NZ735042B2 - Methods of polynucleotide preparation using multivalent cation salt compositions - Google Patents
Methods of polynucleotide preparation using multivalent cation salt compositions Download PDFInfo
- Publication number
- NZ735042B2 NZ735042B2 NZ735042A NZ73504216A NZ735042B2 NZ 735042 B2 NZ735042 B2 NZ 735042B2 NZ 735042 A NZ735042 A NZ 735042A NZ 73504216 A NZ73504216 A NZ 73504216A NZ 735042 B2 NZ735042 B2 NZ 735042B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- polynucleotide
- salt
- composition
- multivalent cation
- counterion
- Prior art date
Links
- 108091033319 polynucleotide Proteins 0.000 title claims abstract description 568
- 102000040430 polynucleotide Human genes 0.000 title claims abstract description 568
- 239000002157 polynucleotide Substances 0.000 title claims abstract description 568
- 239000000203 mixture Substances 0.000 title claims abstract description 218
- 238000000034 method Methods 0.000 title claims abstract description 196
- -1 cation salt Chemical class 0.000 title claims abstract description 104
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 150000003839 salts Chemical class 0.000 claims abstract description 278
- 150000001768 cations Chemical class 0.000 claims abstract description 183
- 239000002777 nucleoside Substances 0.000 claims abstract description 120
- 150000003833 nucleoside derivatives Chemical class 0.000 claims abstract description 108
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 36
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims abstract description 23
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 13
- 239000000047 product Substances 0.000 claims description 50
- 108010017842 Telomerase Proteins 0.000 claims description 47
- 230000000295 complement effect Effects 0.000 claims description 45
- 239000002244 precipitate Substances 0.000 claims description 45
- 239000011777 magnesium Substances 0.000 claims description 42
- 150000002632 lipids Chemical class 0.000 claims description 39
- 229910052749 magnesium Inorganic materials 0.000 claims description 30
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 claims description 26
- 239000011575 calcium Substances 0.000 claims description 23
- 229910052791 calcium Inorganic materials 0.000 claims description 22
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 20
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 20
- 229910052782 aluminium Inorganic materials 0.000 claims description 18
- 229910052739 hydrogen Inorganic materials 0.000 claims description 18
- 239000001257 hydrogen Substances 0.000 claims description 18
- 239000011701 zinc Substances 0.000 claims description 18
- 229910052725 zinc Inorganic materials 0.000 claims description 18
- 108091034117 Oligonucleotide Proteins 0.000 claims description 17
- KDCGOANMDULRCW-UHFFFAOYSA-N Purine Natural products N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 claims description 16
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 16
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 16
- 239000004411 aluminium Substances 0.000 claims description 15
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 claims description 12
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 12
- 125000003545 alkoxy group Chemical group 0.000 claims description 11
- 159000000000 sodium salts Chemical group 0.000 claims description 11
- 125000002091 cationic group Chemical group 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 10
- 229920000642 polymer Polymers 0.000 claims description 10
- 239000002904 solvent Substances 0.000 claims description 10
- 229910052717 sulfur Chemical group 0.000 claims description 10
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 9
- 229910052760 oxygen Inorganic materials 0.000 claims description 9
- 239000012071 phase Substances 0.000 claims description 9
- 125000005415 substituted alkoxy group Chemical group 0.000 claims description 9
- 238000005341 cation exchange Methods 0.000 claims description 8
- 125000001153 fluoro group Chemical group F* 0.000 claims description 8
- 238000010532 solid phase synthesis reaction Methods 0.000 claims description 8
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical group [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 7
- 239000001301 oxygen Substances 0.000 claims description 7
- 239000011593 sulfur Chemical group 0.000 claims description 7
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 6
- 229920001184 polypeptide Polymers 0.000 claims description 6
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 6
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 6
- 125000000561 purinyl group Chemical group N1=C(N=C2N=CNC2=C1)* 0.000 claims description 5
- 150000002431 hydrogen Chemical class 0.000 claims 2
- 150000001875 compounds Chemical class 0.000 description 79
- 230000015572 biosynthetic process Effects 0.000 description 59
- 238000003786 synthesis reaction Methods 0.000 description 52
- LVZYXEALRXBLJZ-ISQYCPACSA-N f60ne4xb53 Chemical compound N1([C@@H]2O[C@@H]([C@H](C2)NP(O)(=S)OC[C@@H]2[C@H](C[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)NP(S)(=O)OC[C@@H]2[C@H](C[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)N)COP(O)(=S)N[C@H]2C[C@@H](O[C@@H]2COP(O)(=S)N[C@H]2C[C@@H](O[C@@H]2COP(O)(=S)N[C@H]2C[C@@H](O[C@@H]2COP(O)(=S)N[C@H]2C[C@@H](O[C@@H]2COP(O)(=S)N[C@H]2C[C@@H](O[C@@H]2COP(O)(=S)N[C@H]2C[C@@H](O[C@@H]2COP(O)(=S)N[C@H]2C[C@@H](O[C@@H]2COP(O)(=S)N[C@H]2C[C@@H](O[C@@H]2COP(O)(=S)N[C@H]2C[C@@H](O[C@@H]2COP(O)(=S)N[C@H]2C[C@@H](O[C@@H]2COP(O)(=S)OCC(O)CNC(=O)CCCCCCCCCCCCCCC)N2C(NC(=O)C(C)=C2)=O)N2C3=NC=NC(N)=C3N=C2)N2C3=C(C(NC(N)=N3)=O)N=C2)N2C3=C(C(NC(N)=N3)=O)N=C2)N2C3=C(C(NC(N)=N3)=O)N=C2)N2C(NC(=O)C(C)=C2)=O)N2C(NC(=O)C(C)=C2)=O)N2C3=NC=NC(N)=C3N=C2)N2C3=C(C(NC(N)=N3)=O)N=C2)N2C3=NC=NC(N)=C3N=C2)C=CC(N)=NC1=O LVZYXEALRXBLJZ-ISQYCPACSA-N 0.000 description 50
- 229950004291 imetelstat Drugs 0.000 description 48
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 34
- 150000002500 ions Chemical class 0.000 description 34
- 239000000243 solution Substances 0.000 description 33
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 32
- 239000003795 chemical substances by application Substances 0.000 description 30
- 125000005647 linker group Chemical group 0.000 description 29
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 28
- 125000006239 protecting group Chemical group 0.000 description 28
- 238000001556 precipitation Methods 0.000 description 24
- 125000001424 substituent group Chemical group 0.000 description 22
- 239000011734 sodium Substances 0.000 description 21
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 20
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 18
- 125000000217 alkyl group Chemical group 0.000 description 18
- 230000008878 coupling Effects 0.000 description 18
- 238000010168 coupling process Methods 0.000 description 18
- 238000005859 coupling reaction Methods 0.000 description 18
- 238000003776 cleavage reaction Methods 0.000 description 17
- 230000007017 scission Effects 0.000 description 17
- 125000003118 aryl group Chemical group 0.000 description 16
- 239000002585 base Substances 0.000 description 16
- 125000000623 heterocyclic group Chemical group 0.000 description 16
- 229910052708 sodium Inorganic materials 0.000 description 16
- 239000013585 weight reducing agent Substances 0.000 description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 15
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 15
- 235000014113 dietary fatty acids Nutrition 0.000 description 15
- 229930195729 fatty acid Natural products 0.000 description 15
- 239000000194 fatty acid Substances 0.000 description 15
- 150000004665 fatty acids Chemical class 0.000 description 15
- 125000001072 heteroaryl group Chemical group 0.000 description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- 125000000392 cycloalkenyl group Chemical group 0.000 description 14
- 150000008300 phosphoramidites Chemical class 0.000 description 14
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 13
- 210000004027 cell Anatomy 0.000 description 13
- IEVORMRANFJJFR-NMZIRJKDSA-A imetelstat sodium Chemical compound [Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].N1([C@@H]2O[C@@H]([C@H](C2)NP([O-])(=S)OC[C@@H]2[C@H](C[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)NP([S-])(=O)OC[C@@H]2[C@H](C[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)N)COP([O-])(=S)N[C@H]2C[C@@H](O[C@@H]2COP([O-])(=S)N[C@H]2C[C@@H](O[C@@H]2COP([O-])(=S)N[C@H]2C[C@@H](O[C@@H]2COP([O-])(=S)N[C@H]2C[C@@H](O[C@@H]2COP([O-])(=S)N[C@H]2C[C@@H](O[C@@H]2COP([O-])(=S)N[C@H]2C[C@@H](O[C@@H]2COP([O-])(=S)N[C@H]2C[C@@H](O[C@@H]2COP([O-])(=S)N[C@H]2C[C@@H](O[C@@H]2COP([O-])(=S)N[C@H]2C[C@@H](O[C@@H]2COP([O-])(=S)N[C@H]2C[C@@H](O[C@@H]2COP([O-])(=S)OCC(O)CNC(=O)CCCCCCCCCCCCCCC)N2C(NC(=O)C(C)=C2)=O)N2C3=NC=NC(N)=C3N=C2)N2C3=C(C(NC(N)=N3)=O)N=C2)N2C3=C(C(NC(N)=N3)=O)N=C2)N2C3=C(C(NC(N)=N3)=O)N=C2)N2C(NC(=O)C(C)=C2)=O)N2C(NC(=O)C(C)=C2)=O)N2C3=NC=NC(N)=C3N=C2)N2C3=C(C(NC(N)=N3)=O)N=C2)N2C3=NC=NC(N)=C3N=C2)C=CC(N)=NC1=O IEVORMRANFJJFR-NMZIRJKDSA-A 0.000 description 13
- 229910001629 magnesium chloride Inorganic materials 0.000 description 13
- 125000003729 nucleotide group Chemical group 0.000 description 13
- 238000000746 purification Methods 0.000 description 13
- 239000007787 solid Substances 0.000 description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 12
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 125000000753 cycloalkyl group Chemical group 0.000 description 12
- 239000000539 dimer Substances 0.000 description 12
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 12
- 125000003835 nucleoside group Chemical group 0.000 description 12
- 239000002773 nucleotide Substances 0.000 description 12
- 125000003277 amino group Chemical group 0.000 description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- 206010028980 Neoplasm Diseases 0.000 description 10
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium trichloride Chemical compound Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 description 10
- 125000000129 anionic group Chemical group 0.000 description 10
- 238000013459 approach Methods 0.000 description 10
- 125000004429 atom Chemical group 0.000 description 10
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 10
- 239000011780 sodium chloride Substances 0.000 description 10
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 description 9
- 238000004128 high performance liquid chromatography Methods 0.000 description 9
- 229930195733 hydrocarbon Natural products 0.000 description 9
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 9
- 239000000178 monomer Substances 0.000 description 9
- 230000002829 reductive effect Effects 0.000 description 9
- 239000007790 solid phase Substances 0.000 description 9
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 8
- 229910021592 Copper(II) chloride Inorganic materials 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 8
- 229910021577 Iron(II) chloride Inorganic materials 0.000 description 8
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 8
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical group [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 8
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 8
- 239000002253 acid Substances 0.000 description 8
- 229910052788 barium Inorganic materials 0.000 description 8
- WDIHJSXYQDMJHN-UHFFFAOYSA-L barium chloride Chemical compound [Cl-].[Cl-].[Ba+2] WDIHJSXYQDMJHN-UHFFFAOYSA-L 0.000 description 8
- 229910001626 barium chloride Inorganic materials 0.000 description 8
- 238000010511 deprotection reaction Methods 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 description 8
- 229910052757 nitrogen Inorganic materials 0.000 description 8
- 102000039446 nucleic acids Human genes 0.000 description 8
- 108020004707 nucleic acids Proteins 0.000 description 8
- 150000007523 nucleic acids Chemical class 0.000 description 8
- PTMHPRAIXMAOOB-UHFFFAOYSA-L phosphoramidate Chemical compound NP([O-])([O-])=O PTMHPRAIXMAOOB-UHFFFAOYSA-L 0.000 description 8
- 229910052698 phosphorus Inorganic materials 0.000 description 8
- 239000012453 solvate Substances 0.000 description 8
- 108010057210 telomerase RNA Proteins 0.000 description 8
- ZMANZCXQSJIPKH-UHFFFAOYSA-O triethylammonium ion Chemical compound CC[NH+](CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-O 0.000 description 8
- 239000011592 zinc chloride Substances 0.000 description 8
- 235000005074 zinc chloride Nutrition 0.000 description 8
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 7
- 125000000304 alkynyl group Chemical group 0.000 description 7
- 239000001110 calcium chloride Substances 0.000 description 7
- 229910001628 calcium chloride Inorganic materials 0.000 description 7
- 235000011148 calcium chloride Nutrition 0.000 description 7
- 125000004432 carbon atom Chemical group C* 0.000 description 7
- 150000002430 hydrocarbons Chemical class 0.000 description 7
- 230000005764 inhibitory process Effects 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 239000011347 resin Substances 0.000 description 7
- 229920005989 resin Polymers 0.000 description 7
- 238000000926 separation method Methods 0.000 description 7
- 125000000547 substituted alkyl group Chemical group 0.000 description 7
- 125000003107 substituted aryl group Chemical group 0.000 description 7
- 125000002252 acyl group Chemical group 0.000 description 6
- DSAJWYNOEDNPEQ-UHFFFAOYSA-N barium atom Chemical compound [Ba] DSAJWYNOEDNPEQ-UHFFFAOYSA-N 0.000 description 6
- 238000011097 chromatography purification Methods 0.000 description 6
- 125000001183 hydrocarbyl group Chemical group 0.000 description 6
- DDUNIBKOBAXULU-UHFFFAOYSA-N nitrosophosphonic acid Chemical compound OP(O)(=O)N=O DDUNIBKOBAXULU-UHFFFAOYSA-N 0.000 description 6
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 6
- 125000006245 phosphate protecting group Chemical group 0.000 description 6
- 239000011574 phosphorus Substances 0.000 description 6
- 239000012266 salt solution Substances 0.000 description 6
- 238000006467 substitution reaction Methods 0.000 description 6
- 125000005309 thioalkoxy group Chemical group 0.000 description 6
- 238000011282 treatment Methods 0.000 description 6
- 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 description 6
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 5
- 239000004215 Carbon black (E152) Substances 0.000 description 5
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 5
- 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 5
- 125000003342 alkenyl group Chemical group 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 5
- 235000011114 ammonium hydroxide Nutrition 0.000 description 5
- 229910052799 carbon Inorganic materials 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 239000005289 controlled pore glass Substances 0.000 description 5
- 125000004093 cyano group Chemical group *C#N 0.000 description 5
- 238000006642 detritylation reaction Methods 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 229910052736 halogen Inorganic materials 0.000 description 5
- 150000002367 halogens Chemical class 0.000 description 5
- 125000003473 lipid group Chemical group 0.000 description 5
- 159000000003 magnesium salts Chemical class 0.000 description 5
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 5
- 230000001590 oxidative effect Effects 0.000 description 5
- 125000005346 substituted cycloalkyl group Chemical group 0.000 description 5
- 238000005987 sulfurization reaction Methods 0.000 description 5
- 150000003573 thiols Chemical class 0.000 description 5
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 4
- MBBZMMPHUWSWHV-BDVNFPICSA-N N-methylglucamine Chemical compound CNC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO MBBZMMPHUWSWHV-BDVNFPICSA-N 0.000 description 4
- 229910019142 PO4 Inorganic materials 0.000 description 4
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 4
- 125000004442 acylamino group Chemical group 0.000 description 4
- 125000004423 acyloxy group Chemical group 0.000 description 4
- 230000002776 aggregation Effects 0.000 description 4
- 238000004220 aggregation Methods 0.000 description 4
- 239000003513 alkali Substances 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- JUHORIMYRDESRB-UHFFFAOYSA-N benzathine Chemical compound C=1C=CC=CC=1CNCCNCC1=CC=CC=C1 JUHORIMYRDESRB-UHFFFAOYSA-N 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 125000004181 carboxyalkyl group Chemical group 0.000 description 4
- 230000004700 cellular uptake Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 238000010494 dissociation reaction Methods 0.000 description 4
- 230000005593 dissociations Effects 0.000 description 4
- 238000004090 dissolution Methods 0.000 description 4
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 4
- 125000005843 halogen group Chemical group 0.000 description 4
- 125000005553 heteroaryloxy group Chemical group 0.000 description 4
- 125000005842 heteroatom Chemical group 0.000 description 4
- 125000004470 heterocyclooxy group Chemical group 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 239000003960 organic solvent Substances 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- 239000010452 phosphate Substances 0.000 description 4
- 150000004713 phosphodiesters Chemical class 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 4
- 238000000108 ultra-filtration Methods 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 3
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 3
- SECXISVLQFMRJM-UHFFFAOYSA-N NMP Substances CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 description 3
- 101710163270 Nuclease Proteins 0.000 description 3
- 239000004793 Polystyrene Substances 0.000 description 3
- RWRDLPDLKQPQOW-UHFFFAOYSA-N Pyrrolidine Chemical compound C1CCNC1 RWRDLPDLKQPQOW-UHFFFAOYSA-N 0.000 description 3
- 125000000266 alpha-aminoacyl group Chemical group 0.000 description 3
- 150000001412 amines Chemical class 0.000 description 3
- 230000000692 anti-sense effect Effects 0.000 description 3
- 229940053200 antiepileptics fatty acid derivative Drugs 0.000 description 3
- 125000004104 aryloxy group Chemical group 0.000 description 3
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- XSCHRSMBECNVNS-UHFFFAOYSA-N benzopyrazine Natural products N1=CC=NC2=CC=CC=C21 XSCHRSMBECNVNS-UHFFFAOYSA-N 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 125000004122 cyclic group Chemical group 0.000 description 3
- ZTHNOZQGTXKVNZ-UHFFFAOYSA-L dichloroaluminum Chemical compound Cl[Al]Cl ZTHNOZQGTXKVNZ-UHFFFAOYSA-L 0.000 description 3
- 150000004683 dihydrates Chemical class 0.000 description 3
- UAOMVDZJSHZZME-UHFFFAOYSA-N diisopropylamine Chemical compound CC(C)NC(C)C UAOMVDZJSHZZME-UHFFFAOYSA-N 0.000 description 3
- 150000002193 fatty amides Chemical class 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 3
- NDJKXXJCMXVBJW-UHFFFAOYSA-N heptadecane Chemical compound CCCCCCCCCCCCCCCCC NDJKXXJCMXVBJW-UHFFFAOYSA-N 0.000 description 3
- DCAYPVUWAIABOU-UHFFFAOYSA-N hexadecane Chemical compound CCCCCCCCCCCCCCCC DCAYPVUWAIABOU-UHFFFAOYSA-N 0.000 description 3
- 238000005342 ion exchange Methods 0.000 description 3
- 208000032839 leukemia Diseases 0.000 description 3
- 229940096405 magnesium cation Drugs 0.000 description 3
- 230000036210 malignancy Effects 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 206010028537 myelofibrosis Diseases 0.000 description 3
- LQERIDTXQFOHKA-UHFFFAOYSA-N nonadecane Chemical compound CCCCCCCCCCCCCCCCCCC LQERIDTXQFOHKA-UHFFFAOYSA-N 0.000 description 3
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 3
- 150000007524 organic acids Chemical class 0.000 description 3
- 125000004043 oxo group Chemical group O=* 0.000 description 3
- IPCSVZSSVZVIGE-UHFFFAOYSA-N palmitic acid group Chemical group C(CCCCCCCCCCCCCCC)(=O)O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 229920002223 polystyrene Polymers 0.000 description 3
- 150000003254 radicals Chemical class 0.000 description 3
- 238000012552 review Methods 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- 239000012607 strong cation exchange resin Substances 0.000 description 3
- 125000005017 substituted alkenyl group Chemical group 0.000 description 3
- 235000000346 sugar Nutrition 0.000 description 3
- 238000001308 synthesis method Methods 0.000 description 3
- 230000009897 systematic effect Effects 0.000 description 3
- BGHCVCJVXZWKCC-UHFFFAOYSA-N tetradecane Chemical compound CCCCCCCCCCCCCC BGHCVCJVXZWKCC-UHFFFAOYSA-N 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 125000005296 thioaryloxy group Chemical group 0.000 description 3
- 125000005404 thioheteroaryloxy group Chemical group 0.000 description 3
- 125000005323 thioketone group Chemical group 0.000 description 3
- UWYZHKAOTLEWKK-UHFFFAOYSA-N 1,2,3,4-tetrahydroisoquinoline Chemical compound C1=CC=C2CNCCC2=C1 UWYZHKAOTLEWKK-UHFFFAOYSA-N 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 2
- FCEHBMOGCRZNNI-UHFFFAOYSA-N 1-benzothiophene Chemical compound C1=CC=C2SC=CC2=C1 FCEHBMOGCRZNNI-UHFFFAOYSA-N 0.000 description 2
- NGNBDVOYPDDBFK-UHFFFAOYSA-N 2-[2,4-di(pentan-2-yl)phenoxy]acetyl chloride Chemical compound CCCC(C)C1=CC=C(OCC(Cl)=O)C(C(C)CCC)=C1 NGNBDVOYPDDBFK-UHFFFAOYSA-N 0.000 description 2
- VKIGAWAEXPTIOL-UHFFFAOYSA-N 2-hydroxyhexanenitrile Chemical compound CCCCC(O)C#N VKIGAWAEXPTIOL-UHFFFAOYSA-N 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- ROSDSFDQCJNGOL-UHFFFAOYSA-N Dimethylamine Chemical compound CNC ROSDSFDQCJNGOL-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerol Natural products OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 2
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 2
- 229910003827 NRaRb Inorganic materials 0.000 description 2
- 206010033661 Pancytopenia Diseases 0.000 description 2
- PCNDJXKNXGMECE-UHFFFAOYSA-N Phenazine Natural products C1=CC=CC2=NC3=CC=CC=C3N=C21 PCNDJXKNXGMECE-UHFFFAOYSA-N 0.000 description 2
- XYFCBTPGUUZFHI-UHFFFAOYSA-N Phosphine Chemical group P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 description 2
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 2
- 239000004695 Polyether sulfone Substances 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- KYQCOXFCLRTKLS-UHFFFAOYSA-N Pyrazine Chemical compound C1=CN=CC=N1 KYQCOXFCLRTKLS-UHFFFAOYSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical compound C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 description 2
- SMWDFEZZVXVKRB-UHFFFAOYSA-N Quinoline Chemical compound N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 description 2
- 244000007853 Sarothamnus scoparius Species 0.000 description 2
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- YTPLMLYBLZKORZ-UHFFFAOYSA-N Thiophene Chemical compound C=1C=CSC=1 YTPLMLYBLZKORZ-UHFFFAOYSA-N 0.000 description 2
- 208000005485 Thrombocytosis Diseases 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical class OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 150000008065 acid anhydrides Chemical class 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 208000017733 acquired polycythemia vera Diseases 0.000 description 2
