CN103201281A - 用于合成含5-羟甲基胞嘧啶的核酸的结构单元和方法 - Google Patents
用于合成含5-羟甲基胞嘧啶的核酸的结构单元和方法 Download PDFInfo
- Publication number
- CN103201281A CN103201281A CN2011800541702A CN201180054170A CN103201281A CN 103201281 A CN103201281 A CN 103201281A CN 2011800541702 A CN2011800541702 A CN 2011800541702A CN 201180054170 A CN201180054170 A CN 201180054170A CN 103201281 A CN103201281 A CN 103201281A
- Authority
- CN
- China
- Prior art keywords
- group
- halogen
- compound
- alkyl
- ribose
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 13
- RYVNIFSIEDRLSJ-UHFFFAOYSA-N 5-(hydroxymethyl)cytosine Chemical compound NC=1NC(=O)N=CC=1CO RYVNIFSIEDRLSJ-UHFFFAOYSA-N 0.000 title abstract description 15
- 230000015572 biosynthetic process Effects 0.000 title abstract description 11
- 238000003786 synthesis reaction Methods 0.000 title abstract description 7
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 15
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 15
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 15
- 229910052736 halogen Inorganic materials 0.000 claims description 32
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 28
- 150000001875 compounds Chemical class 0.000 claims description 26
- 150000002367 halogens Chemical class 0.000 claims description 25
- CKTSBUTUHBMZGZ-SHYZEUOFSA-N 2'‐deoxycytidine Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 CKTSBUTUHBMZGZ-SHYZEUOFSA-N 0.000 claims description 21
- 125000000217 alkyl group Chemical group 0.000 claims description 21
- -1 azido- Chemical class 0.000 claims description 21
- 125000004122 cyclic group Chemical group 0.000 claims description 16
- 150000008300 phosphoramidites Chemical class 0.000 claims description 13
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 10
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 claims description 9
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims description 9
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 125000004432 carbon atom Chemical group C* 0.000 claims description 8
- 238000006555 catalytic reaction Methods 0.000 claims description 8
- 230000000903 blocking effect Effects 0.000 claims description 7
- 229940104302 cytosine Drugs 0.000 claims description 7
- 125000001072 heteroaryl group Chemical group 0.000 claims description 7
- 125000000304 alkynyl group Chemical group 0.000 claims description 6
- 125000005842 heteroatom Chemical group 0.000 claims description 6
- 125000005418 aryl aryl group Chemical group 0.000 claims description 5
- 125000003118 aryl group Chemical class 0.000 claims description 5
- 229910052799 carbon Inorganic materials 0.000 claims description 5
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 claims description 5
- CFHIDWOYWUOIHU-UHFFFAOYSA-N oxomethyl Chemical compound O=[CH] CFHIDWOYWUOIHU-UHFFFAOYSA-N 0.000 claims description 5
- 150000003014 phosphoric acid esters Chemical class 0.000 claims description 5
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 claims description 4
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 claims description 4
- 239000003513 alkali Substances 0.000 claims description 4
- 229910001854 alkali hydroxide Inorganic materials 0.000 claims description 4
- SXADIBFZNXBEGI-UHFFFAOYSA-N phosphoramidous acid Chemical group NP(O)O SXADIBFZNXBEGI-UHFFFAOYSA-N 0.000 claims description 4
- 150000003016 phosphoric acids Chemical class 0.000 claims description 4
- 125000002769 thiazolinyl group Chemical group 0.000 claims description 4
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 claims description 4
- 229910001860 alkaline earth metal hydroxide Inorganic materials 0.000 claims description 3
- 125000001931 aliphatic group Chemical group 0.000 claims description 2
- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical group [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 claims description 2
- 229910052739 hydrogen Inorganic materials 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 125000000962 organic group Chemical group 0.000 claims description 2
