NZ770894A - Nucleosides and nucleotides with 3'-hydroxy blocking groups and their use in polynucleotide sequencing methods - Google Patents
Nucleosides and nucleotides with 3'-hydroxy blocking groups and their use in polynucleotide sequencing methodsInfo
- Publication number
- NZ770894A NZ770894A NZ770894A NZ77089419A NZ770894A NZ 770894 A NZ770894 A NZ 770894A NZ 770894 A NZ770894 A NZ 770894A NZ 77089419 A NZ77089419 A NZ 77089419A NZ 770894 A NZ770894 A NZ 770894A
- Authority
- NZ
- New Zealand
- Prior art keywords
- nucleotide
- nucleoside
- group
- alkyl
- optionally substituted
- Prior art date
Links
- 125000003729 nucleotide group Chemical group 0.000 title claims abstract description 393
- 239000002773 nucleotide Substances 0.000 title claims abstract description 384
- 239000002777 nucleoside Substances 0.000 title claims abstract description 121
- 230000000903 blocking Effects 0.000 title claims abstract description 119
- 229920000023 polynucleotide Polymers 0.000 title claims description 114
- 239000002157 polynucleotide Substances 0.000 title claims description 114
- 125000003835 nucleoside group Chemical group 0.000 title claims description 27
- 150000003833 nucleoside derivatives Chemical class 0.000 claims abstract description 95
- GNVMUORYQLCPJZ-UHFFFAOYSA-M thiocarbamate Chemical compound NC([S-])=O GNVMUORYQLCPJZ-UHFFFAOYSA-M 0.000 claims abstract description 16
- 125000002777 acetyl group Chemical class [H]C([H])([H])C(*)=O 0.000 claims abstract 2
- -1 C1-C6alkoxy Chemical group 0.000 claims description 88
- 238000006243 chemical reaction Methods 0.000 claims description 87
- KDLHZDBZIXYQEI-UHFFFAOYSA-N palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 claims description 78
- 239000000243 solution Substances 0.000 claims description 75
- 239000000203 mixture Substances 0.000 claims description 74
- 125000005647 linker group Chemical group 0.000 claims description 70
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 claims description 56
- 239000003153 chemical reaction reagent Substances 0.000 claims description 50
- 238000010348 incorporation Methods 0.000 claims description 47
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 46
- 238000003776 cleavage reaction Methods 0.000 claims description 40
- 125000004452 carbocyclyl group Chemical group 0.000 claims description 31
- 125000000623 heterocyclic group Chemical group 0.000 claims description 30
- 125000004429 atoms Chemical group 0.000 claims description 26
- 229920000272 Oligonucleotide Polymers 0.000 claims description 24
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims description 24
- 125000003342 alkenyl group Chemical group 0.000 claims description 23
- 230000001809 detectable Effects 0.000 claims description 23
- 230000000295 complement Effects 0.000 claims description 22
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 21
- 102000004190 Enzymes Human genes 0.000 claims description 19
- 108090000790 Enzymes Proteins 0.000 claims description 19
- 239000000126 substance Substances 0.000 claims description 19
- 229910052799 carbon Inorganic materials 0.000 claims description 18
- 229910052736 halogen Inorganic materials 0.000 claims description 18
- 150000002367 halogens Chemical class 0.000 claims description 18
- 239000003054 catalyst Substances 0.000 claims description 17
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 17
- 101700011961 DPOM Proteins 0.000 claims description 16
- 101710029649 MDV043 Proteins 0.000 claims description 16
- 101700061424 POLB Proteins 0.000 claims description 16
- 101700054624 RF1 Proteins 0.000 claims description 16
- 229910052763 palladium Inorganic materials 0.000 claims description 16
- 125000000882 C2-C6 alkenyl group Chemical group 0.000 claims description 15
- 125000003601 C2-C6 alkynyl group Chemical group 0.000 claims description 15
- 239000011780 sodium chloride Substances 0.000 claims description 15
- ASJSAQIRZKANQN-CRCLSJGQSA-N Deoxyribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 claims description 14
- 125000002947 alkylene group Chemical group 0.000 claims description 14
- 125000001188 haloalkyl group Chemical group 0.000 claims description 14
- 125000000171 (C1-C6) haloalkyl group Chemical group 0.000 claims description 13
- HZAXFHJVJLSVMW-UHFFFAOYSA-N ethanolamine Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims description 13
- 125000001072 heteroaryl group Chemical group 0.000 claims description 13
- 125000002467 phosphate group Chemical class [H]OP(=O)(O[H])O[*] 0.000 claims description 12
- XYFCBTPGUUZFHI-UHFFFAOYSA-N phosphine Chemical compound P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 claims description 12
- 229910000073 phosphorus hydride Inorganic materials 0.000 claims description 12
- 125000001424 substituent group Chemical group 0.000 claims description 12
- 125000004737 (C1-C6) haloalkoxy group Chemical group 0.000 claims description 9
- 125000000041 C6-C10 aryl group Chemical group 0.000 claims description 9
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 9
- 150000003839 salts Chemical class 0.000 claims description 9
- KWYHDKDOAIKMQN-UHFFFAOYSA-N Tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 claims description 8
- DHMQDGOQFOQNFH-UHFFFAOYSA-N glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 8
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Tris Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 claims description 7
- 239000007983 Tris buffer Substances 0.000 claims description 7
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 claims description 7
- 150000001642 boronic acid derivatives Chemical class 0.000 claims description 7
- 101710028159 DNTT Proteins 0.000 claims description 6
- 102100002445 DNTT Human genes 0.000 claims description 6
- 125000003710 aryl alkyl group Chemical group 0.000 claims description 6
- 229910052760 oxygen Inorganic materials 0.000 claims description 6
- 150000003512 tertiary amines Chemical class 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 108010092799 EC 2.7.7.49 Proteins 0.000 claims description 5
- 125000004183 alkoxy alkyl group Chemical group 0.000 claims description 5
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 5
- 150000003141 primary amines Chemical class 0.000 claims description 5
- 125000006729 (C2-C5) alkenyl group Chemical group 0.000 claims description 4
- 239000004471 Glycine Substances 0.000 claims description 4
- 239000001301 oxygen Substances 0.000 claims description 4
- 125000003161 (C1-C6) alkylene group Chemical group 0.000 claims description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 3
- ASJSAQIRZKANQN-UHFFFAOYSA-N 3,4,5-trihydroxypentanal Chemical compound OCC(O)C(O)CC=O ASJSAQIRZKANQN-UHFFFAOYSA-N 0.000 claims description 3
- PZIJUXHDLWNNRW-UHFFFAOYSA-N 2-[bis(2-hydroxyethyl)phosphanyl]ethanol Chemical compound OCCP(CCO)CCO PZIJUXHDLWNNRW-UHFFFAOYSA-N 0.000 claims description 2
- 102000033147 ERVK-25 Human genes 0.000 claims 2
- 241000658540 Ora Species 0.000 claims 1
- 125000002228 disulfide group Chemical group 0.000 claims 1
- 125000000467 secondary amino group Chemical class [H]N([*:1])[*:2] 0.000 claims 1
- XEKOWRVHYACXOJ-UHFFFAOYSA-N acetic acid ethyl ester Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 81
- 150000007523 nucleic acids Chemical class 0.000 description 58
- 239000000975 dye Substances 0.000 description 56
- 108020004707 nucleic acids Proteins 0.000 description 56
- 239000002585 base Substances 0.000 description 47
- DGAKHGXRMXWHBX-UHFFFAOYSA-N azoxymethane Chemical compound CN=[N+](C)[O-] DGAKHGXRMXWHBX-UHFFFAOYSA-N 0.000 description 43
- 238000003786 synthesis reaction Methods 0.000 description 43
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 40
- 230000015572 biosynthetic process Effects 0.000 description 38
- 125000004432 carbon atoms Chemical group C* 0.000 description 36
- 235000019439 ethyl acetate Nutrition 0.000 description 36
- YICAEXQYKBMDNH-UHFFFAOYSA-N 3-[bis(3-hydroxypropyl)phosphanyl]propan-1-ol Chemical compound OCCCP(CCCO)CCCO YICAEXQYKBMDNH-UHFFFAOYSA-N 0.000 description 34
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 34
- 230000002194 synthesizing Effects 0.000 description 34
- 150000001875 compounds Chemical class 0.000 description 33
- 230000002829 reduced Effects 0.000 description 33
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 32
- 239000007787 solid Substances 0.000 description 31
- 125000003118 aryl group Chemical group 0.000 description 30
- 150000002500 ions Chemical class 0.000 description 30
- 238000000034 method Methods 0.000 description 30
- 239000001226 triphosphate Substances 0.000 description 29
- 125000000217 alkyl group Chemical group 0.000 description 28
- 238000001514 detection method Methods 0.000 description 28
- 235000011178 triphosphate Nutrition 0.000 description 28
- 229910001868 water Inorganic materials 0.000 description 27
- 125000000304 alkynyl group Chemical group 0.000 description 26
- WYURNTSHIVDZCO-UHFFFAOYSA-N tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 25
- 229920003013 deoxyribonucleic acid Polymers 0.000 description 24
- UNXRWKVEANCORM-UHFFFAOYSA-I triphosphate(5-) Chemical compound [O-]P([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O UNXRWKVEANCORM-UHFFFAOYSA-I 0.000 description 23
- 239000007850 fluorescent dye Substances 0.000 description 22
- 229910052757 nitrogen Inorganic materials 0.000 description 22
- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene dichloride Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 21
- 125000006239 protecting group Chemical group 0.000 description 21
- 150000001241 acetals Chemical class 0.000 description 20
- 238000007792 addition Methods 0.000 description 19
- 230000003321 amplification Effects 0.000 description 19
- 238000003199 nucleic acid amplification method Methods 0.000 description 19
- 125000004122 cyclic group Chemical group 0.000 description 18
- 238000003818 flash chromatography Methods 0.000 description 18
- 239000000758 substrate Substances 0.000 description 17
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 16
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 16
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 16
- 235000019341 magnesium sulphate Nutrition 0.000 description 16
- 239000000741 silica gel Substances 0.000 description 15
- 229910002027 silica gel Inorganic materials 0.000 description 15
- 239000012043 crude product Substances 0.000 description 14
- 238000002360 preparation method Methods 0.000 description 14
- 238000004458 analytical method Methods 0.000 description 13
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 13
- 238000004128 high performance liquid chromatography Methods 0.000 description 13
- 230000000670 limiting Effects 0.000 description 13
- 239000003921 oil Substances 0.000 description 13
- 239000002904 solvent Substances 0.000 description 13
- 239000003039 volatile agent Substances 0.000 description 13
- TYQCGQRIZGCHNB-JLAZNSOCSA-N L-ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(O)=C(O)C1=O TYQCGQRIZGCHNB-JLAZNSOCSA-N 0.000 description 12
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- 125000005843 halogen group Chemical group 0.000 description 12
- 125000004404 heteroalkyl group Chemical group 0.000 description 12
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 12
- 125000000753 cycloalkyl group Chemical group 0.000 description 11
- 125000005842 heteroatoms Chemical group 0.000 description 11
- 229910052739 hydrogen Inorganic materials 0.000 description 11
- 239000001257 hydrogen Substances 0.000 description 11
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 11
- 239000010452 phosphate Substances 0.000 description 11
- 239000000047 product Substances 0.000 description 11
- 239000007858 starting material Substances 0.000 description 11
- CZPWVGJYEJSRLH-UHFFFAOYSA-N 289-95-2 Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 10
- KDCGOANMDULRCW-UHFFFAOYSA-N Purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 10
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 10
- 229960005070 ascorbic acid Drugs 0.000 description 10
- RTZKZFJDLAIYFH-UHFFFAOYSA-N diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 10