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical compound C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 2
- 229960000643 adenine Drugs 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
- 125000001931 aliphatic group Chemical group 0.000 description 2
- 125000000033 alkoxyamino group Chemical group 0.000 description 2
- 125000005210 alkyl ammonium group Chemical group 0.000 description 2
- 150000001408 amides Chemical class 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 150000003863 ammonium salts Chemical class 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 150000001735 carboxylic acids Chemical class 0.000 description 2
- 150000001805 chlorine compounds Chemical class 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000009295 crossflow filtration Methods 0.000 description 2
- 208000024389 cytopenia Diseases 0.000 description 2
- 229940104302 cytosine Drugs 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 2
- AUZONCFQVSMFAP-UHFFFAOYSA-N disulfiram Chemical compound CCN(CC)C(=S)SSC(=S)N(CC)CC AUZONCFQVSMFAP-UHFFFAOYSA-N 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000000921 elemental analysis Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 229910052731 fluorine Inorganic materials 0.000 description 2
- 239000011737 fluorine Substances 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 125000002541 furyl group Chemical group 0.000 description 2
- 230000005021 gait Effects 0.000 description 2
- 125000000592 heterocycloalkyl group Chemical group 0.000 description 2
- 150000004687 hexahydrates Chemical class 0.000 description 2
- 125000002349 hydroxyamino group Chemical group [H]ON([H])[*] 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- CBFCDTFDPHXCNY-UHFFFAOYSA-N icosane Chemical compound CCCCCCCCCCCCCCCCCCCC CBFCDTFDPHXCNY-UHFFFAOYSA-N 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 229910017053 inorganic salt Inorganic materials 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 229910052744 lithium Inorganic materials 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 229960003194 meglumine Drugs 0.000 description 2
- 150000007522 mineralic acids Chemical class 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 125000004433 nitrogen atom Chemical group N* 0.000 description 2
- 239000012011 nucleophilic catalyst Substances 0.000 description 2
- RZJRJXONCZWCBN-UHFFFAOYSA-N octadecane Chemical compound CCCCCCCCCCCCCCCCCC RZJRJXONCZWCBN-UHFFFAOYSA-N 0.000 description 2
- 238000002515 oligonucleotide synthesis Methods 0.000 description 2
- 125000001820 oxy group Chemical group [*:1]O[*:2] 0.000 description 2
- 125000005255 oxyaminoacyl group Chemical group 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 235000019371 penicillin G benzathine Nutrition 0.000 description 2
- YCOZIPAWZNQLMR-UHFFFAOYSA-N pentadecane Chemical compound CCCCCCCCCCCCCCC YCOZIPAWZNQLMR-UHFFFAOYSA-N 0.000 description 2
- RDOWQLZANAYVLL-UHFFFAOYSA-N phenanthridine Chemical compound C1=CC=C2C3=CC=CC=C3C=NC2=C1 RDOWQLZANAYVLL-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- UEZVMMHDMIWARA-UHFFFAOYSA-M phosphonate Chemical compound [O-]P(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-M 0.000 description 2
- SXADIBFZNXBEGI-UHFFFAOYSA-N phosphoramidous acid Chemical group NP(O)O SXADIBFZNXBEGI-UHFFFAOYSA-N 0.000 description 2
- 125000004437 phosphorous atom Chemical group 0.000 description 2
- 208000037244 polycythemia vera Diseases 0.000 description 2
- 229920006393 polyether sulfone Polymers 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- MFDFERRIHVXMIY-UHFFFAOYSA-N procaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 2
- 229960004919 procaine Drugs 0.000 description 2
- 125000002568 propynyl group Chemical group [*]C#CC([H])([H])[H] 0.000 description 2
- 150000003212 purines Chemical class 0.000 description 2
- 125000004076 pyridyl group Chemical group 0.000 description 2
- 150000003230 pyrimidines Chemical class 0.000 description 2
- 229910052705 radium Inorganic materials 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 125000006413 ring segment Chemical group 0.000 description 2
- 229910052701 rubidium Inorganic materials 0.000 description 2
- IXGZXXBJSZISOO-UHFFFAOYSA-N s-(2-phenylacetyl)sulfanyl 2-phenylethanethioate Chemical compound C=1C=CC=CC=1CC(=O)SSC(=O)CC1=CC=CC=C1 IXGZXXBJSZISOO-UHFFFAOYSA-N 0.000 description 2
- 229910001415 sodium ion Inorganic materials 0.000 description 2
- RPACBEVZENYWOL-XFULWGLBSA-M sodium;(2r)-2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate Chemical group [Na+].C=1C=C(Cl)C=CC=1OCCCCCC[C@]1(C(=O)[O-])CO1 RPACBEVZENYWOL-XFULWGLBSA-M 0.000 description 2
- 125000004426 substituted alkynyl group Chemical group 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 125000004568 thiomorpholinyl group Chemical group 0.000 description 2
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 2
- 229940113082 thymine Drugs 0.000 description 2
- 125000004953 trihalomethyl group Chemical group 0.000 description 2
- 229940035893 uracil Drugs 0.000 description 2
- 125000006273 (C1-C3) alkyl group Chemical group 0.000 description 1
- JUDOLRSMWHVKGX-UHFFFAOYSA-N 1,1-dioxo-1$l^{6},2-benzodithiol-3-one Chemical compound C1=CC=C2C(=O)SS(=O)(=O)C2=C1 JUDOLRSMWHVKGX-UHFFFAOYSA-N 0.000 description 1
- 125000005988 1,1-dioxo-thiomorpholinyl group Chemical group 0.000 description 1
- OGYGFUAIIOPWQD-UHFFFAOYSA-N 1,3-thiazolidine Chemical compound C1CSCN1 OGYGFUAIIOPWQD-UHFFFAOYSA-N 0.000 description 1
- MQDLKAADJTYKRH-UHFFFAOYSA-N 1-aminopropane-1,2,3-triol Chemical compound NC(O)C(O)CO MQDLKAADJTYKRH-UHFFFAOYSA-N 0.000 description 1
- WJFKNYWRSNBZNX-UHFFFAOYSA-N 10H-phenothiazine Chemical compound C1=CC=C2NC3=CC=CC=C3SC2=C1 WJFKNYWRSNBZNX-UHFFFAOYSA-N 0.000 description 1
- TZMSYXZUNZXBOL-UHFFFAOYSA-N 10H-phenoxazine Chemical compound C1=CC=C2NC3=CC=CC=C3OC2=C1 TZMSYXZUNZXBOL-UHFFFAOYSA-N 0.000 description 1
- BAXOFTOLAUCFNW-UHFFFAOYSA-N 1H-indazole Chemical compound C1=CC=C2C=NNC2=C1 BAXOFTOLAUCFNW-UHFFFAOYSA-N 0.000 description 1
- KJUGUADJHNHALS-UHFFFAOYSA-N 1H-tetrazole Substances C=1N=NNN=1 KJUGUADJHNHALS-UHFFFAOYSA-N 0.000 description 1
- YKBGVTZYEHREMT-KVQBGUIXSA-N 2'-deoxyguanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](CO)O1 YKBGVTZYEHREMT-KVQBGUIXSA-N 0.000 description 1
- MXHRCPNRJAMMIM-SHYZEUOFSA-N 2'-deoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 MXHRCPNRJAMMIM-SHYZEUOFSA-N 0.000 description 1
- VEPOHXYIFQMVHW-XOZOLZJESA-N 2,3-dihydroxybutanedioic acid (2S,3S)-3,4-dimethyl-2-phenylmorpholine Chemical compound OC(C(O)C(O)=O)C(O)=O.C[C@H]1[C@@H](OCCN1C)c1ccccc1 VEPOHXYIFQMVHW-XOZOLZJESA-N 0.000 description 1
- UYGLXGHUDBKZKX-UHFFFAOYSA-N 2-(chloromethyl)pyridin-1-ium;iodide Chemical compound [I-].ClCC1=CC=CC=[NH+]1 UYGLXGHUDBKZKX-UHFFFAOYSA-N 0.000 description 1
- CFIBTBBTJWHPQV-UHFFFAOYSA-N 2-methyl-n-(6-oxo-3,7-dihydropurin-2-yl)propanamide Chemical compound N1C(NC(=O)C(C)C)=NC(=O)C2=C1N=CN2 CFIBTBBTJWHPQV-UHFFFAOYSA-N 0.000 description 1
- VLRSADZEDXVUPG-UHFFFAOYSA-N 2-naphthalen-1-ylpyridine Chemical compound N1=CC=CC=C1C1=CC=CC2=CC=CC=C12 VLRSADZEDXVUPG-UHFFFAOYSA-N 0.000 description 1
- VHMICKWLTGFITH-UHFFFAOYSA-N 2H-isoindole Chemical compound C1=CC=CC2=CNC=C21 VHMICKWLTGFITH-UHFFFAOYSA-N 0.000 description 1
- KQIGMPWTAHJUMN-UHFFFAOYSA-N 3-aminopropane-1,2-diol Chemical compound NCC(O)CO KQIGMPWTAHJUMN-UHFFFAOYSA-N 0.000 description 1
- CBKDCOKSXCTDAA-UHFFFAOYSA-N 4,5,6,7-tetrahydro-1-benzothiophene Chemical compound C1CCCC2=C1C=CS2 CBKDCOKSXCTDAA-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- CKTSBUTUHBMZGZ-ULQXZJNLSA-N 4-amino-1-[(2r,4s,5r)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-tritiopyrimidin-2-one Chemical compound O=C1N=C(N)C([3H])=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 CKTSBUTUHBMZGZ-ULQXZJNLSA-N 0.000 description 1
- GDRVFDDBLLKWRI-UHFFFAOYSA-N 4H-quinolizine Chemical compound C1=CC=CN2CC=CC=C21 GDRVFDDBLLKWRI-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
- XVMSFILGAMDHEY-UHFFFAOYSA-N 6-(4-aminophenyl)sulfonylpyridin-3-amine Chemical compound C1=CC(N)=CC=C1S(=O)(=O)C1=CC=C(N)C=N1 XVMSFILGAMDHEY-UHFFFAOYSA-N 0.000 description 1
- QQJXZVKXNSFHRI-UHFFFAOYSA-N 6-Benzamidopurine Chemical compound N=1C=NC=2N=CNC=2C=1NC(=O)C1=CC=CC=C1 QQJXZVKXNSFHRI-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 1
- 208000035657 Abasia Diseases 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 108020004394 Complementary RNA Proteins 0.000 description 1
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 1
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 1
- 108091008102 DNA aptamers Proteins 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 206010058314 Dysplasia Diseases 0.000 description 1
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 1
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 description 1
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- WRYCSMQKUKOKBP-UHFFFAOYSA-N Imidazolidine Chemical compound C1CNCN1 WRYCSMQKUKOKBP-UHFFFAOYSA-N 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- BAVYZALUXZFZLV-UHFFFAOYSA-O Methylammonium ion Chemical compound [NH3+]C BAVYZALUXZFZLV-UHFFFAOYSA-O 0.000 description 1
- 208000014767 Myeloproliferative disease Diseases 0.000 description 1
- 150000001204 N-oxides Chemical class 0.000 description 1
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 1
- JCXJVPUVTGWSNB-UHFFFAOYSA-N Nitrogen dioxide Chemical compound O=[N]=O JCXJVPUVTGWSNB-UHFFFAOYSA-N 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 235000021314 Palmitic acid Nutrition 0.000 description 1
- 108091093037 Peptide nucleic acid Proteins 0.000 description 1
- YGYAWVDWMABLBF-UHFFFAOYSA-N Phosgene Chemical compound ClC(Cl)=O YGYAWVDWMABLBF-UHFFFAOYSA-N 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- WTKZEGDFNFYCGP-UHFFFAOYSA-N Pyrazole Chemical compound C=1C=NNC=1 WTKZEGDFNFYCGP-UHFFFAOYSA-N 0.000 description 1
- 108091030071 RNAI Proteins 0.000 description 1
- 208000009527 Refractory anemia Diseases 0.000 description 1
- 208000033501 Refractory anemia with excess blasts Diseases 0.000 description 1
- 206010072684 Refractory cytopenia with unilineage dysplasia Diseases 0.000 description 1
- LKPMZLMOYCJBMK-UHFFFAOYSA-N S=[PH2]SS[PH2]=S Chemical class S=[PH2]SS[PH2]=S LKPMZLMOYCJBMK-UHFFFAOYSA-N 0.000 description 1
- 208000000453 Skin Neoplasms Diseases 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical group OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 1
- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical compound C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 229920006243 acrylic copolymer Polymers 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- MIBQYWIOHFTKHD-UHFFFAOYSA-N adamantane-1-carbonyl chloride Chemical compound C1C(C2)CC3CC2CC1(C(=O)Cl)C3 MIBQYWIOHFTKHD-UHFFFAOYSA-N 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 150000001345 alkine derivatives Chemical class 0.000 description 1
- 150000003973 alkyl amines Chemical class 0.000 description 1
- 125000002877 alkyl aryl group Chemical group 0.000 description 1
- 159000000013 aluminium salts Chemical class 0.000 description 1
- 229910000329 aluminium sulfate Inorganic materials 0.000 description 1
- 125000006242 amine protecting group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 125000003710 aryl alkyl group Chemical group 0.000 description 1
- 238000001479 atomic absorption spectroscopy Methods 0.000 description 1
- HONIICLYMWZJFZ-UHFFFAOYSA-N azetidine Chemical compound C1CNC1 HONIICLYMWZJFZ-UHFFFAOYSA-N 0.000 description 1
- 159000000009 barium salts Chemical class 0.000 description 1
- 239000003637 basic solution Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 125000003785 benzimidazolyl group Chemical class N1=C(NC2=C1C=CC=C2)* 0.000 description 1
- 125000004196 benzothienyl group Chemical group S1C(=CC2=C1C=CC=C2)* 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 125000001246 bromo group Chemical group Br* 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 229910052792 caesium Inorganic materials 0.000 description 1
- TVFDJXOCXUVLDH-UHFFFAOYSA-N caesium atom Chemical compound [Cs] TVFDJXOCXUVLDH-UHFFFAOYSA-N 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 210000003679 cervix uteri Anatomy 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 125000001309 chloro group Chemical group Cl* 0.000 description 1
- AOGYCOYQMAVAFD-UHFFFAOYSA-N chlorocarbonic acid Chemical class OC(Cl)=O AOGYCOYQMAVAFD-UHFFFAOYSA-N 0.000 description 1
- VDANGULDQQJODZ-UHFFFAOYSA-N chloroprocaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1Cl VDANGULDQQJODZ-UHFFFAOYSA-N 0.000 description 1
- 229960002023 chloroprocaine Drugs 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 208000021668 chronic eosinophilic leukemia Diseases 0.000 description 1
- 201000010902 chronic myelomonocytic leukemia Diseases 0.000 description 1
- WCZVZNOTHYJIEI-UHFFFAOYSA-N cinnoline Chemical compound N1=NC=CC2=CC=CC=C21 WCZVZNOTHYJIEI-UHFFFAOYSA-N 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 150000001879 copper Chemical class 0.000 description 1
- 239000007822 coupling agent Substances 0.000 description 1
- 125000001316 cycloalkyl alkyl group Chemical group 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011118 depth filtration Methods 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- MXHRCPNRJAMMIM-UHFFFAOYSA-N desoxyuridine Natural products C1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 MXHRCPNRJAMMIM-UHFFFAOYSA-N 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 150000001993 dienes Chemical class 0.000 description 1
- 229940043279 diisopropylamine Drugs 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000011978 dissolution method Methods 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 150000002192 fatty aldehydes Chemical class 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 125000005456 glyceride group Chemical group 0.000 description 1
- 229940029575 guanosine Drugs 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 208000014951 hematologic disease Diseases 0.000 description 1
- 125000004404 heteroalkyl group Chemical group 0.000 description 1
- 125000005885 heterocycloalkylalkyl group Chemical group 0.000 description 1
- ARBOVOVUTSQWSS-UHFFFAOYSA-N hexadecanoyl chloride Chemical compound CCCCCCCCCCCCCCCC(Cl)=O ARBOVOVUTSQWSS-UHFFFAOYSA-N 0.000 description 1
- 239000012216 imaging agent Substances 0.000 description 1
- 150000002460 imidazoles Chemical class 0.000 description 1
- MTNDZQHUAFNZQY-UHFFFAOYSA-N imidazoline Chemical compound C1CN=CN1 MTNDZQHUAFNZQY-UHFFFAOYSA-N 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- LPAGFVYQRIESJQ-UHFFFAOYSA-N indoline Chemical compound C1=CC=C2NCCC2=C1 LPAGFVYQRIESJQ-UHFFFAOYSA-N 0.000 description 1
- HOBCFUWDNJPFHB-UHFFFAOYSA-N indolizine Chemical compound C1=CC=CN2C=CC=C21 HOBCFUWDNJPFHB-UHFFFAOYSA-N 0.000 description 1
- 125000003406 indolizinyl group Chemical group C=1(C=CN2C=CC=CC12)* 0.000 description 1
- 125000001041 indolyl group Chemical group 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 150000002484 inorganic compounds Chemical class 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 125000002346 iodo group Chemical group I* 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 1
- LSACYLWPPQLVSM-UHFFFAOYSA-N isobutyric acid anhydride Chemical compound CC(C)C(=O)OC(=O)C(C)C LSACYLWPPQLVSM-UHFFFAOYSA-N 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- ZLTPDFXIESTBQG-UHFFFAOYSA-N isothiazole Chemical compound C=1C=NSC=1 ZLTPDFXIESTBQG-UHFFFAOYSA-N 0.000 description 1
- 150000002540 isothiocyanates Chemical class 0.000 description 1
- CTAPFRYPJLPFDF-UHFFFAOYSA-N isoxazole Chemical compound C=1C=NOC=1 CTAPFRYPJLPFDF-UHFFFAOYSA-N 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- TWXDDNPPQUTEOV-FVGYRXGTSA-N methamphetamine hydrochloride Chemical compound Cl.CN[C@@H](C)CC1=CC=CC=C1 TWXDDNPPQUTEOV-FVGYRXGTSA-N 0.000 description 1
- YACKEPLHDIMKIO-UHFFFAOYSA-N methylphosphonic acid Chemical compound CP(O)(O)=O YACKEPLHDIMKIO-UHFFFAOYSA-N 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 150000004682 monohydrates Chemical class 0.000 description 1
- 235000021281 monounsaturated fatty acids Nutrition 0.000 description 1
- 125000002757 morpholinyl group Chemical group 0.000 description 1
- 208000016586 myelodysplastic syndrome with excess blasts Diseases 0.000 description 1
- 208000025113 myeloid leukemia Diseases 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- XBDUZBHKKUFFRH-UHFFFAOYSA-N n-(2-oxo-1h-pyrimidin-6-yl)benzamide Chemical compound OC1=NC=CC(NC(=O)C=2C=CC=CC=2)=N1 XBDUZBHKKUFFRH-UHFFFAOYSA-N 0.000 description 1
- RZCVPJDHJYOZNP-UHFFFAOYSA-N n-(6-oxo-3,7-dihydropurin-2-yl)benzamide Chemical compound N=1C(=O)C=2NC=NC=2NC=1NC(=O)C1=CC=CC=C1 RZCVPJDHJYOZNP-UHFFFAOYSA-N 0.000 description 1
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000000740 n-pentyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000001971 neopentyl group Chemical group [H]C([*])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 125000006574 non-aromatic ring group Chemical group 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 229940038384 octadecane Drugs 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 150000002892 organic cations Chemical class 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 229950000688 phenothiazine Drugs 0.000 description 1
- 150000008298 phosphoramidates Chemical class 0.000 description 1
- LFSXCDWNBUNEEM-UHFFFAOYSA-N phthalazine Chemical compound C1=NN=CC2=CC=CC=C21 LFSXCDWNBUNEEM-UHFFFAOYSA-N 0.000 description 1
- XKJCHHZQLQNZHY-UHFFFAOYSA-N phthalimide Chemical compound C1=CC=C2C(=O)NC(=O)C2=C1 XKJCHHZQLQNZHY-UHFFFAOYSA-N 0.000 description 1
- 125000003386 piperidinyl group Chemical group 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 150000003141 primary amines Chemical class 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011165 process development Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- CPNGPNLZQNNVQM-UHFFFAOYSA-N pteridine Chemical compound N1=CN=CC2=NC=CN=C21 CPNGPNLZQNNVQM-UHFFFAOYSA-N 0.000 description 1
- 150000003217 pyrazoles Chemical class 0.000 description 1
- PBMFSQRYOILNGV-UHFFFAOYSA-N pyridazine Chemical compound C1=CC=NN=C1 PBMFSQRYOILNGV-UHFFFAOYSA-N 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 125000000714 pyrimidinyl group Chemical group 0.000 description 1
- 125000000168 pyrrolyl group Chemical group 0.000 description 1
- JWVCLYRUEFBMGU-UHFFFAOYSA-N quinazoline Chemical compound N1=CN=CC2=CC=CC=C21 JWVCLYRUEFBMGU-UHFFFAOYSA-N 0.000 description 1
- 125000002943 quinolinyl group Chemical group N1=C(C=CC2=CC=CC=C12)* 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 210000001525 retina Anatomy 0.000 description 1
- 208000024725 retina neoplasm Diseases 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
- 235000003441 saturated fatty acids Nutrition 0.000 description 1
- 150000004671 saturated fatty acids Chemical class 0.000 description 1
- 229930195734 saturated hydrocarbon Natural products 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 1
- 239000012265 solid product Substances 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 125000002730 succinyl group Chemical group C(CCC(=O)*)(=O)* 0.000 description 1
- 230000035322 succinylation Effects 0.000 description 1
- 238000010613 succinylation reaction Methods 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 125000000475 sulfinyl group Chemical group [*:2]S([*:1])=O 0.000 description 1
- 125000005420 sulfonamido group Chemical group S(=O)(=O)(N*)* 0.000 description 1
- 125000004434 sulfur atom Chemical group 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- ZTUXEFFFLOVXQE-UHFFFAOYSA-N tetradecanoic acid Chemical compound CCCCCCCCCCCCCC(O)=O.CCCCCCCCCCCCCC(O)=O ZTUXEFFFLOVXQE-UHFFFAOYSA-N 0.000 description 1
- 150000004685 tetrahydrates Chemical class 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 125000003718 tetrahydrofuranyl group Chemical group 0.000 description 1
- 150000003536 tetrazoles Chemical class 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 229930192474 thiophene Natural products 0.000 description 1
- 125000000464 thioxo group Chemical group S=* 0.000 description 1
- 229960002447 thiram Drugs 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 150000005671 trienes Chemical class 0.000 description 1
- 150000005691 triesters Chemical class 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 229960000281 trometamol Drugs 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- JFALSRSLKYAFGM-UHFFFAOYSA-N uranium(0) Chemical compound [U] JFALSRSLKYAFGM-UHFFFAOYSA-N 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/11—Antisense
- C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/314—Phosphoramidates
- C12N2310/3145—Phosphoramidates with the nitrogen in 3' or 5'-position
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/35—Nature of the modification
- C12N2310/351—Conjugate
- C12N2310/3515—Lipophilic moiety, e.g. cholesterol
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/07—Nucleotidyltransferases (2.7.7)
- C12Y207/07049—RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase
Abstract
Aspects of the disclosure include methods for the preparation of a polynucleotide. In some embodiments, the method includes contacting a first polynucleotide composition including: a polynucleotide having a sequence of 7 or more nucleoside subunits and at least two of the nucleoside subunits are joined by a N3'?P5' thiophosphoramidate inter-subunit linkage; and non-target synthetic products and reagents; with a multivalent cation salt to precipitate a polynucleotide salt including at least one multivalent cation counterion; and separating the polynucleotide salt from the contacted first polynucleotide composition to produce a second polynucleotide composition including the polynucleotide salt. In certain embodiments, the method further includes contacting the polynucleotide salt with a reverse phase chromatography support; and eluting from the chromatography support a third polynucleotide composition including the polynucleotide. Also provided are compositions including a salt of the polynucleotide including at least one multivalent cation counterion. ned by a N3'?P5' thiophosphoramidate inter-subunit linkage; and non-target synthetic products and reagents; with a multivalent cation salt to precipitate a polynucleotide salt including at least one multivalent cation counterion; and separating the polynucleotide salt from the contacted first polynucleotide composition to produce a second polynucleotide composition including the polynucleotide salt. In certain embodiments, the method further includes contacting the polynucleotide salt with a reverse phase chromatography support; and eluting from the chromatography support a third polynucleotide composition including the polynucleotide. Also provided are compositions including a salt of the polynucleotide including at least one multivalent cation counterion.