- 150000003290 ribose derivatives Chemical class 0.000 claims 3
- KXDHJXZQYSOELW-UHFFFAOYSA-M Carbamate Chemical compound NC([O-])=O KXDHJXZQYSOELW-UHFFFAOYSA-M 0.000 claims 1
- 125000001424 substituent group Chemical group 0.000 claims 1
- 108091034117 Oligonucleotide Proteins 0.000 description 21
- 239000000872 buffer Substances 0.000 description 13
- 238000005481 NMR spectroscopy Methods 0.000 description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 239000002585 base Substances 0.000 description 11
- 238000013016 damping Methods 0.000 description 11
- 239000012530 fluid Substances 0.000 description 11
- 239000000243 solution Substances 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical class OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 8
- 238000004440 column chromatography Methods 0.000 description 8
- 239000007787 solid Substances 0.000 description 8
- 238000005520 cutting process Methods 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 6
- 230000008878 coupling Effects 0.000 description 6
- 238000010168 coupling process Methods 0.000 description 6
- 238000005859 coupling reaction Methods 0.000 description 6
- 239000012043 crude product Substances 0.000 description 6
- 238000002844 melting Methods 0.000 description 6
- 230000008018 melting Effects 0.000 description 6
- 229910052757 nitrogen Inorganic materials 0.000 description 6
- 239000011541 reaction mixture Substances 0.000 description 6
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 5
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 5
- 238000002372 labelling Methods 0.000 description 5
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 5
- 238000001228 spectrum Methods 0.000 description 5
- 238000010189 synthetic method Methods 0.000 description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 238000006170 formylation reaction Methods 0.000 description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 125000005425 toluyl group Chemical group 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- DHKHKXVYLBGOIT-UHFFFAOYSA-N acetaldehyde Diethyl Acetal Natural products CCOC(C)OCC DHKHKXVYLBGOIT-UHFFFAOYSA-N 0.000 description 3
- 239000000460 chlorine Substances 0.000 description 3
- 238000000354 decomposition reaction Methods 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 235000019439 ethyl acetate Nutrition 0.000 description 3
- 125000000623 heterocyclic group Chemical group 0.000 description 3
- 239000011630 iodine Substances 0.000 description 3
- 229910052740 iodine Inorganic materials 0.000 description 3
- 125000001181 organosilyl group Chemical group [SiH3]* 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 238000004007 reversed phase HPLC Methods 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- VKIGAWAEXPTIOL-UHFFFAOYSA-N 2-hydroxyhexanenitrile Chemical compound CCCCC(O)C#N VKIGAWAEXPTIOL-UHFFFAOYSA-N 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 2
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- 108050008598 Phosphoesterases Proteins 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 2
- 150000001342 alkaline earth metals Chemical class 0.000 description 2
- 125000003342 alkenyl group Chemical group 0.000 description 2
- 150000001408 amides Chemical class 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 229910052786 argon Inorganic materials 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229940126214 compound 3 Drugs 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
- 239000012149 elution buffer Substances 0.000 description 2
- 230000022244 formylation Effects 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 238000001972 liquid chromatography-electrospray ionisation mass spectrometry Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- OKKJLVBELUTLKV-VMNATFBRSA-N methanol-d1 Chemical compound [2H]OC OKKJLVBELUTLKV-VMNATFBRSA-N 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 239000002777 nucleoside Substances 0.000 description 2
- 125000003835 nucleoside group Chemical group 0.000 description 2