- 125000004435 hydrogen atoms Chemical group [H]* 0.000 description 10
- 239000000463 material Substances 0.000 description 10
- 239000012074 organic phase Substances 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 125000006714 (C3-C10) heterocyclyl group Chemical group 0.000 description 9
- 125000001313 C5-C10 heteroaryl group Chemical group 0.000 description 9
- HGCIXCUEYOPUTN-UHFFFAOYSA-N Cyclohexene Chemical compound C1CCC=CC1 HGCIXCUEYOPUTN-UHFFFAOYSA-N 0.000 description 9
- UYTPUPDQBNUYGX-UHFFFAOYSA-N Guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 9
- 239000011324 bead Substances 0.000 description 9
- 239000007853 buffer solution Substances 0.000 description 9
- 235000011180 diphosphates Nutrition 0.000 description 9
- 239000010410 layer Substances 0.000 description 9
- 235000021317 phosphate Nutrition 0.000 description 9
- 238000003756 stirring Methods 0.000 description 9
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 9
- 125000004191 (C1-C6) alkoxy group Chemical group 0.000 description 8
- 229920000160 (ribonucleotides)n+m Polymers 0.000 description 8
- XPPKVPWEQAFLFU-UHFFFAOYSA-J Pyrophosphate Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 8
- 229960005055 SODIUM ASCORBATE Drugs 0.000 description 8
- RWQNBRDOKXIBIV-UHFFFAOYSA-N Thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 8
- 239000000460 chlorine Substances 0.000 description 8
- ZXSBDSGRQIWJPM-UHFFFAOYSA-N dimethylcarbamothioic S-acid Chemical compound CN(C)C(S)=O ZXSBDSGRQIWJPM-UHFFFAOYSA-N 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 239000011521 glass Substances 0.000 description 8
- 239000012044 organic layer Substances 0.000 description 8
- 230000002441 reversible Effects 0.000 description 8
- 239000000377 silicon dioxide Substances 0.000 description 8
- 235000010378 sodium ascorbate Nutrition 0.000 description 8
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 description 8
- 239000011550 stock solution Substances 0.000 description 8
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 7
- 229920005654 Sephadex Polymers 0.000 description 7
- 239000012507 Sephadex™ Substances 0.000 description 7
- LXNAVEXFUKBNMK-UHFFFAOYSA-N acetic acid;palladium Chemical compound [Pd].CC(O)=O.CC(O)=O LXNAVEXFUKBNMK-UHFFFAOYSA-N 0.000 description 7
- 239000007864 aqueous solution Substances 0.000 description 7
- 230000027455 binding Effects 0.000 description 7
- 239000012267 brine Substances 0.000 description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 7
- ROSDSFDQCJNGOL-UHFFFAOYSA-N dimethylamine Chemical compound CNC ROSDSFDQCJNGOL-UHFFFAOYSA-N 0.000 description 7
- PENAXHPKEVTBLF-UHFFFAOYSA-L palladium(2+);prop-1-ene;dichloride Chemical compound [Pd+]Cl.[Pd+]Cl.[CH2-]C=C.[CH2-]C=C PENAXHPKEVTBLF-UHFFFAOYSA-L 0.000 description 7
- HJKYXKSLRZKNSI-UHFFFAOYSA-I pentapotassium;hydrogen sulfate;oxido sulfate;sulfuric acid Chemical compound [K+].[K+].[K+].[K+].[K+].OS([O-])(=O)=O.[O-]S([O-])(=O)=O.OS(=O)(=O)O[O-].OS(=O)(=O)O[O-] HJKYXKSLRZKNSI-UHFFFAOYSA-I 0.000 description 7
- 150000004713 phosphodiesters Chemical class 0.000 description 7
- 230000001603 reducing Effects 0.000 description 7
- 235000017557 sodium bicarbonate Nutrition 0.000 description 7
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 7
- 229910052717 sulfur Inorganic materials 0.000 description 7
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 description 6
- OPTASPLRGRRNAP-UHFFFAOYSA-N Cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 6
- 229940104302 Cytosine Drugs 0.000 description 6
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 6
- JQWHASGSAFIOCM-UHFFFAOYSA-M Sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 6
- 238000004166 bioassay Methods 0.000 description 6
- 230000000875 corresponding Effects 0.000 description 6
- 239000005546 dideoxynucleotide Substances 0.000 description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N edta Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 6
- 125000002534 ethynyl group Chemical group [H]C#C* 0.000 description 6
- 239000000017 hydrogel Substances 0.000 description 6
- AFQIYTIJXGTIEY-UHFFFAOYSA-N hydrogen carbonate;triethylazanium Chemical compound OC(O)=O.CCN(CC)CC AFQIYTIJXGTIEY-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 239000011807 nanoball Substances 0.000 description 6
- 239000012299 nitrogen atmosphere Substances 0.000 description 6
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 6
- JUJWROOIHBZHMG-UHFFFAOYSA-N pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 6
- 238000001712 DNA sequencing Methods 0.000 description 5
- YBBRCQOCSYXUOC-UHFFFAOYSA-N Sulfuryl chloride Chemical compound ClS(Cl)(=O)=O YBBRCQOCSYXUOC-UHFFFAOYSA-N 0.000 description 5
- FPGGTKZVZWFYPV-UHFFFAOYSA-M Tetra-n-butylammonium fluoride Chemical compound [F-].CCCC[N+](CCCC)(CCCC)CCCC FPGGTKZVZWFYPV-UHFFFAOYSA-M 0.000 description 5
- 229940035893 Uracil Drugs 0.000 description 5
- 239000000654 additive Substances 0.000 description 5
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- 125000000392 cycloalkenyl group Chemical group 0.000 description 5
- 230000005284 excitation Effects 0.000 description 5
- 150000002430 hydrocarbons Chemical group 0.000 description 5
- 238000011065 in-situ storage Methods 0.000 description 5
- 229910001496 lithium tetrafluoroborate Inorganic materials 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000006011 modification reaction Methods 0.000 description 5
- 229910000162 sodium phosphate Inorganic materials 0.000 description 5
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 5
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 5
- GJFNRSDCSTVPCJ-UHFFFAOYSA-N 1,8-Bis(dimethylamino)naphthalene Chemical compound C1=CC(N(C)C)=C2C(N(C)C)=CC=CC2=C1 GJFNRSDCSTVPCJ-UHFFFAOYSA-N 0.000 description 4
- 229960000583 Acetic Acid Drugs 0.000 description 4
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Natural products NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 4
- 229960000643 Adenine Drugs 0.000 description 4
- 229960001456 Adenosine Triphosphate Drugs 0.000 description 4
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 4
- XXROGKLTLUQVRX-UHFFFAOYSA-N Allyl alcohol Chemical compound OCC=C XXROGKLTLUQVRX-UHFFFAOYSA-N 0.000 description 4
- ZMANZCXQSJIPKH-UHFFFAOYSA-O CC[NH+](CC)CC Chemical class CC[NH+](CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-O 0.000 description 4
- 102100005410 LINE-1 retrotransposable element ORF2 protein Human genes 0.000 description 4
- 229940113082 Thymine Drugs 0.000 description 4
- IMFACGCPASFAPR-UHFFFAOYSA-N Tributylamine Chemical compound CCCCN(CCCC)CCCC IMFACGCPASFAPR-UHFFFAOYSA-N 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 4
- 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 4
- 125000003545 alkoxy group Chemical group 0.000 description 4
- 125000003277 amino group Chemical group 0.000 description 4
- 235000010323 ascorbic acid Nutrition 0.000 description 4
- 239000011668 ascorbic acid Substances 0.000 description 4
- JMXMXKRNIYCNRV-UHFFFAOYSA-N bis(hydroxymethyl)phosphanylmethanol Chemical compound OCP(CO)CO JMXMXKRNIYCNRV-UHFFFAOYSA-N 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 238000010511 deprotection reaction Methods 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 238000005755 formation reaction Methods 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 4
- 238000004255 ion exchange chromatography Methods 0.000 description 4
- 238000002372 labelling Methods 0.000 description 4
- 239000003446 ligand Substances 0.000 description 4
- 229910052751 metal Inorganic materials 0.000 description 4
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- 102000014961 Protein Precursors Human genes 0.000 description 1
- 108010078762 Protein Precursors Proteins 0.000 description 1
- 229910006024 SO2Cl2 Inorganic materials 0.000 description 1
- 229910006069 SO3H Inorganic materials 0.000 description 1
- AKHNMLFCWUSKQB-UHFFFAOYSA-L Sodium thiosulphate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 1
- 229920000147 Styrene maleic anhydride Polymers 0.000 description 1
- DHXVGJBLRPWPCS-UHFFFAOYSA-N THP Chemical compound C1CCOCC1 DHXVGJBLRPWPCS-UHFFFAOYSA-N 0.000 description 1
- 101700003161 TXLR2 Proteins 0.000 description 1
- 229960004559 Theobromine Drugs 0.000 description 1
- 229940104230 Thymidine Drugs 0.000 description 1
- NHVNXKFIZYSCEB-UHFFFAOYSA-N Thymidine triphosphate Chemical compound O=C1NC(=O)C(C)=CN1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1 NHVNXKFIZYSCEB-UHFFFAOYSA-N 0.000 description 1
- WVLBCYQITXONBZ-UHFFFAOYSA-N Trimethyl phosphate Chemical compound COP(=O)(OC)OC WVLBCYQITXONBZ-UHFFFAOYSA-N 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Trioxopurine Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- ACIAHEMYLLBZOI-ZZXKWVIFSA-N Unsaturated alcohol Chemical compound CC\C(CO)=C/C ACIAHEMYLLBZOI-ZZXKWVIFSA-N 0.000 description 1
- 229940116269 Uric Acid Drugs 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 125000000641 acridinyl group Chemical group C1(=CC=CC2=NC3=CC=CC=C3C=C12)* 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 125000005073 adamantyl group Chemical group C12(CC3CC(CC(C1)C3)C2)* 0.000 description 1
- 150000003838 adenosines Chemical class 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 125000004450 alkenylene group Chemical group 0.000 description 1
- 125000005600 alkyl phosphonate group Chemical group 0.000 description 1
- 150000001343 alkyl silanes Chemical group 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 101710024925 alv Proteins 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 125000006620 amino-(C1-C6) alkyl group Chemical group 0.000 description 1
- 125000004103 aminoalkyl group Chemical group 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 125000002178 anthracenyl group Chemical group C1(=CC=CC2=CC3=CC=CC=C3C=C12)* 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 125000006615 aromatic heterocyclic group Chemical group 0.000 description 1
- 125000005110 aryl thio group Chemical group 0.000 description 1
- 125000004104 aryloxy group Chemical group 0.000 description 1
- 101700053124 asa1 Proteins 0.000 description 1
- 125000002785 azepinyl group Chemical group 0.000 description 1
- IVRMZWNICZWHMI-UHFFFAOYSA-N azide Chemical compound [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 description 1
- 125000003828 azulenyl group Chemical group 0.000 description 1
- 125000003785 benzimidazolyl group Chemical group N1=C(NC2=C1C=CC=C2)* 0.000 description 1
- 125000001164 benzothiazolyl group Chemical group S1C(=NC2=C1C=CC=C2)* 0.000 description 1
- 125000004196 benzothienyl group Chemical group S1C(=CC2=C1C=CC=C2)* 0.000 description 1
- 125000004541 benzoxazolyl group Chemical group O1C(=NC2=C1C=CC=C2)* 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- NYENCOMLZDQKNH-UHFFFAOYSA-K bis(trifluoromethylsulfonyloxy)bismuthanyl trifluoromethanesulfonate Chemical compound [Bi+3].[O-]S(=O)(=O)C(F)(F)F.[O-]S(=O)(=O)C(F)(F)F.[O-]S(=O)(=O)C(F)(F)F NYENCOMLZDQKNH-UHFFFAOYSA-K 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 1
- WKBOTKDWSSQWDR-UHFFFAOYSA-N bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 1
- 230000005587 bubbling Effects 0.000 description 1
- 125000004369 butenyl group Chemical group C(=CCC)* 0.000 description 1
- 125000000480 butynyl group Chemical group [*]C#CC([H])([H])C([H])([H])[H] 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229960001948 caffeine Drugs 0.000 description 1
- 125000000609 carbazolyl group Chemical group C1(=CC=CC=2C3=CC=CC=C3NC12)* 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000000423 cell based assay Methods 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 108091006028 chimera Proteins 0.000 description 1
- 150000001805 chlorine compounds Chemical class 0.000 description 1
- 125000000259 cinnolinyl group Chemical group N1=NC(=CC2=CC=CC=C12)* 0.000 description 1
- 235000019516 cod Nutrition 0.000 description 1
- 230000002596 correlated Effects 0.000 description 1
- 230000001808 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000010779 crude oil Substances 0.000 description 1
- 238000010192 crystallographic characterization Methods 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- 125000000596 cyclohexenyl group Chemical group C1(=CCCCC1)* 0.000 description 1