Description
METHODS OF POLYNUCLEOTIDE PREPARATION USING MULTIVALENT CATION SALT COMPOSITIONS CROSS REFERENCE TO RELATED APPLICATION Pursuant to 35 U.S.C. § 119(e), this application claims priority to the filing date of U.S. provisional application serial No. 62/151,891, filed April 23, 2015, the disclosure of which is herein incorporated by reference.
INTRODUCTION c acid polymer chemistry has played a role in many developing technologies in the pharmaceutical, diagnostic, and analytical fields, and more particularly in the subfields of nse and anti-gene therapeutics, combinatorial try, branched DNA signal amplification, and array-based DNA diagnostics and analysis. Some of this polymer chemistry has been directed to improving the binding strength, icity, and nuclease resistance of natural nucleic acid rs, such as DNA. Peptide nucleic acid (PNAs), phosphorothioate, phosphonate and phosphoramidate internucleoside linkages are examples of some polymer chemistries that have been applied to polynucleotides to provide for one or more desirable properties such as nuclease resistance, cellular uptake and solubility.
Polynucleotide N3'→P5' phosphoramidates can form stable duplexes with complementary DNA and RNA strands, as well as stable triplexes with DNA duplexes, and are resistant to nucleases. Polynucleotide N3'→P5' thiophosphoramidates have found use as potent antisense agents both in vitro and in vivo. Polynucleotide containing compounds that inhibit telomerase activity can be used to treat rase-mediated disorders, such as cancer, since cancer cells express rase activity and normal human somatic cells do not possess rase activity at biologically relevant levels. As such, methods of preparing and isolating such polynucleotides are of st.
SUMMARY Aspects of the sure e methods for the preparation of a polynucleotide. In some embodiments, the method includes contacting a first polynucleotide composition ing: a polynucleotide having a sequence of 7 or more nucleoside subunits where at least two of the nucleoside subunits are joined by a N3′→P5′ thiophosphoramidate inter-subunit linkage; and rget synthetic products and reagents; with a multivalent cation salt to precipitate a first cleotide salt ing at least one multivalent cation counterion; and separating the polynucleotide salt from the contacted first polynucleotide composition to produce a second polynucleotide composition including the first polynucleotide salt. In certain embodiments, the method further includes contacting the first polynucleotide salt with a reverse phase chromatography support; and eluting from the chromatography support a third cleotide composition including a second polynucleotide salt. Also provided are compositions including a salt of the polynucleotide including at least one alent cation counterion. In some embodiments, the at least one multivalent cation counterion is selected from the group consisting of magnesium, zinc, aluminium, and calcium.
In one aspect, there is provided a method of preparing a polynucleotide, the method comprising: a) contacting a first polynucleotide composition with a multivalent cation salt to precipitate a first polynucleotide salt comprising at least one multivalent cation counterion; and b) separating the first polynucleotide salt from the contacted first cleotide composition to produce a second polynucleotide composition comprising the first polynucleotide salt; wherein the first polynucleotide composition comprises: (i) a polynucleotide having a ce of 7 or more nucleoside subunits and at least two of the nucleoside subunits are joined by a N3′→P5′ thiophosphoramidate inter-subunit e; and (ii) soluble non-target synthetic products and reagents.
In another aspect, there is ed a ition comprising: a salt precipitate of a polynucleotide comprising at least one multivalent cation counterion; n the polynucleotide has a sequence of 7 or more nucleoside subunits complementary to the RNA component of human telomerase and at least two of the nucleoside subunits are joined by a N3′→P5′ thiophosphoramidate inter-subunit linkage.
BRIEF DESCRIPTION OF THE FIGURES The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not ed to limit the scope of the present teachings in any way.
Figure 1 shows HPLC chromatograms of Imetelstat-Mg in 1M NaCl solutions at a variety of pH’s.
Figure 2 depicts the results of an elemental analysis of Imetelstat Sodium treated with a variety of salts.
Figure 3 depicts the results of an elemental analysis of Imetelstat Sodium treated with increasing equivalents of magnesium chloride salt.
Figure 4 depicts the results of an elemental is of Imetelstat TEA treated with increasing equivalents of magnesium chloride salt.
DEFINITIONS Before bing exemplary embodiments in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used in the description.
The following terms have the following meanings unless otherwise indicated.
Any undefined terms have their art recognized meanings.
As used herein, the terms polynucleotide and oligonucleotide are used interchangeably to refer to a compound containing a plurality of nucleoside moiety subunits or side residues that are linked by internucleoside bonds or internucleosidic es. Whenever a polynucleotide is represented by a ce of letters, such as "ATGUCCTG," it is understood that the nucleotides are in 5'→3' order from left to right and that "A" denotes deoxyadenosine, "C" s deoxycytidine, "G" denotes deoxyguanosine, "T" s thymidine, and "U" denotes deoxyuridine, unless otherwise noted.
As used herein, "nucleoside" es the natural sides, including 2'- deoxy and 2'-hydroxyl forms, e.g. as bed in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). "Analogs" in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g. described generally by Scheit, Nucleotide Analogs (John Wiley, New York, 1980). Such analogs include synthetic sides designed to enhance binding properties, e.g. stability, specificity, or the like, such as disclosed by Uhlmann and Peyman (Chemical Reviews, 90:543-584, 1990). In some ments, a nucleoside or side analog includes a 3’-hydroxyl group or a 3’-amino group.
The terms "base" and "nucleobase" are used hangeably and defined herein to include (i) conventional DNA and RNA bases (uracil, thymine, adenine, guanine, and cytosine), and (ii) modified bases or base analogs (e.g., 5-methyl-cytosine, -bromouracil, or inosine). A base analog is a chemical whose molecular structure mimics that of a conventional DNA or RNA base.
As used herein, "pyrimidine" means the pyrimidines ing in natural nucleosides, including cytosine, thymine, and uracil, and common analogs thereof, such as those containing oxy, methyl, propynyl, methoxy, hydroxyl, amino, thio, halo, and like, substituents. The term as used herein further includes pyrimidines with common protecting groups attached, such as N4-benzoylcytosine. r pyrimidine protecting groups of interest include but are not d to, those protecting groups are disclosed by Beaucage and Iyer Tetrahedron 48: 2223-2311 (1992).
As used herein, e" means the purines occurring in natural nucleosides, including adenine, guanine, and hypoxanthine, and common s thereof, such as those containing oxy, methyl, propynyl, y, hydroxyl, amino, thio, halo, and like, substituents. The term as used herein further includes purines with common protection groups attached, such as N2-benzoylguanine, N2-isobutyrylguanine, N6-benzoyladenine, and the like. Further common purine protection groups are disclosed by Beaucage and Iyer Tetrahedron 48: 2223-2311 (1992). As used herein, the term "-protected-" as a component of a chemical name refers to art-recognized protection groups for a particular moiety of a compound, e.g. "5'-protected- hydroxyl" in reference to a nucleoside includes triphenylmethyl (i.e., trityl), p-anisyldiphenylmethyl (i.e., monomethoxytrityl or MMT), di-p-anisylphenylmethyl (i.e., dimethoxytrityl or DMT), and the like; and a protected nucleobase in reference to a nucleobase including a atom protected with a group such as a ylaminoformamidine (DMF), l (Bz), isobutyryl, and the like. Art- recognized protecting groups include those described in the following references: Gait, editor, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984); th and Broom, al Reviews, 77:183-217, 1977; Pon et al., hniques, 6:768-775, 1988; Ohtsuka et al., Nucleic Acids ch, 10:6553-6570, 1982; Eckstein, editor, Oligonucleotides. and Analogues: A Practical Approach (IRL Press, Oxford, 1991), Greene and Wuts, Protective Groups in Organic Synthesis, Second Edition, (John Wiley & Sons, New York, 1991), Narang, editor, Synthesis and Applications of DNA and RNA (Academic Press, New York, 1987), Beaucage and Iyer Tetrahedron 48: 2223- 2311 (1992), and like references.
As used herein, "polynucleotide N3'→P5' thiophosphoramidate" means an oligomer, usually linear, of nucleoside subunits linked by at least one N3'→P5' thiophosphoramidate linkage. In general terms, the nucleoside subunits comprise nucleosides or nucleoside analogs, but may also se more general moieties having compatible chemistry, such as abasic sugars and other hydrocarbon moieties, such as described in the following references: Newton et al., Nucleic Acids Research, 21: 1155- 1162 (1993); Griffin et al., J. Am. Chem. Soc., 114: 7976-7982 (1992); Jaschke et al., Tetrahedron Letters, 34: 301-304 (1992); Ma et al., International application PCT/CA92/00423; Zon et al., International application PCT/US90/06630; Durand et al., Nucleic Acids Research, 18: 6353-6359 (1990); Salunkhe et al., J. Am. Chem. Soc., 114: 772 (1992); and the like. In some instances, the term means a polynucleotide where all ucleosidic linkages are replaced by N3'→P5' thiophosphoramidate linkages. As such, the term hends partially as well as fully "amidated" oligomers.
In some instances, the term means a polynucleotide where all the internucleosidic linkages are replaced by N3'→P5' thiophosphoramidate linkages and wherein the nucleoside subunits are the l sides or s thereof. A subject polynucleotide N3'→P5' thiophosphoramidate in which every e is an N3'→P5' thiophosphoramidate linkage ("fully amidated") may be imbedded in or attached to other oligonucleotides or polynucleotides to form a larger oligomer which is "partially amidated." A subject polynucleotide N3'→P5' thiophosphoramidate may include any convenient 3’ and/or 5’ terminal groups. In some ments, the polynucleotide N3'→P5' thiophosphoramidate es a 3’-hydroxyl terminal group or a no terminal group.
As used herein, the terms "phosphate" and "phosphate group" are meant to encompass a thiophosphate group and an oxophosphate group.
As used herein, the term "phosphoramidite amino group" refers to the amino group, --NR4R5, attached to the phosphorus atom of a phosphoramidite group, and the term "phosphoramidite nitrogen" refers to the nitrogen atom of the oramidite amino group.
"Alkyl" refers to monovalent saturated aliphatic hydrocarbyl groups having from 1 to 10 carbon atoms and such as 1 to 6 carbon atoms (e.g., "an alkyl of 1 to 6 carbons atoms"), or 1 to 5 (e.g., "an alkyl of 1 to 5 carbons atoms"), or 1 to 4 (e.g., "an alkyl of 1 to 4 carbons atoms"), or 1 to 3 carbon atoms (e.g., "an alkyl of 1 to 3 carbons atoms"). This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH- ), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), tyl ((CH3)(CH3CH2)CH- ), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl 3CCH2-).
The term ituted alkyl" refers to an alkyl group as defined herein n one or more carbon atoms in the alkyl chain have been optionally replaced with a heteroatom such as -O-, -N-, -S-, -S(O)n- (where n is 0 to 2), -NR- (where R is hydrogen or alkyl) and having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, y, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, teroaryl, -SO2-alkyl, -SO2-aryl, -SO2- heteroaryl, and -NRaRb, wherein Ra and Rb may be the same or ent and are chosen from hydrogen, optionally tuted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic. In some instances, a "substituted alkyl" refers to an alkyl group as defined herein having from 1 to 5 substituents selected from the group consisting of , cycloalkyl, lkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, noacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, thiol, thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, sulfonamido, and -NRaRb, wherein Ra and Rb may be the same or different and are chosen from hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.
"Alkoxy" refers to the group –O-alkyl, wherein alkyl is as defined herein.
Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. The term "alkoxy" also refers to the groups alkenyl-O-, cycloalkyl-O-, lkenyl-O-, and alkynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.
The term "substituted alkoxy" refers to the groups substituted alkyl-O-, tuted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as d herein.
"Acyl" refers to the groups H-C(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, l-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, aryl-C(O)-, substituted heteroaryl-C(O)-, cyclyl-C(O)-, and substituted heterocyclyl-C(O)-, wherein alkyl, substituted alkyl, alkenyl, substituted l, alkynyl, substituted l, cycloalkyl, substituted lkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted aryl, heterocyclic, and substituted heterocyclic are as defined herein. For example, acyl includes the "acetyl" group CH3C(O)- The term ituted amino" refers to the group -NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, l, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, tuted alkynyl, aryl, heteroaryl, and heterocyclyl provided that at least one R is not hydrogen.
"Halo" or "halogen" refers to fluoro, chloro, bromo, and iodo.
"Hydroxy" or "hydroxyl" refers to the group –OH.
"Heteroaryl" refers to an aromatic group of from 1 to 15 carbon atoms, such as from 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a single ring (such as, pyridinyl, olyl or furyl) or multiple condensed rings in a ring system (for example as in groups such as, indolizinyl, quinolinyl, uran, idazolyl or benzothienyl), wherein at least one ring within the ring system is aromatic, provided that the point of attachment is through an atom of an aromatic ring. In certain embodiments, the nitrogen and/or sulfur ring atom(s) of the heteroaryl group are ally ed to provide for the e (N→O), sulfinyl, or yl moieties. This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, enyl, and furanyl. Unless otherwise constrained by the definition for the heteroaryl substituent, such aryl groups can be optionally substituted with 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, aryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, lamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, - SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2- heteroaryl, and trihalomethyl. In such cases, a heteroaryl group that is substituted with from 1 to 5 substituents (e.g., as bed herein) is referred to as a "substituted heteroaryl".
"Heterocycle," "heterocyclic," "heterocycloalkyl," and "heterocyclyl" refer to a saturated or unsaturated group having a single ring or multiple condensed rings, including fused bridged and spiro ring systems, and having from 3 to 20 ring atoms, including 1 to 10 hetero atoms. These ring atoms are selected from the group consisting of nitrogen, sulfur, or oxygen, wherein, in fused ring systems, one or more of the rings can be cycloalkyl, aryl, or heteroaryl, provided that the point of attachment is h the non-aromatic ring. In certain embodiments, the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, -S(O)-, or –SO2- moieties.
Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, ole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, noline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, ole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7- tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to as thiamorpholinyl), 1,1- dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.
Unless otherwise constrained by the definition for the heterocyclic substituent, such heterocyclic groups can be optionally substituted with 1 to 5, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, tuted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, cyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, terocyclooxy, thiol, koxy, substituted thioalkoxy, aryl, aryloxy, aryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, kyl, -SO- substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2- aryl, -SO2-heteroaryl, and fused heterocycle.
"Nitro" refers to the group –NO2.
"Oxo" refers to the atom (=O).
"Thiol" refers to the group -SH.
"Thioxo" or the term "thioketo" refers to the atom (=S).
In addition to the disclosure herein, the term ituted," when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one r, replaced with the same or different substituent groups as defined below.
In addition to the groups disclosed with respect to the individual terms , substituent groups for substituting for one or more ens (any two hydrogens on a single carbon can be replaced with =O, =NR70, =N-OR70, =N2 or =S) in the specified group or radical are, unless otherwise specified, -R60, halo, =O, -OR70, -SR70, -NR80R80, trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R70, -SO2O– M+, -SO2OR70, -OSO2R70, -OSO2O–M+, -OSO2OR70, O–)2(M+)2, -P(O)(OR70)O– M+, -P(O)(OR70) 2, -C(O)R70, -C(S)R70, -C(NR70)R70, -C(O)O– M+, -C(O)OR70, R70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, - OC(O)O-M+, -OC(O)OR70, OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2– M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70 and -NR70C(NR70)NR80R80, where R60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, kyl, heteroaryl and arylalkyl, each R70 is independently hydrogen or R60; each R80 is independently R70 or alternatively, two R80’s, taken er with the nitrogen atom to which they are bonded, form a 5-, 6- or 7-membered heterocycloalkyl which may optionally include from 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N and S, of which N may have -H or C1-C3 alkyl substitution; and each M+ is a counter ion with a net single positive charge. Each M+ may independently be, for example, an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as +N(R60) 2+] 2+] 2+] 4; or an alkaline earth ion, such as [Ca 0.5, [Mg 0.5, or [Ba 0.5 ("subscript 0.5 means that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound of the invention and the other a counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized nd of the invention can serve as the counter ion for such divalent alkali earth ions). As specific examples, -NR80R80 is meant to include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazinyl and N-morpholinyl.
In addition to the disclosure , in a certain embodiment, a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.
It is understood that in all substituted groups defined above, polymers arrived at by defining tuents with further substituents to lves (e.g., substituted aryl having a substituted aryl group as a substituent which is itself tuted with a substituted aryl group, which is further substituted by a substituted aryl group, etc.) are not ed for inclusion herein. In such cases, the maximum number of such substitutions is three. For example, serial substitutions of substituted aryl groups specifically contemplated herein are limited to tuted aryl-(substituted aryl)- substituted aryl.
Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the al portion of the functionality followed by the adjacent functionality toward the point of attachment.
As to any of the groups disclosed herein which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution ns which are sterically impractical and/or synthetically non-feasible.
In addition, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.
The term "pharmaceutically acceptable salt" means a salt which is acceptable for administration to a patient, such as a mammal (salts with counterions having acceptable mammalian safety for a given dosage regime). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically able salt" refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and nic counter ions well known in the art and include, by way of example only, sodium, and the like; and when the molecule contains a basic onality, salts of organic or nic acids, such as hydrochloride, and the like. Pharmaceutically acceptable salts of interest include, but are not d to, aluminium, ammonium, arginine, barium, benzathine, calcium, cholinate, ethylenediamine, lysine, lithium, magnesium, meglumine, procaine, potassium, sodium, hamine, N- methylglucamine, N,N′-dibenzylethylene-diamine, chloroprocaine, diethanolamine, ethanolamine, piperazine, zinc, ropylamine, triethylamine, diisopropylethylamine and triethanolamine salts.
The term "salt thereof" means a compound formed when a proton of an acid is replaced by a cation, such as a metal cation or an organic cation and the like. Where applicable, the salt is a pharmaceutically acceptable salt, gh this is not required for salts of intermediate compounds that are not intended for administration to a patient. By way of example, salts of the present compounds include those wherein the compound is protonated by an inorganic or organic acid to form a , with the conjugate base of the nic or organic acid as the anionic component of the salt. Salts of interest include, but are not limited to, aluminium, ammonium, arginine, barium, benzathine, calcium, cesium, cholinate, ethylenediamine, lithium, magnesium, meglumine, ne, N-methylglucamine, piperazine, potassium, sodium, tromethamine, zinc, N,N′- dibenzylethylene-diamine, procaine, diethanolamine, ethanolamine, piperazine, diisopropylamine, triethylamine, diisopropylethylamine and triethanolamine salts. It is understood that for any of the polynucleotide structures depicted herein that include a backbone of internucleoside linkages, such cleotides may also include any convenient salt forms. In some embodiments, acidic forms of the internucleoside linkages are depicted for simplicity. In some ces, the salt of the subject compound is a monovalent cation salt. In certain instances, the salt of the subject compound is a divalent cation salt. In some instances, the salt of the subject compound is a trivalent cation salt.
"Solvate" refers to a complex formed by ation of solvent molecules with molecules or ions of the solute. The solvent can be an organic compound, an inorganic compound, or a mixture of both. Some examples of ts include, but are not limited to, methanol, N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.
"Stereoisomer" and "stereoisomers" refer to compounds that have same atomic connectivity but different atomic arrangement in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.
"Tautomer" refers to alternate forms of a molecule that differ only in electronic bonding of atoms and/or in the position of a , such as enol-keto and imine-enamine tautomers, =S)(OH)-O- and -NH-P(=O)(SH)-O-, or the tautomeric forms of heteroaryl groups containing a -N=C(H)-NH- ring atom ement, such as pyrazoles, imidazoles, benzimidazoles, les, and tetrazoles. A person of ry skill in the art would recognize that other eric arrangements of the groups described herein are possible. For example, it is understood that a polynucleotide described by the following structure: H OH N O P O T O SH O P SH O A O P SH O [ G n p s G n p s G n p s T n p s T n p s A n p s G n p s A n p s C n p s A n p s ] A NH 2 also encompasses the following structure showing one le alternate tautomeric arrangement of linkage groups: H OH N O P O T O OH S P O H O A S P O H O [ G n p s G n p s G n p s T n p s T n p s A n p s G n p s A n p s C n p s A n p s ] A NH 2 where "nps" ents a osphoramidate linkage (—NH—P(═O)(SH)—O— or — NH—P(═S)(OH)—O—) connecting the 3'-carbon of one nucleoside to the 5'-carbon of the adjacent nucleoside. It is understood that all tautomeric forms of a subject compound are encompassed by a structure where one possible tautomeric arrangement of the groups of the compound is described, even if not specifically indicated. Any convenient tautomeric arrangement of the groups of the subject compounds may be utilized in describing the compounds.
It will be iated that the term "or a salt or solvate or stereoisomer f" is intended to include all permutations of salts, solvates and stereoisomers, such as a solvate of a pharmaceutically acceptable salt of a stereoisomer of subject compound.
It is understood that the term "or a salt thereof" is intended to include all permutations of salts. It is understood that the term "or a pharmaceutically acceptable salt thereof" is intended to include all permutations of salts. It is understood that the term "or a solvate thereof" is intended to include all permutations of solvates. It is understood that the term "or a stereoisomer thereof" is intended to include all permutations of stereoisomers. It is understood that the term "or a tautomer thereof" is intended to include all permutations of tautomers. Thus for e it follows that it is intended to e a solvate of a pharmaceutically acceptable salt of a tautomer of a stereoisomer of subject compound.
As used herein the term "isolated" is meant to describe a compound of interest that is in an environment different from that in which the compound naturally occurs.
"Isolated" is meant to include compounds that are within samples that are substantially ed for the compound of interest and/or in which the compound of interest is partially or substantially purified.
As used herein, the term "substantially purified" refers to a compound that is removed from its natural environment and is at least 60% free, at least 75% free, at least 80% free, at least 81% free, at least 82% free, at least 83% free, at least 84% free, at least 85% free, at least 86% free, at least 87% free, at least 88% free, at least 89% free, at least 90% free, at least 91% free, at least 92% free, at least 93% free, at least 94% free, at least 95% free, at least 96% free, at least 97% free, at least 98% free, at least 99% free, or more than 99% free, from other components with which it is naturally associated.
The term ological conditions" is meant to encompass those conditions compatible with living cells, e.g., predominantly aqueous conditions of a temperature, pH, salinity, etc. that are compatible with living cells.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates ise, between the upper and lower limit of that range and any other stated or ening value in that stated range, is encompassed within the ion. The upper and lower limits of these smaller ranges may independently be ed in the smaller , and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
It must be noted that as used herein and in the appended claims, the ar forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such ive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.
Other definitions of terms may appear throughout the specification.
DESCRIPTION OF ARY EMBODIMENTS As summarized above, aspects of the disclosure e methods for the preparation of a polynucleotide. In some embodiments, the method includes contacting a first polynucleotide composition ing: a cleotide having a sequence of 7 or more nucleoside subunits where at least two of the nucleoside subunits are joined by a N3′→P5′ thiophosphoramidate subunit linkage; and non-target synthetic products and reagents; with a multivalent cation salt to precipitate a first polynucleotide salt including at least one multivalent cation counterion; and separating the first polynucleotide salt from the ted first polynucleotide composition to produce a second cleotide composition including the first polynucleotide salt. In certain ments, the method further includes contacting the polynucleotide salt with a reverse phase chromatography support; and eluting from the chromatography support a third polynucleotide composition including the polynucleotide. In some instances, third polynucleotide composition includes a second polynucleotide salt. Also provided are compositions including a salt of the polynucleotide including at least one multivalent cation counterion. In some ments, the at least one multivalent cation counterion is selected from the group consisting of magnesium, zinc, aluminium, and calcium.