- 239000012044 organic layer Substances 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 108010062513 snake venom phosphodiesterase I Proteins 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- BCNZYOJHNLTNEZ-UHFFFAOYSA-N tert-butyldimethylsilyl chloride Chemical compound CC(C)(C)[Si](C)(C)Cl BCNZYOJHNLTNEZ-UHFFFAOYSA-N 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 150000003672 ureas Chemical class 0.000 description 2
- 230000004304 visual acuity Effects 0.000 description 2
- NXLNNXIXOYSCMB-UHFFFAOYSA-N (4-nitrophenyl) carbonochloridate Chemical compound [O-][N+](=O)C1=CC=C(OC(Cl)=O)C=C1 NXLNNXIXOYSCMB-UHFFFAOYSA-N 0.000 description 1
- JBWYRBLDOOOJEU-UHFFFAOYSA-N 1-[chloro-(4-methoxyphenyl)-phenylmethyl]-4-methoxybenzene Chemical compound C1=CC(OC)=CC=C1C(Cl)(C=1C=CC(OC)=CC=1)C1=CC=CC=C1 JBWYRBLDOOOJEU-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- ABZSPJVXTTUFAA-UHFFFAOYSA-N 4-acetamido-N-(2-amino-5-thiophen-2-ylphenyl)benzamide Chemical compound C1=CC(NC(=O)C)=CC=C1C(=O)NC1=CC(C=2SC=CC=2)=CC=C1N ABZSPJVXTTUFAA-UHFFFAOYSA-N 0.000 description 1
- MJEQLGCFPLHMNV-UHFFFAOYSA-N 4-amino-1-(hydroxymethyl)pyrimidin-2-one Chemical compound NC=1C=CN(CO)C(=O)N=1 MJEQLGCFPLHMNV-UHFFFAOYSA-N 0.000 description 1
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 240000006439 Aspergillus oryzae Species 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- QOPYYEHPKRREQO-UHFFFAOYSA-O CC(C)C1=[N+](C(C)C)NN=N1.N Chemical compound CC(C)C1=[N+](C(C)C)NN=N1.N QOPYYEHPKRREQO-UHFFFAOYSA-O 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 235000014493 Crataegus Nutrition 0.000 description 1
- 241001092040 Crataegus Species 0.000 description 1
- 241000271527 Crotalus adamanteus Species 0.000 description 1
- 230000008836 DNA modification Effects 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 1
- BDAGIHXWWSANSR-UHFFFAOYSA-N Formic acid Chemical compound OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 229930003756 Vitamin B7 Natural products 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 125000002777 acetyl group Chemical class [H]C([H])([H])C(*)=O 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 150000008044 alkali metal hydroxides Chemical class 0.000 description 1
- 150000001345 alkine derivatives Chemical class 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000005102 attenuated total reflection Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 125000002619 bicyclic group Chemical group 0.000 description 1
- 238000010876 biochemical test Methods 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 1
- 229910052794 bromium Inorganic materials 0.000 description 1
- 125000004106 butoxy group Chemical group [*]OC([H])([H])C([H])([H])C(C([H])([H])[H])([H])[H] 0.000 description 1
- 150000001720 carbohydrates Chemical group 0.000 description 1
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 150000001733 carboxylic acid esters Chemical class 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 229940125898 compound 5 Drugs 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 125000004093 cyano group Chemical group *C#N 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 238000007872 degassing Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 125000006575 electron-withdrawing group Chemical group 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000003818 flash chromatography Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007306 functionalization reaction Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 125000005843 halogen group Chemical group 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 150000004678 hydrides Chemical class 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 150000002460 imidazoles Chemical class 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000001869 matrix assisted laser desorption--ionisation mass spectrum Methods 0.000 description 1
- 238000001254 matrix assisted laser desorption--ionisation time-of-flight mass spectrum Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 1
