) -2'-deoxyadenosine>
[Synthesis Example 10-1: Synthesis of 8- (2-formylphenyl) -2′-deoxyadenosine]

8-Bromo-2'-deoxyadenosine was synthesized according to a known document (Japanese Patent Laid-Open No. 2011-37796). 2.20 g (6.66 mmol) 8-bromo-2'-deoxyadenosine (molecular weight 330.14), 2.00 g (13.3 mmol) 2-formylbenzeneboronic acid (molecular weight 149.94) and 150 mg (0.666 mmol) palladium acetate (molecular weight) 224.51) and 525 mg (2.00 mmol) of triphenylphosphine (molecular weight 262.29) and 1.84 g (13.3 mmol) of potassium carbonate were added to an eggplant flask, and 40 mL of acetonitrile and 20 mL of water were added thereto at 80 ° C. Stir for 4 hours. The solvent of the reaction solution was distilled off under reduced pressure, 200 mL of methanol was added and filtered. The residue was washed with methanol. The filtrate was collected, the solvent was distilled off under reduced pressure, and the target substance was purified with a silica gel column (developing solvent was 10% MeOH / CH ₂ Cl ₂ ). The solvent of the objective fraction was distilled off under reduced pressure, dissolved in a small amount of acetonitrile, methylene chloride was added, and the precipitate formed by adding hexane was filtered and dried under reduced pressure. 1.81 g (5.09 mmol, yield 76%) of the target substance (molecular weight 355.35) was obtained as a white powder.

¹ H NMR (400 MHz, DMSO-d6) δ9.93 (s, 1H), 8.18 (s, 1H), 8.09-8.07 (m, 1H), 7.90-7.80 (m, 2H), 7.74-7.72 (m , 1H), 7.46 (br, 2H), 5.90 (dd, J = 8.5, 6.2 Hz, 1H), 5.49 (dd, J = 8.3, 4.1 Hz, 1H), 5.17 (d, J = 4.1 Hz, 1H) , 4.39-4.35 (m, 1H), 3.82-3.79 (m, 1H), 3.65-3.59 (m, 1H), 3.50-3.44 (m, 1H), 3.19 (ddd, J = 12.8, 8.7, 6.0 Hz, 1H), 2.06 (ddd, J = 13.0, 6.2, 1.8 Hz, 1H); ¹³ C NMR (100 MHz, DMSO-d6) δ191.7, 156.2, 152.3, 149.6, 147.4, 135.4, 133.8, 131.5, 131.4, 130.8, 129.5, 119.3, 88.3, 85.7, 71.3, 62.2, 37.4; HRMS (ESI): Calculated for C ₁₇ H ₁₈ N ₅ O ₄ : 356.1359 [M + H] ⁺ ; Found: 356.1364.
[Synthesis Example 10-2: Synthesis of 6-N- {N ′, N′-di (n-butyl) formamidine} -8- (2-formylphenyl) -2′-deoxyadenosine]

1.55 g (4.36 mmol) of 8- (2-formylphenyl) -2'-deoxyadenosine (molecular weight 355.35) was dissolved in 20 mL of methanol, and 8.14 mL (34.8 mmol) of N, N- Dialkylformamidines: depurination resistant N6-protecting group for deoxyadenosine. Froehler, BC and Matteucci, MD Tetrahedron Letters 1983, 11, p. 8031-8036 ) Was added and stirred at 25 ° C. for 2 hours. The solvent was distilled off under reduced pressure, and the target substance was purified with a silica gel column (developing solvent was 2 to 5% MeOH / CH ₂ Cl ₂ ). The solvent of the desired fraction was distilled off under reduced pressure, a small amount of methylene chloride was added, diethyl ether was added, and the precipitate formed by adding hexane was filtered and dried under reduced pressure. 1.40 g (2.83 mmol, yield 65%) of the target substance (molecular weight 494.59) was obtained as a white powder.