Before the various embodiments are described, it is to be understood that the teachings of this disclosure are not limited to the particular ments bed, and as such can, of course, vary. It is also to be understood that the ology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present teachings will be limited only by the appended claims.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter bed in any way. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, cations, and equivalents, as will be iated by those of skill in the art.
Unless defined otherwise, all cal and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or g of the present invention, methods and materials of st are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present claims are not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication ed can be different from the actual publication dates which can be independently confirmed.
It is appreciated that certain features of the invention, which are, for clarity, described in the t of separate embodiments, may also be provided in combination in a single ment. sely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace subject matter that are, for example, compounds that are stable nds (i.e., compounds that can be made, isolated, characterized, and tested for biological activity). In on, all mbinations of the various embodiments and elements thereof (e.g., elements of the chemical groups listed in the embodiments describing such variables) are also ically embraced by the present ion and are disclosed herein just as if each and every such sub-combination was individually and itly disclosed herein.
All patents and publications, including all sequences disclosed within such patents and publications, ed to herein are expressly incorporated by reference.
In further describing the subject invention, methods of preparing a polynucleotide are described first in greater detail. Next, polynucleotide compositions of interest for practicing the subject methods are reviewed.
METHODS OF PREPARATION Aspects of the present disclosure include methods for the preparation of a polynucleotide. In some embodiments, the method includes contacting a first polynucleotide composition including a polynucleotide (e.g., as described herein) and non-target sis products and agents, with a multivalent cation salt to precipitate a polynucleotide salt including at least one multivalent cation counterion. Precipitation of the cleotide salt using the subject methods provides for removal of all soluble nontarget synthesis products and agents. In some embodiments, the method includes separating the polynucleotide salt from the contacted first polynucleotide composition to produce a second cleotide composition including the polynucleotide salt. In n embodiments, the first polynucleotide composition, the polynucleotide salt and the second polynucleotide composition each include a target polynucleotide having a sequence of 7 or more nucleoside subunits where at least two of the nucleoside subunits are joined by a ′ thiophosphoramidate inter-subunit linkage (e.g., as described herein).
The second polynucleotide composition may have a reduced amount of nontarget synthesis ts and agents as compared to the first polynucleotide composition.
By reduced amount of non-target synthesis products and agents is meant that there is a % or more by weight ion of the non-target synthesis products and agents in the second polynucleotide ition as compared to the first polynucleotide composition, such as a 15% or more by weight reduction, 20% or more by weight reduction, 25% or more by weight reduction, 30% or more by weight reduction, 35% or more by weight reduction, 40% or more by weight reduction, 45% or more by weight reduction, 50% or more by weight reduction, 55% or more by weight reduction, 60% or more by weight reduction, 65% or more by weight ion, 70% or more by weight reduction, 75% or more by weight reduction, 80% or more by weight reduction, 85% or more by weight reduction, 90% or more by weight reduction, or 95% or more by weight reduction. As such, the subject methods may provide for selective itation of target polynucleotide over non-target synthesis products and agent. In certain embodiments, the subject methods provide for improved selectivity of precipitation as compared to a control method of polynucleotide precipitation using an organic solvent, such as neat ethanol or an ethanol solution (see e.g., Crouse J, Amorese D (1987). "Ethanol Precipitation: um Acetate as an Alternative to Sodium Acetate". Focus 9 (2): 3–5). By improved selectivity of precipitation is meant that 5% or more by weight of non-target synthesis products and agents are removed from the second polynucleotide composition as compared to a control composition, such as 10% or more by weight, 15% or more by weight, 20% or more by weight, 25% or more by weight, 30% or more by weight, 35% or more by weight, 40% or more by weight, 45% or more by weight, 50% or more by weight, 55% or more by weight, 60% or more by weight, 65% or more by weight, 70% or more by weight, 75% or more by weight, 80% or more by weight, 85% or more by weight, 90% or more by weight, or 95% or more by weight of non-target synthesis ts and agents are removed. The reduced amount of non-target synthesis products and agents as compared to the first polynucleotide composition may be determined using any convenient methods, for example using HPLC methods.
As used herein, the terms "target tic cleotide" and t polynucleotide" are used hangeably and refer to a polynucleotide having a particular desired sequence of nucleotides that is synthesized on a support via any convenient stepwise solid phase polynucleotide synthesis method (e.g., as described herein), and where the polynucleotide is devoid of any protecting groups that are utilized solely for purposes of executing the synthetic strategy of the target polynucleotide. Such ting groups may be d from a polynucleotide in the final steps of solid phase synthesis, e.g., during final ection and cleavage of the polynucleotide from a t to produce the target polynucleotide. As used herein, the term "non-target" refers to any ient component, e.g., a nd, a polynucleotide or tive thereof, an agent, etc., or mixtures thereof that is not the desired target product of a synthesis.
The target polynucleotide can include any convenient number of nucleoside subunits, such as between 7 and 500 nucleoside subunits, between 7 and 100 side subunits, between 7 and 75 nucleoside subunits, between 7 and 50 nucleoside subunits, between 7 and 40 nucleoside subunits, between 7 and 30 nucleoside subunits, between 7 and 20 nucleoside subunits, between 7 and 15 nucleoside subunits, between 10 and 15 nucleoside subunits, or between 13 and 15 nucleoside subunits. In some instances, the target polynucleotide has between 7 and 100 nucleoside subunits, such as between 7 and 50 side subunits, between 10 and 50 side subunits, between 10 and 40 nucleoside subunits, between 10 and 30 nucleoside subunits, between 10 and 25 nucleoside subunits, between 10 and 20 nucleoside subunits, n 12 and 18 nucleoside subunits, or between 12 and 16 nucleoside subunits. In certain cases, the target polynucleotide has 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24 or 25 nucleoside ts.
As used herein, the term "non-target synthesis products and agents" refers collectively to a variety of non-target components that may be present in a crude synthetic product of solid phase polynucleotide synthesis, including but not limited to: rget polynucleotide products of the synthesis, such as truncated polynucleotides, capped polynucleotide fragments (i.e., sequences that were capped after a failed subunit coupling), polynucleotides including deletion(s) (i.e., missing one or more target side monomers or dimers, e.g., as described herein) and derivatized polynucleotides (e.g., polynucleotide sequences that undergo an undesirable side reaction during synthesis or cleavage); and agents such as cleaved linkers, products of deprotection, e.g., removed protecting group products such as phosphorus protecting groups products and base protecting group products (e.g., exocyclic amine protecting group products), cleavage reagents and/or cleavage scavengers and residual sis reagents, such as monomers, dimers, coupling, g or deprotection ts.
In certain embodiments, the methods provide for selective precipitation of target polynucleotide over non-target sis products and agents that e polynucleotides having 6 nucleoside subunits or less, such as 5 or less, 4 or less, 3 or less or 2 nucleoside subunits. In n cases, all non-target synthesis products and agents which are not polynucleotides remain soluble during the selective itation step of the subject methods and may thus be easily removed from the resulting polynucleotide salt precipitate.
The subject methods may include precipitation and separation of the target polynucleotide from a crude tic ation to produce a polynucleotide composition that has several desirable properties, such as a reduced amount of non-target synthesis products and agents (e.g., synthesis ts, cleavage reagents, scavengers, removed protecting groups, cleavage side products (linkers, capping , etc.), and small polynucleotide fragments).
In some embodiments, the subject methods include precipitating the polynucleotide from a crude synthetic ation as a multivalent cation salt prior to chromatography purification. In certain cases, the subject methods are methods of purification of a target polynucleotide. Precipitation of the crude polynucleotide composition using a alent cation salt produces a polynucleotide salt precipitate including at least one multivalent cation counterion. In some cases, the polynucleotide salt precipitate includes a mixture of monovalent and multivalent cation counterions which form ion pairs with the polyanionic polynucleotide backbone. As used here, the terms "multivalent cation salt" and "multivalent salt" when used in reference to a polynucleotide are used interchangeably to refer to a polynucleotide salt that includes at least one multivalent cation counterion that is ion paired to an anionic inter-subunit linkage group of the polynucleotide backbone. In some instances, the multivalent cation salt of the polynucleotide includes a mixture of monovalent and multivalent cations. In some embodiments, the multivalent cation may provide for ation of the target cleotide by ion pairing to anionic inter-subunit linkage groups of two or more polynucleotide backbones. In certain instances, a divalent cation ion pairs with two distinct cleotides to form a dimer. In some cases, further ation of the polynucleotides may be achieved by additional multivalent interactions mediated by additional multivalent cations. As such, in some cases, the subject methods may provide for selective aggregation and precipitation of target polynucleotides over non-target synthetic products and agents.
In some embodiments of the method, the at least one multivalent cation counterion is divalent. In n embodiments, the at least one multivalent cation counterion is selected from the group consisting of magnesium, zinc and calcium. In some embodiments, the at least one multivalent cation counterion is trivalent. In certain embodiments, the at least one multivalent cation counterion is aluminium. In some embodiments, the polynucleotide salt further includes a lent cation counterion. In such cases, the polynucleotide salt is a mixed salt, e.g., a salt including two or more different cation rions.
Any convenient methods of precipitating a polynucleotide may find use in the t methods. The step of contacting the first polynucleotide ition with a multivalent cation salt to precipitate a polynucleotide salt including at least one multivalent cation counterion may be achieved using any convenient s. Any convenient multivalent cations and salts thereof (e.g., as described herein) may be utilized in the contacting step to produce the itate. In certain instances, a salt of a polynucleotide including at least one multivalent cation counterion is produced in a solution phase, e.g., via the addition of a multivalent cation salt to a solution including the polynucleotide. Once the multivalent cation salt has been added to the solution the precipitate may then form. In some cases, a salt of a polynucleotide including at least one multivalent cation counterion may be formed on an ion exchange support. Any convenient ion exchange supports may be utilized in the ting step. In some cases, the ion exchange support is a strong cation exchange resin. In some embodiments of the method, the contacting step includes eluting the first polynucleotide composition from a cation exchange support that includes multivalent cation counterions. As used herein, the term "cation exchange t" refers to a t which is itself anionic and is capable of ion pairing with a cationic analyte, such as a multivalent cation of interest. Any convenient eluant may be ed for the step of eluting from the cation exchange support. In some instances, the precipitate forms in the eulate after the polynucleotide salt has been eluted from the cation exchange support.
The subject methods may be performed on any convenient crude synthetic preparation of a target synthetic polynucleotide. In some instances, the first polynucleotide ition is a crude synthetic preparation of a target synthetic polynucleotide. In certain embodiments, the first polynucleotide composition is a composition that is the product of cleavage of a target polynucleotide from a t, post synthesis. As such, the first polynucleotide ition may include a variety of rget synthetic products and agents. The subject methods provide for selective itation of the polynucleotide salt over non-target synthesis products and agents, which remain in solution and thus can be easily removed from the resulting precipitate.
Any convenient methods of synthesis (e.g., as described herein) may be utilized to synthesize the target polynucleotide. Following synthesis, the target polynucleotide is cleaved from the support on which se synthesis is performed.
Following cleavage, the full length target polynucleotide may be purified to remove undesirable synthesis and cleavage reagents and to remove non-target polynucleotide fragments, and derivatives f. The subject methods including precipitation of the polynucleotide salt including at least one alent cation counterion may be performed at any convenient stage of the preparation of a target polynucleotide, such as post synthesis and prior to reverse phase chromatography purification.
As used herein, the terms "crude synthetic preparation", "crude composition" and "crude polynucleotide" refer to a composition including the synthetic products of solid phase polynucleotide synthesis that are ted post synthesis via cleavage from a solid phase synthesis t, where the composition is unpurified, i.e., no chromatography purification has been performed on the composition. Chromatography purification refers to any ient purification method that includes absorption of target polynucleotide to a chromatography support and subsequent elution and resolution of the target cleotide from non-target polynucleotides. In some cases, chromatography purification refers to reverse phase tography cation.
In some embodiments, the method further includes providing a first polynucleotide composition, where the composition is produced via post synthesis cleavage from a solid phase synthesis support. Any convenient additional steps such as evaporation, dilution, or concentration steps may also be performed on the crude synthetic preparation prior to ing the resulting composition in the subject methods.
In some instances, the method further includes synthesizing the target polynucleotide (e.g., as described herein on a solid phase synthesis support). In certain embodiments, the method further includes ng the polynucleotide from a support to e the first polynucleotide composition.
A solid precipitate including the polynucleotide salt may be separated from the first polynucleotide composition that is contacted with the multivalent salt (i.e., the contacted first polynucleotide composition) using any convenient method. Separation methods of interest include, but are not d to, centrifugation, filtration, decanting, and the like.
In some instances, tion of the itate including the polynucleotide salt is achieved by centrifugation where the ation of a centrifugal force to the contacted first polynucleotide composition, e.g., in a centrifuge, causes the precipitate to form a pellet, e.g., at the bottom of the container. The formation of a pellet via centrifugation may be referred to as spinning down the precipitate. In certain embodiments of the , the separating step includes centrifuging the contacted first polynucleotide composition to spin down the polynucleotide salt itate. The supernatant liquid may then be decanted from the tube without disturbing the precipitate, or withdrawn from the container, e.g., with a Pasteur pipette. The centrifugation process can be repeated with a wash solution.
In some instances, separation of the precipitate including the cleotide salt is achieved by filtration. In some embodiments of the method, the separating step includes filtering the polynucleotide salt from the contacted first polynucleotide. Any ient filters and filter media may be utilized in the t methods. In certain cases, the separation is achieved by depth filtration using a filter media that is selected according to the target polynucleotide.
In some embodiments, the method includes: ting a first polynucleotide composition including: a polynucleotide having a sequence of 7 or more nucleoside subunits and at least two of the nucleoside subunits are joined by a N3′→P5′ thiophosphoramidate inter-subunit linkage; and non-target synthetic products and agents; with a multivalent cation salt to precipitate a first polynucleotide salt including at least one multivalent cation counterion; and ting the first polynucleotide salt from the ted first polynucleotide composition to produce a second polynucleotide composition including the polynucleotide salt.
Separating the precipitate from the contacted first polynucleotide composition produces a second polynucleotide ition including the first polynucleotide salt. In some cases, selective precipitation of the first polynucleotide salt using the multivalent cation salt via the subject methods es a second cleotide composition that includes a reduced amount of non-target synthetic products and agents.
After selective precipitation, the subject polynucleotide salts may then be converted into a soluble polynucleotide salt by cation exchange of the at least one multivalent cation counterion away from the polynucleotide and replacement with another cation counterion of interest (e.g., as described herein). As such, the subject methods provide for ible formation of a first polynucleotide salt including at least one multivalent cation counterion. As used herein, the terms "reversible formation" and "reversible exchange" are used interchangeably and refer to the preparation of a polynucleotide salt by, e.g., selective precipitation (e.g., as described herein), where the salt formed may also be subsequently dissociated to exchange away the at least one multivalent cation salt from the salt. In some cases, polynucleotide salts which are insoluble in any solvent may be referred to as irreversibly formed salts. In some ments, the method includes exchanging the at least one multivalent cation counterion away from the first polynucleotide salt to e a soluble second cleotide salt, where the ging includes dissociating the multivalent cation counterion and ion pairing with a e salt cation of interest. In n instances, the soluble second polynucleotide salt is a monovalent salt. In certain instances, the soluble second polynucleotide salt is a sodium salt. In certain instances, the e second polynucleotide salt is a triethylammonium salt. In some instances, the first and second polynucleotide are distinct from each other, i.e., include different cation counterions. The dissociation of the subject polynucleotide salts and exchange of the at least one multivalent cation counterion may be achieved using any convenient methods. In certain instances, dissociation is achieved using reverse phase chromatography, e.g., as described herein. In some cases, ion exchange chromatography may be utilized to achieve dissociation. In certain embodiments, dissociation of the first polynucleotide salt is achieved by dissolution of the salt in a t including a cation counterion of interest.
After the separation, further cation steps may be performed on the second polynucleotide composition. In some embodiments, the method further includes: ting the first polynucleotide salt with a reverse phase chromatography support; and eluting from the tography support a third polynucleotide composition including the polynucleotide. In certain embodiments, the third polynucleotide composition includes a second polynucleotide salt. Any convenient reverse phase chromatography methods may be utilized to purify the polynucleotide salt. Reverse phase chromatography methods and supports of interest include, but are not limited to, chromatographic purification using ir reversed-phase chromatography, C18 ed-phase chromatography and those methods and supports described by Chen et al., Journal of Chromatography A, Volume 1288, 3 May 2013, Pages 73–81; and Zimmermann et al., Journal of Chromatography A, Volume 1354, 8 August 2014, Pages 43–55. In some embodiments, the second polynucleotide ition is loaded directly onto the reverse phase chromatography support. By loaded directly on the support is meant that the second polynucleotide composition produced using the subject method is added directly, e.g., as an isolated solid precipitate, to the e phase tography support. In some instances, the reverse phase chromatography support is a resin configured as a column and the polynucleotide composition is added to the top of the resin bed. In certain embodiments, the method further es dissolving the second polynucleotide composition in a solvent. Any convenient solvents may be ed, ing but not limited to, s buffers, organic solvents miscible with water and mixtures thereof. In such cases, a solution of the second polynucleotide ition may be contacted with the reverse phase chromatography support to absorb the polynucleotide to the support prior to elution.
In some cases, the contacting includes absorbing the polynucleotide onto the reverse phase chromatography support and subsequently eluting the polynucleotide to provide for chromatographic resolution of the target polynucleotide from non-target polynucleotide and residual synthetic agents that are present in the composition. The eluate containing target polynucleotide is collected. Any convenient eluants may be utilized to elute the polynucleotide from the reverse phase chromatography support. The eluant may be selected according to a variety of factors, such as the nature of the e phase chromatography support, the target oligonucleotide, particular desired salts of the target cleotide, etc. In some instances, the at least one multivalent cation counterion of the first polynucleotide salt is ion exchanged on the reverse phase chromatography support with another distinct cation counterion of interest that is included in the eluant. In such cases, when the polynucleotide is eluted from the reverse phase chromatography support, it is in a different salt form (i.e., a second polynucleotide salt) that when it was loaded because the at least one alent cation counterion is been exchanged away from the polynucleotide. In certain ces, the salt form of the polynucleotide that is eluted from the support in the third polynucleotide composition is more water soluble than the first polynucleotide salt including at least one multivalent cation counterion.
In n embodiments, the third polynucleotide composition includes a second polynucleotide salt that is a pharmaceutically acceptable salt of the polynucleotide. In certain instances, the third ition includes includes a second polynucleotide salt that is a monovalent cation salt of the polynucleotide. In certain cases, the third composition includes a second polynucleotide salt that is a triethylammonium salt of the polynucleotide. In n cases, the third composition includes a second cleotide salt that is a sodium salt of the polynucleotide. It is understood that after the polynucleotide is purified by reverse phase tography, any number of further cation counterion exchange steps may be performed on the polynucleotide salt to produce a desired salt form of the polynucleotide. In some embodiments, the method further includes ion ging cation rions from the second polynucleotide salt to produce a third polynucleotide salt. In certain ments, the third cleotide salt is a pharmaceutically acceptable salt of the polynucleotide. In certain instances, the third cleotide salt is a monovalent cation salt of the cleotide. In certain instances, the third polynucleotide salt is a sodium salt of the polynucleotide (e.g., as bed herein).
In certain instances, the first composition includes a monovalent cation salt of the polynucleotide. In certain cases, the monovalent cation salt is selected from the group consisting of sodium, ammonium and alkyl ammonium. In certain instances, the alkyl ammonium is selected from the group consisting of dimethylammonium, methylammonium, mmonium and triethylammonium. In certain cases, the first composition includes an ammonium salt of the polynucleotide. In certain cases, the first composition includes an alkyl um salt of the polynucleotide. In certain cases, the first composition includes a triethylammonium salt of the polynucleotide. In certain cases, the first composition includes a sodium salt of the polynucleotide. The first polynucleotide composition may be contacted with a multivalent cation salt to itate a first polynucleotide salt including at least one multivalent cation counterion. As such, in certain embodiments, the contacted first polynucleotide composition includes the first polynucleotide salt including at least one multivalent cation counterion.
Considered to be embraced within the scope of this invention are embodiments of any of the above-indicated embodiments of the method, where the polynucleotide is as described herein.
Methods of Synthesis Any convenient cleotide synthesis methods, strategies and tries may be utilized to prepare the crude synthetic product polynucleotide compositions which find use in the subject methods of preparation. Polynucleotide synthesis chemistries and methods of interest that may be adapted for use in the subject methods include, but are not limited to, phosphoramidite, H-phosphonate, phosphodiester, phosphotriester, ite triester. The polynucleotide components of the invention compounds may be synthesized by adapting any conventional protocols for the type of chemistry selected. Methods of interest for the synthesis of ucleotides having N3′→P5′ thiophosphoramidate chemistries include, but are not limited to, those methods described in U.S. 793, y et al., (1997) Tetrahedron Letters, 38:207-210; Pongracz & Gryaznov, (1999) Tetrahedron Letters, 1-7664; US 6,835,826, US 7,494,982, US 7,485,717 and US 143.
In some cases, a polynucleotide of interest is synthesized via sequential couplings starting from the 5’-terminal and proceeding to the 3’-terminal of the target polynucleotide sequence. In n cases, a cleotide of interest is synthesized via sequential couplings starting from the 3’-terminal and ding to the 5’-terminal of the target polynucleotide sequence. In some embodiments, the polynucleotide is sized by sequential couplings of monomer phosphoramidites to the growing terminal of the polynucleotide. The 5’-terminal nucleoside subunit may be attached to any convenient solid support via an optional linking group or 5’-terminal group. Once the first subunit is ed to the solid support, the subunit may be deprotected to produce a free, immobilized 3’-terminal group. Then, subunit couplings to the growing oligonucleotide chain may be achieved. In some instances, the method includes coupling a support bound 3’-terminal group with a tected-nucleotide-5'-phosphoramidite monomer. In certain embodiments, the 3’-terminal group is a 3’-hydroxyl group. In certain embodiments, the 3’-terminal group is a no group.
In some instances, the method of polynucleotide synthesis includes the steps of: (a) deprotecting the protected 3'-amino group of a terminal nucleoside attached to a solid phase support, the deprotecting forming a free 3'-amino group; (b) contacting the free 3'-amino group with a 3'-protected amino-nucleoside-5'-phosphoramidite monomer in the ce of a nucleophilic catalyst to form an internucleoside N3'→P5' oramidite linkage; and (c) oxidizing the linkage to produce a N3'→P5' thiophosphoramidate linkage. In some ments, the method includes (d) repeating steps (a) through (c) until the polynucleotide is synthesized.
In some cases, the method includes coupling a support bound 3’-terminal group with a 3’-protected-dinucleotide-5'-phosphoramidite dimer. Polynucleotide synthesis methods of interest include, but are not limited to, those methods of solid phase synthesis ing at least one coupling of a dinucleotide dimer as described in PCT Publication No. /168310 which application claims the benefit of U.S.
Provisional Application Serial No. 61/987,396. The target polynucleotide sequence may be synthesized via a retrosynthetic strategy that includes sequential couplings of both dimer and r subunits to the 3’terminal group of the growing ucleotide chain. In some embodiments, the polynucleotide is synthesized using a method including at least one coupling of a dinucleotide dimer to the free 3’ terminal group of a growing polynucleotide chain.