- 230000004059 degradation Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- RAFNCPHFRHZCPS-UHFFFAOYSA-N di(imidazol-1-yl)methanethione Chemical compound C1=CN=CN1C(=S)N1C=CN=C1 RAFNCPHFRHZCPS-UHFFFAOYSA-N 0.000 description 1
- 125000000723 dihydrobenzofuranyl group Chemical group O1C(CC2=C1C=CC=C2)* 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 125000005879 dioxolanyl group Chemical group 0.000 description 1
- VDCSGNNYCFPWFK-UHFFFAOYSA-N diphenylsilane Chemical compound C=1C=CC=CC=1[SiH2]C1=CC=CC=C1 VDCSGNNYCFPWFK-UHFFFAOYSA-N 0.000 description 1
- CVKCKLNGVYHFAX-UHFFFAOYSA-L dipotassium;4-[phenyl-(4-sulfonatophenyl)phosphanyl]benzenesulfonate;dihydrate Chemical compound O.O.[K+].[K+].C1=CC(S(=O)(=O)[O-])=CC=C1P(C=1C=CC(=CC=1)S([O-])(=O)=O)C1=CC=CC=C1 CVKCKLNGVYHFAX-UHFFFAOYSA-L 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000005670 electromagnetic radiation Effects 0.000 description 1
- 239000012039 electrophile Substances 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 125000003700 epoxy group Chemical group 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 239000012065 filter cake Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000003260 fluorescence intensity Methods 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 125000002541 furyl group Chemical group 0.000 description 1
- 230000005251 gamma ray Effects 0.000 description 1
- 230000002068 genetic Effects 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 239000003228 hemolysin Substances 0.000 description 1
- 125000004475 heteroaralkyl group Chemical group 0.000 description 1
- 125000004446 heteroarylalkyl group Chemical group 0.000 description 1
- 125000006038 hexenyl group Chemical group 0.000 description 1
- 125000005980 hexynyl group Chemical group 0.000 description 1
- 101700027420 hly Proteins 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-O hydron;urea Chemical compound NC([NH3+])=O XSQUKJJJFZCRTK-UHFFFAOYSA-O 0.000 description 1
- 230000002209 hydrophobic Effects 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 125000002632 imidazolidinyl group Chemical group 0.000 description 1
- 125000002636 imidazolinyl group Chemical group 0.000 description 1
- 125000002883 imidazolyl group Chemical group 0.000 description 1
- 125000003387 indolinyl group Chemical group N1(CCC2=CC=CC=C12)* 0.000 description 1
- 125000001041 indolyl group Chemical group 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- 125000004594 isoindolinyl group Chemical group C1(NCC2=CC=CC=C12)* 0.000 description 1
- 125000000904 isoindolyl group Chemical group C=1(NC=C2C=CC=CC12)* 0.000 description 1
- 125000003253 isopropoxy group Chemical group [H]C([H])([H])C([H])(O*)C([H])([H])[H] 0.000 description 1
- 125000001786 isothiazolyl group Chemical group 0.000 description 1
- 125000003965 isoxazolidinyl group Chemical group 0.000 description 1
- 125000003971 isoxazolinyl group Chemical group 0.000 description 1
- 125000000842 isoxazolyl group Chemical group 0.000 description 1
- 239000011967 lanthanide triflate Substances 0.000 description 1
- 101710030587 ligN Proteins 0.000 description 1
- 101700077585 ligd Proteins 0.000 description 1
- 238000011068 load Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229910052987 metal hydride Inorganic materials 0.000 description 1
- 150000004681 metal hydrides Chemical class 0.000 description 1
- BAVYZALUXZFZLV-UHFFFAOYSA-N methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- YNAVUWVOSKDBBP-UHFFFAOYSA-N morpholine Chemical compound C1COCCN1 YNAVUWVOSKDBBP-UHFFFAOYSA-N 0.000 description 1
- 229940113083 morpholine Drugs 0.000 description 1
- 125000002757 morpholinyl group Chemical group 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
- 239000002086 nanomaterial Substances 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- 125000001326 naphthylalkyl group Chemical group 0.000 description 1
- 238000007481 next generation sequencing Methods 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 125000006574 non-aromatic ring group Chemical group 0.000 description 1
- 238000003499 nucleic acid array Methods 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- JMJRYTGVHCAYCT-UHFFFAOYSA-N oxan-4-one Chemical compound O=C1CCOCC1 JMJRYTGVHCAYCT-UHFFFAOYSA-N 0.000 description 1
- 125000000160 oxazolidinyl group Chemical group 0.000 description 1
- 125000005968 oxazolinyl group Chemical group 0.000 description 1
- 125000002971 oxazolyl group Chemical group 0.000 description 1
- 125000003551 oxepanyl group Chemical group 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001590 oxidative Effects 0.000 description 1
- 125000000466 oxiranyl group Chemical group 0.000 description 1
- 125000004043 oxo group Chemical group O=* 0.000 description 1
- HXNFUBHNUDHIGC-UHFFFAOYSA-N oxoallopurinol Chemical compound O=C1NC(=O)N=C2NNC=C21 HXNFUBHNUDHIGC-UHFFFAOYSA-N 0.000 description 1
- 125000001820 oxy group Chemical group [*:1]O[*:2] 0.000 description 1
- 239000005022 packaging material Substances 0.000 description 1
- PIBWKRNGBLPSSY-UHFFFAOYSA-L palladium(II) chloride Chemical compound Cl[Pd]Cl PIBWKRNGBLPSSY-UHFFFAOYSA-L 0.000 description 1
- 230000036961 partial Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000002161 passivation Methods 0.000 description 1
- 125000002255 pentenyl group Chemical group C(=CCCC)* 0.000 description 1
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 1
- 125000005981 pentynyl group Chemical group 0.000 description 1
- 230000000737 periodic Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- NJRWNWYFPOFDFN-UHFFFAOYSA-L phosphonate(2-) Chemical compound [O-][P]([O-])=O NJRWNWYFPOFDFN-UHFFFAOYSA-L 0.000 description 1
- 125000004592 phthalazinyl group Chemical group C1(=NN=CC2=CC=CC=C12)* 0.000 description 1
- GLUUGHFHXGJENI-UHFFFAOYSA-N piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 1
- 125000004193 piperazinyl group Chemical group 0.000 description 1
- NQRYJNQNLNOLGT-UHFFFAOYSA-N piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 1
- 101700005116 plc Proteins 0.000 description 1
- 125000003367 polycyclic group Chemical group 0.000 description 1
- 229920000867 polyelectrolyte Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- HVAHYVDBVDILBL-UHFFFAOYSA-M potassium;oxidooxy hydrogen sulfate Chemical compound [K+].OS(=O)(=O)OO[O-] HVAHYVDBVDILBL-UHFFFAOYSA-M 0.000 description 1
- OZAIFHULBGXAKX-UHFFFAOYSA-N precursor Substances N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 description 1
- 230000001681 protective Effects 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 125000003373 pyrazinyl group Chemical group 0.000 description 1
- 125000003072 pyrazolidinyl group Chemical group 0.000 description 1
- 125000002755 pyrazolinyl group Chemical group 0.000 description 1
- 125000003226 pyrazolyl group Chemical group 0.000 description 1
- 125000002098 pyridazinyl group Chemical group 0.000 description 1
- 125000004076 pyridyl group Chemical group 0.000 description 1
- 125000000714 pyrimidinyl group Chemical group 0.000 description 1
- 125000000719 pyrrolidinyl group Chemical group 0.000 description 1
- 125000004929 pyrrolidonyl group Chemical group N1(C(CCC1)=O)* 0.000 description 1
- 125000000168 pyrrolyl group Chemical group 0.000 description 1
- 125000002943 quinolinyl group Chemical group N1=C(C=CC2=CC=CC=C12)* 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 230000000717 retained Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 239000002342 ribonucleoside Substances 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 229910052701 rubidium Inorganic materials 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007841 sequencing by ligation Methods 0.000 description 1
- 101710010431 shlA Proteins 0.000 description 1
- 238000004557 single molecule detection Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 235000019345 sodium thiosulphate Nutrition 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000000037 tert-butyldiphenylsilyl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1[Si]([H])([*]C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- ODGCEQLVLXJUCC-UHFFFAOYSA-N tetrafluoroborate Chemical compound F[B-](F)(F)F ODGCEQLVLXJUCC-UHFFFAOYSA-N 0.000 description 1
- 125000003718 tetrahydrofuranyl group Chemical group 0.000 description 1
- 125000001412 tetrahydropyranyl group Chemical group 0.000 description 1
- 125000003507 tetrahydrothiofenyl group Chemical group 0.000 description 1
- 125000004632 tetrahydrothiopyranyl group Chemical group S1C(CCCC1)* 0.000 description 1
- 125000001113 thiadiazolyl group Chemical group 0.000 description 1
- 125000001984 thiazolidinyl group Chemical group 0.000 description 1
- 125000002769 thiazolinyl group Chemical group 0.000 description 1
- 125000000335 thiazolyl group Chemical group 0.000 description 1
- 125000001544 thienyl group Chemical group 0.000 description 1
- 125000001583 thiepanyl group Chemical group 0.000 description 1
- 125000004568 thiomorpholinyl group Chemical group 0.000 description 1
- 125000004306 triazinyl group Chemical group 0.000 description 1
- 125000001425 triazolyl group Chemical group 0.000 description 1
- 125000000876 trifluoromethoxy group Chemical group FC(F)(F)O* 0.000 description 1
- SOBHUZYZLFQYFK-UHFFFAOYSA-K trisodium;hydroxy-[[phosphonatomethyl(phosphonomethyl)amino]methyl]phosphinate Chemical class [Na+].[Na+].[Na+].OP(O)(=O)CN(CP(O)([O-])=O)CP([O-])([O-])=O SOBHUZYZLFQYFK-UHFFFAOYSA-K 0.000 description 1
- 238000010626 work up procedure Methods 0.000 description 1
- 229940075420 xanthine Drugs 0.000 description 1
Abstract
Embodiments of the present disclosure relate to nucleotide and nucleoside molecules with acetal or thiocarbamate 3ʹ-OH blocking groups. Also provided herein are methods to prepare such nucleotide and nucleoside molecules, and the uses of fully functionalized nucleotides containing the 3ʹ-OH blocking group for sequencing applications. group for sequencing applications.
Description
W0 2020/ 136170 PCT/EP2019/086926
NUCLEOSIDES AND NUCLEOTIDES WITH 3'-HYDROXY BLOCKING GROUPS
Background
[0001] The present disclosure generally relates to nucleotides, nucleosides, or
oligonucleotides comprising 3’-hydroxy protecting groups and their use in polynucleotide
sequencing methods. Methods of preparing the 3'-hydroxy protected nucleotides,
nucleosides, or oligonucleotides are also disclosed.
Description of the Related Art
[0002] Advances in the study of molecules have been led, in part, by improvement in
technologies used to characterize the molecules or their biological reactions. In particular, the
study of the nucleic acids DNA and RNA has benefited from developing technologies used
for sequence analysis and the study of hybridization events.
[0003] An example of the technologies that have improved the study of nucleic acids is the
development of fabricated arrays of immobilized nucleic acids. These arrays consist typically
of a high-density matrix of polynucleotides immobilized onto a solid support material. See,
e.g., Fodor et al., Trends Biotech. 12: 19-26, 1994, which describes ways of assembling the
nucleic acids using a chemically sensitized glass surface protected by a mask, but exposed at
defined areas to allow attachment of suitably modified nucleotide phosphoramidites.
Fabricated arrays can also be manufactured by the technique of “spotting” known
polynucleotides onto a solid support at predetermined positions (e. g., Stimpson et al., Proc.
Natl. Acad. Sci. 92: 6379-6383, 1995).
[0004] One way of determining the nucleotide sequence of a nucleic acid bound to an array is
called “sequencing by synthesis” or “SBS”. This technique for determining the sequence of
DNA ideally requires the controlled (i.e., one at a time) incorporation of the correct
complementary nucleotide opposite the nucleic acid being sequenced. This allows for
accurate sequencing by adding nucleotides in multiple cycles as each nucleotide residue is
sequenced one at a time, thus preventing an uncontrolled series of incorporations from
occurring. The incorporated nucleotide is read using an appropriate label attached thereto
before removal of the label moiety and the subsequent next round of sequencing.