¹ H NMR (400 MHz, DMSO-d6) δ9.92 (s, 1H), 8.91 (s, 1H), 8.45 (s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.91-7.82 ( m, 2H), 7.74 (d, J = 7.3 Hz, 1H), 5.94 (dd, J = 7.8, 6.8 Hz, 1H), 5.28 (dd, J = 7.6, 4.4 Hz, 1H), 5.18 (d, J = 4.1 Hz, 1H), 4.40-4.36 (m, 1H), 3.81-3.77 (m, 1H), 3.64-3.41 (m, 6H), 3.25-3.18 (m, 1H), 2.09-2.04 (m, 1H ), 1.62-1.52 (m, 4H), 1.33-1.22 (m, 4H), 0.94-0.83 (m, 6H); ¹³ C NMR (100 MHz, DMSO-d6) δ191.8, 159.5, 157.9, 151.7, 151.6, 149.3, 135.5, 133.8, 131.5, 131.3, 130.9, 129.7, 125.8, 88.2, 85.5, 71.2, 62.1, 50.9, 44.4, 37.2, 30.4, 28.7, 19.6, 19.1,13.7, 13.5; HRMS (ESI): C Calculated for ₂₆ H ₃₅ N ₆ O ₄ : 495.2720 [M + H] ⁺ ; Found: 495.2718.
[Synthesis Example 10-3: 6-N- {N ′, N′-di (n-butyl) formamidine} -8- (2-formylphenyl) -5′-O-dimethoxytrityl-2′-deoxyadenosine Synthesis]

1.31 g (2.64 mmol) 6-N- {N ', N'-di (n-butyl) formamidine} -8- (2-formylphenyl) -2'-deoxyguanosine (molecular weight 494.59) in 10 mL Dissolved in pyridine. To the obtained solution, 991 mg (2.92 mmol) of 4,4′-dimethoxytrityl chloride (molecular weight: 338.83) was added and stirred at 25 ° C. for 1 hour. About 10 mL of methanol was added, and the solvent was distilled off under reduced pressure. A small amount of methylene chloride and hexane were added to the residue, and the mixture was azeotroped three times. Then, the target substance was purified with a silica gel column (developing solvent was 2% Et ₃ N, 2% MeOH / CH ₂ Cl ₂ ). The solvent of the objective fraction was distilled off under reduced pressure, and a small amount of methylene chloride and hexane were added and azeotroped. 1.24 g (1.56 mmol, 59% yield) of the desired product (molecular weight 796.95) was obtained as a white powder.

¹ H NMR (400 MHz, DMSO-d6) δ9.89 (s, 1H), 8.88 (s, 1H), 8.25 (s, 1H), 8.06-8.04 (m, 1H), 7.88-7.78 (m, 3H ), 7.33-7.31 (m, 2H), 7.21-7.15 (m, 7H), 6.81-6.73 (m, 4H), 5.98 (t, J = 6.9 Hz, 1H), 5.25 (d, J = 4.6 Hz, 1H), 4.57-4.53 (m, 1H), 3.94-3.90 (m, 1H), 3.71 (s, 3H), 3.69 (s, 3H), 3.59-3.53 (m, 2H), 3.44-3.32 (m, 3H), 3.20-3.12 (m, 2H), 2.11 (ddd, J = 13.0, 7.3, 4.3 Hz, 1H), 1.62-1.52 (m, 4H), 1.34-1.23 (m, 4H), 0.90-0.87 ( m, 6H); ¹³ C NMR (100 MHz, DMSO-d6) δ 191.6, 159.2, 157.9, 157.9, 157.7, 151.8, 149.5, 145.0, 135.7, 135.60, 135.55, 133.6, 131.7, 131.4, 130.7, 129.7, 129.5, 129.1, 127.6, 126.5, 125.6, 113.0, 112.9, 85.8, 85.2, 84.8, 71.0, 63.6, 54.94, 54.90, 50.9, 44.4, 36.3, 30.4, 28.7, 19.6, 19.1, 13.7, 13.5; HRMS (ESI) : Calculated for C ₄₇ H ₅₃ N ₆ O ₆ : 797.4027 [M + H] ⁺ ; Found: 797.4046.
[Synthesis Example 10-4: 6-N- {N ′, N′-di (n-butyl) formamidine} -8- (2-formylphenyl) -5′-O-dimethoxytrityl-2′-deoxyadenosine Synthesis of -3'-N, N-diisopropyl (cyanoethyl) phosphoramidite]