- 238000001906 matrix-assisted laser desorption--ionisation mass spectrometry Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- POPACFLNWGUDSR-UHFFFAOYSA-N methoxy(trimethyl)silane Chemical compound CO[Si](C)(C)C POPACFLNWGUDSR-UHFFFAOYSA-N 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 125000005574 norbornylene group Chemical group 0.000 description 1
- 230000000269 nucleophilic effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 229940046166 oligodeoxynucleotide Drugs 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- ZRSNZINYAWTAHE-UHFFFAOYSA-N p-methoxybenzaldehyde Chemical compound COC1=CC=C(C=O)C=C1 ZRSNZINYAWTAHE-UHFFFAOYSA-N 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- HGPWSFNGQIFXOV-UHFFFAOYSA-N propan-2-yloxyphosphonamidous acid Chemical compound CC(C)OP(N)O HGPWSFNGQIFXOV-UHFFFAOYSA-N 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 150000003222 pyridines Chemical class 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 229910052701 rubidium Inorganic materials 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 239000003998 snake venom Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000017105 transposition Effects 0.000 description 1
- 239000011735 vitamin B7 Substances 0.000 description 1
- 235000011912 vitamin B7 Nutrition 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
- C07D498/04—Ortho-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/24—Heterocyclic radicals containing oxygen or sulfur as ring hetero atom
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Saccharide Compounds (AREA)
Abstract
本发明涉及用于有效合成含5-羟甲基胞嘧啶的核酸(如DNA或RNA)的结构单元和方法。
Description
描述
本发明涉及用于有效合成含5-羟甲基胞嘧啶的核酸(如DNA或RNA)的结构单元和方法。
从四个典型碱基dA、dC、dG和dT构建遗传材料。将dC碱基进一步接受外成的修饰。在真核生物中,dC碱基常常在C5位置是甲基化的,以产生5-甲基胞嘧啶(5-MedC)1。最近发现了一种新的基于dC的修饰,其在位置C5替代甲基而含有羟基亚甲基(图1)2-3。其他人和我们能够表明羟甲基胞嘧啶是大脑中广泛分布的DNA修饰并且其水平根据组织类型而改变4-5。新的“第六个”碱基5-HOMedC的功能目前尚不清楚。然而,显示出特定的酮戊二酸依赖性TET(十至十一易位)氧化酶负责其形成3,6。这些酶将5-MedC的5-甲基特异性地氧化,以产生5-HOMedC。最近,发现了这些TET酶的缺失产生了功能错误的干细胞,这提供了碱基5-HOMedC的形成与细胞发育之间的联系。7
为了促进5-HOMedC依赖性生物过程的生化研究,需要含5-HOMedC的寡核苷酸(ODNs)的有效合成。目前用于含5-HOMedC的DNA链合成的结构单元必需通过不稳定的溴-胸腺嘧啶中间产物的相当冗长的化学合成8-9。此外,包埋的5-HOMedC单体的去保护需要用浓缩的氨将合成的寡核苷酸在60℃下加热60小时9,这阻碍了寡核苷酸的任何衍生,因此对于许多生化实验通常需要用荧光或生物素标记。
本申请涉及从稳定且可购得的起始材料,例如,5-卤脱氧胞苷,优选5-碘脱氧胞苷,产生新的5-OHMedC结构单元,特别是一些合成步骤中可用的亚磷酰胺结构单元。发现了该结构单元能够使用标准亚磷酰胺化学合成含5-OHMedC的核酸,并且具有极好的偶联产率。
根据本发明,作为环氨基甲酸酯保护了5-OHMedC碱基的氨基和羟基。该基团极好地灭活了5-HOMedC的两个亲核基团并且是最小的可能的保护基团之一,因此使得在DNA合成仪中有效偶联。此外,可以通过在一个步骤中的简单碱处理,将其容易地去保护同时将DNA链与树脂切割,例如,通过在温和条件下用稀释的碱金属氢氧化物溶液处理,例如,室温下12小时。
因此,本发明的第一个方面涉及具有结构式(Ia)或(Ib)的化合物
其中R1是具有至多20个碳原子,优选至多10个碳原子的直链或环状有机基团,其任选含有杂原子,并且Z是H或环状基团。
通式(Ia)或(Ib)的化合物是受保护的5-羟甲基胞嘧啶化合物,并且优选是受保护的5-羟甲基胞苷化合物。
在化合物(Ia)或(Ib)中,Z优选是5-或6-元环或杂环基团,特别是呋喃酰或吡喃酰基团,更特别是核糖、修饰的核糖或脱氧核糖基团,其中核糖、修饰的核糖或脱氧核糖基团的3'-OH基团可以被含磷的基团,例如,磷酸盐、磷酸酯或亚磷酰胺基团所取代,并且其中核糖、修饰的核糖或脱氧核糖基团的5'-OH基团可以被保护基团,例如,羟基保护基团,如三苯基甲基基团,优选二甲氧基三苯基甲基基团(DMT)所取代。3'-OH和5'-OH基团的连接点可以颠倒,用于逆DNA合成中。
更优选,Z是具有结构式(II)的基团:
其中R2是H、OH、卤素、叠氮基、CN、-(O)C1-6(卤)烷基、-(O)C2-6(卤)烯基、-(O)C2-6(卤)炔基或N(R5)2,其中R5在每种情况下独立地是H、C1-6(卤)烷基或苯基,R3是H、羟基保护基团,例如,如上所述,或是磷酸盐、磷酸酯、亚磷酰胺或H-膦酸酯基团,优选式(III)的亚磷酰胺基团
并且R4是如上所示的羟基保护基团,优选三苯基甲基基团,如二甲氧基三苯基甲基(DMT)基团。R3和R4的连接点可以颠倒,用于逆DNA合成中。
通式(Ib)中的基团R1优选是含有至多6个C-原子且任选含有至多2个杂原子(如N或O)的脂肪族直链或环状基团,例如,直链C1-6(卤)烷基,或环状C3-6(杂)烷基;或者是C5-10芳基或杂芳基基团,例如,苯基或甲苯甲酰,其任选被OH、卤素、CN、(O)C1-6(卤)烷基、甲硅烷基或N(R5)2取代,其中R5如以上所定义。R1的特定实例是甲基、乙基、丙基、异丙基、2-三氟乙基、2氰基-乙基、2-(三甲基甲硅烷基)乙基、苯基或甲苯甲酰。
此外,本发明还涉及甲酰基或羧基-保护的胞嘧啶或胞苷衍生物,其可以用作含5-羟甲基胞嘧啶的核酸合成的结构单元或结构单元中间体。
优选的甲酰基保护的胞嘧啶或胞苷衍生物具有结构式(IVa)、(IVb)或(IVc),
其中R6是C1-6(卤)烷基,例如,甲基或乙基,或C5-10芳基或杂芳基,例如,苯基或甲苯甲酰,其任选被OH、卤素、CN、(O)C1-6(卤)烷基或N(R5)2取代,其中R5如以上所定义,并且Z如以上所定义(包括其优选的实施方案)。
优选的羧基保护的胞嘧啶或胞苷衍生物具有结构式(Va)、(Vb)或(Vc):
其中R6是C1-6(卤)烷基,例如,甲基或乙基,或C5-10芳基或杂芳基,例如,苯基或甲苯甲酰,其任选被OH、卤素、CN1(O)C1-6(卤)烷基或N(R5)2取代,其中R5如以上所定义,并且Z如以上所定义(包括其优选的实施方案),并且R7是C1-6(卤)烷基或C5-10芳基或杂芳基,其任选被CN、甲硅烷基或芳基,如苯基取代,如甲基、乙基、丙基、2-三氟乙基、2-三甲基甲硅烷基-乙基、苯基或苄基,并且Z如以上所定义(包括其优选的实施方案)。
如在此所用的,短语“任选取代的”意思是未取代的或取代的。术语“取代的”意思是除去了氢原子并且被取代基替代。
术语“烷基”指的是具有1-6个,优选1-4个碳原子的直链或支链烃基。术语“烯基”和“炔基”指的是具有2-6个碳原子,优选2-4个碳原子和至少一个CC双键或三键的直链或支链烃基。每个烷基、烯基或炔基可以被至少一个卤素原子取代。
术语“卤素”和“卤”指的是氟、氯、溴和碘。
术语“O烷基、O烯基或O炔基”意思是结合O原子的烷基、烯基或炔基,如甲氧基、乙氧基、丙氧基、丁氧基等。