In some instances, the method of polynucleotide synthesis es the steps of: (a) deprotecting the protected 3'-amino group of a al nucleoside attached to a solid phase support, the deprotecting forming a free 3'-amino group; (b) contacting the free 3'-amino group with a 3'-protected amino-dinucleotide thiophosphoramidate or phosphoramidite-5'-phosphoramidite dimer in the presence of a nucleophilic catalyst to form an internucleoside N3'→P5' phosphoramidite linkage; and (c) oxidizing the linkage a N3'→P5' thiophosphoramidate linkage. In some embodiments, the method includes (d) repeating steps (a) through (c) until the polynucleotide is sized, where is step (b) a 3'-protected amino-dinucleotide thiophosphoramidate-5'-phosphoramidite dimer or 3'- protected amino-nucleotide-5'-phosphoramidite monomer may be utilized.
Any convenient protecting group strategies may be utilized in the t s to protect the base, phosphoramidite, oramidate, 5’, 2’ and/or 3’groups of the polynucleotide. Protecting groups of interest include, but are not limited to, those protecting groups described by Ohkubo et al., Org. Lett., 2010, 12 (11), pp 2496–2499; and ge and Iyer, Tetrahedron 48: 2223-2311 (1992).
As used herein, the term "phosphate protecting group" refers to a protecting group that may be attached to a phosphorus-containing intersubunit linkage of an oligonucleotide. When present, a phosphate protecting group may prevent (i.e., block) reaction of the phosphorus-containing linkage at the location where the phosphate ting group is attached. Any ient phosphorus-containing intersubunit linkages (e.g., P(III) and P(V) es) may be protected by the subject phosphate protecting groups, including, but not limited to, phosphoramidite, oxophosphoramidate, thiophosphoramidate, phosphate ester, thiophosphate ester, phosphodiester linkages and the like. The phosphate protecting group may be attached to an available oxygen atom of the phosphorus-containing intersubunit linkage. Any convenient protecting groups may be utilized as a phosphate protecting group. In certain ments, a phosphate protecting group is methyl, or β-cyanoethyl.
In some instances, the 3’-terminal group of the growing cleotide chain may include a 3’-hydroxyl, a 3’-amino group or a protected version thereof. Any convenient hydroxyl and/or amino protecting groups may be utilized at the 3’-terminal group during cleotide sis. In some embodiments, the 3’terminal group is a protected 3’-amino group and the method includes deprotecting or removing the protecting group to produce a free 3’amino group. As used herein, the term "free amino group" means an amino group available for reacting with the phosphoramidite group of an incoming monomer or dimer. In some embodiments, a free amino group is a primary amine. After the ection (e.g., detritylation) step, the amino group may be in the form of a salt (e.g., the salt of a conjugate base of the acid used for detritylation). This salt may be optionally neutralized with a basic solution such as 2% triethylamine or pyridine in acetonitrile after the detritylation step. 3’-Protection of the incoming subunit phosphoramidites prevents undesirable polymerization of the chain. In some embodiments, the 3’-terminal group is a protected 3’-hydroxyl group and the method includes ecting or removing the protecting group to e a free 3’-hydroxyl group. In some embodiments, the 3’-terminal group is a protected 3’-amino group and the method includes deprotecting or removing the protecting group to produce a free 3’-amino group. The protected 3’-amino or 3’- hydroxyl group may be protected with a trityl protecting group. In certain embodiments, the trityl protecting group is triphenylmethyl (Tr or Trt, Ph3C-). In certain embodiments, the trityl protecting group is imethoxytrityl (DMT). Deprotection of the 3’-terminal amino or hydroxyl group may be achieved using any convenient methods. Methods of interest e, but are not d to, those methods described by Beaucage and Iyer, Tetrahedron 48: 2223-2311 (1992). In some cases, deprotection of the protected 3' amino group of a terminal nucleoside includes detritylation to produce a free 3’terminal group, e.g., acid-catalyzed ylation. In some cases, the dimer or monomer subunit phosphoramidites include a protected 3’-hydroxyl or 3’-amino group that is the same as the minal group of the terminal nucleoside attached to the solid t.
Any convenient solid phase supports may be used for the synthesis of polynucleotides according to the subject methods. Solid supports of st include, but are not limited to, microparticles made of controlled pore glass (CPG), highly cross- linked polystyrene (e.g., NittoPhase HL 400 or GE Primer 350), acrylic copolymers, cellulose, nylon, dextran, latex, polyacrolein, and the like, such as those disclosed in the ing exemplary references: Meth. Enzymol., Section A, pages11-147, vol.44 (Academic Press, New York, 1976); U.S. Pat. Nos. 4,678,814; 4,413,070; and 4,046;720; and Pon, Chapter 19, in Agrawal, , Methods in Molecular Biology, Vol.20, (Humana Press, Totowa, N.J., 1993). Further supports of interest include polystyrene beads; yrene d with polyethylene glycol (e.g., TentaGel™, Rapp Polymere, Tubingen Germany); and the like. Selection of the support characteristics, such as material, porosity, size, shape, and the like, and the type of linking moiety employed depends on a variety of factors, such as protection groups employed, length of final product, quantity of final product, and the like. Exemplary linking moieties are disclosed in Pon et al., Biotechniques, 6:768-775 (1988); Webb , U.S. Pat. No. 4,659,774; Barany et al., International patent application PCT/US91/06103; Brown et al., J. Chem. Soc.
Commun., 1989: 891-893; Damha et al., Nucleic Acids Research, 18: 3813-3821(1990); Beattie et al., Clinical Chemistry, 39: 719-722 (1993); Maskos and Southern, Nucleic Acids Research, 20: 1679-1684 (1992); and the like.
In some embodiments, the solid supports that find use in the subject methods include CPG and polystyrene grafted with polyethylene glycol and possessing a terminal amino group (e.g., TentaGel-NH2 ™, Rapp Polymere, Tubingen Germany). The aminopropyl group may be used as a spacer between CPG and the side linkage. In some cases, the linkage to the roxyl of the first nucleoside is a succinyl group which es a base-labile ester linkage that may be cleaved after sis with aqueous ammonia.
Following deprotection, the support-bound nucleoside is e of reacting with a dimer or monomer subunit phosphoramidite to form an internucleoside linkage. It is understood that the support-bound nucleoside may refer to a single e ed to a solid support or may refer to the terminal residue of an oligonucleotide chain that is attached to the support. Any convenient coupling chemistry, coupling reagents and methods may be utilized in the t methods. Any convenient selections ning coupling conditions, protecting , solid phase supports, linking groups, deprotection reagents, reagents to cleave products from solid phase supports, purification of product, and the like, may be made in the context of the subject methods according to the guidance of, e.g. Gait, editor, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984); Amarnath and Broom, Chemical Reviews, Vol. 77, pgs. 183-217 (1977); Pon et al., Biotechniques, Vol. 6, pgs. 5 (1988); Ohtsuka et al., Nucleic Acids Research, Vol. 10, pgs. 6553-6570 (1982); Eckstein, editor ucleotides. and Analogues: A Practical Approach (IRL Press, Oxford, 1991), Greene and Wuts "Protective Groups in c Synthesis", Third edition, Wiley, New York 1999, Narang, editor, Synthesis and Applications of DNA and RNA (Academic Press, New York, 1987), Beaucage and Iyer, Tetrahedron 48: 2223-2311 (1992), and like references.
In some ces, after ng, unreacted 3'-amino groups of a supportbound growing chain of the polynucleotide may be optionally capped with a convenient capping agent before the next deprotection step (e.g., detritylation step) to render them inert to uent coupling steps. This capping step may e the HPLC profile of the preparation to make purification more facile, and may also improve the overall yield of product. Capping reagents useful in the subject s include electrophilic reagents such as acetic anhydride and isobutyric anhydride, acid chlorides such as tyl carbonyl chloride, pivaoyl chloride, and the like, isothiocyanates, chloroformates, etc.
Also useful are oramidites in ction with an activator and followed by oxidation, and H-phosphonate salts such as triethylammonium isopropyl-H-phosphonate used in ction with an acid chloride such as pivaoyl chloride or adamantyl carbonyl chloride.
In some embodiments, the method includes ing an internucleoside N3'→P5' phosphoramidite linkage. As used herein, the terms "oxidize," "oxidation," "oxidizing", and the like, in reference to a phosphorus-containing internucleosidic linkage means a process or treatment for converting the phosphorus atom of the linkage from a phosphorus (III) form to a phosphorus (V) form. ion of the internucleotide linkages may be performed at any convenient point in the synthesis using any convenient methods. In some embodiments, oxidation is performed in a stepwise , e.g., during every coupling cycle. In other embodiments, oxidation of multiple internucleotide linkages is performed at the end of the synthesis. In some instances, oxidizing a N3'→P5' phosphoramidite linkage (e.g., using an iodine/water based oxidizing agent) produces an osphoramidate linkage. In other instances, oxidizing a N3'→P5' phosphoramidite linkage includes sulfurization to produce a N3'→P5' thiophosphoramidate linkage.
Sulfurization may be performed using any convenient s. Sulfurization methods of interest include those described by nov et al. in WO2001018015 and US6,114,519. Sulfurizing agents of interest include, but are not limited to, elemental sulfur, thiuram disulfides such as tetraethyl thiuram disulfide, acyl disulfides such as phenacyldisulfide, phenyl acetyl disulfide, phosphinothioyl disulfides such as S-Tetra™, and 1,1-dioxo-3H-1,2-benzodithiolone. In some embodiments, sulfurization may be performed using phenyl acetyl disulfide in tidine. In certain embodiments, sulfurization may be performed using Beaucage reagent, using methods as described by Iyer et al., J. Organic Chemistry 55:4693-4699, 1990.
Cleavage of the polynucleotide from the solid phase sis support may be achieved using any convenient methods and reagents, which may be selected depending on a variety of factors, such as the nature of the support, linker chemistry and the protecting group strategy utilized during synthesis. The selections made in the sis and cleavage of a target polynucleotide may determine the identities of the get synthesis ts and agent present in the first polynucleotide composition.
In some embodiments, prior to cleavage, the phosphorus protecting groups of the polynucleotide are d to avoid the formation of any potential rable adducts of the cleaved protecting group (e.g., the -cyanoethyl protecting group) with the polynucleotide. Methods of interest that may be adapted for use in deprotecting and cleaving polynucleotides include those described in 9,236. In some embodiments, the polynucleotide is cleaved from the support using an ammonia solution to remove any base protecting groups (e.g., exocyclic amino protecting groups) and any remaining orus protecting groups. Any convenient ions may be utilized in the polynucleotide ge reaction. In some cases, the cleavage is performed at a temperature in the range of 40-60oC. In some instances, the cleavage is performed over an extended period of time, such as a time in the range of 12-24 hours. Post ge of the polynucleotide, the t may then be removed by filtration and rinsed. The combined filtrate and rinse solutions, which now contain the crude synthetic preparation of polynucleotide, may be utilized in the subject methods of preparation, before being carried forward to further purification steps. In some cases, purification of a polynucleotide solution includes preparative Reversed High Performance Liquid Chromatography LC) RP HPLC, e.g., using Kromasil C18 at 45-55oC. In some instances, the polynucleotide compositions of the subject methods may undergo any number of convenient desalting and concentration steps, e.g., by using a Tangential Flow Filtration (TFF) apparatus equipped with polyethersulfone membranes with a pore diameter cut-off size of 1,000 Da.
POLYNUCLEOTIDE COMPOSITIONS ] Aspects of the t disclosure include polynucleotide salt compositions including multivalent cation counterions. In some embodiments, the composition includes: a salt of a polynucleotide including at least one multivalent cation counterion, where the cleotide has a sequence of 7 or more nucleoside subunits and at least two of the side subunits are joined by a N3′→P5′ thiophosphoramidate inter- subunit linkage. In certain ments, the polynucleotide has a sequence of 7 or more nucleoside subunits mentary to the RNA component of human telomerase.
Multivalent cation counterions Any convenient multivalent cations may find use as a counterion in the subject polynucleotide salts. As such, a multivalent cation may form an ion pair with an anionic site on a polynucleotide backbone in the subject polynucleotide itions.
Polynucleotides may include nucleoside subunits linked by phosphorus-containing intersubunit linkages (e.g., P(V) linkages) such as phosphoramidate, thiophosphoramidate, phosphate ester, odiester linkages and the like. It is understood that the intersubunit linkages of the polynucleotide may be negatively charged (e.g., in an aqueous solution) and ion paired with a cationic counterion. Such ubunit linkages may be referred to as anionic groups of the cleotide backbone.
As used herein, the term alent cation refers to a cation capable of forming multiple ion pairs, e.g., a multiply charged cation, such as a double charged or a triply charged cation. Any convenient multivalent cations may find use in the subject cleotide salt compositions. In some embodiments, a multivalent cation ion pairs to two or more adjacent anionic groups of the polynucleotide backbone. In some embodiments, a alent cation ion pairs to one anionic group of the cleotide ne. In some embodiments, the multivalent cation counterion is divalent. Divalent cation counterions of interest include, but are not limited to, magnesium, zinc and calcium. In some ments, the multivalent cation counterion is trivalent. Trivalent cation counterions of interest include, but are not limited to, aluminium. In certain embodiments of the composition, the at least one multivalent cation counterion is selected from the group consisting of magnesium, zinc, aluminium and calcium. In certain embodiments of the ition, the at least one multivalent cation counterion is magnesium. In certain embodiments of the composition, the at least one multivalent cation counterion is zinc. In certain embodiments of the composition, the at least one multivalent cation rion is ium. In certain embodiments of the composition, the at least one multivalent cation counterion is calcium.
It is understood that the number of cation counterions that are t in a polynucleotide salt is dependent on a variety of factors, such as the length of the polyanionic backbone, the valency of the cations in the salts, the pH of the solution, aggregation of polynucleotides in the composition, etc. The subject compositions may include at least one multivalent cation counterion to the polyanionic polynucleotide backbone in the subject polynucleotide compositions, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, 100 or more, or even more multivalent cation rions. In certain embodiments, a polynucleotide having n side subunits may include between 1 and (n-1)/2 (if n is an odd integer) divalent cation counterion(s) or between 1 and (n-2)/2 (if n is an even integer) divalent cation counterion(s). In some instances, a polynucleotide salt that includes at least one multivalent cation, may further include a variety of other cation counterions, which may be monovalent, divalent or trivalent. In certain ces, n is in the range of 7 to 50, such as 7 to 40, 10 to 40, 10 to 30, 10 to 25, 10 to 20, or in the range of 12 to 15 nucleoside subunits.
In some embodiments of the composition, the polynucleotide salt may include 3 mol% or more of the multivalent cation counterion relative to a polyanionic backbone of the polynucleotide (i.e., relative to a theoretical maximum inclusion of cation counterions along the polyanionic backbone), such as 4 mol% or more, 5 mol% or more, 6 mol% or more, 7 mol% or more, 8 mol% or more, 9 mol% or more, 10 mol% or more, 11 mol% or more, 12 mol% or more, 13 mol% or more, 14 mol% or more, 15 mol% or more, 16 mol% or more, 17 mol% or more, 18 mol% or more, 19 mol% or more, 20 mol% or more, 25 mol% or more, 30 mol% or more, 35 mol% or more, 40 mol% or more, 45 mol% or more, 50 mol% or more, 55 mol% or more, 60 mol% or more, or even more, of the multivalent cation rion relative to a ionic backbone of the polynucleotide. In some embodiments of the subject itions, the cleotide may include 10 mol% or more of the multivalent cation counterion relative to a polyanionic backbone of the polynucleotide. For e, a polynucleotide salt that includes a ionic backbone of 10 internucleoside subunit linkages and includes one divalent cation counterion ion pairing to two of the linkages, is described as including 20 mol% of the divalent cation counterion. If the one divalent cation counterion ion pairs to only one of the linkages instead of two, the polynucleotide salt is described as including mol% of the divalent cation counterion. As such, the mol% value refers to a level of occupation of the polyanionic polynucleotide backbone by the multivalent cation counterions that are present in the polynucleotide salt. For example, one Mg2+ cation in a 13-mer polynucleotide salt having 12 internucleoside subunit linkages gives 16.7 mol% tion of the backbone. It is understood that in some embodiments, the polynucleotide salt may include additional ion g sites at the terminals of the polynucleotide (e.g., a 5’-thiophosphate group), and if present, such sites should be included in the mol% value of the compound.
In some embodiments of the composition, the polynucleotide salt includes 90 mol% or less of the multivalent cation counterion relative to a polyanionic backbone of the polynucleotide, such as 70 mol% or less, 65 mol% or less, 60 mol% or less, 50 mol% or less, or even less of the multivalent cation counterion.
In n embodiments of the composition, the polynucleotide salt includes 3 to 90 mol% of the multivalent cation counterion relative to a polyanionic backbone of the polynucleotide, such as 3 to 65 mol% (e.g., 6 to 50 mol%, 10 to 50 mol% or 10 to 40 mol%), 3 to 50 mol%, 3 to 40 mol%, 3 to 30 mol%, 3 to 20 mol% or 3 to 15 mol% of the multivalent cation rion ve to a polyanionic backbone of the polynucleotide.
In certain instances of the composition, the polynucleotide salt includes 3 to 60 mol% of a divalent cation counterion relative to a polyanionic backbone of the polynucleotide, such as 3 to 50 mol% (e.g., 5 to 50 mol%), 3 to 40 mol%, 3 to 30 mol%, 3 to 20 mol%, 3 to 15 mol%, such as 3-12 mol% of a divalent cation counterion, In certain instances of the composition, the polynucleotide salt includes 3 to 60 mol% of a magnesium cation counterion relative to a polyanionic backbone of the polynucleotide, such as magnesium, 5-50 mol%, 5-40 mol%, 10-40 mol% or 20-40 mol% of a magnesium cation counterion.
In certain instances of the composition, the polynucleotide salt includes 10 to 70 mol% of a trivalent cation counterion relative to a ionic backbone of the polynucleotide, such as 10 to 60 mol%, 20 to 60 mol%, 20 to 50 mol% or 30 to 50 mol% of a trivalent cation counterion.In some embodiments of the composition, the polynucleotide salt includes 0.5 % or more by weight of the multivalent cation counterion (e.g., magnesium), such as 0.6 % or more, 0.7 % or more, 0.8 % or more, 0.9 % or more, 1.1 % or more, 1.2 % or more, 1.3 % or more, 1.4 % or more, 1.5 % or more, 1.6 % or more, 1.7 % or more, 1.8 % or more, 1.9 % or more, 2.0 % or more, 2.1 % or more, 2.2 % or more, 2.3 % or more 2.4 % or more, 2.5 % or more, 2.6 % or more, 2.7 % or more, 2.8 % or more, 2.9 % or more, 3.0 % or more by weight of the multivalent cation counterion.
The polynucleotide salt is a mixed salt that includes a mixture of multivalent and monovalent cation counterions. In n embodiments of the composition, the polynucleotide salt includes a ratio of alent cation counterion to monovalent cation counterion of at least 0.05 or more by molarity, such as 0.10 or more, 0.15 or more, 0.20 or more, 0.25 or more, 0.30 or more, 0.35 or more, 0.40 or more, 0.45 or more, 0.50 or more, 0.55 or more, 0.60 or more, 0.65 or more, 0.70 or more by molarity, or even more of multivalent cation counterion to monovalent cation counterion.
] In some instances, the polynucleotide salt includes a ratio of multivalent to monovalent cation counterion of 1:12 by ty. In some instances, the cleotide salt includes a ratio of multivalent to monovalent cation counterion of 1:11 by molarity.
In some instances, the polynucleotide salt includes a ratio of multivalent to monovalent cation counterion of 1:10 by molarity. In some instances, the polynucleotide salt includes a ratio of multivalent to monovalent cation counterion of 1:9 by molarity. In some ces, the polynucleotide salt includes a ratio of multivalent to monovalent cation counterion of 1:8 by molarity. In some instances, the polynucleotide salt es a ratio of multivalent to lent cation counterion of 1:7 by molarity. In some ces, the polynucleotide salt includes a ratio of multivalent to monovalent cation counterion of 1:6 by molarity. In some instances, the polynucleotide salt includes a ratio of multivalent to monovalent cation counterion of 1:5 by molarity. In some instances, the polynucleotide salt includes a ratio of multivalent to monovalent cation counterion of 1:4 by molarity. In some instances, the polynucleotide salt es a ratio of multivalent to monovalent cation counterion of 2:9 by molarity. In some instances, the polynucleotide salt es a ratio of multivalent to monovalent cation counterion of 3:7 by ty. In some instances, the polynucleotide salt includes a ratio of multivalent to monovalent cation counterion of 4:5 by molarity. In some instances, the polynucleotide salt includes a ratio of multivalent to monovalent cation counterion of 5:3 by molarity.
In certain instances of the mixed polynucleotide salt, the alent cation counterion is magnesium and the monovalent cation counterion is sodium. In certain instances of the mixed polynucleotide salt, the multivalent cation counterion is magnesium and the monovalent cation counterion is ammonium. In certain instances of the mixed polynucleotide salt, the multivalent cation rion is magnesium and the monovalent cation counterion is triethylammonium. In certain instances of the mixed cleotide salt, the multivalent cation rion is aluminium. In certain instances of the mixed cleotide salt, the multivalent cation counterion is zinc. In certain instances of the mixed polynucleotide salt, the multivalent cation counterion is calcium.
In certain instances of the mixed polynucleotide salt, the monovalent cation counterion is sodium. In certain ces of the mixed polynucleotide salt, the monovalent cation counterion is ammonium. In n instances of the mixed polynucleotide salt, the monovalent cation counterion is triethylammonium.In certain embodiments, the polynucleotide salt es one multivalent cation counterion. In certain embodiments, the polynucleotide salt includes 2 multivalent cation counterions. In certain embodiments, the polynucleotide salt includes 3 multivalent cation counterions. In certain embodiments, the polynucleotide salt includes 4 multivalent cation counterions. In certain embodiments, the cleotide salt includes 5 multivalent cation counterions. In certain embodiments, the polynucleotide salt includes 6 multivalent cation counterions. In certain embodiments, the polynucleotide salt includes 7 multivalent cation counterions. In certain embodiments, the cleotide salt includes 8 multivalent cation counterions. In certain embodiments, the polynucleotide salt includes 9 multivalent cation counterions. In certain embodiments, the polynucleotide salt includes 10 multivalent cation counterions.
In on to a target polynucleotide, a variety of non-target polynucleotide synthesis ts may be produced during cleotide synthesis. Minor products that may be present in polynucleotide preparations include, but are not limited to, deletion products (e.g., products lacking one or more nucleoside es), products that include one or more protecting groups, terminated products (e.g., products that include a capped polynucleotide chain), products that lack one or more nucleobases, products that include partially oxidized phosphoramidite linkages and products that include lly ized linkages.
] The subject methods provide for compositions that include an improved purity of target polynucleotide in the composition. In some embodiments, the composition includes 20% or more by weight of the target polynucleotide, such as 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or even 95% or more by weight of the target polynucleotide. In certain embodiments, the composition includes 50% or more by weight of the target polynucleotide. In certain embodiments, the ition includes 55% or more by weight of the target polynucleotide. In certain embodiments, the composition includes 60% or more by weight of the target polynucleotide. In certain embodiments, the composition includes 65% or more by weight of the target polynucleotide. In certain embodiments, the composition includes 70% or more by weight of the target polynucleotide. In n embodiments, the composition includes 75% or more by weight of the target cleotide. In n ments, the composition includes 80% or more by weight of the target polynucleotide. In certain embodiments, the composition includes 85% or more by weight of the target polynucleotide. In certain embodiments, the composition es 90% or more by weight of the target cleotide. In certain ments, the ition includes 95% or more by weight of the target polynucleotide.