159 mg (0.200 mmol) 6-N- {N ', N'-di (n-butyl) formamidine} -8- (2-formylphenyl) -5'-O-dimethoxytrityl-2'-deoxyadenosine (Molecular weight 796.95) was placed in an eggplant flask, 5 mL of CH ₃ CN and 223 μL (1.600 mmol) of triethylamine (molecular weight 101.19, specific gravity 0.726 g / mL) were added, stirred, and 178 μL (0.800 mmol) of 2-cyanoethyl N , N, N ′, N′-Tetraisopropylphosphorodiamidite (molecular weight 236.68, specific gravity 1.061 g / mL) was added, and the mixture was stirred at room temperature for 30 minutes. 25 mL of AcOEt and 25 mL of saturated saline were added, and the mixture was separated. The organic layer was further washed twice with saturated brine. The organic layer was dried over anhydrous magnesium sulfate and filtered through a filter, and the solvent of the filtrate was distilled off under reduced pressure. After azeotroping with acetonitrile three times, 10 mL of acetonitrile was added and dissolved, and a small amount of molecular sieve was added. Attached to an automatic DNA synthesizer and used for DNA synthesis

[Synthesis Example 10-5: Synthesis of DNA oligomer containing 8- (2-formylphenyl) -2'-deoxyadenosine (fPA)]

The DNA was synthesized by the phosphoramidite method with DMTr ON using a DNA synthesis protocol of a normal automatic DNA synthesizer with 1 μmol scale. Cutout was performed with 28% aqueous ammonia, and deprotection was performed by standing at 25 ° C. for 16 hours. After deprotection, the ammonia was distilled off under reduced pressure using a centrifugal concentrator, the solution containing DNA was passed through a 0.45 μm filter, and the main peak was purified by reverse-phase HPLC (0-50% CH ₃ CN / 50 mM formic acid. Ammonium aqueous solution, 20 minutes, detected at 254 nm), ammonium formate and solvent were distilled off by centrifugal concentrator and lyophilization. To the obtained residue, 100 μL of 80% aqueous acetic acid was added, mixed well, and allowed to stand at 25 ° C. for 30 minutes. Thereafter, distilled water was added to the mixture so as to be about 1 mL, passed through a 0.45 μm filter, and then the main peak eluted at a retention time of 13-14 minutes was purified by reverse phase HPLC (5- 20% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, detected at 254 nm), ammonium formate and the solvent were distilled off by centrifugal concentrator and lyophilization. The obtained DNA oligomer was identified by MALDI TOF-MS measurement. The synthesized DNA oligomer was made into an aqueous solution by adding about 500 μL of distilled water.

In the following, 8- (2-formylphenyl) -2′-deoxyadenosine:

Is indicated by “fPA”.

  The concentration of DNA was determined by calculating the molar extinction coefficient at 260 nm by approximating fPA to C, and calculating the concentration from the UV absorption at 260 nm.

  69 and 70 show reverse phase HPLC chromatograms of the synthesized DNA oligomers.

  The retention time in the reverse phase HPLC of the synthesized DNA oligomer is as follows.

14.9 minutes for 5'-TTTTTT (fPA) TTTTTT-3 '(SEQ ID NO: 32)
13.8 minutes for 5'-TTTTCTT (fPA) TCCTTTT-3 '(SEQ ID NO: 33)
The MALDI TOF-MS data of the synthesized DNA oligomer is as follows.

5'-TTTTTT (fPA) TTTTTT- 3 ' molecular formula _{C 137 H 173 N 29 O 88} P 12 about, [MH] ^- Calculated 4004.7, Found 4005.2 (SEQ ID NO: 32)
For 5′-TTTTCTT (fPA) TCCTTTT-3 ′, the molecular formula C ₁₅₄ H ₁₉₆ N ₃₆ O ₉₉ P ₁₄ , calculated value of [MH] ⁻ 4568.0, measured value 4568.5 (SEQ ID NO: 33)
<20. Use Example 10: DNA oligomer containing 8- (2-formylphenyl) -2'-deoxyadenosine (fPA)>
[Use Example 10-1: Cleavage reaction of methyl oligomer containing 8- (2-formylphenyl) -2'-deoxyadenosine (fPA) with methylamine and identification of the product]
Add 300 µL of 5'-T ₆ (fPA) T ₆ -3 'or 5'-TTTTCTT (fPA) TCCTTTT-3' aqueous solution (strand concentration 100 µM) and 300 µL of methylamine (40% aqueous solution) to the screw tube. Heated in a water bath at 70 ° C. for 6 hours. After the reaction, methylamine was distilled off under reduced pressure with a centrifugal concentrator and analyzed by reverse phase HPLC to fractionate the product (5-20% CH ₃ CN / 50 mM ammonium formate aqueous solution, 20 minutes, at 254 nm. detection). The molecular weight of the fractionated product was measured by MALDI TOF-MS.