术语“环状基团”指的是包括全饱和或不饱和的3-6元单环或8-10元双环系统,如芳香族或非芳香族环状基团,其可以具有至少一个杂原子,例如,选自氮原子、氧原子和/或硫原子。
术语“呋喃酰”和“吡喃酰”指的是5-或6-元环状碳水化合物基团。
术语“芳基”指的是苯基或奈基,特别是苯基。术语“杂芳基”指的是5-10-元杂环系统,其包括1-4个选自N、S和/或O的杂原子。
图2中描绘了化合物(Ia)合成的优选方法。起点是5-碘去氧胞苷110,其可以与TBS-Cl反应来保护羟基。可以没有使用OH-保护,替换地进行进一步的合成,然而以下反应的产率较低并且纯化时间更长。为了插入羟甲基,利用了与CO的Pd-催化甲酰化反应。该反应非常有效,即使是在未保护的环外氨基的存在下,以高于95%的产率提供了2。接着,用NaBH4还原所获得的C5处的甲酰基,以获得化合物3。对于该步骤,Luche条件的应用是绝对关键的11。没有加入CeCl3,推测将氢化物添加至碱基非常亲电子的C6位置,导致起始材料2的分解。为了引入环氨基甲酸酯,可以用4-硝基-苯基氯甲酸酯12处理化合物3,以非常好的产率获得受保护的化合物4。可以用吡啶中的HF实现甲硅烷基的去保护。在作为溶剂的乙酸乙酯中,在完成去保护后,二醇5沉淀,使得可以通过简单离心来分离。接着,可以使用标准程序13,将化合物5转化成5-HOMedC亚磷酰胺结构单元6。
图3中显示了用于合成化合物(Ia)、(IVa)和(Vb)的备选方法。
图4中显示了用于制造化合物(Ia)的更多备选合成方法。
可以通过与R1-三甲氧基乙缩醛(其中R1如以上所定义)的缩合以及随后按照对(Ia)所述的转化成亚磷酰胺来合成化合物(Ib)。可以通过Pd催化的甲酰化和随后作为酰胺或DMF乙缩醛的保护来合成化合物(IVb)和(IVc)。步骤的顺序可以颠倒。可以按照对(Ia)所述的来实现转化成亚磷酰胺。可以用R7-OH(其中R7如以上所定义)通过Pd催化的酯化并且随后作为酰胺或DMF乙缩醛的保护来合成化合物(Va)和(Vc)。步骤的顺序可以颠倒。可以按照对(Ia)所述的来实现转化成亚磷酰胺。这些合成方法显示于图7A、7B和7C。
根据本发明,发现了Pd催化的使用CO的5-卤脱氧胞苷的甲酰化反应,优选5-碘脱氧胞苷,以高产率获得了5-甲酰脱氧胞苷。因此,本发明的再一个方面涉及将甲酰基取代基引入胞嘧啶或胞苷化合物的第5位的方法,包括在Pd的催化下将5-卤素-取代的起始化合物、5-卤胞嘧啶、5-卤胞苷、5-卤脱氧胞苷或其受保护的衍生物与CO反应。
本发明的结构单元可以用于在核酸(如DNA或RNA或修饰的核酸,例如,糖和/或磷酸酯修饰的核酸)中引入5-羟甲基胞嘧啶结构单元。可以使用标准程序,例如,标准固相化学合成程序,如亚磷酰胺程序,来进行核酸合成。
本发明的再一个主题是结合了如上所述的作为保护的5-羟甲基胞嘧啶结构单元的至少一种化合物的核酸分子,例如,化合物(Ia)、(Ib)、(IVa)、(IVb)、(IVc)、(Va)、(Vb)和(Vc)。可以在碱性条件下去除保护基团,优选在碱或碱土金属氢氧化物溶液的存在下,例如,在0.01-1mol/l的浓度下。碱和碱土金属可以选自Li、Na、K、Rb和Mg。优选,使用Na。
因此,本发明还涉及除去化合物(Ia)、(Ib)、(IVa)、(IVb)、(IVc)、(Va)、(Vb)、(Vc)或掺加到少一种所示化合物的核酸分子上的环氨基甲酸酯保护基团或备选的甲酰基或羧酸酯保护基团的方法,其包括用水或水/醇碱或碱土金属氢氧化物溶液处理。
如上所述的新5-HOMedC结构单元可以与炔或降冰片烯结构单元一起掺加到DNA和RNA中,用于进一步的click修饰,优选通过与携带标记基团的功能化叠氮化合物反应。这将可以合成标记的含有寡核苷酸的5-HOMedC,特别是使用荧光素标记的生物素。
此外,甲酰-dC结构单元自身可以通过结合携带标记基团的含肼或羟胺化合物快速修饰寡核苷酸,例如,如上所述的标记基团。
此外,将通过以下的附图和实施例更详细地描述本发明。
附图
图1:哺乳动物基因组中存在的核苷。
图2:环氨基甲酸酯保护的胞嘧啶亚磷酰胺结构单元6和ODN1的核酸序列的合成(C*=5-OHMedC)。
图3:备选的合成方法。
图4:更多的备选合成方法。
图5:使用标准的基于NH3的条件,在寡核苷酸去保护后,获得了核苷7和8。然而,用NaOH去保护,只产生5-HOMedC。
图6:A)从树脂切割后直接的反相HPLC色谱(45分钟内0-50%缓冲液B)。B)DMT基团切割和纯化后的反相HPLC色谱(45分钟内0-20%缓冲液B)。C)纯化链ODN1的MALDI谱。D)纯化DNA链ODN1的消化。
图7A、B和C:化合物(Ib)、(IVb)、(IVc)、(Va)、(Vc)的合成方法。
实施例
1.通用方法
在干氮的气氛下,使用火焰干燥或烘干的玻璃器皿进行了所有非水反应。将来自Sigma-Aldrich或Acros的商业试剂作为公认的来使用,除非另外指出。在氮气下使用注射器或套管转移非水试剂。将溶液在Heidolph旋转蒸发器上在真空下浓缩。在Merck Geduran Si60(40-63μM)硅胶(正常相)或Fluka硅胶100C18-反相(15-35μm)上,使用快速柱色谱来完成产品的色谱纯化。在Merck60(硅胶F254)平板上进行了薄层色谱(TLC)。使用荧光淬灭或茴香醛染色进行了所产生色谱的观察。在Bruker ARX300、Varian VXR400S、Varian Inova400和Bruker AMX600光谱仪上在氘化溶剂中记录1H和13C NMR光谱并且校准至残余的溶剂峰。多重性缩写如下:s=单,d=双,t=三重,q=四重,m=多重。在质谱仪Thermo Finnigan LTQ FT-ICR上获得了ESI光谱和高分辨率ESI光谱。用于HPLC-ESI-MS分析的乙腈购自VWR,HPLC梯度级。HCOOH购自Fluka,p.a.,用于质谱。在Bruker AutoflexII光谱仪上记录了MALDI光谱。在具有钻石-ATR(Attenuated TotalReflection)设置的Perkin Elmer Spectrum BX FT-IR光谱仪(PerkinElmer)上进行了IR测量。使用Büchi Melting Point B540测定了熔点。
2.寡核苷酸合成
在Expedite8909Nucleic Acid Synthesis System(PerSeptiveBiosystems)上,使用标准DNA合成条件(规模:1μM)进行了寡核苷酸合成。用于dA、dC、dG、dT和CPG载体的亚磷酰胺获自GlenResearch。合成后将末端DMT保护基团保持在寡核苷酸上并且在从树脂切割后去除(参见去保护和纯化)。除了5-HOMedC,使用了标准偶联条件。对于5-HOMedC,偶联时间加倍,以确保良好的产率。
3.寡核苷酸的去保护和纯化
用4:1MeOH/H2O中的0.4M NaOH溶液进行了寡核苷酸从CPG的去保护和切割,在室温下进行12小时。使用来自Machery-Nagel的Nucleosil柱(250*4mm,C18ec,颗粒大小3μm或250*10mm,C18ec,5μm),在Waters2695分析HPLC和制备性HPLC Merck Hitachi(L-7150泵,L-7420检测仪)上进行了DNA纯化。所用的缓冲液为水中的0.1M三乙基醋酸铵(缓冲液A)和80%含水MeCN中的0.1M三乙基醋酸铵(缓冲液B)。通过分析HPLC和MALDI-MS检测了级分的纯度。使用Christα2-4LD plus冻干仪浓缩纯化的寡核苷酸。通过加入100μL80%醋酸溶液,将仍然含有三苯甲基的寡核苷酸去保护。在室温下培养20分钟后,将100μL水与60μL3M醋酸钠溶液一起加入。按照如上所述的,通过制备性HPLC纯化寡核苷酸。
4.酶消化
对于酶消化,将100μL H2O中的1nmol ODN1与缓冲液A(10μL,300mM醋酸铵,100mM CaCl2,1mM ZnSO4,pH5.7)和核酸酶S1(80单位,米曲霉)混合,并在37℃下培养3小时。加入缓冲液B(12μL,500mM Tris-HCl,1mM EDTA)、南极磷酸酯酶(10单位)、蛇毒磷酸二酯酶I(0.2单位,Crotalus adamanteus蛇毒),并且在37℃下再培养3小时,以完成消化。将样品离心(12100g,15分钟)并且通过HPLC分析(Waters2695,柱子:来自Interchim的Uptisphere120-3HDO)。洗脱缓冲液为缓冲液A(H2O中的2mM NH4HCOO(pH5.5))和缓冲液B(H2O/MeCN20/80中的2mM NH4HCOO)。梯度为0→12分钟;0%→3%缓冲液B;12→60分钟;3%→60%缓冲液B;60→62分钟;60%→100%缓冲液B;62→70分钟;100%缓冲液B;70→85分钟;100→0%缓冲液B;85→95分钟;0%缓冲液B。在260nm处监控洗脱。
5.LC-ESI-MS