[0005] In order to ensure that only a single incorporation occurs, a structural modification
(“protecting group” or “blocking group”) is included in each labeled nucleotide that is added
to the growing chain to ensure that only one nucleotide is incorporated. After the nucleotide
W0 2020/ 136170 PCT/EP2019/086926
with the protecting group has been added, the protecting group is then removed, under
reaction conditions which do not interfere with the integrity of the DNA being sequenced.
The sequencing cycle can then continue with the incorporation of the next protected, labeled
nucleotide.
[0006] To be useful in DNA sequencing, nucleotides, which are usually nucleotide
triphosphates, generally require a 3'-hydroxy protecting group so as to prevent the
polymerase used to incorporate it into a polynucleotide chain from continuing to replicate
once the base on the nucleotide is added. There are many limitations on the types of groups
that can be added onto a nucleotide and still be suitable. The protecting group should prevent
additional nucleotide molecules from being added to the polynucleotide chain whilst
simultaneously being easily removable from the sugar moiety without causing damage to the
polynucleotide chain. Furthermore, the modified nucleotide needs to be compatible with the
polymerase or another appropriate enzyme used to incorporate it into the polynucleotide
chain. The ideal protecting group must therefore exhibit long-term stability, be efficiently
incorporated by the polymerase enzyme, cause blocking of secondary or further nucleotide
incorporation, and have the ability to be removed under mild conditions that do not cause
damage to the polynucleotide structure, preferably under aqueous conditions.
[0007] Reversible protecting groups have been described previously. For example, Metzker
er al., (Nucleic Acids Research, 22 (20): 4259-4267, 1994) discloses the synthesis and use of
eight 3'-modified 2-deoxyribonucleoside 5’-triphosphates (3'-modified dNTPs) and testing in
two DNA template assays for incorporation activity. WO 2002/029003 describes a
sequencing method which may include the use of an allyl protecting group to cap the 3’-OH
group on a growing strand of DNA in a polymerase reaction.
[0008] In addition, the development of a number of reversible protecting groups and methods
of deprotecting them under DNA compatible conditions was previously reported in
International Application Publication Nos. WO 2004/018497 and W0 2014/ 139596, each of
which is hereby incorporated by reference in its entirety.
Summary
W0 2020/ 136170 PCT/EP2019/086926
[0009] Some embodiments of the present disclosure relate to a nucleotide or nucleoside
comprising a ribose or deoxyribose having a removable 3'-OH protecting or blocking group
R1 a R23
. E10/[LEO/l;R3
forming a structure R
each R” and Rlb is independently H, C1-C6 alkyl, C1.C6 haloalkyl, C1-C6 Qlkoxy, C1.C6
covalently attached to the 3'-carbon atom, wherein:
haloalkoxy, cyano, halogen, optionally substituted phenyl, or optionally substituted aralkyl;
each R23 and Rzb is independently H, C1-C6 alkyl, C1-Ce haloalkyl, cyano, or halogen,
alternatively, R” and R23 together with the atoms to which they are attached form an
optionally substituted five to eight membered heterocyclyl group;
R3 is H, optionally substituted C2-C6 alkenyl, optionally substituted C3-C7 cycloalkenyl,
optionally substituted C2-C6 alkynyl, or optionally substituted (Ci.C6 alkylene)Si(R4)3, and
each R4 is independently H, C1-C6 alkyl, or optionally substituted C6-C10 aryl. In some
embodiments, when each R” and R“’ is H or C1-Cs alkyl, both R23 and Rzb are H, then R3 is
substituted C2-C6 alkenyl, optionally substituted C3-C7 cycloalkenyl, optionally substituted C2.
C5 alkynyl, or optionally substituted (C1-Cs alkylene)Si(R4)3. In some embodiments, when
each R13, R“’, R23 and Rzb is H, then R3 is not H.
[0010] Some embodiments of the present disclosure relate to a nucleoside or nucleotide
comprising a ribose or deoxyribose having a removable 3'-OH blocking group forming a
JV\IIV\:
structure NR5R6 covalently attached to the 3'-carbon atom, wherein:
each of R5 and R6 is independently H, C1-C6 alkyl, C2.C6 alkenyl, C2.C6 alkynyl, C1.C6
haloalkyl, C2-Cs alkoxyalkyl, optionally substituted —(CH2)m—phenyl, optionally substituted
—(CH2)n—(5 or 6 membered heteroaryl), optionally substituted —(CH2)1<~C3-C7 carbocyclyl, or
optionally substituted —(CH2)p—(3 to 7 membered heterocyclyl);
each of —(CH2)m—, —(CH2)n~, —(CH2)k—, and —(CH2)p— is optionally substituted; and
each of m, n, k, and p is independently O, 1, 2, 3, or 4.
[0011] Some embodiments of the present disclosure relate to an oligonucleotide or
polynucleotide comprising a 3’-OH blocked nucleotide molecule described herein.
[0012] Some embodiments of the present disclosure relate to a method of preparing a
growing polynucleotide complementary to a target single-stranded polynucleotide in a
sequencing reaction, comprising incorporating a nucleotide molecule described herein into
the growing complementary polynucleotide, wherein the incorporation of the nucleotide
W0 2020/ 136170 PCT/EP2019/086926
prevents the introduction of any subsequent nucleotide into the growing complementary
polynucleotide. In some embodiments, the incorporation of the nucleotide is accomplished by
a polymerase, a terminal deoxynucleotidyl transferase (TdT), or a reverse transcriptase. In
one embodiment, the incorporation is accomplished by a polymerase (e.g., a DNA
polymerase).
[0013] Some further embodiments of the present disclosure relate to a method for
determining the sequence of a target single-stranded polynucleotide, comprising:
(a) incorporating a nucleotide comprising a 3’-OH blocking group and a detectable label as
described herein into a copy polynucleotide strand complementary to at least a portion of the
target polynucleotide strand,
(b) detecting the identity of the nucleotide incorporated into the copy polynucleotide strand;
(c) chemically removing the label and the 3’-OH blocking group from the nucleotide
incorporated into the copy polynucleotide strand.
[0014] In some embodiments, the sequencing method further comprises (d) washing the
chemically removed label and the 3’ blocking group away from the copy polynucleotide
strand. In some embodiment, such washing step also removes the unincorporated nucleotides.
In some such embodiments, the 3' blocking group and the detectable label of the incorporated
nucleotide are removed prior to introducing the next complementary nucleotide. In some
further embodiments, the 3' blocking group and the detectable label are removed in a single
step of chemical reaction. In some embodiments, the sequential incorporation described
herein is performed at least 50 times, at least 100 times, at least 150 times, at least 200 times,
or at least 250 times.
[0015] Some further embodiments of the present disclosure relate to kits comprising a
plurality of nucleotide or nucleoside molecules described herein, and packaging materials
therefor. The nucleotides, nucleosides, oligonucleotides, or kits that are set forth herein may
be used to detect, measure, or identify a biological system (including, for example, processes
or components thereof). Exemplary techniques that can employ the nucleotides,
oligonucleotides, or kits include sequencing, expression analysis, hybridization analysis,
genetic analysis, RNA analysis, cellular assay (e.g., cell binding or cell function analysis), or
protein assay (e.g., protein binding assay or protein activity assay). The use may be on an
automated instrument for carrying out a particular technique, such as an automated
sequencing instrument. The sequencing instrument may contain two or more lasers operating
at different wavelengths to distinguish between different detectable labels.
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Brief Description of the Drawings
[0016] is a line chart illustrating the stability of Various 3' blocked nucleotides as a
function of time in a buffer solution at 65 °C.
[0017] is a line chart illustrating the percentage (%) of remaining nucleotide
(starting material) as a fi,1I1CtlOI1 of time comparing the deblocking rate of nucleotide with 3'-
AOM blocking group to nucleotide with the 3’-O-azidomethyl (-CH2N3) blocking group in
solution.
[0018] is a line chart illustrating the percentage (%) of 3' deblocked nucleotides as a
function of time comparing the deblocking rate of 3’ blocked nucleotides with Various acetal
blocking groups in solution.
[0019] FIGs. 3A and 3B illustrate the sequencing results on Illumina MiniSeq® instrument
using fiilly fiinctionalized nucleotides (ffNs) with 3'-AOM blocking group in the
incorporation mix.
[0020] illustrates the sequencing error rate using fully functionalized nucleotides
(ffNs) with 3'-AOM blocking group in the incorporation mix as compared to the standard
ffN s with 3'-O-azidometyl blocking group.
[0021] FIGs. 4A and 4B each illustrates comparison of the primary sequencing metrics
including phasing, pre-phasing and error rate using fully functionalized nucleotides with 3'-
AOM and 3'-O-azidomethyl blocking groups using two different DNA polymerases (Pol 812
and Pol 1901) respectively.
[0022] is a line chart illustrating the sequencing stability of fully functionalized
nucleotides with 3'-AOM or 3'-O-azidomethyl blocking groups as a fiinction of time in a
buffer solution at 45 °C.
[0023] is a line chart illustrating the stability of nucleosides with Various 3’ blocking
groups as a function of time in a buffer solution at 65 °C.
[0024] is a line chart illustrating the percentage (%) of remaining 3' blocked
nucleotide as a fiinction of time, comparing the cleavage (deblocking) rate of a thiocarbamate
3' blocking group dimethylthiocarbamate (DMTC) under two different conditions (Oxone®
or NaIO4) to that of the 3'-O-azidomethyl (3 ’-O-CH2N3) blocking group.
Detailed Description
[0025] Embodiments of the present disclosure relate to nucleosides and nucleotides with 3'-
OH acetal or thiocarbamate blocking groups for sequencing applications, for example,
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sequencing-by-synthesis (SBS). These blocking groups offer better stability in solution
compared to those known in the art. In particular, the 3’-OH blocking groups have improved
stability during the synthesis of the fitlly functionalized nucleotides (ffNs) and also great
stability in solution during formulation, storage and operation on the sequencing instruments.
In addition, the 3’-OH blocking groups described herein may also achieve low pre-phasing,
lower signal decay for improved data quality, which enables longer reads from the
sequencing applications.
Definitions
[0026] Unless defined otherwise, all technical and scientific terms used herein have the same
meaning as is commonly understood by one of ordinary skill in the art. The use of the term
“including” as well as other forms, such as “include”, “includes,” and “included,” is not
limiting. The use of the term “having” as well as other forms, such as “have”, “has,” and
“had,” is not limiting. As used in this specification, whether in a transitional phrase or in the
body of the claim, the terms “comprise(s)” and “comprising” are to be interpreted as having
an open-ended meaning. That is, the above terms are to be interpreted synonymously with
the phrases “having at least” or “including at least.” For example, when used in the context
of a process, the term “comprising” means that the process includes at least the recited steps,
but may include additional steps. When used in the context of a compound, composition, or
device, the term “comprising” means that the compound, composition, or device includes at
least the recited features or components, but may also include additional features or
components.
[0027] As used herein, common organic abbreviations are defined as follows:
°C Temperature in degrees Centigrade
dATP Deoxyadenosine triphosphate
dCTP Deoxycytidine triphosphate
dGTP Deoxyguanosine triphosphate
dTTP Deoxythymidine triphosphate
ddNTP Dideoxynucleotide triphosphate
ffN Fully functionalized nucleotide
RT Room temperature
SBS Sequencing by Synthesis
SM Starting material
W0 2020/ 136170 PCT/EP2019/086926
[0028] As used herein, the term “array” refers to a population of different probe molecules
that are attached to one or more substrates such that the different probe molecules can be
differentiated from each other according to relative location. An array can include different
probe molecules that are each located at a different addressable location on a substrate.
Alternatively, or additionally, an array can include separate substrates each bearing a
different probe molecule, wherein the different probe molecules can be identified according
to the locations of the substrates on a surface to which the substrates are attached or
according to the locations of the substrates in a liquid. Exemplary arrays in which separate
substrates are located on a surface include, without limitation, those including beads in wells
as described, for example, in U.S. Patent No. 6,355,431 B1, US 2002/0102578 and PCT
Publication No. WO O0/63437. Exemplary formats that can be used in the invention to
distinguish beads in a liquid array, for example, using a microfluidic device, such as a
fluorescent activated cell sorter (FACS), are described, for example, in US Pat. No.
6,524,793. Further examples of arrays that can be used in the invention include, without
limitation, those described in U.S. Pat Nos. 5,429,807; 5,436,327; 5,561,071, 5,583,211,
,658,734, 5,837,858; 5,874,219, 5,919,523, 6,136,269, 6,287,768, 6,287,776, 6,288,220,
6,297,006, 6,291,193, 6,346,413, 6,416,949, 6,482,591, 6,514,751 and 6,610,482, and W0
93/17126; W0 95/1 1995, WO 95/35505, EP 742 287; and EP 799 897.