  71 and 72 show reverse phase HPLC chromatograms after the reaction of each synthesized DNA oligomer.

The MALDI TOF-MS data of products 1 and 2 in each DNA oligomer are as follows. In the following, “R ^Nu ” means a part derived from fPA that is generated along with the cleavage reaction.

5'-TTTTTT (fPA) TTTTTT- 3 ' product for 1: pTTTTTT molecular formula _{C 60 H 80 N 12 O 43} P 6 FW 1843.2 [MH] - 1842.2 Found 1842.0 and 4019.5 (the larger the latter molecular weight peak)
HPLC retention time 12.1 min product 2: TTTTTTpR ^Nu molecular formula _{C 66 H 91 N 13 O 45} P 6 FW 1972.3 [MH] - 1971.3 Found 1971.2
HPLC retention time 13.9 minutes
5'-TTTTCTT (fPA) TCCTTTT- 3 ' product for 1: pTCCTTTT molecular formula _{C 68 H 91 N 16 O 48} P 7 FW 2117.3 [MH] - 2116.3 Found 2116.2
HPLC retention time 11.1 min product 2: TTTTCTTpR ^Nu molecular formula _{C 75 H 103 N 16 O 51} P 7 FW 2261.5 [MH] - 2260.5 Found 2260.6
HPLC retention time 13.2 minutes From the molecular weight of the product, for 5'-T ₆ (fPA) T ₆ -3 ', it is expected that another product was eluted at the same retention time as product 1 . The aldehyde on methylamine and adenosine analogues, creation of a stable imine (molecular formula _{C 138 H 176 N 30 O 87} P 12, molecular weight 4018.7) is expected.

Degradation reaction of DNA oligomer by methylamine is shown below. In the following reaction formula, the product on the left corresponds to the product 2, and the product on the right corresponds to the product 1.

In the above reaction formula, A and C have the same meaning as in formula I defined above. Based on the molecular weight of the product, R ^Nu is expected to be formed by adding one molecule of nucleophile to the degraded nucleotide and removing the unnatural base.

The oligodeoxynucleotide containing fPG or fPA is considered to be cleaved by elimination of a portion derived from an unnatural base while causing the following intramolecular cyclization reaction. The following reaction formula is for oligodeoxynucleotides containing fPG, but similar reactions are thought to proceed in the case of oligodeoxynucleotides containing fPA.

<21. Use Example 11: Amplification and cleavage of relatively long DNA by polymerase chain reaction using a DNA oligomer containing 5-ethynyl-2′-deoxyuridine (Pa) and ligation of amplification products>
[Use Example 11-1: Amplification of DNA by polymerase chain reaction using a DNA oligomer containing 5-ethynyl-2′-deoxyuridine (Pa)]
A DNA oligomer containing 5-ethynyl-2′-deoxyuridine (Pa) having the following base sequence was synthesized in the same procedure as in Synthesis Example 3.