通过Thermo Finnigan LTQ Orbitrap XL上的LC-ESI-MS,分析了样品(100μL注射体积),并且通过Dionex Ultimate3000HPLC系统进行了色谱,在来自Interchim的Uptisphere120-3HDO柱上使用了0.15mL/分钟的流速。将柱温维持在30℃。洗脱缓冲液为缓冲液C(H2O中的2mM HCOONH4(pH5.5))和缓冲液D(H2O/MeCN20/80中的2mM HCOONH4(pH5.5))。梯度为0→12分钟;0%→3%缓冲液D;12→60分钟;3%→60%缓冲液D;60→62分钟;60%→100%缓冲液D;62→70分钟;100%缓冲液D;70→85分钟;100→0%缓冲液D;85→95分钟;0%缓冲液D。在260nm处监控洗脱(Dionex Ultimate3000二极管矩阵检测仪)。将色谱洗脱液直接注入离子源中,没有之前的分解。在m/z200-1000全扫描范围内,使用30.000的分辨率,通过使用正极性模式扫描离子。使用缓冲液C中的新鲜混合的腺苷溶液(5μM)调整质谱仪的参数。该部分中所用的参数为鞘气流速,16arb;辅助气流速,11arb;尾气流速,4arb;喷雾电压,5.0kV;毛细管温度,200℃;毛细管电压,12V,镜筒透镜60V。
6.亚磷酰胺结构单元的合成程序
5-(卤)脱氧胞苷(1)
在火焰干燥的圆底烧瓶中,将10.0g dC(44.0mmol,1.0当量)、7.70g碘(26.4mmol,0.6当量)和11.4g mCPBA(70%,46.2mmol,1.05当量)溶解于120mL DMF中。将反应混合物在室温下搅拌2小时,并且随后蒸发至干。(在随后的柱色谱过程中,少量的DMF是容许的。)通过柱色谱(DCM/MeOH/H2O/NH3190:10:0.6:0.6→90:10:0.6:0.6)的纯化产生了9.71g(63%)橙色固体的1。
1H NMR(400MHz,CDCl3/MeOD)δ(ppm)=8.46(s,1H),6.13(t,3J=6.0,1H),4.34(dt,3J=4.7,3J=6.3,1H),3.93(dt,3J=3.0,3J=4.3,1H),3.84(dd,3J=3.0Hz,2J=12.1,1H),3.72(dd,3J=3.2,2J=12.1,1H),2.39(ddd,3J=4.8,3J=6.3,2J=13.7,1H),2.20-2.09(m,1H)。13C NMR(101MHz,MeOD)δ(ppm)=163.9,153.9,150.9,89.5,88.3,71.5,62.2,56.2,42.5。对C9H13IN3O4 +[M+H]+计算HRMS(ESI+):353.9945,发现:353.9944。熔化范围:133°C-135°C(分解)。IR(ATR):3191(w),1718(m),1642(s),1286(m),1087(s),957(s),750(m)。
3',5'-(叔丁基二甲基甲硅烷基)-5-(碘)脱氧胞苷(9)
在火焰干燥的圆底烧瓶中,将5.00g1(13.5mmol,1.0当量)、4.16g咪唑(60.5mmol,4.5当量)和6.24g(40.4mmol,3.0当量)TBS-Cl溶解于80mL DMF中,并且在室温下搅拌16小时。随后,通过加入150mL饱和NaHCO3停止反应,并且用300mL CHCl3萃取。用300mLH2O洗涤有机层,通过MgSO4干燥,并且在真空下除去溶剂。通过柱色谱(DCM/MeOH99:1→49:1)纯化粗产物,产生了6.25g(80%)淡黄色固体的9。
1H NMR(400MHz,CDCl3)δ(ppm)=8.06(s,1H),6.25-6.19(m,1H),4.34(dt,3J=2.9,3J=5.9,1H),3.97(q,3J=2.6,1H),3.87(dd,3J=2.6,2J=11.4,1H),3.74(dd,3J=2.6,2J=11.4,1H),2.44(ddd,3J=3.0,3J=5.9,2J=13.3,1H),2.00-1.90(m,1H),0.92(s,9H),0.87(s,9H),0.13(s,3H),0.12(s,3H),0.06(s,3H),0.05(s,3H)。13C NMR(101MHz,CDCl3)δ(ppm)=163.2,154.3,146.7,88.3,86.8,72.2,62.8,56.2,42.6,26.1,25.7,18.5,18.0,-4.6,-4.9,-5.2,-5.3。HRMS(ESI+):计算C21H41IN3O4Si2 +[M+H]+:582.1675,发现:582.1683。熔化范围:196°C-198°C。IR(ATR):2929(w),2857(w),1649(m),1470(m),1256(m),1086(m),829(s),776(s)。
3',5'-(叔丁基二甲基甲硅烷基)-5-(甲酰基)脱氧胞苷(2)
在高压玻璃高压容器中,将3.50g9(6.02mmol,1.0当量)、947mgPPh3(3.61mmol,0.6当量)和623mg Pd2(dba)3*CHCl3(0.60mmol,0.1当量)溶解于90mL甲苯中。用CO冲刷高压容器两次,以除去残留的空气,并且随后在60℃下在3.5巴的CO压力下搅拌反应。使用注射泵,将2.02mL Bu3SnH(7.22mmol,1.2当量)通过隔膜以0.3mL/小时加入。完成添加后,将反应混合物在60℃下再搅拌12小时。随后,排出CO,并且在真空下蒸发溶剂。通过柱色谱(iHex/EtOAc4:1→2:1→1:1)纯化粗产物,以产生2.84g黄色固体(97%)的2。
1H NMR(300MHz,CDCl3)δ(ppm)=9.51(s,1H),8.57(s,1H),8.37(s,1H),7.46(s,1H),6.19(t,3J=6.1,1H),4.40-4.32(m,1H),4.08-4.02(m,1H),3.95(dd,3J=2.7,2J=11.7,1H),3.78(dd,3J=2.6,2J=11.6,1H),2.59(ddd,3J=3.6,3J=5.8,2J=10.3,1H),2.20-2.08(m,1H),0.89(s,9H),0.88(s,9H),0.10(s,3H),0.08(s,6H),0.07(s,3H)。13C NMR(75MHz,CDCl3)δ(ppm)=187.1,162.1,153.1,152.6,104.9,88.8,87.9,71.5,62.6,42.8,25.9,25.7,18.4,17.9,-4.5,-4.9,-5.2,-5.4。HRMS(ESI+):计算C22H42N3O5Si2 +[M+H]+:484.2658,发现:484.2654。熔化范围:150-152°C。IR(ATR):3365(w),2952(w),2929(w),2857(w),1651(s),1245(m),1083(s),829(s),776(s)。
3',5'-(叔丁基二甲基甲硅烷基)-5-(羟基亚甲基)脱氧胞苷(3)
在火焰干燥的圆底烧瓶中,将300mg2(0.62mmol,1.0当量)和707mg CeCl3*7H2O(1.86mmol,3.0当量)溶解于30mL甲醇中。向此溶液中加入24mg NaBH4(0.62mmol,1.0当量)并且将混合物在室温下搅拌30分钟。通过加入100mL饱和NH4Cl停止反应并且用100mL EtOAc萃取。此后,用100mL NH4Cl洗涤有机层两次,用MgSO4干燥,蒸发至干,并且通过柱色谱(DCM/MeOH19:1,干法填充的)纯化粗产物,以产生184mg(61%)无色油的3。
1H NMR(599MHz,CDCl3)δ(Ppm)=7.59(s,1H),6.13(t,3J=6.4,1H),4.39(d,2J=13.1,1H),4.36(d,2J=13.1,1H),4.30(dt,3J=3.5,3J=6.6,1H),3.90(q,3J=3.1,1H),3.81(dd,3J=3.2,2J=11.2,1H),3.72(dd,3J=3.0,2J=11.3,1H),2.36(ddd,3J=3.6,3J=6.1,2J=13.3,1H),1.93(dt,3J=6.5,2J=13.2,1H),0.88(s,9H),0.87(s,9H),0.08(s,3H),0.07(s,3H),0.05(s,3H),0.04(s,3H).13CNMR(151MHz,CDCl3)δ(ppm)=165.2,156.2,138.6,106.0,87.8,86.2,71.7,62.7,59.5,42.2,25.9,25.8,18.4,18.0,-4.6,-4.9,-5.3,-5.4.HRMS(ESl+):calculated for C22H44N3OsSi2 +[M+H]+:486.2814,found:484.2815.IR(ATR):3193(m),3060(m),2950(m),2928(m),2857(m),1663(s)1485(s),1378(m),1291(s),1100(s),829(s),776(s).
3',5'-(叔丁基二甲基甲硅烷基)-4,5-(1,3-[3H,6H]嗪-2-酮)脱氧胞苷(4)
在火焰干燥的圆底烧瓶中,将12mg(0.02mmol,1.0当量)3溶解于5mL THF中,并且随后加入5mg(0.02mmol,1.0当量)4-硝基苯基氯甲酸酯。将混合物在室温下搅拌90分钟。加入9μL(0.05mmol,2.0当量)DIPEA,并且将溶液再搅拌90分钟。此后,将反应混合物蒸发至干,并且通过柱色谱(DCM/MeOH99:1)纯化粗产物,以产生11mg(87%)无色固体的4。