The subject methods e for compositions including a d amount of non-target synthesis products and agents. By reduced amount is meant that the amount by weight of the non-target synthesis products and agents in the composition is reduced relative to a control method. In some embodiments, the subject compositions include non-target synthesis products and agents in an amount of 50% or less of the total nontarget polynucleotides in the composition, such as 40% or less, 30% or less, 25% or less, % or less, 15% or less, 10% or less or even 5% or less of the non-target synthesis products and agents.
Any of a wide variety of polynucleotide compositions can be prepared using the methods described herein. A variety of classes and types of polynucleotides are of interest for preparation using the subject methods (e.g., as described herein).
Polynucleotides suitable for preparation according to the subject methods include, but are not limited to, anti-sense polynucleotides, RNA polynucleotides, siRNA polynucleotides, RNAi polynucleotides, DNA aptamers, micro RNA and the like.
In some embodiments, the polynucleotide is bed by Formula (I): Formula (I) wherein: each B is independently a , a ted purine, a pyrimidine or a protected pyrimidine, or an analog thereof; each X is independently oxygen or sulfur; each R3 is independently hydrogen, fluoro, hydroxyl, an alkoxy, a substituted alkoxy or a protected hydroxyl; R6 is amino, hydroxyl, a protected amino, a protected hydroxy, -O-T-Z or –NH-TZ each T is independently an optional linker; each Z is independently H, a lipid, a carrier, an oligonucleotide, a polymer, a polypeptide, a detectable label, or a tag; and n is an integer of 1 to 1000. It is understood that the oligonucleotides of Formula (I), may exist in a salt form. As such, the internucleoside linkages of Formula (I) may be in a salt form that includes any convenient counterion. Such forms are intended to be included within the scope of the t disclosure. It is understood that other tautomeric arrangements of the internucleoside linkages of the polynucleotide described in Formula (I) may be possible. Such forms are ed to be included within the scope of the present disclosure.
In some embodiments of Formula (I), each R3 is hydrogen. In some embodiments of a (I), each R3 is fluoro. In some embodiments of a (I), each R3 is hydroxyl. In some embodiments of a (I), R6 is amino. In certain embodiments of Formula (I), R6 is hydroxyl. In some embodiments of Formula (I), Z is H. In some ments of Formula (I), Z is a lipid (e.g., as described herein). In certain cases, the lipid is a fatty acid (e.g., as described herein). In some embodiments of Formula (I), Z is a carrier. In some embodiments of Formula (I), Z is an oligonucleotide.
In some embodiments of Formula (I), Z is a r. In certain cases, the polymer is a PEG. In some embodiments of Formula (I), Z is a polypeptide. In some embodiments of Formula (I), Z is a detectable label. In some embodiments of Formula (I), Z is a tag. In some embodiments of Formula (I), T is absent. In some embodiments, each B is independently selected from A, C, G, T and U.
] In certain embodiments of Formula (I), n is an integer of between 7 and 500, such as between 7 and 100, n 7 and 75, between 7 and 50, between 7 and 40, between 7 and 30, n 7 and 20, between 7 and 15, n 10 and 15, or n 13 and 15. In certain embodiments, n is an integer of between 7 and 100, such as between 7 and 50, n 10 and 50, between 10 and 40, between 10 and 30, between 10 and 25, between 10 and 20, between 12 and 18, or between 12 and 16. In certain embodiments, n is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24 or 25. cleotides complementary to RNA component of Telomerase s of the disclosure e compounds and compositions including polynucleotides complementary to the RNA component of human telomerase, and methods for preparing the same. The compounds may inhibit telomerase activity in cells with a high potency and have cellular uptake characteristics.
In certain instances, the polynucleotide includes a sequence of 7 or more nucleoside subunits complementary to the RNA component of human telomerase, such as 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 20 or more, 30 or more, 50 or more nucleoside subunits complementary to the RNA component of human telomerase.
In some embodiments, the polynucleotide includes between 3 and 50 contiguous nucleoside subunits complementary to the RNA ent of human telomerase, such as between 5 and 40, between 7 and 40, 10 and 40, between 10 and 30, between 10 and 25, between 10 and 20, or between 12 and 15 nucleoside subunits. In certain embodiments, the polynucleotide includes a ce of 7 or more uous nucleoside subunits complementary to the RNA component of human telomerase, such as or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 20 or more, 30 or more, 50 or more contiguous nucleoside subunits complementary to the RNA component of human telomerase.
In some embodiments, the polynucleotide is a compound described by the formula: O-(x-L)n where O represents the cleotide including a sequence of nucleoside subunits complementary to the RNA component of human telomerase, x is an al linker group, L represents a lipid moiety and n is an integer from 1-5. In some ces, n is 5. In some instances, n is 4. In some instances, n is 3. In some instances, n is 2. In some instances, n is 1. Design of the compounds therefore requires the selection of two entities, O and L, and the determination of the structural linkage(s) between these entities, which may involve the optional linker group x.
In some embodiments, the polynucleotide nd may be described by the formula: O-(x-L)n where O represents the polynucleotide including a ce of nucleoside subunits complementary to the RNA component of human rase, x is an optional linker group, L represents the lipid moiety and n is 1, such as a polynucleotide of a (I), or a salt thereof, wherein in a (I), Z is the lipid moiety, T is the optional linker (e.g., as described herein) and the B groups correspond to the sequence of nucleoside subunits complementary to the RNA component of human telomerase.
The polynucleotide component O may be regarded as the "effector" component of the compound in that it is this component that effects inhibition of the telomerase enzyme by binding to the RNA component of telomerase. Thus, the sequence of O is selected such that it includes a region that is mentary to the ce of the telomerase RNA, which is shown in SEQ ID NO:1. The region that is complementary to the telomerase RNA component may in theory be targeted to any portion of the rase RNA, but particular regions of the telomerase RNA are preferred targets for inhibitory polynucleotides. One red target region is the region spanning nucleotides -67 of SEQ ID NO:1, which includes the ate region," an 11 nucleotide region of sequence 5′-CUAACCCUAAC-3′ (SEQ ID NO: 21) that spans nucleotide 46-56 of SEQ ID NO: 1. The template region functions to specify the sequence of the telomeric repeats that telomerase adds to the chromosome ends and is essential to the activity of the telomerase enzyme (see Chen et al., Cell 3-514, 2000; Kim et al., Proc. Natl.
Acad. Sci., USA 98(14):7982-7987, 2001). Compounds of interest that contain a polynucleotide moiety including a sequence mentary to all or part of the template region are thus of interest. Another target region of interest is the region spanning nucleotides 137-179 of hTR (see Pruzan et al., Nucl. Acids Research, 30:559-588, 2002).
Within this , the sequence spanning 141-153 is a preferred target. PCT publication WO 98/28442 describes the use of polynucleotides of at least 7 nucleotides in length to inhibit telomerase, where the polynucleotides are designed to be complementary to accessible portions of the hTR sequence outside of the template region, including nucleotides 137-196, 290-319, and 350-380 of hTR.
The region of O that is targeted to the hTR sequence is in some cases exactly complementary to the corresponding hTR sequence. While ches may be tolerated in n instances, they are expected to decrease the specificity and activity of the resultant polynucleotide conjugate. In some embodiments, the base sequence of the polynucleotide O is thus selected to include a sequence of at least 5 nucleotides y complementary to the telomerase RNA, and enhanced telomerase inhibition may be obtained if increasing lengths of complementary sequence are employed, such as at least 6, at least 7, at least 8, at least 10, at least 12, at least 13 or at least 15 nucleotides exactly complementary to the telomerase RNA. In other ments, the sequence of the polynucleotide includes a sequence of from at least 7 to 20, from at least 8 to 20, from at least 10 to 20 or from at least 10 to 15 nucleotides exactly complementary to the telomerase RNA sequence. Optimal telomerase inhibitory activity may be obtained when the full length of the polynucleotide O is selected to be complementary to the telomerase RNA. However, it is not necessary that the full length of the polynucleotide component be exactly complementary to the target sequence, and the cleotide sequence may include regions that are not complementary to the target sequence. Such regions may be added, for example, to confer other ties on the compound, such as sequences that facilitate purification. If the polynucleotide component O is to include regions that are not complementary to the target sequence, such regions may be oned at one or both of the 5′ or 3′ termini. In instances where the region of exact complementarity is targeted to the te region, ive telomerase inhibition may be achieved with a short (5-8 nucleotide) region of exact complementarity to which a telomerase-like (G-rich) sequence is joined at the 5′ end.
Exemplary sequences that are complementary to the human telomerase RNA and which may be included as part of the polynucleotide ent O, or which may be used as the entire polynucleotide component O e the ing: hTR complementary sequences (regions of cleotide sequence SEQ ID NO:1 of U.S. Publication 2012329858); GGGUUGCGGA GGGUGGGCCU GGGAGGGGUG GUGGCCAUUU UUUGUCUAAC CCUAACUGAG AAGGGCGUAG GCGCCGUGCU UUUGCUCCCC GCGCGCUGUU UUUCUCGCUG ACUUUCAGCG GGCGGAAAAG CCUCGGCCUG CCGCCUUCCA CCGUUCAUUC UAGAGCAAAC AAAAAAUGUC AGCUGCUGGC GCCC CUCCCGGGGA CCUGCGGCGG GUCGCCUGCC CAGCCCCCGA ACCCCGCCUG GAGGCCGCGG UCGGCCCGGG GCUUCUCCGG AGGCACCCAC UGCCACCGCG AAGAGUUGGG CUCUGUCAGC CGCGGGUCUC GCGA GGGCGAGGUU CAGGCCUUUC AGGCCGCAGG AAGAGGAACG GUCC CCGCGCGCGG CGCGAUUCCC UGAGCUGUGG GACGUGCACC CAGGACUCGG CAUG C (SEQ ID NO: 1) GCTCTAGAATGAACGGTGGAAGGCGGCAGG 137-166 (SEQ ID NO: 2) GTGGAAGGCGGCAGG 137-151 (SEQ ID NO: 6) GGAAGGCGGCAGG 137-149 (SEQ ID NO: 7) GTGGAAGGCGGCA 139-151 (SEQ ID NO: 8) GTGGAAGGCGG 141-151 (SEQ ID NO: 9) CGGTGGAAGGCGG 141-153 (SEQ ID NO: 10) ACGGTGGAAGGCG 142-154 (SEQ ID NO: 11) AACGGTGGAAGGCGGC 143-155 (SEQ ID NO: 12) ATGAACGGTGGAAGGCGG 8 (SEQ ID NO: 13) ACATTTTTTGTTTGCTCTAG 160-179 (SEQ ID NO: 14) TAGGGTTAGACAA 42-54 (SEQ ID NO: 3) GTTAGGGTTAG 46-56 (SEQ ID NO: 4) GTTAGGGTTAGAC 44-56 (SEQ ID NO: 15) GTTAGGGTTAGACAA 42-56 (SEQ ID NO: 16) GGGTTAGAC 44-52 (SEQ ID NO: 19) CAGTTAGGG 50-58 (SEQ ID NO: 20) CCCTTCTCAGTT 54-65 (SEQ ID NO: 17) CGCCCTTCTCAG 56-67 (SEQ ID NO: 18) In some embodiments, the polynucleotide includes a sequence selected from the group ting of: GTTAGGGTTAG (SEQ ID NO:4); TAGGGTTAGACAA (SEQ ID NO:3); and CAGTTAGGGTTAG (SEQ ID NO:5).
The choice of the type of inter-nucleoside linkages used in the sis of the O component may be made from any of the available polynucleotide chemistries, including but not limited to, phosphodiester, phosphotriester, methylphosphonate, P3′→N5′ phosphoramidate, N3′→P5′ phosphoramidate, N3′→P5′ thiophosphoramidate, and phosphorothioate linkages. In some embodiments, the polynucleotide ent O has at least one N3′→P5′ thiophosphoramidate linkage. In certain embodiments, the side subunits complementary to the RNA component of human telomerase are all joined by N3′→P5′ thiophosphoramidate inter-subunit linkages. In certain cases, the N3′→P5′ thiophosphoramidate inter-subunit linkage has the following structure: 3′—NH—P(S)(OR)—O—5’ where R is hydrogen, or a salt thereof. It is understood that for any of the polynucleotide components O described herein that include such an inter-subunit linkage, such polynucleotide components O may also include any convenient salt forms of the e. As such, the inter-subunit linkage may be in a salt form that includes any convenient rion.
In some embodiments, at least two of the nucleoside subunits are joined by a N3′→P5′ thiophosphoramidate inter-subunit linkage, and the other inter-subunit linkages each independently are selected from N3′→P5′ osphoramidate and N3′→P5′ thiophosphoramidate inter-subunit es. In some embodiments, the nucleoside subunits are joined by subunit es each independently selected from N3′→P5′ oxo-phosphoramidate and N3′→P5′ thiophosphoramidate subunit linkages. In some embodiments, the nucleoside subunits are joined by inter-subunit linkages each independently ed from N3′→P5′ oxo-phosphoramidate and N3′→P5′ thiophosphoramidate inter-subunit linkages; provided that at least two of the nucleoside subunits are joined by a N3′→P5′ thiophosphoramidate inter-subunit linkage. In some embodiments, the nucleoside subunits are joined by are all joined by N3′→P5′ thiophosphoramidate inter-subunit linkages.
In some embodiments, the polynucleotide component O has the sequence TAGGGTTAGACAA (SEQ ID NO:3), and the nucleoside subunits are joined by inter- subunit linkages comprising at least one N3′→P5′ thiophosphoramidate linkage. In some embodiments, the polynucleotide component O has the sequence TAGGGTTAGACAA (SEQ ID NO:3), and the nucleoside subunits are joined by inter-subunit linkages comprising at least two N3′→P5′ thiophosphoramidate linkages. In some embodiments, the polynucleotide component O has the sequence TAGACAA (SEQ ID NO:3), and the nucleoside subunits are joined by inter-subunit linkages comprising at least three N3′→P5′ thiophosphoramidate es. In some ments, the polynucleotide component O has the sequence TAGGGTTAGACAA (SEQ ID NO:3), and the nucleoside ts are joined by inter-subunit linkages comprising at least four ′ thiophosphoramidate linkages. In some embodiments, the polynucleotide component O has the sequence TAGGGTTAGACAA (SEQ ID NO:3), and the nucleoside subunits are joined by inter-subunit linkages comprising at least five N3′→P5′ thiophosphoramidate linkages. In some embodiments, the polynucleotide ent O has the sequence TAGGGTTAGACAA (SEQ ID NO:3), and the nucleoside subunits are joined by subunit linkages comprising at least six N3′→P5′ thiophosphoramidate linkages. In some embodiments, the polynucleotide component O has the sequence TAGGGTTAGACAA (SEQ ID NO:3), and the nucleoside subunits are joined by ubunit linkages comprising at least seven N3′→P5′ thiophosphoramidate linkages. In some embodiments, the polynucleotide component O has the sequence TAGACAA (SEQ ID NO:3), and the nucleoside subunits are joined by intersubunit linkages comprising at least eight N3′→P5′ osphoramidate linkages. In some embodiments, the polynucleotide component O has the sequence TAGGGTTAGACAA (SEQ ID NO:3), and the nucleoside subunits are joined by intersubunit linkages comprising at least nine N3′→P5′ thiophosphoramidate linkages. In some embodiments, the polynucleotide component O has the sequence TAGGGTTAGACAA (SEQ ID NO:3), and the nucleoside subunits are joined by intersubunit linkages comprising at least ten N3′→P5′ thiophosphoramidate linkages. In some embodiments, the polynucleotide component O has the sequence TAGGGTTAGACAA (SEQ ID NO:3), and the nucleoside subunits are joined by inter-subunit linkages comprising at least eleven N3′→P5′ thiophosphoramidate linkages. In some embodiments, the polynucleotide component O has the sequence TAGGGTTAGACAA (SEQ ID NO:3), and the nucleoside subunits are joined by inter-subunit linkages each independently selected from N3′→P5′ oxo-phosphoramidate and N3′→P5′ thiophosphoramidate inter-subunit linkages. In some embodiments, the cleotide component O has the sequence TAGGGTTAGACAA (SEQ ID NO:3), and the nucleoside subunits are joined by inter-subunit linkages each independently selected from N3′→P5′ oxo-phosphoramidate and ′ thiophosphoramidate inter-subunit linkages; provided that at least two of the nucleoside subunits are joined by a ′ thiophosphoramidate inter-subunit e. In some embodiments, the polynucleotide component O has the sequence TAGGGTTAGACAA (SEQ ID NO:3), and the nucleoside subunits are all joined by N3′→P5′ thiophosphoramidate inter-subunit linkages.
In all ments hereinbefore and hereinafter, N3′→P5′ thiophosphoramidate inter-subunit linkages in particular are —NH—P(═O)(SH)—O— or a tautomer thereof, or a salt thereof; and N3′→P5′ oxo-phosphoramidate intersubunit linkages in particular are —NH—P(═O)(OH)—O— or a tautomer thereof, or a salt f. More in particular, in all embodiments hereinbefore and hereinafter, N3′→P5′ thiophosphoramidate inter-subunit linkages in particular are —NH— P(═O)(SH)—O— or a tautomer thereof, or a sodium salt thereof; and N3′→P5′ oxo- phosphoramidate inter-subunit linkages in ular are —NH—P(═O)(OH)—O— or a tautomer thereof, or a sodium salt thereof.
In one of the embodiments, the invention s to any one of the specific structures described herein wherein optionally one or more, in particular one, N3′→P5′ osphoramidate inter-subunit linkages are ed by N3′→P5′ oxophosphoramidate inter-subunit linkages. In one of the embodiments, the invention relates to any one of the specific structures described herein wherein one or more, in particular one, N3′→P5′ thiophosphoramidate inter-subunit es are replaced by N3′→P5′ oxophosphoramidate subunit linkages.
In some cases, the subject nds are more effective in producing telomerase tion in cells than corresponding polynucleotides that are not conjugated to lipid components. The lipid component L is believed to function to enhance cellular uptake of the compound, particularly in facilitating passage through the cellular membrane. While the ism by which this occurs has not been fully elucidated, one possibility is that the lipid component may facilitate binding of the compound to the cell membrane as either a single molecule, or an aggregate (micellar) form, with subsequent alization. However, understanding of the precise mechanism is not required for the subject compounds to be utilized.
The lipid component may be any lipid or lipid derivative that provides enhanced cellular uptake compared to the unmodified polynucleotide. Lipids of interest include, but are not limited to, hydrocarbons, fats (e.g., ides, fatty acids and fatty acid derivatives, such as fatty amides) and s. Where the lipid component is a hydrocarbon, the L component may be a substituted or unsubstituted cyclic hydrocarbon or an aliphatic straight chain or branched hydrocarbon, which may be ted or unsaturated. Examples include straight chain unbranched hydrocarbons that are fully saturated or polyunsaturated. The length of the hydrocarbon chain may vary from C2- C30, but optimal telomerase inhibition may be obtained with carbon chains that are C8- C22. Examples of saturated hydrocarbons (alkanes) of interest are listed below: Systematic name / Carbon chain Tetradecane C14H30 Pentadecane C15H32 Hexadecane C16H34 Heptadecane C17H36 Octadecane C18H38 Nonadecane C19H40 Eicosane C20H42 Mono- and poly-unsaturated forms (alkenes and es, such as alkadienes and alkatrienes) of arbons may also be selected, with compounds having one to three double bonds being of interest, gh compound having more double bonds may be employed. Alkynes (containing one or more triple bonds) and alkenynes e ) and double bond(s)) may also be utilized.
Substituted forms of hydrocarbons may be employed in the subject compounds, with substituent groups that are inert in vivo and in vitro being of interest. In some cases, the substituent is fluorine. Exemplary generic ures of polyfluorinated hydrocarbons include: CF3(CF2)n—(CH2)m- where m is at least 1, in some cases at least 2, and n is 1 to 30, such as fluorotridecane: CF3(CF2)9(CH2)3; and CH3(CH2)a(CF2)b(CH2)c- where a, b and c are ndently 1-30.
Other le lipid components of interest include, but are not limited to, simple fatty acids and fatty acid derivatives, glycerides and more complex lipids such as sterols, for example cholesterol. Fatty acids and their derivatives of interest may be fully saturated or mono- or poly-unsaturated. The length of the carbon chain may vary from , but optimal telomerase inhibition may be obtained with carbon chains that are . Examples of saturated fatty acids of interest are listed below: Systematic name /Trivial name / Carbon chain Tetradecanoic myristic 14:0 canoic palmitic 16:0 Octadecanoic stearic 18:0 Eicosanoic arachidic 20:0 ] Mono- and poly-unsaturated forms of fatty acids may also be employed, with compounds having one to three double bonds being of interest, although compounds having more double bonds may also be employed. Examples of common mono- and poly-unsaturated fatty acids of interest that may be employed include: Systematic name / l name / Carbon chain Cishexadecanoic palmitoleic 16:1(n-7) Cisoctadecanoic petroselinic 18:1 (n-12) Cisoctadecanoic oleic 18:1 (n-9) 9,12-octadecadienoic linoleic 18:2 (n-6) 6,9,12-octadecatrienoic gamma-linoleic 18:3 (n-6) -octadecatrienoic alpha-linoleic 18:3 (n-3) ,8,11,14-eicosatetraenoic arachidonic 20:4 (n-6) Fatty acids with one or more triple bonds in the carbon chain, as well as branched fatty acids may also be employed in the subject compounds. Substituted forms of fatty acids may be employed in the subject compounds. As with the hydrocarbon groups, substituent groups that are inert in vivo and in vitro are of interest, such as fluorine. ary generic structures of polyfluorinated derivatives of fatty acids suitable for use in the invention are: CF3(CF2)n—(CH2)mCO— where m is at least 1, preferably at least 2, and n is 1 to 30, and CH3(CH2)a(CF2)b(CH2)cCO— where a, b and c are independently 1-30.
In some cases, between one and five L components (n is 1, 2, 3, 4 or 5) are covalently linked to the O component, via an optionally linker. In some cases, one or two L components are utilized (n=1 or 2). Where more than one L component is linked to the O component, each L component is independently selected.
It will be appreciated that nds of the invention described as having a specified arbon as the L moiety and compounds described as having a ied fatty acid (with the same number of carbon atoms as the specified hydrocarbon) are closely related and differ in structure only in the nature of the bond that joins the L moiety to the polynucleotide, which in turn is a result of the synthesis ure used to produce the compound. For example, and as described in more detail below, when compounds are synthesized having the L moiety conjugated to the 3′-amino terminus of a polynucleotide (having phosphoramidate or thiophosphoramidate internucleoside linkages), the use of the aldehyde form of a fatty acid (a fatty aldehyde) as the starting material results in the formation of an amine linkage between the lipid chain and the polynucleotide, such that the lipid group appears as a hydrocarbon. In contrast, use of the ylic acid, acid anhydride or acid chloride forms of the same fatty acid results in the formation of an amide linkage, such that the lipid group appears as a fatty acid derivative, specifically in this instance a fatty amide (as noted in the definitions n above, for the sake of simplicity, the term "fatty acid" when describing the conjugated L group is used broadly herein to include fatty acid derivatives, including fatty amides). This is illustrated in the following schematics which depict the 3′-amino terminus of a phosphoramidate polynucleotide joined to a C14 lipid component. In tic A, L is tetradecanoic acid (myristic acid), in which the connection between L and O groups is an amide. In schematic B, L is ecane, and the connection between the L and O groups is an amine.
The linkage n the O and L components may be a direct linkage, or may be via an al linker moiety, e.g., x or optional linker T of Formula (I). The linker group may serve to facilitate the chemical synthesis of the compounds. Whether or not a linker group is used to mediate the conjugation of the O and L components, there are le sites on the polynucleotide ent O to which the L component(s) may be conveniently conjugated. Suitable linkage points include the 5′ and 3′ termini, one or more sugar rings, the internucleoside backbone and the nucleobases of the polynucleotide. In some cases, the L moiety is attached to the 3′ or 5′ terminus of the polynucleotide.