LL1: 5'-AGG (Pa) GCTTTATGACTCTGCCG-3 '(SEQ ID NO: 34)
LL2: 5'-AGGTGCTTTA (Pa) GACTCTGCCG-3 '(SEQ ID NO: 35)
LL3: 5'-ACCGAAGCG (Pa) GGTGTTTACGAAGG-3 '(SEQ ID NO: 36)
LL4: 5'-AGCG (Pa) GGTGTTTACGAAGG-3 '(SEQ ID NO: 37)
LL5: 5'-ACGC (Pa) TCGGTATCGCTCTG-3 '(SEQ ID NO: 38)
LL6: 5'-ACGCTTCGG (Pa) ATCGCTCTG-3 '(SEQ ID NO: 39)
LL7: 5'-ATAAAGCACC (Pa) CGGTGGTGAAAGG-3 '(SEQ ID NO: 40)
LL8: 5'-ACC (Pa) CGGTGGTGAAAGGGCAG-3 '(SEQ ID NO: 41)
Polymerase chain reaction was performed using λDNA (catalog number N3011 manufactured by New England BioLabs) as a template DNA, KOD FX Neo (catalog number KFX-201 manufactured by TOYOBO) as an enzyme, and the DNA oligomer as a primer. . Strand concentration: 10 μM 2 μL of primer, 1 μL of 1 ng / μL λDNA, 10 μL of 2 mM dNTPs, 1 μL (1 U) of KOD Fx neo (Toyobo KFX-201), and buffer supplied with the product (× 2) of 25 μL was added to the polymerase chain reaction tube. Attach the above reaction tube to TaKaRa PCR Thermal Cycler Dice (registered trademark) Gradient (thermal cycler manufactured by Takara, catalog number TP600), 94 ° C, 2 minutes → [98 ° C, 10 seconds → 60 ° C, 30 seconds → 68 ° C., 2 minutes] The reaction was carried out at a temperature cycle of 30 × 4 ° C. to amplify DNA of about 1.5 kb and about 2.3 kb. By using a primer set of LL1 or LL2 and LL3 or LL4, DNA of about 1.5 kb was amplified, and by using a primer set of LL5 or LL6 and LL7 or LL8, DNA of about 2.3 kb was amplified. . The reaction solution (0.5 μL) after the polymerase chain reaction was analyzed by 1% agarose gel electrophoresis. The gel after electrophoresis was stained with ethidium bromide to detect the fluorescence of DNA. FIG. 73 shows the gel after ethidium bromide staining. In the photo in the figure, black and white are reversed.

  In FIG. 73, the bands in lane 1 are DNA markers (100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 and 2072 bp, bands that appeared strongly. Are DNA of 600, 1500 and 2072 bp). The lane 2 band shows the amplification product DNA when LL1 and LL3 are used as the primer set, and the lane 3 band shows the amplification product DNA when LL2 and LL4 are used as the primer set. The lane 4 band shows the amplification product DNA when LL5 and LL8 are used as the primer set, and the lane 5 band shows the amplification product DNA when LL6 and LL7 are used as the primer set. Show.

[Use Example 11-2: Cleavage reaction of DNA amplification product containing 5-ethynyl-2′-deoxyuridine (Pa) and ligation reaction of the obtained 5 ′ terminal phosphorylated DNA fragment (1)]
Two kinds of DNA amplification products prepared in Use Example 11-1 were mixed in an equivalent amount. One volume of 40% methylamine aqueous solution was added to the resulting mixture and mixed well. The resulting mixture was allowed to stand at 25 ° C. for 2 days (48 hours). Using a centrifugal concentrator, methylamine was removed from the solution without heating. Thereafter, water was added to prepare the same volume as the original reaction solution. 2 μL of this solution, 6.5 μL of water, 0.5 μL of Taq DNA ligase (New England BioLabs catalog number M0208S), and 1 μL of the buffer (× 10) attached to the product were added to the polymerase chain reaction tube. The reaction tube is attached to a TaKaRa PCR Thermal Cycler Dice (registered trademark) Gradient (Takara thermal cycler, catalog number TP600), and [80 ° C., 1 minute → 45 ° C., 5 minutes] × 10, 20 or The DNA was ligated by reaction at a temperature cycle of 30 → 4 ° C. The reaction solution (4 μL) after the ligation reaction was analyzed by 1% agarose gel electrophoresis. The gel after electrophoresis was stained with ethidium bromide to detect the fluorescence of DNA. FIG. 74 shows the gel after ethidium bromide staining. In the photo in the figure, black and white are reversed.

  In FIG. 74, the band in lane 1 shows DNA markers (0.5, 1.0, 1.5, 2, 3, 4, 6, 8, 10, and 12 kb). The bands in lanes 2 and 3 show the DNA of the ligation product when the ligation reaction was performed in 10 temperature cycles, and the bands in lanes 4 and 5 were ligated in 20 temperature cycles. In this case, the ligation reaction product DNA is shown, and the bands in lanes 6 and 7 show the ligation reaction product DNA when the ligation reaction is performed in 30 temperature cycles. The bands in lanes 2, 4 and 6 were ligated with the amplification product DNA when LL1 and LL3 were used as a primer set and the amplification product DNA when LL6 and LL7 were used as a primer set. The ligation reaction product DNA is shown, and the bands in lanes 3, 5 and 7 are the amplification product DNA when LL2 and LL4 are used as a primer set, and the bands when LL6 and LL7 are used as a primer set. The DNA of the ligation product when the ligation reaction is performed with the DNA of the amplification product is shown.