[0029] As used herein, the term “covalently attached” or “covalently bonded” refers to the
forming of a chemical bonding that is characterized by the sharing of pairs of electrons
between atoms. For example, a covalently attached polymer coating refers to a polymer
coating that forms chemical bonds with a functionalized surface of a substrate, as compared
to attachment to the surface via other means, for example, adhesion or electrostatic
interaction. It will be appreciated that polymers that are attached covalently to a surface can
also be bonded via means in addition to covalent attachment.
[0030] As used herein, any “R” group(s) represent substituents that can be attached to the
indicated atom. An R group may be substituted or unsubstituted. If two “R” groups are
described as “together with the atoms to which they are attached” forming a ring or ring
system, it means that the collective unit of the atoms, intervening bonds and the two R groups
are the recited ring. For example, when the following substructure is present:
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and R1 and R2 are defined as selected from the group consisting of hydrogen and alkyl, or R1
and R2 together with the atoms to which they are attached form an aryl or carbocyclyl, it is
meant that R1 and R2 can be selected from hydrogen or alkyl, or alternatively, the
substructure has structure:
where A is an aryl ring or a carbocyclyl containing the depicted double bond.
[0031] It is to be understood that certain radical naming conventions can include either a
mono-radical or a di-radical, depending on the context. For example, where a substituent
requires two points of attachment to the rest of the molecule, it is understood that the
substituent is a di-radical. For example, a substituent identified as alkyl that requires two
points of attachment includes di-radicals such as —CH2—, —CH2CH2—, —CH2CH(CH3)CH2—,
and the like. Other radical naming conventions clearly indicate that the radical is a di-radical
such as “alkylene” or “alkenylene.”
[0032] The term “halogen” or “halo,” as used herein, means any one of the radio-stable
atoms of column 7 of the Periodic Table of the Elements, e. g., fluorine, chlorine, bromine, or
iodine, with fluorine and chlorine being preferred.
[0033] As used herein, “Ca to C1,” in which “a” and “b” are integers refer to the number of
carbon atoms in an alkyl, alkenyl or alkynyl group, or the number of ring atoms of a
cycloalkyl or aryl group. That is, the alkyl, the alkenyl, the alkynyl, the ring of the
(C 77
cycloalkyl, and ring of the aryl can contain from a to “b” inclusive, carbon atoms. For
example, a “C1 to C4 alkyl” group refers to all alkyl groups having from 1 to 4 carbons, that
is, CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)- and
(CH3)3C-, a C3 to C4 cycloalkyl group refers to all cycloalkyl groups having from 3 to 4
carbon atoms, that is, cyclopropyl and cyclobutyl. Similarly, a “4 to 6 membered
heterocyclyl” group refers to all heterocyclyl groups with 4 to 6 total ring atoms, for example,
azetidine, oxetane, oxazoline, pyrrolidine, piperidine, piperazine, morpholine, and the like. If
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no “a” and “b” are designated with regard to an alkyl, alkenyl, alkynyl, cycloalkyl, or aryl
group, the broadest range described in these definitions is to be assumed. As used herein, the
term “C1-C6” includes C1, C2, C3, C4, C5 and C6, and a range defined by any of the two
numbers, For example, C1-C6 alkyl includes C1, C2, C3, C4, C5 and C6 alkyl, C2-C6 alkyl, C1-
C3 alkyl, etc. Similarly, C2-C6 alkenyl includes C2, C3, C4, C5 and C6 alkenyl, C2-C5 alkenyl,
C3-C4 alkenyl, etc, and C2-C6 alkynyl includes C2, C3, C4, C5 and C6 alkynyl, C2-C5 alkynyl,
C3-C4 alkynyl, etc. C3-Cs cycloalkyl each includes hydrocarbon ring containing 3, 4, 5, 6, 7
and 8 carbon atoms, or a range defined by any of the two numbers, such as C3-C7 cycloalkyl
or C5-C6 cycloalkyl.
[0034] As used herein, “alkyl” refers to a straight or branched hydrocarbon chain that is fiilly
saturated (i.e., contains no double or triple bonds). The alkyl group may have 1 to 20 carbon
atoms (whenever it appears herein, a numerical range such as “1 to 20” refers to each integer
in the given range, e.g., “l to 20 carbon atoms” means that the alkyl group may consist of 1
carbon atom, 2 carbon atoms, 3 carbon atoms, eZc., up to and including 20 carbon atoms,
although the present definition also covers the occurrence of the term “alkyl” where no
numerical range is designated). The alkyl group may also be a medium size alkyl having 1 to
9 carbon atoms. The alkyl group could also be a lower alkyl having 1 to 6 carbon atoms.
The alkyl group may be designated as “C1-C4alkyl” or similar designations. By way of
example only, “C1-C6 alkyl” indicates that there are one to six carbon atoms in the alkyl
chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso-
propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are in no
way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl,
and the like.
[0035] As used herein, “alkoxy” refers to the formula —OR wherein R is an alkyl as is
defined above, such as “C1-C9 alkoxy”, including but not limited to methoxy, ethoxy, n-
propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy,
and the like.
[0036] As used herein, “alkenyl” refers to a straight or branched hydrocarbon chain
containing one or more double bonds. The alkenyl group may have 2 to 20 carbon atoms,
although the present definition also covers the occurrence of the term “alkenyl” where no
numerical range is designated. The alkenyl group may also be a medium size alkenyl having
2 to 9 carbon atoms. The alkenyl group could also be a lower alkenyl having 2 to 6 carbon
atoms. The alkenyl group may be designated as “C2-C6 alkenyl” or similar designations. By
way of example only, “C2-C6 alkenyl” indicates that there are two to six carbon atoms in the
W0 2020/ 136170 PCT/EP2019/086926
alkenyl chain, i.e., the alkenyl chain is selected from the group consisting of ethenyl, propen-
l-yl, propen-2-yl, propen-3-yl, buten-l-yl, buten-2-yl, buten-3-yl, buten-4-yl, l-methyl-
propen-l-yl, 2-methyl-propen-l-yl, 1-ethyl-ethen-1-yl, 2-methyl-propen-3-yl, buta-l,3-
dienyl, buta-1,2,-dienyl, and buta-1,2-dien-4-yl. Typical alkenyl groups include, but are in no
way limited to, ethenyl, propenyl, butenyl, pentenyl, and hexenyl, and the like.
[0037] As used herein, “alkynyl” refers to a straight or branched hydrocarbon chain
containing one or more triple bonds. The alkynyl group may have 2 to 20 carbon atoms,
although the present definition also covers the occurrence of the term “alkynyl” where no
numerical range is designated. The alkynyl group may also be a medium size alkynyl having
2 to 9 carbon atoms. The alkynyl group could also be a lower alkynyl having 2 to 6 carbon
atoms. The alkynyl group may be designated as “C2-C6 alkynyl” or similar designations. By
way of example only, “C2.C6 alkynyl” indicates that there are two to six carbon atoms in the
alkynyl chain, i.e., the alkynyl chain is selected from the group consisting of ethynyl, propyn-
l-yl, propyn-2-yl, butyn-l-yl, butyn-3-yl, butyn-4-yl, and 2-butynyl. Typical alkynyl groups
include, but are in no way limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl, and
the like.
[0038] As used herein, “heteroalkyl” refers to a straight or branched hydrocarbon chain
containing one or more heteroatoms, that is, an element other than carbon, including but not
limited to, nitrogen, oxygen and sulfur, in the chain backbone. The heteroalkyl group may
have 1 to 20 carbon atoms, although the present definition also covers the occurrence of the
term “heteroalkyl” where no numerical range is designated. The heteroalkyl group may also
be a medium size heteroalkyl having 1 to 9 carbon atoms. The heteroalkyl group could also
be a lower heteroalkyl having 1 to 6 carbon atoms. The heteroalkyl group may be designated
as “C1.Cs heteroalkyl” or similar designations. The heteroalkyl group may contain one or
more heteroatoms. By way of example only, “C4.C6 heteroalkyl” indicates that there are four
to six carbon atoms in the heteroalkyl chain and additionally one or more heteroatoms in the
backbone of the chain.
[0039] The term “aromatic” refers to a ring or ring system having a conjugated pi electron
system and includes both carbocyclic aromatic (e.g., phenyl) and heterocyclic aromatic
groups (e.g., pyridine). The term includes monocyclic or fused-ring polycyclic (i.e., rings
which share adjacent pairs of atoms) groups provided that the entire ring system is aromatic.
[0040] As used herein, “aryl” refers to an aromatic ring or ring system (i.e., two or more
fused rings that share two adjacent carbon atoms) containing only carbon in the ring
backbone. When the aryl is a ring system, every ring in the system is aromatic. The aryl
W0 2020/ 136170 PCT/EP2019/086926
group may have 6 to 18 carbon atoms, although the present definition also covers the
occurrence of the term “aryl” where no numerical range is designated. In some
embodiments, the aryl group has 6 to 10 carbon atoms. The aryl group may be designated as
“C6-C1o aryl,” “C6 or C10 aryl,” or similar designations. Examples of aryl groups include, but
are not limited to, phenyl, naphthyl, azulenyl, and anthracenyl.
[0041] An “aralkyl” or “arylalkyl” is an aryl group connected, as a substituent, Via an
alkylene group, such as “C744 aralkyl” and the like, including but not limited to benzyl, 2-
phenylethyl, 3-phenylpropyl, and naphthylalkyl. In some cases, the alkylene group is a lower
alkylene group (i.e., a C1-C6 alkylene group).
[0042] As used herein, “heteroaryl” refers to an aromatic ring or ring system (i.e., two or
more fused rings that share two adjacent atoms) that contain(s) one or more heteroatoms, that
is, an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur, in
the ring backbone. When the heteroaryl is a ring system, every ring in the system is aromatic.
The heteroaryl group may have 5-18 ring members (ie., the number of atoms making up the
ring backbone, including carbon atoms and heteroatoms), although the present definition also
covers the occurrence of the term “heteroaryl” where no numerical range is designated. In
some embodiments, the heteroaryl group has 5 to 10 ring members or 5 to 7 ring members.
The heteroaryl group may be designated as “5-7 membered heteroaryl,” “5-10 membered
heteroaryl,” or similar designations. Examples of heteroaryl rings include, but are not limited
to, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl,
isothiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,
quinolinyl, isoquinlinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl,
and benzothienyl.
[0043] A “heteroaralkyl” or “heteroarylalkyl” is heteroaryl group connected, as a substituent,
Via an alkylene group. Examples include but are not limited to 2-thienylmethyl, 3-
thienylmethyl, furylmethyl, thienylethyl, pyrrolylalkyl, pyridylalkyl, isoxazollylalkyl, and
imidazolylalkyl. In some cases, the alkylene group is a lower alkylene group (i.e., a C1-C6
alkylene group).
[0044] As used herein, “carbocyclyl” means a non-aromatic cyclic ring or ring system
containing only carbon atoms in the ring system backbone. When the carbocyclyl is a ring
system, two or more rings may be joined together in a fiised, bridged or spiro-connected
fashion. Carbocyclyls may have any degree of saturation provided that at least one ring in a
ring system is not aromatic. Thus, carbocyclyls include cycloalkyls, cycloalkenyls, and
cycloalkynyls. The carbocyclyl group may have 3 to 20 carbon atoms, although the present
W0 2020/ 136170 PCT/EP2019/086926
definition also covers the occurrence of the term “carbocyclyl” where no numerical range is
designated. The carbocyclyl group may also be a medium size carbocyclyl having 3 to 10
carbon atoms. The carbocyclyl group could also be a carbocyclyl having 3 to 6 carbon
atoms. The carbocyclyl group may be designated as “C3.C6 carbocyclyl” or similar
designations. Examples of carbocyclyl rings include, but are not limited to, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicycle[2.2.2]octanyl,
adamantyl, and spiro[4.4]nonanyl.
[0045] As used herein, “cycloalkyl” means a fully saturated carbocyclyl ring or ring system.
Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
[0046] As used herein, “heterocyclyl” means a non-aromatic cyclic ring or ring system
containing at least one heteroatom in the ring backbone. Heterocyclyls may be joined
together in a fused, bridged or spiro-connected fashion. Heterocyclyls may have any degree
of saturation provided that at least one ring in the ring system is not aromatic. The
heteroatom(s) may be present in either a non-aromatic or aromatic ring in the ring system.