If the L component is to be attached to the 3′ terminus, the attachment may be directly to the 3′ substituent, which in the case of the preferred phosphoramidate and thiophosphoramidate polynucleotides is the 3′-amino group, and in other instances, such as conventional phosphodiester polynucleotides, is a 3-hydroxy group. Alternatively, the L moiety may be linked via a 3′-linked phosphate group, in which a hexadecane hydrocarbon is linked to the 3′ phosphate of a thiophosphoramidate polynucleotide through an O-alkyl linker. If the L moiety is to be linked to the 5′ terminus, it may be attached through a 5′-linked phosphate group. Attachment to a base on the O moiety may through any le atom, for example to the N2 amino group of guanosine. Where n>1 such that a plurality of lipid moieties is to be attached to the O component, the individually selected L components may be attached at any convenient site(s). For example, one L group may be attached to each us, various L groups may be ed to the bases, or two or more L groups may be attached at one terminus.
The optional linker component x may be used to join the O and L components of the compounds. It is understood that the optional linker (e.g., x, or T of a (I)) may be attached to the polynucleotide (e.g., O) through a terminal phosphate group, e.g., a 3’-linked or a 5’-linked phosphate group. If a linker is to be employed, it is incorporated into the synthesis procedures as bed herein. Examples of suitable linker groups include amino glycerol and O-alkyl glycerol-type linkers which respectively can be depicted by the generic structures: wherein R′ is H, OH, NH2 or SH; Y is O, S or NR; R is H, an alkyl or a substituted alkyl; and n and m are each independently integers between 1-18. es of suitable linkers of interest are the aminoglycerol linker in which R′ is OH, Y is O, and m and n are each 1: the bis-aminoglycerol linker, in which R′ is OH, Y is NH, and m and n are each 1: and the O-alkyl glycerol linker in which R is H: Exemplary lipid-modified polynucleotides that may be ed ing to the subject methods include those nds described in Figure 1 (e.g., Figures 1A- 1DD) of U.S. Application US20120329858 to Gryaznov et al. "Modified oligonucleotides for telomerase inhibition", the disclosure of which is herein incorporated by reference in its entirety.
In certain embodiments, the composition includes a compound described by the structure: H OH N O P O T O SH O P SH O A O P SH O [G n p sG n p sG n p sT n p sT n p sA n p sG n p sA n p sC n p sA n p s] A NH 2 or a salt thereof, where "nps" represents a thiophosphoramidate linkage (e.g., —NH— P(═O)(SH)—O— or a tautomer thereof, or a salt thereof), connecting the 3'-carbon of one nucleoside to the 5'-carbon of the adjacent nucleoside. It is understood that the compound described in the formula above may exist in a salt form. Such forms in so far as they may exist, are intended to be included within the scope of the present disclosure.
In certain embodiments, the composition es a pharmaceutically able salt of the compound. In certain instances, the composition includes a sodium salt of the compound. In certain embodiments, the composition includes a divalent cation salt of the compound, such as a magnesium salt of the compound. In certain embodiments, the composition includes a trivalent cation salt of the compound, such as an aluminium salt of the compound.
] In certain embodiments, the composition es a compound described by the following structure: (M x +) n where each Mx+ is independently hydrogen or any convenient counterion of a salt, each x is independently 1, 2 or 3 and n is an integer from 5 to 13. In some instances, n is 5, 6, 7, 8, 9, 10, 11, 12 or 13. In certain instances, each x is independently 1, 2 or 3 and n is an integer from 5 to 12. In certain instances, n is 13. In certain instances, each x is 1. In certain instances, each x is independently 1 or 2. In certain instances, each x is independently 1 or 3. In certain instances, each Mx+ is independently a cationic rion. In certain instances, each Mx+ is ndently a cationic counterion, each x is independently 1, 2 or 3 and n is an r from 5 to 12. In certain instances, each Mx+ is independently en or any convenient cationic counterion, each x is independently 1, 2 or 3 and n is an integer from 5 to 12. In certain ces, Mx+ is hydrogen. In some embodiments, (Mx+)n is (Mg2+)(M+)11. In some embodiments, (M x+) 2+) +) n is (Mg 2(M 9. In some embodiments, (Mx+)n is (Mg2+)2(M+)9. In some embodiments, (Mx+)3 is (Mg2+)3(M+)7. In some embodiments, (Mx+)n is (Mg2+)4(M+)5. In some embodiments, (Mx+)n is (Mg2+)5(M+)3. In some embodiments, (Mx+)n is (Mg2+)6(M+). In some embodiments, (Mx+)n is (Mg2+)(M+)12, where the Mg2+ counterion may form an additional ion pair to the anionic backbone of another oligonucleotide. In some embodiments, (Mx+)n is (Mg2+)2(M+)11, where the Mg2+ counterions may form two an additional ion pairs to the anionic backbone(s) of one or two other oligonucleotide(s). In certain instances, the M+ counterion of the mixed magnesium salt is sodium. In certain instances, the M+ counterion of the mixed ium salt is ammonium. In certain instances, the M+ counterion of the mixed ium salt is triethylammonium.
In certain embodiments, the composition includes a compound described by the following ure and may include any convenient ic counterions of a salt: In n embodiments, the composition includes a compound described by the structure: Lipid modified polynucleotides A variety of synthetic approaches can be used to conjugate a lipid moiety L to the cleotide, depending on the nature of the linkage selected, ing the approaches described in Mishra et al., (1995) Biochemica et Biophysica Acta, 1264:229- 237, Shea et al., (1990) Nucleic Acids Res. 18:3777-3783, and Rump et al., (1998) Bioconj. Chem. 9:341-349. The synthesis of compounds in which the lipid moiety is ated at the 5′ or 3′ us of the polynucleotide can be achieved through use of suitable functional groups at the appropriate terminus, in some cases an amino group or a yl group, which can be reacted with carboxylic acids, acid chlorides, anhydrides and active esters. Thiol groups may also be used as onal groups (see Kupihar et al., (2001) Bioorganic and Medicinal Chemistry 9:1241-1247). Both amino- and thiolmodifiers of different chain s are commercially available for polynucleotide synthesis. Polynucleotides having N3′→P5′thiophosphoramidate linkages n 3′- amino groups (rather than 3′-hydroxy found in most conventional polynucleotide chemistries), and hence these cleotides e a unique opportunity for conjugating lipid groups to the 3′-end of the polynucleotide.
Various approaches can be used to attach lipid groups to the termini of polynucleotides with the N3′→P5′ thiophosphoramidate chemistry (e.g., a palmitoylamidoO-(4,4’-dimethoxytrityl)O-succinyl ediol linker). For attachment to the 3′ terminus, the conjugated compounds can be synthesized by reacting the free 3′-amino group of the fully protected solid support bound polynucleotide with the corresponding acid anhydride followed by deprotection with ammonia and purification.
Alternatively, coupling of carboxylic acids of lipids to the free 3′-amino group of the support bound cleotide using coupling agents such as carbodiimides, HBTU (N,N,N′,N′-tetramethyl-O-(1H-benzotriazolyl)uronium hexafluorophosphate) or 2- chloromethylpyridinium iodide can be used to conjugate the lipid groups. These two methods form an amide bond between the lipid and the polynucleotide. Lipids may also be attached to the polynucleotide chain using a phosphoramidite derivative of the lipid coupled to the polynucleotides during chain elongation. This approach yields a phosphoramidate (e.g., osphoramidate) linkage connecting the lipid and the polynucleotide (exemplified by propyl-palmitoyl and 2-hydroxy-propyl-palmitoyl compounds). Still another approach involves reaction of the free 3′-amino group of the fully protected support bound polynucleotide with a suitable lipid aldehyde, followed by reduction with sodium cyanoborohydride, which produces an amine linkage.
For attachment to the 5′ terminus, the polynucleotide can be synthesized using a modified, lipid-containing solid support, ed by synthesis of the polynucleotide in the 5’ to 3’ direction as described in Pongracz & Gryaznov (1999). An example of the modified support is provided below. In the instance where n=14, the fatty acid is palmitic acid: reaction of 3-amino-1, 2-propanediol with palmitoyl chloride, ed by dimethoxytritylation and succinylation provided the intermediate used for coupling to the solid support. In some instances, R may be long chain alkyl amine controlled pore glass.
In n instances, R is a polymeric solid support.
UTILITY The methods and compositions of the invention, e.g., as described above, find use in a variety of applications. Applications of interest include, but are not d to: therapeutic applications, diagnostic ations, research applications, and screening applications, as reviewed in greater detail below.
The subject compounds find use in a variety of therapeutic applications. In some embodiments, the methods of producing a cleotide are applied to prepare polynucleotides that provide for a therapeutic benefit. The types of diseases which are treatable using the itions of the present invention are limitless. For example, the compositions may be used for treatment of a number of genetic diseases. In some embodiments, the subject methods and compositions have antisense applications. In some embodiments, the subject methods and compositions have antigene ations. In n ments, the subject methods and itions have telomerase inhibition applications, such as those described in U.S. Patent 6,835,826, and U.S. Publication 20120329858, the disclosures of which are herein incorporated by reference in their entirety.
The present disclosure provides compounds that can specifically and potently inhibit telomerase activity, and which may therefore be used to inhibit the proliferation of telomerase-positive cells, such as tumor cells. A very wide y of cancer cells have been shown to be telomerase-positive, including cells from cancer of the skin, connective tissue, adipose, breast, lung, stomach, pancreas, ovary, cervix, uterus, kidney, bladder, colon, prostate, l nervous system (CNS), retina and hematologic tumors (such as myeloma, leukemia and lymphoma). Cancers of interest include, but are not limited to, myelofibrosis, thrombocythemia, myelodysplasic syndrome and myelogenous leukemia.
] The subject compounds can be used to treat logic malignancies and myeloproliferative disorders, including but not limited to, ial thrombocythemia (ET), polycythemia vera (PV) chronic myelogenous leukemia (CML), myelofibrosis (MF), chronic philic leukemia, chronic eosinophilic leukemia, and acute enous leukemia (AML). The subject compounds can be used to treat myelodysplastic mes, which e such disease as refractory anemia, refractory anemia with excess blasts, refractory cytopenia with multilineage sia, tory cytopenia with unilineage dysplasia, and chronic myelomonocytic leukemia (CMML).
The subject compounds can be used to treat hematological diseases, such as those described in PCT patent application No. PCT/US13/070437 filed November 15, 2013, the disclosure of which is incorporated herein by nce in its entirety.
Accordingly, the compounds provided herein are broadly useful in treating a wide range of malignancies. In some instances, the subject compounds can be effective in providing treatments that discriminate between malignant and normal cells to a high degree, avoiding many of the deleterious ffects present with most current herapeutic regimens which rely on agents that kill dividing cells indiscriminately.
Moreover, in some cases, the subject lipid modified compounds are more potent than equivalent unconjugated oligonucleotides, which means that they can be administered at lower doses, providing enhanced safety and significant reductions in cost of ent. rase inhibitors may be employed in conjunction with other cancer treatment approaches, including surgical removal of primary tumors, chemotherapeutic agents and radiation treatment. Hence, the invention relates to compounds and compositions provided herein for use as a medicament. The invention also relates to compounds and compositions provided herein for use in treating or ting any one of the malignancies mentioned hereinbefore.
The subject compounds and methods find use in a variety of diagnostic applications, including but not limited to, the development of clinical diagnostics, e.g., in vitro stics or in vivo tumor imaging agents. Such applications are useful in diagnosing or confirming diagnosis of a disease condition, or susceptibility thereto. The methods are also useful for monitoring disease progression and/or response to treatment in patients who have been previously diagnosed with the disease.
EXAMPLES ] Example 1: Summary These examples describe experiments to prepare various divalent or trivalent forms of Imetelstat, such as Ca, Ba, Mg, Al, Fe, Cu, and Zn from the sodium salt form of Imetelstat. In these experiments, improvements in purity using methods of preparation involving the formation and isolation of salts of the bi-dentate or ntate cations that can bind with one, two or three phosphate groups of Imetelstat were ted. The solubility and osmolality of ing salt forms were also studied.
The preparation of Imetelstat Calcium, Imetelstat Barium, Imetelstat Magnesium, stat Aluminum, Fe (II or III) Imetelstat, and Cupric Imetelstat salts were investigated using CaCl2, MgCl2, BaCl2, CuCl2, ZnCl2, AlCl3, FeCl2, and FeCl3.
Three s for salt exchange were studied: use of a strong cationexchange resin (FINEX MFG 210), precipitation, and simple dissolution. When the Imetelstat Sodium solution was passed through a resin exchanged with CaCl2, BaCl2 or MgCl2, the eluate ons contained fine powders, indicating that sodium counterions were sfully exchanged from the Imetelstat backbone and replaced with calcium, barium or magnesium counterions. For the other five reagents (CuCl2, ZnCl2, AlCl3, FeCl2, FeCl3) which were equilibrated with the cation exchange resin, the top part of resin in the column became aggregated when Imetelstat on was passed through, also ting that sodium counterions were successfully exchanged from the Imetelstat backbone.
Precipitation and dissolution methods were also tested using an excess of salt ts. When a large excess of salt reagent (e.g. 900 lents) was treated with Imetelstat Sodium, a precipitate was formed. The precipitates were isolated by filtration. uent tests indicate that seven to fifty equivalents of nic salt reagents were necessary to convert all of the Imetelstat to a precipitate.
Five equivalents of the three inorganic salts (Mg, Ba or Ca) were each treated with either Imetelstat TEA hylammonium) salt form or Imetelstat Na salt form. It was confirmed that precipitation did not occur and the solutions were desalted and freezedried.
The analysis of freeze-dried powder by Flame AA (Atomic Absorption) showed that some of the sodium counterions of stat were exchanged.
An additional experiment was performed with MgCl2 using one to nine equivalents of magnesium cation to the Imetelstat form. The sodium counterions were partially exchanged to Mg counterions with the highest exchange occurring at nine equivalents of MgCl2, with the resulting compositions showing 1.2% by weight of Na and 1.1% by weight of Mg.
Example 2: Materials and Equipments The inorganic reagents, organic solvents, and other materials used for the study are listed in Table 1. Imetelstat Sodium (CAS #10073805) of Lot # of G163/LG-13002 ed by Geron was used for the study. stat ammonium is a crude composition derived from cleavage of Imetelstat from a solid phase synthesis support using ammonia and ethanol (e.g., as described by Gryaznov et al. in US 29858) and was obtained from the manufacturer’s stock. Imetelstat TEA (triethylammonium form) is a composition derived from an HPLC purification column eluate where a triethylammonium acetate (TEAA) containing mobile phase is used (e.g., as described by ov et al. in US 20120329858) and was obtained from the manufacturer’s stock obtained from various process development studies. The ultrafiltration was performed using a Stirred iltration Cell (Amicon 8400, ore) with 1KD PES membranes.
The lyophilization was conducted using a Speed Vacuum Concentrator (ScanSpeed 40, LaboGene).
] Example 3: Procedure Exchange By Ion-Exchange Resin Column A column of strong cation exchange resin, FINEX MFG 210, was prepared having a column volume of 200 mL (4.6 cm x 12 cm) and the resin was washed with 1M NaOH and water. The column was then equilibrated with a 1M solution of each salt of interest.
In total, eight 1M salt solutions were prepared and used , MgCl2, BaCl2, CuCl2, ZnCl2, AlCl2, FeCl2, and FeCl3) in these experiments. A 50 mL solution of Imetelstat sodium at 100 mg/mL was added to the column.
In case of the CuCl2, ZnCl2, AlCl2, FeCl2, and FeCl3 equilibrated s, aggregation of Imetelstat on the resin was observed in the top part of column when Imetelstat sodium was loaded onto the .
The three s equilibrated with CaCl2, MgCl2 and BaCl2, salt solutions did not result in any Imetelstat aggregation on the column and Imetelstat was recovered from the column , which were observed as cloudy solutions. Fine powders were recovered from these s by fugation (4000 rpm, 20 min). After centrifugation, it was confirmed that the supernatant did not contain any Imetelstat by HPLC analysis.
This indicates that the precipitation and separation of calcium, magnesium and barium salts of Imetelstat was successfully achieved.
By Precipitation The llization or precipitation of divalent or trivalent forms of Imetelstat was igated using a large excess of inorganic salts of interest (900 equivalents, weight base). 1M salt solutions CaCl2, MgCl2, BaCl2, CuCl2, ZnCl2, AlCl2, FeCl2, and FeCl3 were prepared. Three types of Imetelstat solution: crude Imetelstat solution (ammonium salt), purified Imetelstat (triethylammonium (TEA) salt form), and Imetelstat sodium (Na salt form), were mixed with each salt solution.
All mixed solutions showed precipitates of Imetelstat, which were isolated easily by filtration with an Advantec 2 filter paper. This result indicates that the precipitation and separation of multivalent salts of Imetelstat was successfully achieved.
The solubilities of the precipitates isolated under the conditions of large excess of salt regent were initially investigated using the following solvents: water, acetonitrile, MeOH, EtOH, IPA (isopropyl alcohol), 0.1M NaOH, 0.1M HCl, 1M NaCl, and NMP.
For salts precipitated in a large excess of salt t, calcium, barium, and magnesium salts of Imetelstat were soluble in a 0.1M NaOH and 1M NaCl solution. (see Table 2). Solubility studies of Imetelstat precipitate obtained from a large excess of magnesium salt reagent were conducted in 1M NaCl solutions at different concentrations (2mg/mL to 6 mg/mL) and under different pH conditions (pH 8, 9, 10, 11, 12) and analyzed by HPLC (see tograms of Figure 1). The Imetelstat precipitate was observed to be soluble at 6 mg/mL and pH 11 to pH 12. The compound also showed stability up to pH 12 t any precipitates (Figure 1).
By Dissolution 30 to 50 equivalents salt reagent The number of equivalents of salt reagents of interest that could achieve a complete precipitation of Imetelstat was igated by adding the salt reagent of st step by step. The complete formation of precipitate was observed in the range of 7 to 50 lents of added salt reagent for the eight salts listed in Table 3. As more equivalents of salt reagents were added, a trend towards gel formation with precipitation was observed for all salts.
Three types of Imetelstat on were used: crude Imetelstat ammonium (crude form), Imetelstat triethylammonium (purified TEA salt form), and Imetelstat sodium (Na salt form), were mixed with each salt solution. The Imetelstat ammonium salt was used as either a NH4OH solution or a solution in water. The Imetelstat ammonium and Imetelstat TEA solutions required approximately 50 equivalents or 30 equivalents of Mg salt t, respectively, to e complete itation.
] The solubility of precipitates formed from the Imetelstat TEA solution and the Imetelstat ammonium solution were investigated under various pH conditions from pH 8 to pH 12. After leaving the mixed solutions for 6 hrs at RT, the solubility of the Imetelstat-Mg precipitates was analyzed by UV absorbance at 260 nm. Both precipitates obtained from the Imetelstat ammonium and the Imetelstat TEA showed a similar trend in that more Imetelstat salt dissolved in 1M NaCl solution at high pH (see Table 3).
This result suggests that when the number of equivalents of salt reagent of interest relative to Imetelstat is controlled, complete precipitation of Imetelstat salt may be achieved by any convenient method to produce a itate that may be successfully redissolved. 5 equivalents salt reagent Imetelstat Sodium solution (100mg in 1mL of water) was mixed with 5 equivalents of eight salt reagents and each solution was desalted by iltration using a Stirred Ultrafiltration Cell and 1KD membrane. The ultrafiltered solution was then lyophilized. The resulting powder was analyzed for the content of Na and each metal counterion of interest by Flame AA (atomic absorption spectroscopy. As show in Figure 2 and 3, the highest metal counterion content was 1.1% by weight for Zn, Al, and Mg, with Na contents of 2.6%, 1.7%, and 2.6%, respectively. ] 6 to 9 equivalents salt t Addition of 6 to 9 equivalents of ium salt reagent to Imetelstat Sodium solution was made and the subsequent ultrafiltration and lzation provided the solid product which was completely soluble in water. The analysis of sodium and magnesium t was performed (see results in Figure 3). Addition of nine equivalents of MgCl2 to Imetelstat Sodium solution, produce a ition where the Na and Mg counterion content is 1.1% and 1.2% by weight, respectively. 1 to 10 lents salt reagent To investigate the exchange of Mg with TEA counterions in Imetelstat TEA salt as compared to Imetelstat sodium salt, another set of experiments was designed and performed. One to ten equivalents of MgCl2 in aqueous solutions were mixed with Imetelstat TEA salt solution (purity > 90% by HPLC). An analysis of the Mg counterion t was performed after ultrafiltration and lyophilization. The results are shown in Figure 4. The addition of up to 10 equivalents of MgCl2 reagent produced a composition having 1.6% of Mg by weight.
Example 4: Conclusion The preparation of divalent and ent salt forms of Imetelstat was achieved including calcium, ium, zinc, aluminium, barium, iron(II), iron (III) and copper salts. When a controlled excess of selected inorganic salt reagents was used (see Table 2 and 3) to precipitate the cleotide, precipitates were formed which could be subsequently olved, and which show improved purity with t to fast g impurities using HPLC analysis.
The use of a magnesium salt reagent produced a soluble solid precipitate of Imetelstat after the exchange step. Precipitates were produced which ed a 1.2 % by weight of magnesium counterion relative to 1.1 % by weight of sodium counterion.
The precipitation of Imetelstat using divalent or trivalent salts provides for the removal of non-target synthetic products and reagents which remain in solution. The l of such impurities present in crude Imetelstat ons provides several advantages for subsequent tography purification steps of Imetelstat, such as reduced column loading, improved resolution, reduced number of chromatography purification steps and improved lifetime of chromatography columns, decreased purification costs and faster purifications.