1H NMR(599MHz,CDCl3)δ(ppm)=8.17(s,1H),6.22(t,3J=6.0,1H),5.12(d,2J=13.2,1H),5.09(d,2J=13.3,1H),4.34(dd,3J=4.1,3J=9.8,1H),4.04-3.98(m,1H),3.93(dd,3J=2.4,2J=11.6,1H),3.77(dd,3J=2.2,2J=11.5,1H),2.57(ddd,3J=4.6,3J=6.0,2J=13.4,1H),2.10-2.00(m,1H),0.91(s,9H),0.88(s,9H),0.11(s,3H),0.10(s,3H),0.07(s,3H),0.06(s,3H).13CNMR(75MHz,CDCl3)δ(ppm)=159.7,154.4,149.8,138.4,96.2,88.2,87.4,71.1,64.6,62.3,42.4,25.8,25.7,18.3,17.9,-4.6,-5.0,-5.42,-5.44。HRMS(ESI+):计算C23H42N3O6Si2 +[M+H]+:512.2607,发现:512.2611。熔化范围:96°C-97°C。IR(ATR):2929(w),2857(w),1758(m),1667(m),1562(m),1251(m),1066(m),829(s),776(s)。
在聚丙烯管中,将187mg4(0.37mmol,1.0当量)溶解于25mL EtOAc中,随后加入147μL吡啶(1.83mmol,5.0当量)和157μL HF*吡啶(70%HF,5.48mml,15.0当量),并且将反应混合物在室温下搅拌14小时。在该时间过程中,沉淀出白色固体。加入500μL TMSOMe,并且将反应混合物再搅拌30分钟。随后,通过离心(6000rpm,15分钟)收集固体。将上清液蒸发至干,并且按照以上所述的再次处理。反应产生了88mg(85%)无色固体的5。
1H NMR(400MHz,CD3OD)δ(ppm)=8.39(t,5J=1.1,1H),6.20(t,3J=6.2,1H),5.21(dd,5J=0.9,2J=13.2,1H),5.18(dd,5J=0.9,2J=13.2,1H),4.37(dt,3J=3.9,3J=6.3,1H),4.00(dd,3J=3.7,3J=7.2,1H),3.84(dd,3J=3.2,2J=12.2,1H),3.75(dd,3J=3.8,2J=12.2,1H),2.49(ddd,3J=4.2,3J=6.2,2J=13.7,1H),2.17(dt,3J=6.3,2J=13.7,1H)。13C NMR(101MHz,CD3OD)δ(ppm)=162.0,157.8,153.0,140.4,99.4,89.6,88.9,71.7,66.2,62.5,42.6。HRMS(ESI+):计算C11H14N3O6 +[M+H]+:284.0877,发现:284.0877。熔化范围:>200°C分解。IR(ATR):3320(m),1745(m),1668(s),1626(s),1499(s),1276(s),1103(s)872(s)。
在火焰干燥的圆底烧瓶中,将85mg(0.30mmol,1.0当量)5和105mg DMT-Cl(0.30mmol,1.0当量)溶解于10mL吡啶中。将反应混合物在室温下搅拌17小时,并且随后蒸发至干。通过柱色谱(DCM/MeOH99:1→49:1;0.1%NEt3)纯化粗产物,以产生75mg(43%)无色油的10。
1H NMR(300MHz,CDCl3)δ(ppm)=8.32(s,1H),7.33-7.14(m,9H),6.77(d,3J=8.9,4H),6.24(t,3J=5.8,1H),4.64(m,1H),4.14(dd,3J=2.8,3J=6.6,1H),4.08-4.01(m,1H),3.72(s,6H),3.43(dd,3J=2.8,2J=10.8,1H),3.37(dd,3J=2.8,2J=10.7,1H),2.76-2.65(m,2H),2.35-2.23(m,2H)。13C NMR(75MHz,CDCl3)δ(ppm)=159.5,158.64,158.60,158.4,154.9,149.9,144.1,139.0,135.0,134.9,130.0,129.9,128.0,127.9,127.1,113.2,96.7,87.1,86.8,86.3,70.3,63.8,62.6,55.13,55.05,42.1。HRMS(ESI+):计算C32H30N3O8 -[M-H]-:584.2038,发现:584.2033。
3'-(二异丙基氰基乙基膦基)-5'-(二甲氧基三苯甲基)-4,5-(1,3-[3H,6H]嗪-2-酮)脱氧胞苷(6)
在火焰干燥的圆底烧瓶中,将86mg(0.15mmol,1.0当量)10,13mg(0.07mmol,0.5当量)二异丙基四唑铵和57μL(0.18mmol,1.2当量)2-氰基乙氧基-N,N,N',N'-四异丙基亚磷酰胺溶解于严格脱气的DCM中(冷冻、抽吸、融化)。使溶液在室温下搅拌15小时,并且随后在氩气氛下浓缩至干。通过柱色谱(DCM/MeOH49:1,0.1%NEt3)纯化粗产物。将纯的级分在氩气氛下蒸发至干,以产生58mg(50%)无色泡沫的6。
化合物是空气敏感的,因此直接用于固相DNA合成。通过成功掺加到DNA中明确地证明了其特性。
7.寡核苷酸合成
使用亚磷酰胺6(C*)制备了寡核苷酸ODN1(图2)。使用6的偶联时间加倍,使其有效地掺加到寡核苷酸链中。使用标准实验方案(室温下的浓缩氨过夜)使链去保护的最初尝试提供了含5-HOMedC的寡核苷酸。然而,形成了作为主要副产物的脲衍生物7和氨基甲基-dC核碱基8(图5)。为了防止这些不理想的副反应,将4:1MeOH/H2O中的0.4M NaOH作为去保护溶液在室温下过夜。在这些条件下实现了DNA链从固体支持物上的切割以及包括环氨基甲酸酯的所有碱基的去保护,产生了只含有5-HOMedC的DNA链。令人感兴趣地,证明了相关的脲衍生物在DNA和去保护过程中是稳定的14。
图6A描绘了在DNA切割和去保护后直接获得的原始HPLC色谱。该谱显示出结构单元6在合成仪中的DNA装备过程中真正地高效偶联。
图3B显示了纯化的含5-HOMedC的寡核苷酸的反相HPLC色谱,与MALDI-TOF质谱(图6C)一起验证了5-HOMedC正确地掺加到DNA链中。这是值得注意的,因为我们观察到了5-HOMedC的假-苄型位置的SN2-型反应,尤其是在酸性条件下或者当氧原子被吸电子基团衍生时。主要OH基团的不正常高反应性最初阻碍了我们作为双-醋酸酯来保护5-HOMedC的尝试。这种反应性也解释了与NH4OH的反应中副产物8的形成。
为了获得只形成5-HOMedC的更多证据,我们进行了酶消化研究。为此,我们首先用核酸酶S1在37℃下将所获得的寡核苷酸ODN1处理3小时,接着用南极磷酸酯酶和蛇毒磷酸二酯酶在37℃下再培养3小时。通过HPLC-ESI-MS分析了所获得的消化物。色谱描绘于图6D中,并且显示出除了四种典型碱基dA、dC、dG和dT以外的其他信号,其反映出5-HOMedC的正确分子量。高分辨率的MS数据支持对目标化合物预期的分子式C10H15N3O5。
8.小结
我们报道了一种新的5-HOMedC亚磷酰胺结构单元的短而有效的合成。合成中的关键步骤是Pd(0)催化的甲酰化,以及同时伯羟基与环外氨基在杂环上作为环氨基酸酯的保护。使用NaOH溶液在温和条件下方便地实现了该单元的去保护,现在能够合成含有其他修饰(如,荧光团或生物素标记)的5-HOMedC寡核苷酸。为了这些目的,在此报道的化学与新的进行DNA和RNA的Cu(I)催化的click修饰或无Cu修饰15-16能力的结合应当是特别合适的。
文献
1Law,J.A.;Jacobsen,S.E.Nat.Rev.Genet.2010,11,204-220.
2Kriaucionis,S.;Heintz,N.ScienCe2009,324,929-930.
3Tahiliani,M.;Koh,K.P.;Shen,Y.H.;Pastor,W.A.;Bandukwala,H.;Brudno,Y.;Agarwal,S.;lyer,L.M.;Liu,D.R.;Aravind,L.;Rao,A.Science2009,324,930-935.
4Münzel,M.;Globisch,D.;Brückl,T.;Wagner,M.;Welzmiller,V.;Michalakis,S.;Müller,M.;Biel,M.;Carell,T.AngeW.Chem.lnt.Ed.2010,49,5375-5377.
5Szwagierczak,A.;Bultmann,S.;Schmidt,C.S.;Spada,F.;Leonhardt,H.NUc/eic Acids Res.2010,38,e181.
6Loenarz,C.;Schofield,C.J.Chem.Biol.2009,16,580-583.
7lto,S.;D'Alessio,A.C.;Taranova,O.V.;Hong,K.;Sowers,L.C.;Zhang,Y.Nature2O10,466,1129-1133.
8Shiau,G.T.;Schinazi,R.F.;Chen,M.S.;Prusoff,W.H.J.Med.Chem.1980,23,127-133.
9Tardy-Planechaud,S.;Fujimoto,J.;Lin,S.S.;Sowers,L.C.Nuc/eic AcidsReS.1997,25,553-558.
10Hwang,C.H.;Park,J.S.;Won,J.H.;Kim,J.N.;Ryu,E.K.Arch.Pharm.Res.1992,15,69-72.
11Luche,J.L.J.Am.Chem.Soc.1978,100,2226-2227.
12Sammet,B.;Syn/ett2009,3050-3051.