The heterocyclyl group may have 3 to 20 ring members (i.e., the number of atoms making up
the ring backbone, including carbon atoms and heteroatoms), although the present definition
also covers the occurrence of the term “heterocyclyl” where no numerical range is
designated. The heterocyclyl group may also be a medium size heterocyclyl having 3 to 10
ring members. The heterocyclyl group could also be a heterocyclyl having 3 to 6 ring
members. The heterocyclyl group may be designated as “3-6 membered heterocyclyl” or
similar designations. In preferred six membered monocyclic heterocyclyls, the heteroatom(s)
are selected from one up to three of O, N or S, and in preferred five membered monocyclic
heterocyclyls, the heteroatom(s) are selected from one or two heteroatoms selected from O,
N, or S. Examples of heterocyclyl rings include, but are not limited to, azepinyl, acridinyl,
carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl,
oxepanyl, thiepanyl, piperidinyl, piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl,
pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxanyl, 1,4-
dioxinyl, 1,4-dioxanyl, 1,3-oxathianyl, 1,4-oxathiinyl, 1,4-oxathianyl, 2H-1,2-oxazinyl,
trioxanyl, hexahydro-1,3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxolanyl, 1,3-dithiolyl, 1,3-
dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazolidinonyl,
thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, indolinyl, isoindolinyl, tetrahydrofuranyl,
tetrahydropyranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl,
thiamorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, and tetrahydroquinoline.
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[0047] An “O-carboxy” group refers to a “-OC(=O)R” group in which R is selected from
hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3.C7 carbocyclyl, C6-C10 aryl, 5-10
membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.
[0048] A “C-carboxy” group refers to a “-C(=O)OR” group in which R is selected from the
group consisting of hydrogen, C1-C6 alkyl, C2-C5 alkenyl, C2-C6 alkynyl, C3.C7 carbocyclyl,
C6.C1o aryl, 5-l0 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.
A non-limiting example includes carboxyl (i.e., -C(=O)OH).
[0049] A “sulfonyl” group refers to an “-SO2R” group in which R is selected from hydrogen,
C1-C6 alkyl, C2.C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclyl, C6.C10 aryl, 5-10 membered
heteroaryl, and 3-10 membered heterocyclyl, as defined herein.
[0050] A “sulfino” group refers to a “-S(=O)OH” group.
[0051] A “S-sulfonamido” group refers to a “-SO2NRARB” group in which RA and R3 are
each independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7
carbocyclyl, C6-C10 aryl, 5-l0 membered heteroaryl, and 3-10 membered heterocyclyl, as
defined herein.
[0052] An “N-sulfonamido” group refers to a “-N(RA)SO2RB” group in which RA and Rb are
each independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7
carbocyclyl, C6-C10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as
defined herein.
[0053] A “C-amido” group refers to a “-C(=O)NRARB” group in which RA and R3 are each
independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2.C6 alkynyl, C3.C7
carbocyclyl, C6-C10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as
defined herein.
[0054] An “N-amido” group refers to a “-N(RA)C(=O)RB” group in which RA and Rs are
each independently selected from hydrogen, Cl-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7
carbocyclyl, C6—C10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as
defined herein.
[0055] An “amino” group refers to a “-NRARB” group in which RA and R3 are each
independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2.C6 alkynyl, C3.C7
carbocyclyl, C6-C10 aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as
defined herein. A non-limiting example includes free amino (i.e., -NH2).
[0056] An “aminoalkyl” group refers to an amino group connected Via an alkylene group.
[0057] An “alkoxyalkyl” group refers to an alkoxy group connected Via an alkylene group,
such as a “C2-Cg alkoxyalkyl” and the like.
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[0058] As used herein, a substituted group is derived from the unsubstituted parent group in
which there has been an exchange of one or more hydrogen atoms for another atom or group.
Unless otherwise indicated, when a group is deemed to be “substituted,” it is meant that the
group is substituted with one or more substituents independently selected from C1-C6 alkyl,
C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 heteroalkyl, C3-C7 carbocyclyl (optionally substituted
with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), C3-C7-
carbocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6
haloalkyl, and C1-C6 haloalkoxy), 3-10 membered heterocyclyl (optionally substituted with
halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 3-10 membered
heterocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6
haloalkyl, and C1-C6 haloalkoxy), aryl (optionally substituted with halo, C1-C6 alkyl, C1-C6
alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), aryl(C1-C6)alkyl (optionally substituted with
halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 5-10 membered
heteroaryl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and
C1-C6 haloalkoxy), 5-10 membered heteroaryl(C1-C6)alkyl (optionally substituted with halo,
C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), halo, -CN, hydroxy, C1-
C6 éilkoxy, C1-C6 §lkoxy(C1-C6)alkyl (i.e., ether), aryloxy, sulfhydryl (mercapto), halo(C1-
C6)alkyl (e.g., —CF3), halo(C1-C6)all<oxy (e.g., —OCF3), C1-C6 alkylthio, arylthio, amino,
amino(C1-C6)alkyl, nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-
amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, acyl, cyanato,
isocyanato, thiocyanato, isothiocyanato, sulfinyl, sulfonyl, -SO3H, sulfino, -OSO2C1—4alkyl,
and oxo (=0). Wherever a group is described as “optionally substituted” that group can be
substituted with the above substituents.
[0059] The term “hydroxy” as used herein refers to a —OH group.
[0060] The term “cyano” group as used herein refers to a “-CN” group.
[0061] The term “azido” as used herein refers to a —N3 group.
[0062] As used herein, a “nucleotide” includes a nitrogen containing heterocyclic base, a
sugar, and one or more phosphate groups. They are monomeric units of a nucleic acid
sequence. In RNA, the sugar is a ribose, and in DNA a deoxyribose, tie. a sugar lacking a
hydroxyl group that is present in ribose. The nitrogen containing heterocyclic base can be
purine or pyrimidine base. Purine bases include adenine (A) and guanine (G), and modified
derivatives or analogs thereof Pyrimidine bases include cytosine (C), thymine (T), and uracil
(U), and modified derivatives or analogs thereof. The C-l atom of deoxyribose is bonded to
N-1 of a pyrimidine or N-9 of a purine.
W0 2020/ 136170 PCT/EP2019/086926
[0063] As used herein, a “nucleoside” is structurally similar to a nucleotide, but is missing
the phosphate moieties. An example of a nucleoside analogue would be one in which the
label is linked to the base and there is no phosphate group attached to the sugar molecule. The
term “nucleoside” is used herein in its ordinary sense as understood by those skilled in the
art. Examples include, but are not limited to, a ribonucleoside comprising a ribose moiety and
a deoxyribonucleoside comprising a deoxyribose moiety. A modified pentose moiety is a
pentose moiety in which an oxygen atom has been replaced with a carbon and/or a carbon has
been replaced with a sulfur or an oxygen atom. A “nucleoside” is a monomer that can have a
substituted base and/or sugar moiety. Additionally, a nucleoside can be incorporated into
larger DNA and/or RNA polymers and oligomers.
[0064] The term “purine base” is used herein in its ordinary sense as understood by those
skilled in the art, and includes its tautomers. Similarly, the term “pyrimidine base” is used
herein in its ordinary sense as understood by those skilled in the art, and includes its
tautomers. A non-limiting list of optionally substituted purine-bases includes purine,
adenine, guanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (eg. 7-
methylguanine), theobromine, caffeine, uric acid and isoguanine. Examples of pyrimidine
bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil and 5-
alkylcytosine (e. g., 5-methylcytosine).
[0065] As used herein, when an oligonucleotide or polynucleotide is described as
“comprising” a nucleoside or nucleotide described herein, it means that the nucleoside or
nucleotide described herein forms a covalent bond with the oligonucleotide or
polynucleotide. Similarly, when a nucleoside or nucleotide is described as part of an
oligonucleotide or polynucleotide, such as “incorporated into” an oligonucleotide or
polynucleotide, it means that the nucleoside or nucleotide described herein forms a covalent
bond with the oligonucleotide or polynucleotide. In some such embodiments, the covalent
bond is formed between a 3’ hydroxy group of the oligonucleotide or polynucleotide with the
' phosphate group of a nucleotide described herein as a phosphodiester bond between the 3'
carbon atom of the oligonucleotide or polynucleotide and the 5’ carbon atom of the
nucleotide.
[0066] As used herein, “derivative” or “analogue” means a synthetic nucleotide or nucleoside
derivative having modified base moieties and/or modified sugar moieties. Such derivatives
and analogs are discussed in, e.g., Scheit, Nucleotide Analogs (John Wiley & Son, 1980) and
Uhlman er al., Chemical Reviews 90:543-584, 1990. Nucleotide analogs can also comprise
modified phosphodiester linkages, including phosphorothioate, phosphorodithioate, alkyl-
W0 2020/ 136170 PCT/EP2019/086926
77 CC
phosphonate, phosphoranilidate and phosphoramidate linkages. “Derivative , analog” and
"modified" as used herein, may be used interchangeably, and are encompassed by the terms
“nucleotide” and “nucleoside” defined herein.
[0067] As used herein, the term “phosphate” is used in its ordinary sense as understood by
those skilled in the art, and includes its protonated forms (for example,
0' and OH ). As used herein, the terms “monophosphate,”
“diphosphate,” and “triphosphate” are used in their ordinary sense as understood by those
skilled in the art, and include protonated forms.
[0068] The terms “protecting group” and “protecting groups” as used herein refer to any
atom or group of atoms that is added to a molecule in order to prevent existing groups in the
molecule from undergoing unwanted chemical reactions. Sometimes, “protecting group” and
“blocking group” can be used interchangeably.
[0069] As used herein, the prefixes “photo” or “photo-” mean relating to light or
electromagnetic radiation. The term can encompass all or part of the electromagnetic
spectrum including, but not limited to, one or more of the ranges commonly known as the
radio, microwave, infrared, visible, ultraviolet, X-ray or gamma ray parts of the spectrum.
The part of the spectrum can be one that is blocked by a metal region of a surface such as
those metals set forth herein. Altematively, or additionally, the part of the spectrum can be
one that passes through an interstitial region of a surface such as a region made of glass,
plastic, silica, or other material set forth herein. In particular embodiments, radiation can be
used that is capable of passing through a metal. Alternatively, or additionally, radiation can
be used that is masked by glass, plastic, silica, or other material set forth herein.
[0070] As used herein, the term “phasing” refers to a phenomenon in SBS that is caused by
incomplete removal of the 3’ terminators and fluorophores, and failure to complete the
incorporation of a portion of DNA strands within clusters by polymerases at a given
sequencing cycle. Pre-phasing is caused by the incorporation of nucleotides without effective
3' terminators, wherein the incorporation event goes 1 cycle ahead due to a termination
failure. Phasing and pre-phasing cause the measured signal intensities for a specific cycle to
consist of the signal from the current cycle as well as noise from the preceding and following
cycles. As the number of cycles increases, the fraction of sequences per cluster affected by
phasing and pre-phasing increases, hampering the identification of the correct base. Pre-
phasing can be caused by the presence of a trace amount of unprotected or unblocked 3’-OH
W0 2020/ 136170 PCT/EP2019/086926
nucleotides during sequencing by synthesis (SBS). The unprotected 3’-OH nucleotides could
be generated during the manufacturing processes or possibly during the storage and reagent
handling processes. Accordingly, the discovery of nucleotide analogues which decrease the
incidence of pre-phasing is surprising and provides a great advantage in SBS applications
over existing nucleotide analogues. For example, the nucleotide analogues provided can
result in faster SBS cycle time, lower phasing and pre-phasing values, and longer sequencing
read lengths.
3’-Hydroxv Acetal Blocking Groups
[0071] Some embodiments of the present disclosure relate to a nucleotide or nucleoside
molecule comprising a ribose or deoxyribose having a removable 3’-OH protecting or
R13 R23
}{ /i\ /i\ 3
o Rmo R2bR
atom, wherein:
each R13 and Rlb is independently H, C1-C6 alkyl, C1.Cs haloalkyl, C1-Cs alkoxy, C1-C6
blocking group forming a structure covalently attached to the 3'-carbon
haloalkoxy, cyano, halogen, optionally substituted phenyl, or optionally substituted aralkyl,
each R23 and Rzb is independently H, C1-Cs alkyl, Cl-C6 haloalkyl, cyano, or halogen,
alternatively R13 and R2‘‘ together with the atoms to which they are attached form an
optionally substituted five to eight membered heterocyclyl group;
R3 is H, optionally substituted C2-C6 alkenyl, optionally substituted C3-C7 cycloalkenyl,
optionally substituted C2.Cs alkynyl, or optionally substituted (C1.Cs alkylene)Si(R4)3; and
each R4 is independently H, C1-C6 alkyl, or optionally substituted C6-C10 aryl, provided that
when each R1“, RH’, R23‘ and R2” is H, then R3 is not H.