Table 1. Inorganic Salts, Organic Solvents, and Other als Molecular Formula Grade or Material (Molecular Purity Weight) Calcium Chloride CaCl2∙2H2O ≥ 99 % dihydrate (MW 147.01) Magnesium chloride MgCl2∙H2O ≥ 99 % monohydrate (MW 203.30) Barium chloride BaCl2∙2H2O ≥ 99 % dihydrate (MW 244.26) Copper(II) chloride CuCl2∙2H2O ≥ 99 % dihydrate (MW ) ZnCl2 Zinc chloride ≥ 98 % (MW 136.30) Aluminum chloride AlCl3∙6H2O ≥ 95 % hexahydrate (MW 241.43) Iron (II) chloride FeCl2∙4H2O ≥ 98 % tetrahydrate (MW 198.81) Iron (III) chloride FeCl3∙6H2O ≥ 98 % hexahydrate (MW 270.30) NaCl Sodium chloride USP grade (MW 58.4) Table 2 (O: Yes, X: No, "-" means not performed) Imetelstat Method Test Performed CaCl2 MgCl2 BaCl2 CuCl2 ZnCl2 AlCl3 FeCl2 FeCl3 Tested Column O O O X X X X X Pass through itation in Sodium Ion solution after O O O - - - - - (Na form, Exchange column 100mg Resin Filtration of /mL) X X X - - - - - precipitates Solubility* of X X X - - - - - Precipitate Precipitates O O O O O O O O (Filterable) Sodium Solubility** of (Na form, X X X X X X X X itate 100mg Solubility of /mL) Precipitate in O O O X X X X X 1M NaOH Precipitat- Precipitates O O O O O O O O ion (Filterable) (900 TEA Solubility** of X X X X X X X X equivalent) (35mg Precipitate /mL) Solubility of Precipitate in O O X X X X X X 1M NaOH Precipitates O O O O O O O O Crude (Filterable) (in NH4OH) Solubility** of X X X - - - - - Precipitate *Tested in Acetonitrile, MeOH, EtOH, IPA, Water, NMP, 1M HCl, 1M NaCl, 1M NaOH ** Tested in Acetonitrile, MeOH, EtOH, IPA, Water, NMP, 1M HCl, 1M NaCl Table 3 Sodium 9 15 12 7 50 50 10 11 Inorganic Salt TEA 15 30 30 10 50 10 50 10 Equivalents to Dissolution Crude get complete precipitation - >50 - - - - - - (in NH4OH) Crude - - - - - (in water) Solubility Imetelstat-Mg ppt pH 8 pH 9 pH 10 pH 11 pH 12 1M NaCl 6mg (After 64 hours) 28 OD 31 OD 70 OD 290 OD 434 OD (1 mL) Crude stat-Mg ppt pH 8 pH 9 pH 10 pH 11 pH 12 (in water) 6mg (After 6 hours) 10 OD 7 OD 17 OD 111 OD 377 OD Notwithstanding the appended claims, the disclosure set forth herein is also defined by the following clauses: 1. A method of ing a polynucleotide, the method comprising: contacting a first polynucleotide composition comprising: a polynucleotide having a sequence of 7 or more nucleoside subunits and at least two of the nucleoside subunits are joined by a N3′→P5′ thiophosphoramidate intersubunit linkage; and non-target synthetic products and ts; with a multivalent cation salt to precipitate a first polynucleotide salt comprising at least one multivalent cation counterion; and separating the first polynucleotide salt from the contacted first polynucleotide composition to produce a second polynucleotide composition sing the first polynucleotide salt. 2. The method of clause 1, further comprising: contacting the first polynucleotide salt with a reverse phase chromatography support; and g from the chromatography support a third polynucleotide composition comprising a second polynucleotide salt. 3. The method of any one of clauses 1-2, n the polynucleotide comprises a sequence comprising 13 or more nucleoside subunits complementary to the RNA component of human telomerase. 4. The method of any one of clauses 1-3, wherein the polynucleotide comprises between 10 and 50 contiguous side subunits complementary to the RNA component of human telomerase. 5. The method of any one of clauses 3-4, n the nucleoside subunits complementary to the RNA component of human telomerase are all joined by N3′→P5′ thiophosphoramidate subunit linkages. 6. The method of any one of clauses 1-5 wherein the polynucleotide comprises a sequence selected from the group ting of: GTTAGGGTTAG (SEQ ID NO:4), TAGGGTTAGACAA (SEQ ID NO:3) and GGGTTAG (SEQ ID NO:5). 7. The method of any one of clauses 1-6, wherein the polynucleotide is conjugated to a lipid moiety via an optional linker. 8. The method of any one of clauses 2-7, wherein the second polynucleotide salt has the structure: (Mx+)n wherein each Mx+ is independently hydrogen or a cationic counterion, each x is independently 1, 2 or 3 and n is an integer from 5 to 13. 9. The method of any one of clauses 2-8, wherein the second polynucleotide salt is a pharmaceutically acceptable salt of the polynucleotide. 10. The method of any one of clauses 2-9, wherein the second polynucleotide salt is a monovalent cation salt of the polynucleotide. 11. The method of any one of clauses 2-10, wherein the second cleotide salt is a sodium salt of the polynucleotide. 12. The method of any one of clauses 1-5, further comprising cleaving the cleotide from a support to produce the first polynucleotide composition. ] 13. The method of any one of clauses 1-12, wherein the first composition comprises a monovalent cation salt of the polynucleotide. 14. The method of any one of clauses 1-13, wherein the contacting step comprises eluting the first polynucleotide composition from a cation exchange support. 15. The method of any one of clauses 1-14, wherein the separating step comprises centrifuging the contacted first polynucleotide composition to spin down the polynucleotide salt itate. 16. The method of any one of clauses 1-15, wherein the separating step ses filtering the polynucleotide salt from the contacted first polynucleotide. 17. The method of clause 2, n the second polynucleotide composition is loaded directly onto the reverse phase chromatography support. 18. The method of any one of s 1-17, further sing dissolving the second polynucleotide composition in a solvent. 19. The method of any one of clauses 1-18, wherein the at least one alent cation counterion is divalent. 20. The method of clause 19, wherein the at least one alent cation counterion is selected from the group consisting of magnesium, zinc and calcium. 21. The method of any one of clauses 1-18, wherein the at least one multivalent cation rion is trivalent. 22. The method of clause 21, wherein the at least one multivalent cation counterion is aluminium. 23. The method of any one of clauses 1-22, wherein the polynucleotide salt further ses a monovalent cation counterion. 24. A composition comprising: a salt of a polynucleotide comprising at least one multivalent cation counterion; wherein the polynucleotide has a sequence of 7 or more side subunits mentary to the RNA component of human telomerase and at least two of the nucleoside ts are joined by a N3′→P5′ thiophosphoramidate subunit linkage. 25. The composition of clause 24, wherein the at least one alent cation counterion is divalent. 26. The composition of clause 25, wherein the at least one multivalent cation counterion is selected from the group consisting of ium, zinc and calcium. 27. The composition of any one of clauses 24-26, n the at least one multivalent cation counterion is magnesium. 28. The composition of clause 24, wherein the at least one multivalent cation counterion is trivalent. 29. The composition of clause 28, wherein the at least one multivalent cation counterion is aluminium. 30. The composition of any one of clauses 24-29, wherein the polynucleotide comprises 3 mol % or more of the multivalent cation counterion relative to a polyanionic backbone of the polynucleotide. 31. The composition of any one of clauses 24-29, wherein the polynucleotide comprises 1.0 % by weight or more of the multivalent cation counterion relative to the polynucleotide. 32. The composition of any one of clauses 24-31, wherein the composition is a precipitate. 33. The composition of any one of clauses 24-32, wherein the polynucleotide comprises a sequence comprising 13 or more nucleoside subunits complementary to the RNA component of human telomerase. 34. The composition of any one of clauses 24-33, wherein the polynucleotide ses between 10 and 50 contiguous side ts complementary to the RNA component of human telomerase. 35. The composition of any one of clauses 24-34, wherein the nucleoside subunits complementary to the RNA component of human telomerase are all joined by N3′→P5′ thiophosphoramidate inter-subunit linkages. 36. The composition of any one of clauses 24-35, wherein the polynucleotide ses a sequence selected from the group consisting of: GTTAGGGTTAG (SEQ ID NO:4), TAGGGTTAGACAA (SEQ ID NO:3) and CAGTTAGGGTTAG (SEQ ID NO:5). 37. The composition of any one of clauses 24-36, wherein the polynucleotide is conjugated to a lipid moiety via an al linker. 38. The ition of any one of clauses 24-37, wherein the polynucleotide has the structure: (M x +) n wherein each Mx+ is independently a cationic counterion, each x is 1, 2 or 3 and n is 5 to 12.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the ngs of this invention that certain s and modifications may be made thereto without departing from the spirit or scope of the appended claims.
Accordingly, the preceding merely illustrates the principles of the invention.
It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the ples of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited es and conditions. er, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples f, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both tly known lents and equivalents ped in the future, i.e., any ts developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the embodiments shown and described herein.
Rather, the scope and spirit of present invention is embodied by the appended embodiments. All possible combinations of the above-indicated embodiments are considered to be embraced within the scope of this invention.
What is claimed is: 1. A method of preparing a cleotide, the method comprising: a) contacting a first cleotide composition with a multivalent cation salt to precipitate a first polynucleotide salt comprising at least one multivalent cation counterion; and b) separating the first polynucleotide salt from the contacted first polynucleotide composition to produce a second polynucleotide composition comprising the first polynucleotide salt; n the first polynucleotide composition comprises: (i) a polynucleotide having a sequence of 7 or more nucleoside subunits and at least two of the nucleoside subunits are joined by a N3′→P5′ thiophosphoramidate inter-subunit linkage; and (ii) soluble non-target synthetic products and reagents. 2. The method of claim 1, further comprising: c) contacting the second polynucleotide composition comprising the first polynucleotide salt from step (b) with a e phase chromatography support; and d) g from the reverse phase chromatography t a third polynucleotide composition comprising a second polynucleotide salt. 3. The method of claim 1 or 2, wherein the polynucleotide is described by Formula (I): wherein: each B is independently a purine, a protected purine, a pyrimidine or a protected pyrimidine, or an analog thereof; each X is independently oxygen or sulfur; each R3 is ndently hydrogen, fluoro, hydroxyl, an alkoxy, a substituted alkoxy or a protected yl; R6 is amino, hydroxyl, a protected amino, a protected hydroxy, -O-T-Z or -NH-T-Z; each T is ndently an al linker; each Z is independently H, a lipid, a carrier, an oligonucleotide, a polymer, a polypeptide, a detectable label, or a tag; and n is an integer of 7 to 100. 4. The method of any one of claims 1-3, wherein the polynucleotide comprises a sequence comprising 13 or more nucleoside subunits complementary to the RNA component of human telomerase.
. The method of any one of claims 1-4, wherein the polynucleotide ses between 10 and 50 uous nucleoside subunits complementary to the RNA component of human telomerase. 6. The method of any one of claims 4-5, wherein the nucleoside subunits complementary to the RNA component of human telomerase are all joined by ′ thiophosphoramidate inter-subunit linkages. 7. The method of any one of claims 1-6 wherein the polynucleotide comprises a sequence selected from the group consisting of: GTTAGGGTTAG (SEQ ID NO:4), TAGGGTTAGACAA (SEQ ID NO:3) and CAGTTAGGGTTAG (SEQ ID NO:5). 8. The method of any one of claims 1-7, wherein the cleotide is conjugated to a lipid moiety via an optional linker. 9. The method of any one of claims 2-8, wherein the second polynucleotide salt has the structure: (Mx+)n wherein each Mx+ is independently hydrogen or a cationic counterion, each x is independently 1, 2 or 3 and n is an integer from 5 to 13.
. The method of any one of claims 2-9, wherein after the eluting step d) the second cleotide salt is a pharmaceutically acceptable salt of the polynucleotide. 11. The method of any one of claims 2-10, wherein after the g step d) the second polynucleotide salt is a monovalent cation salt of the polynucleotide. 12. The method of any one of claims 2-11, wherein after the eluting step d) the second polynucleotide salt is a sodium salt of the polynucleotide. 13. The method of any one of claims 1-6, further comprising, prior to the contacting step a), cleaving the polynucleotide from a solid phase synthesis support to produce the first polynucleotide composition as a crude synthetic preparation of the polynucleotide. 14. The method of any one of claims 1-13, wherein prior to the ting step a) the first polynucleotide composition comprises a monovalent cation salt of the polynucleotide.
. The method of any one of claims 1-14, wherein the contacting step a) comprises g and eluting the first polynucleotide composition from a cation exchange t. 16. The method of any one of claims 1-15, wherein the separating step b) comprises centrifuging the contacted first polynucleotide composition to spin down the first polynucleotide salt precipitate. 17. The method of any one of claims 1-16, n the separating step b) comprises filtering the first polynucleotide salt from the contacted first polynucleotide composition. 18. The method of claim 2, wherein the second polynucleotide composition of step b) is loaded directly onto the reverse phase chromatography support. 19. The method of any one of claims 1-18, further comprising, prior to the contacting step c), dissolving the second polynucleotide composition in a solvent.
. The method of any one of claims 1-19, wherein the at least one multivalent cation rion is nt. 21. The method of claim 20, wherein the at least one multivalent cation counterion is selected from the group consisting of magnesium, zinc and calcium. 22. The method of any one of claims 1-19, wherein the at least one multivalent cation counterion is trivalent. 23. The method of claim 22, wherein the at least one multivalent cation counterion is aluminium. 24. The method of any one of claims 1-23, wherein the first polynucleotide salt further comprises a monovalent cation counterion.
. A composition comprising: a salt precipitate of a polynucleotide comprising at least one multivalent cation counterion; wherein the polynucleotide has a sequence of 7 or more nucleoside subunits complementary to the RNA component of human rase and at least two of the nucleoside subunits are joined by a N3′→P5′ thiophosphoramidate inter-subunit linkage. 26. The ition of claim 25, wherein the at least one multivalent cation counterion is nt. 27. The composition of claim 26, wherein the at least one multivalent cation rion is selected from the group consisting of magnesium, zinc and calcium. 28. The composition of any one of claims 25-27, wherein the at least one multivalent cation counterion is magnesium. 29. The composition of claim 25, wherein the at least one multivalent cation counterion is trivalent.
. The composition of claim 29, wherein the at least one multivalent cation counterion is aluminium. 31. The ition of any one of claims 25-30, wherein the polynucleotide salt comprises 3 mol % or more of the multivalent cation counterion. 32. The ition of any one of claims 25-30, wherein the polynucleotide salt ses 1.0 % by weight or more of the multivalent cation counterion. 33. The composition of any one of claims 25-32, wherein the composition is a precipitate. 34. The composition of any one of claims 25-33, wherein the polynucleotide is described by Formula (I): wherein: each B is independently a purine, a ted purine, a pyrimidine or a protected pyrimidine, or an analog thereof; each X is independently oxygen or sulfur; each R3 is independently hydrogen, fluoro, yl, an alkoxy, a substituted alkoxy or a protected hydroxyl; R6 is amino, hydroxyl, a protected amino, a protected y, -O-T-Z or -NH-T-Z; each T is independently an optional ; each Z is independently H, a lipid, a carrier, an oligonucleotide, a polymer, a polypeptide, a detectable label, or a tag; and n is an integer of 7 to 100.
. The composition of any one of claims 25-34, wherein the polynucleotide comprises a sequence comprising 13 or more nucleoside ts complementary to the RNA component of human telomerase. 36. The composition of any one of claims 25-35, wherein the polynucleotide comprises between 10 and 50 contiguous nucleoside subunits complementary to the RNA component of human rase. 37. The ition of any one of claims 25-36, wherein the nucleoside subunits complementary to the RNA component of human telomerase are all joined by N3′→P5′ thiophosphoramidate inter-subunit linkages. 38. The composition of any one of claims 25-37, wherein the polynucleotide comprises a sequence selected from the group consisting of: GTTAGGGTTAG (SEQ ID NO:4), TAGGGTTAGACAA (SEQ ID NO:3) and CAGTTAGGGTTAG (SEQ ID NO:5). 39. The composition of any one of claims 25-38, wherein a 5′ or 3′ terminus of the polynucleotide is conjugated to a lipid moiety via an optional linker. 40. The composition of any one of claims 25-39, wherein the polynucleotide has the structure: (Mx+)n n each Mx+ is independently a cationic counterion, each x is 1, 2 or 3 and n is 5 to WO 72346 Uniipidated i Spades 2:3. 30 min I ' 20 30 ‘ ..
Q).C3 ' Z 20 30 SUBSTITUTE SHEET (RULE 26) 0 A A A ..A ....A A ......A C8032 F6032 CUCEQ BaCiE ZnCEZ FECEB AECES MG. 2 SUBSTITUTE SHEET (RULE 26) WO 72346 ....... iaqu. 2mm. Beau. 4€£§LL 59cm. 6 saw. 7 mu. 8 equ. 9 equ.
MG. 3 SUBSTITUTE SHEET (RULE 26) WO 72346 MG. 4 SUBSTITUTE SHEET (RULE 26)
Claims (40)
1. A method of preparing a cleotide, the method comprising: a) contacting a first cleotide composition with a multivalent cation salt to precipitate a first polynucleotide salt comprising at least one multivalent cation counterion; and b) separating the first polynucleotide salt from the contacted first polynucleotide composition to produce a second polynucleotide composition comprising the first polynucleotide salt; n the first polynucleotide composition comprises: (i) a polynucleotide having a sequence of 7 or more nucleoside subunits and at least two of the nucleoside subunits are joined by a N3′→P5′ thiophosphoramidate inter-subunit linkage; and (ii) soluble non-target synthetic products and reagents.
2. The method of claim 1, further comprising: c) contacting the second polynucleotide composition comprising the first polynucleotide salt from step (b) with a e phase chromatography support; and d) g from the reverse phase chromatography t a third polynucleotide composition comprising a second polynucleotide salt.
3. The method of claim 1 or 2, wherein the polynucleotide is described by Formula (I): wherein: each B is independently a purine, a protected purine, a pyrimidine or a protected pyrimidine, or an analog thereof; each X is independently oxygen or sulfur; each R3 is ndently hydrogen, fluoro, hydroxyl, an alkoxy, a substituted alkoxy or a protected yl; R6 is amino, hydroxyl, a protected amino, a protected hydroxy, -O-T-Z or -NH-T-Z; each T is ndently an al linker; each Z is independently H, a lipid, a carrier, an oligonucleotide, a polymer, a polypeptide, a detectable label, or a tag; and n is an integer of 7 to 100.
4. The method of any one of claims 1-3, wherein the polynucleotide comprises a sequence comprising 13 or more nucleoside subunits complementary to the RNA component of human telomerase.
5. The method of any one of claims 1-4, wherein the polynucleotide ses between 10 and 50 uous nucleoside subunits complementary to the RNA component of human telomerase.
6. The method of any one of claims 4-5, wherein the nucleoside subunits complementary to the RNA component of human telomerase are all joined by ′ thiophosphoramidate inter-subunit linkages.
7. The method of any one of claims 1-6 wherein the polynucleotide comprises a sequence selected from the group consisting of: GTTAGGGTTAG (SEQ ID NO:4), TAGGGTTAGACAA (SEQ ID NO:3) and CAGTTAGGGTTAG (SEQ ID NO:5).
8. The method of any one of claims 1-7, wherein the cleotide is conjugated to a lipid moiety via an optional linker.
9. The method of any one of claims 2-8, wherein the second polynucleotide salt has the structure: (Mx+)n wherein each Mx+ is independently hydrogen or a cationic counterion, each x is independently 1, 2 or 3 and n is an integer from 5 to 13.
10. The method of any one of claims 2-9, wherein after the eluting step d) the second cleotide salt is a pharmaceutically acceptable salt of the polynucleotide.
11. The method of any one of claims 2-10, wherein after the g step d) the second polynucleotide salt is a monovalent cation salt of the polynucleotide.
12. The method of any one of claims 2-11, wherein after the eluting step d) the second polynucleotide salt is a sodium salt of the polynucleotide.
13. The method of any one of claims 1-6, further comprising, prior to the contacting step a), cleaving the polynucleotide from a solid phase synthesis support to produce the first polynucleotide composition as a crude synthetic preparation of the polynucleotide.
14. The method of any one of claims 1-13, wherein prior to the ting step a) the first polynucleotide composition comprises a monovalent cation salt of the polynucleotide.
15. The method of any one of claims 1-14, wherein the contacting step a) comprises g and eluting the first polynucleotide composition from a cation exchange t.
16. The method of any one of claims 1-15, wherein the separating step b) comprises centrifuging the contacted first polynucleotide composition to spin down the first polynucleotide salt precipitate.
17. The method of any one of claims 1-16, n the separating step b) comprises filtering the first polynucleotide salt from the contacted first polynucleotide composition.
18. The method of claim 2, wherein the second polynucleotide composition of step b) is loaded directly onto the reverse phase chromatography support.
19. The method of any one of claims 1-18, further comprising, prior to the contacting step c), dissolving the second polynucleotide composition in a solvent.
20. The method of any one of claims 1-19, wherein the at least one multivalent cation rion is nt.
21. The method of claim 20, wherein the at least one multivalent cation counterion is selected from the group consisting of magnesium, zinc and calcium.
22. The method of any one of claims 1-19, wherein the at least one multivalent cation counterion is trivalent.
23. The method of claim 22, wherein the at least one multivalent cation counterion is aluminium.
24. The method of any one of claims 1-23, wherein the first polynucleotide salt further comprises a monovalent cation counterion.
25. A composition comprising: a salt precipitate of a polynucleotide comprising at least one multivalent cation counterion; wherein the polynucleotide has a sequence of 7 or more nucleoside subunits complementary to the RNA component of human rase and at least two of the nucleoside subunits are joined by a N3′→P5′ thiophosphoramidate inter-subunit linkage.
26. The ition of claim 25, wherein the at least one multivalent cation counterion is nt.
27. The composition of claim 26, wherein the at least one multivalent cation rion is selected from the group consisting of magnesium, zinc and calcium.
28. The composition of any one of claims 25-27, wherein the at least one multivalent cation counterion is magnesium.
29. The composition of claim 25, wherein the at least one multivalent cation counterion is trivalent.
30. The composition of claim 29, wherein the at least one multivalent cation counterion is aluminium.
31. The ition of any one of claims 25-30, wherein the polynucleotide salt comprises 3 mol % or more of the multivalent cation counterion.
32. The ition of any one of claims 25-30, wherein the polynucleotide salt ses 1.0 % by weight or more of the multivalent cation counterion.
33. The composition of any one of claims 25-32, wherein the composition is a precipitate.
34. The composition of any one of claims 25-33, wherein the polynucleotide is described by Formula (I): wherein: each B is independently a purine, a ted purine, a pyrimidine or a protected pyrimidine, or an analog thereof; each X is independently oxygen or sulfur; each R3 is independently hydrogen, fluoro, yl, an alkoxy, a substituted alkoxy or a protected hydroxyl; R6 is amino, hydroxyl, a protected amino, a protected y, -O-T-Z or -NH-T-Z; each T is independently an optional ; each Z is independently H, a lipid, a carrier, an oligonucleotide, a polymer, a polypeptide, a detectable label, or a tag; and n is an integer of 7 to 100.
35. The composition of any one of claims 25-34, wherein the polynucleotide comprises a sequence comprising 13 or more nucleoside ts complementary to the RNA component of human telomerase.
36. The composition of any one of claims 25-35, wherein the polynucleotide comprises between 10 and 50 contiguous nucleoside subunits complementary to the RNA component of human rase.
37. The ition of any one of claims 25-36, wherein the nucleoside subunits complementary to the RNA component of human telomerase are all joined by N3′→P5′ thiophosphoramidate inter-subunit linkages.
38. The composition of any one of claims 25-37, wherein the polynucleotide comprises a sequence selected from the group consisting of: GTTAGGGTTAG (SEQ ID NO:4), TAGGGTTAGACAA (SEQ ID NO:3) and CAGTTAGGGTTAG (SEQ ID NO:5).
39. The composition of any one of claims 25-38, wherein a 5′ or 3′ terminus of the polynucleotide is conjugated to a lipid moiety via an optional linker.
40. The composition of any one of claims 25-39, wherein the polynucleotide has the structure:
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201562151891P | 2015-04-23 | 2015-04-23 | |
US62/151,891 | 2015-04-23 | ||
PCT/US2016/028657 WO2016172346A1 (en) | 2015-04-23 | 2016-04-21 | Methods of polynucleotide preparation using multivalent cation salt compositions |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ735042A NZ735042A (en) | 2021-11-26 |
NZ735042B2 true NZ735042B2 (en) | 2022-03-01 |
Family
ID=
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11441144B2 (en) | Methods of polynucleotide preparation using multivalent cation salt compositions | |
JP7308309B2 (en) | Oligonucleotide composition and method for producing same | |
NZ735042B2 (en) | Methods of polynucleotide preparation using multivalent cation salt compositions | |
OA18769A (en) | Methods of polynucleotide preparation using multivalent cation salt compositions. | |
NZ724764B2 (en) | Oligonucleotide compositions and methods of making the same | |
OA18106A (en) | Oligonucleotide compositions and methods of making the same | |
OA19860A (en) | Oligonucleotide compositions and methods of making the same. | |
NZ763996B2 (en) | Oligonucleotide compositions and methods of making the same |