  As shown in FIG. 74, a 3.8 kb DNA band was confirmed by ligation reaction of the 1.5 kb and 2.3 kb source DNA. This suggests that the DNA was cleaved by methylamine treatment, and hybridization of two DNA strands having one nucleotide gap (deletion site) occurred during the heating and cooling cycles for ligation. Ligase is thought to cause a covalent bond between the two DNAs. However, it is unclear only from the results of analysis by agarose gel electrophoresis how often the two DNAs were covalently linked by ligase. When hybridized DNA is introduced into E. coli or the like, nicks (cuts) that are not linked are generally repaired.

  In order to compare the DNA before ligation with the DNA after ligation, these DNAs were analyzed together by 1% agarose gel electrophoresis. The gel after electrophoresis was stained with ethidium bromide to detect the fluorescence of DNA. The gel after ethidium bromide staining is shown in FIG. In the photo in the figure, black and white are reversed.

  In FIG. 75, the band in lane 1 shows DNA markers (0.5, 1.0, 1.5, 2, 3, 4, 6, 8, 10, and 12 kb). The bands in lanes 2 and 3 show the DNA of the cleavage reaction product when the DNA cleavage reaction was performed with methylamine treatment for 9 hours, and the bands in lanes 4 and 5 were subjected to methylamine treatment for 24 hours. The DNA of the cleavage reaction product when the DNA cleavage reaction is performed is shown, and the bands in lanes 6 and 7 indicate the DNA of the cleavage reaction product when the DNA cleavage reaction is performed for 48 hours with methylamine treatment. Is shown. The bands in lanes 8 and 9 indicate the DNA of the ligation product when the ligation reaction was performed with 30 temperature cycles after methylamine treatment for 48 hours to cleave the DNA. The bands in lanes 2, 4, 6, and 8 cleave the amplification product DNA when LL1 and LL3 are used as a primer set and the amplification product DNA when LL6 and LL7 are used as a primer set. Shows the DNA of the cleavage reaction product in the case of ligation or the DNA of the ligation reaction product in the case of the cleavage reaction and the ligation reaction, and the bands of lanes 3, 5, 7, and 9 used LL2 and LL4 as the primer set DNA of the amplification product in the case of, and DNA of the cleavage reaction product when the DNA of the amplification product in the case of using LL6 and LL7 as a primer set or the reaction of the ligation product in the case of the cleavage reaction and the ligation reaction Shows DNA.

  In the cleavage reaction of the DNA amplification product, when methylamine was added to the DNA amplification product and heated, the DNA was alkali-denatured and no clear band was detected. In contrast, as shown in FIGS. 74 and 75, it was confirmed that when the DNA amplification product was reacted at 25 ° C. in the presence of methylamine, the alkaline denaturation of DNA was suppressed. Further, as shown in FIG. 75, it was confirmed that in the state before the ligation reaction, even when the raw material DNA was mixed, a 3.8 kb DNA band corresponding to the ligation product was not generated. This is because the short DNA fragments produced by the cleavage are bound to the terminal portions of the DNA after cleavage, and it is difficult for the DNA end portions to hybridize with each other simply by mixing the DNA after the cleavage. It suggests.

  5 position substituted 2'-deoxyuridine derivatives are known to be suitable as substrates or templates for DNA replication reactions, and report of DNA replication reactions using 5 position substituted 2'-deoxyuridine triphosphates. Are many (for example, non-patent documents 74 to 76). A DNA replication and amplification reaction using 5- (1-propynyl) -2'-deoxyuridine triphosphate having a structure similar to Pa has also been reported, and the replication reaction proceeds efficiently with a relatively high degree of loyalty. It is known (Non-patent Document 77). Until now, there has been no report of DNA replication reaction using Pa as a template. In this use example, DNA ligation was confirmed, suggesting that DNA elongation occurred using Pa as a template.