[0072] Some further embodiments of the present disclosure relate to a compound having the
structure of Formula (I):
R'O B
RuR2a
o 0 R3
R13 R“) Rzb (I), wherein R’ is H, monophosphate, di-phosphate, tri-phosphate,
thiophosphate, a phosphate ester analog, -0- attached to a reactive phosphorous containing
group, or —O— protected by a protecting group; R" is H or OH; B is a nucleobase; each of R”,
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<’N I \”
N N;
R11’, R23, R21’ and R3 is defined above. In some further embodiment, B is“"~'«» ,
NH NW
NH’ \2 O 0 J11: N NH 1N N NH2
\N | N NH NH < | Y 2 | Y
/ I ,) N’1*o 1 /K 1 /K \N N“ \ NH
N N | N O N O
*4» “’I""‘ “LN “In” , O or 0 . In some
7 7 7
further embodiments, the nucleobase is covalently bounded to a detectable label (e.g., a
Dye‘
L NH2
/ I /J
fluorescent dye), optionally through a linker, for example, B is "‘''w ,
O Pye
NH O L
Dye\L 2 Dye\L
N’1*o NAG H2NJ\\N N\
“V” , «w» ,or NW" .In some such embodiments, R’ is
triphosphate. In some such embodiment, R" is H.
[0073] In some embodiments of the acetal blocking group described herein, at least one of
R1’ and R11’ is H. In some such embodiments, each R1’ and R11’ is H. In some other
embodiments, at least one of R1’ and R11’ is C1-C6 alkyl, for example, methyl, ethyl, isopropyl
or t-butyl. In some embodiments, each of R21‘ and R21’ is independently H, halogen or C1-C6
alkyl. In some such embodiments, at least one of R2’ and R21’ is H or C1-C6 alkyl. In some
such embodiment, each R23 and R21’ is H. In some such embodiments, each R2“ and R21’ is C1-
C6 alkyl, for example methyl, ethyl, isopropyl or t-butyl. In one embodiment, each R2’ and
R21’ is methyl. In some such embodiments, each R23 and R21’ is independently C1-C6 alkyl or
halogen. In some such embodiments, R2“ is H, and R21’ is halogen or C1-C6 alkyl.
[0074] In some embodiments of the acetal blocking group described herein, R3 is optionally
substituted C2-Cs alkenyl. In some such embodiments, R3 is C2-C6 alkenyl (for example, vinyl,
propenyl) optionally substituted with one or more substituents independently selected from
the group consisting of halogen, C1-C6 alkyl, C1-Cs haloalkyl, and combinations thereof. In
If /
31/ ;e:/\, ;e‘\/\/’ 3:/\F,
some further embodiments, R3 is
W0 2020/ 136170 PCT/EP2019/086926
F C" ~ 25/ 2% ;s / ;s /
};\%\C|, ;"‘\2\F ,}}\2\C|, F , CI , \K\F, d/\C|,
F CI F
}<%\F }5‘\K\C| 3‘{W2\F
, or F . In some other embodiments, R3 is optionally
substituted C2-C6 alkynyl. In some such embodiments, R3 is C2-C6 alkynyl (e.g., ethynyl,
propynyl) optionally substituted with one or more substituents independently selected from
the group consisting of halogen, C1-C6 alkyl, C1'C6 haloalkyl, and combinations thereof. In
one embodiment, R3 is optionally substituted ethynyl (231%). In some other embodiments,
R3 is optionally substituted (C1.C6 alkylene)Si(R4)3. In some such embodiments, at least one
of R4 is Ci-4 alkyl. In some further embodiments, each one of R4 is Ci.C4 alkyl, for example,
methyl, ethyl, isopropyl or t-butyl. In one embodiment, R3 is
—(CH2)-SiMe3. In some alternative embodiments, R3 is C1-C6 alkyl.
[0075] In some alternative embodiments, R” and R3” together with the atoms to which they
are attached form a five to seven membered heterocyclyl. In some such embodiments, R13 and
R33 together with the atoms to which they are attached form a six membered heterocyclyl. In
some such embodiments, the six membered heterocyclyl group has the
0 R . In some further embodiments, at least one of each R"’, R31’ and R3
structureR1b
is H. In some other embodiments, at least one of each Rlb, R3" and R3 is C1-C6 alkyl. In one
embodiment, each R“’, R31’ and R3 is H.
[0076] In some further embodiments, the compound of Formula (I) is also represented by
Formula (Ia):
R'O B
Ru R C
O O /
R13 R1b 2b R281 2C 2d_ .
R (Ia), where each R and R is independently H, halogen (e.g., fluoro,
chloro), C1-C6 alkyl (e.g., methyl, ethyl, or isopropyl), or C1-C5 haloalkyl (e.g., -CHF2,
-CH2F, or —CF3). In some such embodiments, one of R” and Rlb is H. In some such
embodiments, each R” and R” is H. In some other embodiments, at least one of R13 and R”
is Ci-C6 alkyl, for example, methyl, ethyl, isopropyl or t-butyl. In some embodiments, each of
W0 2020/ 136170 PCT/EP2019/086926
R” and R21’ is independently H, halogen or C1-C6 alkyl. In some such embodiment, each R”
and R21’ is H. In some such embodiments, each of R” and R“ is independently H, halogen or
C1-C6 alkyl. In some such embodiments, each R“ and R2“ is C1-C6 alkyl, for example methyl,
ethyl, isopropyl or t-butyl. In one embodiment, each R” and R2“ is methyl. In some such
embodiments, each R2° and R2“ is independently halogen. In some such embodiments, RZC is
H, and R2“ is H, halogen (fluoro, chloro) or Cl'C6 alkyl (e.g., methyl, ethyl, isopropyl or t-
butyl). In further embodiments, each R” and R1” is H; R“ is H; Rzb is H, halogen or methyl;
R” is H, and R2“ is H, halogen, methyl, ethyl, isopropyl or t-butyl.
[0077] Non-limiting embodiments of the blocking groups described herein including those
having the structure selected from the group consisting of:
'9{O/\O/\]/
j\O/\O/\/ (ADM), ',iO/\O/\/\/ (eAOM)’ GAOM)’
50/\O/\%(Pr0M), \O/\O 7(DPr0M), ‘£0 0 (THP), and
S" M
§‘O/\O/\/ K e)3 (SEM), covalently attached to the 3’-carbon of the ribose or
deoxyribose.
3'-H drox Thiocarbamate Blockin Grou s
[0078] Some additional embodiments of the present disclosure relate to a nucleoside or
nucleotide comprising a ribose or deoxyribose having a removable 3’-OH blocking group
JV\IIV\o
forming a structure NR5R6 covalently attached to the 3'-carbon atom, wherein:
each of R5 and R6 is independently H, C1-C6 alkyl, C2-Cs alkenyl, C2-Cs alkynyl, C1-Cs
haloalkyl, C2.Cs alkoxyalkyl, optionally substituted —(CH2)m—phenyl, optionally substituted
—(CH2)n—(5 or 6 membered heteroaryl), optionally substituted —(CH2)k~C3-C7 carbocyclyl, or
optionally substituted —(CH2)p—(3 to 7 membered heterocyclyl);
alternatively, R5 and R6 together with the atoms to which they are attached form an optionally
substituted five to seven membered heterocyclyl,
each of —(CH2)m—, —(CH2)n—, —(CH2)k—, and —(CH2)p— is optionally substituted; and
each of m, n, k, and p is independently O, 1, 2, 3, or 4.
[0079] Some additional embodiments relate to a compound of Formula (II):
W0 2020/ 136170 PCT/EP2019/086926
R'O B
S 0 RH
NR5R6 (II), wherein R’ is H, monophosphate, di-phosphate, tri-phosphate,
thiophosphate, a phosphate ester analog, -0- attached to a reactive phosphorous containing
group, or —O— protected by a protecting group; R" is H or OH; B is a nucleobase, each of R5
NH2 NH2 (Ml
N \ \
N / N
(NE: W0
and R6 is defined above. In some further embodiment,Bis"”"n» ,“‘'*''I» , “‘“I"" ,
O 0 if N NH if
N 2 N N NH
I NH N NH <\ | Y I Y 2
/K /K N NH \ NH
'l' O 'T‘ 0
NW NW 0 or O . In some further embodiments,
the nucleobase is covalently bounded to a detectable label (e.g., a fluorescent dye), optionally
Dye‘ NH O
L NH2 Dye\L 2
,N N’ W0 N 0
through a linker, for example, B is M» , N.“ , «w» or
*1 » \
H2N \N N\
M“ . In some such embodiments, R’ is triphosphate. In some such
embodiment, R" is H.
[0080] In some embodiments of the thiocarbamate blocking group described herein, at least
one of R5 and R6 is H. In some such embodiments, each R5 and R6 is H. In some such
embodiments, R5 is H and R6 is C1-C5 alkyl, for example, methyl, ethyl, isopropyl or t-butyl.
In some such embodiments, R5 is H and R6 is C2-C5 alkenyl (for example, Vinyl or allyl) or
C2-C6 alkynyl (for example, ethynyl or propynyl). In some such embodiments, R5 is H and R2
is optionally substituted —(CH2)m—phenyl, optionally substituted —(CH2)n—(5 or 6 membered
heteroaryl), optionally substituted —(CH2)k~C3-C7 carbocyclyl, or optionally substituted —
(CH2)p—(3 to 7 membered heterocyclyl). In some further embodiment, the C3.C7 carbocyclyl
group may be a C3.C7 cycloalkyl or C3-C7 cycloalkenyl. The 3 to 7 membered heterocyclyl
group may comprise zero or one double bond in the ring structure. In further embodiments,
W0 2020/ 136170 PCT/EP2019/086926
R5 is H and R6 is optionally substituted —(CH2)m—phenyl, optionally substituted —(CH2)n~6
membered heteroaryl, optionally substituted —(CH2)k~C5 or C6 carbocyclyl, or optionally
substituted —(CH2)p—(5 or 6 membered heterocyclyl). In some embodiments, m, n, k, or p is
O. In other embodiments, m, n, k or p is l or 2. In some other embodiments, at least one of
R5 and R6 is C1-C5 alkyl, for example, methyl, ethyl, isopropyl or t-butyl. In some further
embodiments, both R5 and R6 is are C1'C6 alkyl. In one embodiment, both R5 and R6 is are
methyl.
[0081] In some alternative embodiments, R5 and R6 together with the atoms to which they are
attached form an optionally substituted five to seven membered heterocyclyl. In some such
embodiments, R5 and R6 together with the atoms to which they are attached form an
optionally substituted piperidinyl.
[0082] Non-limiting embodiments of the 3'-O-thiocarbamate blocking groups described
herein including those having the structure selected from the group consisting of:
JL /
SOJL ndgo N
I (DMTC) covalently attached to the 3'-carbon of the
gO)kN/
2, H
ribose or deoxyribose.
NH a
[0083] Additional embodiments of the present disclosure relate to an oligonucleotide or a
polynucleotide comprising a nucleoside or nucleotide described herein.
[0084] In any of the embodiments of the blocking groups described herein, when a group is
described as “optionally substituted” it may be either unsubstituted or substituted.
[0085] In any embodiments of the nucleotides or nucleosides with the 3' hydroxy blocking
group described herein, the nucleoside or nucleotide may be covalently attached to a
detectable label (for example, a fluorophore), optionally via a linker. The linker may be
cleavable or non-cleavable. In some such embodiments, the detectable label (e.g.,
fluorophore) is covalently attached to the nucleobase of the nucleoside or nucleotide via a
cleavable linker. In some other embodiments, the detectable label (e.g., fluorophore) is
covalently attached to the 3’oxygen of the nucleoside or nucleotide via a cleavable linker. In
some further embodiments, such cleavable linker may comprise an azido moiety or a
disulfide moiety, an acetal moiety, or a thiocarbamate moiety. In some embodiments, the 3'
hydroxy blocking group and the cleavable linker (and the attached label) may be removed
under the same or substantially same chemical reaction conditions, for example, the blocking
group and the detectable label may be removed in a single chemical reaction. In other
W0 2020/ 136170 PCT/EP2019/086926
embodiments, the blocking group and the detectable labeled are removed in two separate
steps.