13Caruthers,M.H.Acc.Chem.Res.1991.24,278-284.
14·Miyata,K.;Tamamushi,R.;Ohkubo,A.;Taguchi,H.;Seio,K.;Santa,T.;Sekine,M.Org.Lett.2006,8,1545-1548.
15Gierlich,J.;Burley,G.A.;Gramlich,P.M.E.;Hammond,D.M.;Carell,T.Org.Lett.2006,8,3639-3642.
16Gramiich,P.M.E.;Wirges,C.T.;Manetto,A.;Carell,T.Angew.Chem./nt.Ed.2008,47,8350-8358.
Claims (9)
2.权利要求1的化合物,其中Z是5-或6-元环基团,特别是核糖、核糖类似物或脱氧核糖基团,其中核糖、核糖类似物或脱氧核糖基团的3'-OH基团可以被含磷的基团,例如磷酸盐、磷酸酯或亚磷酰胺基团所取代,并且其中核糖、核糖类似物或脱氧核糖基团的5'-OH基团可以被保护基团取代。
4.权利要求1-3任一项的化合物,其中R1是含有至多6个C-原子且任选含有至多2个杂原子如N或O的脂肪族直链或环状基团,例如,C1-6(卤)烷基,或C3-6(杂)烷基;或者是C5-10芳基或杂芳基基团,该芳基或杂芳基基团任选被OH、卤素、CN、(O)C1-6(卤)烷基或N(R5)2取代,其中R5如权利要求1中所定义。
5.一种在胞嘧啶、胞苷或脱氧胞苷的第5位引入甲酰基取代基的方法,包括在Pd的催化下,将5-卤素取代的起始化合物与CO反应。
6.权利要求1-5任一项的化合物作为核酸合成的结构单元的用途。
7.权利要求6的用途,其中核酸合成通过亚磷酰胺程序进行。
8.一种核酸分子,其结合了至少一种权利要求1-5任一项的化合物。
9.一种除去权利要求1-5任一项的化合物或权利要求8的核酸分子上的环状氨基甲酸酯保护基团的方法,包括用水或水/醇碱或碱土金属氢氧化物溶液处理。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP10191078.4 | 2010-11-12 | ||
EP10191078 | 2010-11-12 | ||
PCT/EP2011/069954 WO2012062907A1 (en) | 2010-11-12 | 2011-11-11 | Nucleic acidsbuilding blocks and methods for the synthesis of 5-hydroxymethylcytosine-containing |
Publications (1)
Publication Number | Publication Date |
---|---|
CN103201281A true CN103201281A (zh) | 2013-07-10 |
Family
ID=44913329
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN2011800541702A Pending CN103201281A (zh) | 2010-11-12 | 2011-11-11 | 用于合成含5-羟甲基胞嘧啶的核酸的结构单元和方法 |
Country Status (4)
Country | Link |
---|---|
US (1) | US20130237697A1 (zh) |
EP (1) | EP2638048A1 (zh) |
CN (1) | CN103201281A (zh) |
WO (1) | WO2012062907A1 (zh) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2580963B (en) * | 2019-02-01 | 2021-03-31 | Hemispherian As | Cancer therapies |
WO2024039516A1 (en) * | 2022-08-19 | 2024-02-22 | Illumina, Inc. | Third dna base pair site-specific dna detection |
WO2024044375A2 (en) * | 2022-08-26 | 2024-02-29 | Regents Of The University Of Minnesota | Antiviral compounds |
-
2011
- 2011-11-11 WO PCT/EP2011/069954 patent/WO2012062907A1/en active Application Filing
- 2011-11-11 EP EP11779721.7A patent/EP2638048A1/en not_active Withdrawn
- 2011-11-11 CN CN2011800541702A patent/CN103201281A/zh active Pending
- 2011-11-11 US US13/884,007 patent/US20130237697A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
WO2012062907A1 (en) | 2012-05-18 |
EP2638048A1 (en) | 2013-09-18 |
US20130237697A1 (en) | 2013-09-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP0466773B1 (en) | Coumarin derivatives for use as nucleotide crosslinking reagents | |
ES2587512T3 (es) | Derivados de 2'-O-aminooximetil nucleósido para su uso en la síntesis y modificación de nucleósidos, nucleótidos y oligonucleótidos | |
CN112533892B (zh) | 烷氧基苯基衍生物、核苷保护体和核苷酸保护体、寡核苷酸制造方法以及取代基除去方法 | |
EP0767657A1 (en) | Novel method of preparation of known and novel 2'-modified nucleosides by intramolecular nucleophilic displacement | |
IL99451A (en) | Method for linking nucleosides by the siloxane bridge in the presence of a spatially inhibited base catalyst | |
KR20080059323A (ko) | 폴리뉴클레오티드 표지 시약 | |
US20090062521A1 (en) | Amidite for synthesizing modified nucleic acid and method for synthesizing modified nucleic acid | |
CN114907370B (zh) | 高纯度的噻吩并嘧啶化合物及其制备方法 | |
JP2001522860A (ja) | 標識化結合パートナーのためのピリミジン誘導体 | |
Hari et al. | Synthesis and properties of thymidines with six-membered amide bridge | |
CN103201281A (zh) | 用于合成含5-羟甲基胞嘧啶的核酸的结构单元和方法 | |
Srivastava et al. | 1, N6-etheno deoxy and ribo adenoGine and 3, N4-etheno deoxy and ribo cytidine phosphoramidites. Strongly fluorescent structures for selective introduction in defined sequence DNA and RNA molecules | |
Sugizaki et al. | Facile synthesis of hydroxymethylcytosine-containing oligonucleotides and their reactivity upon osmium oxidation | |
US7723495B2 (en) | Amidite for nucleic acid synthesis and nucleic acid synthesizing method | |
US20230212178A1 (en) | Method of producing photoreactive nucleotide analog | |
US5606049A (en) | Method of preparing 2'-O-methyl cytidine monomers useful in oligomer synthesis | |
EP1538154B1 (en) | Quencher composition comprising anthraquinone moieties | |
Milecki et al. | 5-Fluoro-4-thiouridine phosphoramidite: New synthon for introducing photoaffinity label into oligodeoxynucleotides | |
Hancox et al. | Some reactions of 4′-thionucleosides and their sulfones | |
EP1308452B1 (en) | Oligonucleotide labeling reactants based on acyclonucleosides and conjugates derived thereof | |
Efimov et al. | N-azidomethylbenzoyl blocking group in the phosphotriester synthesis of oligoribonucleotides | |
US7329515B2 (en) | Solid support for the synthesis of 3′-amino oligonucleotides | |
Ferrer et al. | Synthesis of oligodeoxynucleotides containing 5-aminouracil and itsN-acetyl derivative | |
EP4116313A1 (en) | Cap analog for the 5'-end of eukaryotic messenger rnas | |
US20030236397A1 (en) | Process for preparing beta-L-2'deoxy-thymidine |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130710 |