[Use Example 11-3: Cleavage reaction of a DNA amplification product containing 5-ethynyl-2′-deoxyuridine (Pa) and ligation reaction of the obtained 5′-terminal phosphorylated DNA fragment (2)]
In Use Example 11-2, a combination of DNA amplification products was selected so that only one end of two types of DNA amplification products was completely complementary and ligated. In this use example, the combination of DNA amplification products was selected so that the ends on both sides of the two types of DNA amplification products were complementary and ligated. 2 or 4 μL of methylamine-treated DNA mixed solution in the same procedure as in Use Example 11-2, 6.5 or 4.5 μL of water, Taq DNA ligase (New England BioLabs, catalog number M0208S) or T4 DNA ligase (New England 0.5 μL of BioLabs catalog number M0202S) and 1 μL of the buffer (× 10) attached to the product were added to the polymerase chain reaction tube. When Taq DNA ligase is used, the reaction tube is attached to TaKaRa PCR Thermal Cycler Dice (registered trademark) Gradient (Takara Thermal Cycler, Catalog No. TP600), [80 ° C, 1 minute → 45 ° C, 5 ° C] The reaction was carried out at a temperature cycle of [minute] × 30 → 4 ° C. to ligate the DNA. When T4 DNA ligase was used, the reaction tubes were allowed to stand at 25 ° C. for 18 hours to ligate DNA. The reaction solution (4 or 2 μL) after the ligation reaction was analyzed by 1% agarose gel electrophoresis. The gel after electrophoresis was stained with ethidium bromide to detect the fluorescence of DNA. The gel after ethidium bromide staining is shown in FIG. In the photo in the figure, black and white are reversed.

  In FIG. 76, the band in lane 1 shows DNA markers (0.5, 1.0, 1.5, 2, 3, 4, 6, 8, 10, and 12 kb). The bands in lanes 2, 3, 6, and 7 were obtained by ligating the amplification product DNA when LL1 and LL4 were used as a primer set with the amplification product DNA when LL5 and LL8 were used as a primer set. In this case, the ligation reaction product DNA is shown, and the bands in lanes 4, 5, 8 and 9 are the amplification product DNA when LL2 and LL3 are used as the primer set, and LL6 and LL7 as the primer set. The DNA of the ligation product in the case of ligation reaction with the DNA of the amplification product in this case is shown. The bands in lanes 2 to 5 show the DNA of the ligation product when the DNA is ligated using T4 DNA ligase, and the bands in lanes 6 to 9 ligate the DNA using Taq DNA ligase. The DNA of the ligation reaction product is shown. The bands in lanes 2, 4, 6, and 8 show the DNA of the ligation product when the DNA is ligated using a mixed solution of 2 μL of the amplified product DNA, and lanes 3, 5, Bands 7 and 9 indicate the DNA of the ligation product when the DNA is ligated using 4 μL of the amplification product DNA mixed solution.

  As shown in FIG. 76, when DNA having a long protruding end formed by the cleavage reaction of the present invention was ligated using Taq DNA ligase (lanes 8 and 9), DNA was most efficiently hybridized or It was confirmed that they were ligated to produce very long DNA.

  By the method of the present invention, a DNA strand is cleaved by a chemical reaction at a low cost and with a simple experimental operation, and compared with an enzymatic method such as polynucleotide kinase or a terminal phosphorylation method of synthetic DNA using phosphorylated amidite. It is possible to obtain oligodeoxynucleotides whose ends are phosphorylated with higher yield.

  All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (7)

Nucleophiles and formula I:
(A) _n -BC
[Where:
A is an oligodeoxynucleotide,
B is a deoxynucleoside containing a non-natural base having a moiety E removable by a nucleophile;
C is an oligodeoxynucleotide,
n is 0 or 1,
When n is 1, the 3 ′ end of A and the 5 ′ position of B are linked by a phosphodiester bond,
The 3 ′ position of B and the 5 ′ end of C are linked by a phosphodiester bond,
The phosphodiester bond between B and C is such that the oligodeoxynucleotide part phosphorylated at the 5 'end of C from the 3' position of B with E elimination induced by nucleophile attack. Disconnected by detachment]
By contacting the oligodeoxynucleotide represented by the following formula, the phosphodiester bond between B and C is released from the 3 'position of B by phosphorylation of the 5' end of C. An oligodeoxynucleotide which has been cleaved to phosphorylate the 5 ′ end of C, and a compound of formula II:
(A) _n -B '
[Where:
A and n have the same meaning as defined in Formula I,
B ′ is the residue part of B that is formed with the elimination of E by nucleophile attack]
A method for producing an oligodeoxynucleotide, comprising a 5′-terminal phosphorylated oligodeoxynucleotide forming step, which obtains an oligodeoxynucleotide represented by the formula: Formula II:
(A) _n -B '
[Where:
A and B ′ have the same meaning as in claim 1,
n is 1]
By reacting with oligodeoxynucleotide represented by Formula IIa:
A-PO ₃ H ₂
The method according to claim 1, further comprising a step of forming a 3'-terminal phosphorylated oligodeoxynucleotide, wherein an oligodeoxynucleotide phosphorylated at the 3 'end represented by: The deoxynucleoside B containing the unnatural base is:

[Where:
R is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, Substituted or unsubstituted heteroarylalkyl or carbonyl;
R ′ is hydrogen or substituted or unsubstituted alkyl, alkenyl or alkynyl;
r is an integer from 0 to 4;
R ″ is halogen or substituted or unsubstituted alkyl, alkenyl or alkynyl;
R ′ ″ is —COZ, —C≡CZ or —CN;
Z is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, Or substituted or unsubstituted heteroarylalkyl]
The method of Claim 1 or 2 selected from the deoxynucleoside represented by these.   Before the 5′-terminal phosphorylated oligodeoxynucleotide forming step, deoxynucleoside B containing a non-natural base having a moiety E removable by a nucleophile or a derivative thereof, and one or more containing a natural base The method according to any one of claims 1 to 3, further comprising an oligodeoxynucleotide synthesis step of synthesizing an oligodeoxynucleotide represented by the formula I using a substrate containing deoxynucleoside or a derivative thereof. Formula I:
(A) _n -BC
[Where:
A is an oligodeoxynucleotide,
B is a deoxynucleoside containing a non-natural base having a moiety E removable by a nucleophile;
C is an oligodeoxynucleotide,
n is 0 or 1,
When n is 1, the 3 ′ end of A and the 5 ′ position of B are linked by a phosphodiester bond,
The 3 ′ position of B and the 5 ′ end of C are linked by a phosphodiester bond,
The phosphodiester bond between B and C is such that the oligodeoxynucleotide part phosphorylated at the 5 'end of C from the 3' position of B with E elimination induced by nucleophile attack. Disconnected by detachment]
A DNA fragment amplification step of amplifying a linear double-stranded DNA fragment using the oligodeoxynucleotide represented by
By contacting the nucleophile with the amplified linear double-stranded DNA fragment, the phosphodiester bond between B and C becomes an oligodeoxynucleotide moiety phosphorylated at the 5 'end of C. It is cleaved by detaching from the 3 ′ position of B, the 5 ′ end of C contained in the linear double-stranded DNA fragment is phosphorylated, and at least the primer part represented by Formula I Forming a linear double-stranded DNA fragment having a 3 ′ protruding end containing a sequence complementary to a part thereof; and a linear double-stranded DNA fragment having the 3 ′ protruding end; And a ligation step of ligating another double-stranded DNA fragment;
A method for ligating double-stranded DNA. A kit for use in the method for producing an oligodeoxynucleotide according to any one of claims 1 to 4 or the method for ligating double-stranded DNA according to claim 5, comprising the formula I:
(A) _n -BC
[Where:
A is an oligodeoxynucleotide,
B is a deoxynucleoside containing a non-natural base having a moiety E removable by a nucleophile;
C is an oligodeoxynucleotide,
n is 0 or 1,
When n is 1, the 3 ′ end of A and the 5 ′ position of B are linked by a phosphodiester bond,
The 3 ′ position of B and the 5 ′ end of C are linked by a phosphodiester bond,
The phosphodiester bond between B and C is such that the oligodeoxynucleotide part phosphorylated at the 5 'end of C from the 3' position of B with E elimination induced by nucleophile attack. Disconnected by detachment]
Or at least a deoxynucleoside B containing a non-natural base or a derivative thereof having a moiety E removable by a nucleophile. The following formula:

[Where:
R is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, Substituted or unsubstituted heteroarylalkyl or carbonyl;
R ′ is methyl;
r is an integer from 0 to 4;
R ″ is halogen or substituted or unsubstituted alkyl, alkenyl or alkynyl;
R ′ ″ is —COZ, —C≡CZ or —CN;
Z is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, Or substituted or unsubstituted heteroarylalkyl]
A deoxynucleoside comprising an unnatural base represented by

Images