[0086] In some embodiments, the nucleotides or nucleosides described herein comprises 2’
deoxyribose. In some further aspects, the 2' deoxyribose contains one, two or three phosphate
groups at the 5’ position of the sugar ring. In some further aspect, the nucleotides described
herein are nucleotide triphosphate.
[0087] In some embodiments, the 3’ blocked nucleotides or nucleosides described herein
provide superior stability in solution during storage, or reagent handling during sequencing
applications, compared to the same nucleotides or nucleosides protected with a standard 3’-
OH blocking group disclosed in the prior art, for example, the 3'-O-azidomethyl protecting
group. For example, the acetal or thiocarbamate blocking groups disclosed herein may confer
at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%,
500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000%, 2500%, or 3000% improved
stability compare to an azidomethyl protected 3'-OH at the same condition for the same
period of time, thereby reducing the pre-phasing values and resulting in longer sequencing
read lengths. In some embodiments, the stability is measured at ambient temperature or a
temperature below ambient temperature (such as 4-10°C). In other embodiments, the
stability is measured at an elevated temperature, such as 40°C, 45°C, 50°C, 55°C, 60°C or
65°C. In some such embodiments, the stability is measured in solution in a basic pH
environment, e.g., at pH 9.0, 9.2, 9.4, 9.6, 9.8. or 10.0. In some such embodiments, the
stability is measured with or without the presence of an enzyme, such as a polymerase (e. g., a
DNA polymerase), a terminal deoxynucleotidyl transferase, or a reverse transcriptase.
[0088] In some embodiments, the 3’ blocked nucleotides or nucleosides described herein
provide superior deblocking rate in solution during the chemical cleavage step of the
sequencing applications, compared to the same nucleotides or nucleosides protected with a
standard 3'-OH blocking group disclosed in the prior art, for example, the 3’-O-azidomethyl
protecting group. For example, the acetal or thiocarbamate blocking groups disclosed herein
may confer at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%,
200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, or 2000% improved
deblocking rate compare to an azidomethyl protected 3'-OH using the standard deblocking
reagent (such as tris(hydroxypropyl)phosphine), thereby reducing the overall time for a
sequencing cycle. In some embodiments, the deblocking rate is measured at ambient
temperature or a temperature below ambient temperature (such as 4-10°C). In other
embodiments, the deblocking rate is measured at an elevated temperature, such as 40°C,
W0 2020/ 136170 PCT/EP2019/086926
45°C, 50°C, 55°C, 60°C or 65°C. In some such embodiments, the deblocking rate is
measured in solution in a basic pH environment, e.g., at pH 9.0, 9.2, 9.4, 9.6, 9.8. or 10.0. In
some such embodiments, the molar ratio of the deblocking reagent to substrate (ie., 3’
blocked nucleoside or nucleotide) is about 10: 1, about 5: 1, about 2:1 or about 1:1.
[0089] In some embodiments, a palladium deblocking reagent (e.g., Pd(0) is used to remove
the 3’ acetal blocking groups (e.g., AOM blocking group). Pd may forms a chelation complex
with the two oxygen atoms of the AOM group, as well as the double bond of the allyl group,
allowing the deblocking reagent in direct vicinity of the functionality to be removed and may
result in accelerated deblocking rate.
Deprotection of the 3’-OH Blocking Groups
[0090] The 3’-acetal blocking groups described herein may be removed or cleaved under
R13 R23
various chemical conditions. For acetal blocking groups R that contain a
vinyl or alkenyl moiety, non-limiting cleaving condition includes a Pd(II) complex, such as
Pd(OAc)2 or allylPd(II) chloride dimer, in the presence of a phosphine ligand, for example
tris(hydroxymethyl)phosphine (TH1\/IP), or tris(hydroxylpropyl)phosphine (THP or THPP),
For those blocking groups containing an alkynyl group (e.g., an ethynyl), they may also be
removed by a Pd(II) complex (e.g., Pd(OAc)2 or allyl Pd(II) chloride dimer) in the presence
of a phosphine ligand (e.g., THP or TH1\/IP).
Palladium Cleavage Reagents
[0091] In some embodiments, the acetal blocking group described herein may be cleaved by
a palladium catalyst. In some such embodiments, the Pd catalyst is water soluble. In some
such embodiments, is a Pd(0) complex (e. g., Tris(3,3 ’,3 "-
phosphinidynetris(benzenesulfonato)palladium(0) nonasodium salt nonahydrate). In some
instances, the Pd(0) complex may be generated in situ from reduction of a Pd(II) complex by
reagents such as alkenes, alcohols, amines, phosphines, or metal hydrides. Suitable
palladium sources include Na2PdCl4, Pd(CH3CN)2Cl2, (PdCl(C3H5))2, [Pd(C3H5)(THP)]Cl,
[Pd(C3H5)(THP)2]Cl, Pd(OAc)2, Pd(Ph3)4, Pd(dba)2, Pd(Acac)2, PdCl2(COD), and Pd(TFA)2.
In one such embodiment, the Pd(0) complex is generated in situ from Na2PdCl4. In another
embodiment, the palladium source is allyl palladium(II) chloride dimer [(PdCl(C3H5))2]. In
W0 2020/ 136170 PCT/EP2019/086926
some embodiments, the Pd(0) complex is generated in an aqueous solution by mixing a Pd(II)
complex with a phosphine. Suitable phosphines include water soluble phosphines, such as
tris(hydroxypropyl)phosphine (THP), tris(hydroxymethyl)phosphine (THIVIP), 1,3,5-triaza-7-
phosphaadamantane (PTA), bis(p-sulfonatophenyl)phenylphosphine dihydrate potassium salt,
tris(carboxyethyl)phosphine (TCEP), and triphenylphosphine-3,3’,3"-trisulfonic acid
trisodium salt.
[0092] In some embodiments, the Pd(0) is prepared by mixing a Pd(II) complex
[(PdCl(C3H5))2] with THP in situ. The molar ratio of the Pd(II) complex and the THP may be
about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some further embodiments, one or more
reducing agents may be added, such as ascorbic acid or a salt thereof (e.g., sodium ascorbate).
In some embodiments, the cleavage mixture may contain additional buffer reagents, such as a
primary amine, a secondary amine, a tertiary amine, a carbonate salt, a phosphate salt, or a
borate salt, or combinations thereof. In some further embodiments, the buffer reagent
comprises ethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine, sodium
carbonate, sodium phosphate, sodium borate, 2-dimethyalaminomethanol (DMEA), 2-
diethyalaminomethanol (DEEA), N,N,N',N'-tetramethylethylenediamine(TEMED), or
N,N,N’,N'-tetraethylethylenediamine (TEEDA), or combinations thereof In one embodiment,
the buffer reagent is DEEA. In another embodiment, the buffer reagent contains one or more
inorganic salts such as a carbonate salt, a phosphate salt, or a borate salt, or combinations
thereof. In one embodiment, the inorganic salt is a sodium salt.
[0093] Alternatively, alkynyl moiety containing blocking groups may also be cleaved in the
presence of (NH4)2MoS4. Other non-limiting cleaving condition for alkynyl moiety includes a
Cu(II) complex with THPTA ligand (tris(3-hydroxypropyltriazolylmethyl)amine), and
ascorbate. Non-limiting cleaving conditions for blocking groups containing a six-membered
heterocycle (e.g., tetrahydropyran) include cyclodextrin or Ln(OTf)3 (lanthanide triflate).
Non-limiting cleaving condition for blocking groups containing an alkylsilane group (e.g., -
CH2SiMe3) includes LiBF4 (lithium tetrafluoroborate). Other acetal blocking groups such as —
O(CH2)O-C1-C6 alkyl may be removed by LiBF4 or Bi(OTf)3 (bismuth triflate). Non-limiting
exemplary conditions for cleaving the described various blocking groups are illustrated in
Scheme 1 below.
Scheme 1. Illustration of 3’-Deblocking Conditions
W0 2020/ 136170 PCT/EP2019/086926
Dye+Linker
O» OH I H
0“ IP\_O‘F'>-o N o
lP\-O o .-
-o OH 0
3'-P OM-ffT .
r Ovoj 3—DPrOM—f’r'|' OVOV
Pd(OAc)2 or Pd(OAc)2 gr”
(||),THPTA,
THP (NH4)2MoS4 THP Ascorbate
ROH ROH ROH
0 o o
O\ Dye;L|_i|nker | NH O‘ Dye-I(-DL|_i|nker | NH O‘ Dye-IC-)L|_i|nker | NH
Q‘ \'P\_O‘||°—O N’i§o Q‘ \'P\_O‘|-'|’—O N’i§o Q‘ /P\_O‘F'>-o N)“
/P\-O 0' F /P\-O 0' F R-0 0' F
-0 OH 0 -0 OH 0 -0 OH 0
o o o
3'-THP-ftT 3'-SEM-ffT 3'-MOM-ffT
o o ovo oVo\
c I d xt' 0
W0 e ""9 LiBF4 M938‘ LiBF4 or Bi(OTf)3
Ln(OTf)3
ROH ROH ROH
R is the unprotected labeled nucleotide triphosphate with 3' OH
[0094] The 3’-O-thiocarbamate blocking groups described herein may be removed or cleaved
under various chemical conditions. Non-limiting exemplary conditions for cleaving the
thiocarbamate blocking groups described herein include NaIO4 and OXone® (potassium
peroxymonosulfate).
[0095] In addition, the azido group in —CH2N3 can be converted to an amino group by
phosphine. Alternatively, the azido group in —CH2N3 may be converted to an amino group by
contacting such molecules with the thiols, in particular water-soluble thiols such as
dithiothreitol (DTT). In one embodiment, the phosphine is THP.
Compatibility with Linearization
[0096] In order to maximize the throughput of nucleic acid sequencing reactions it is
advantageous to be able to sequence multiple template molecules in parallel. Parallel
processing of multiple templates can be achieved with the use of nucleic acid array
W0 2020/ 136170 PCT/EP2019/086926
technology. These arrays typically consist of a high-density matrix of polynucleotides
immobilized onto a solid support material.
[0097] WO 98/44151 and W0 00/ 18957 both describe methods of nucleic acid amplification
which allow amplification products to be immobilized on a solid support in order to form
arrays comprised of clusters or “colonies” formed from a plurality of identical immobilized
polynucleotide strands and a plurality of identical immobilized complementary strands.
Arrays of this type are referred to herein as “clustered arrays.” The nucleic acid molecules
present in DNA colonies on the clustered arrays prepared according to these methods can
provide templates for sequencing reactions, for example as described in WO 98/44152. The
products of solid-phase amplification reactions such as those described in WO 98/44151 and
W0 00/ 18957 are so-called “bridged” structures formed by annealing of pairs of immobilized
polynucleotide strands and immobilized complementary strands, both strands being attached
to the solid support at the 5' end. In order to provide more suitable templates for nucleic acid
sequencing, it is preferred to remove substantially all or at least a portion of one of the
immobilized strands in the “bridged” structure in order to generate a template which is at
least partially single-stranded. The portion of the template which is single-stranded will thus
be available for hybridization to a sequencing primer. The process of removing all or a
portion of one immobilized strand in a “bridged” double-stranded nucleic acid structure is
referred to as “linearization.” There are various ways for linearization, including but not
limited to enzymatic cleavage, photo-chemical cleavage, or chemical cleavage. Non-limiting
examples of linearization methods are disclosed in PCT Publication No. WO 2007/010251,
U.S. Patent Publication No. 2009/0088327, U.S. Patent Publication No. 2009/0118128, and
U.S. Appl. 62/671,816, which are incorporated by reference in their entireties.
[0098] In some embodiments, the condition for deprotecting or removal of the 3'-OH
blocking groups is also compatible with the linearization processes. In some further
embodiments, the deprotection condition is compatible with a chemical linearization process
which comprises the use of a Pd complex and a phosphine, for example Pd(OAc)2 and THP.
In some embodiments, the Pd complex is a Pd(II) complex, which generates Pd(0) in situ in
the presence of the phosphine.
[0099] Unless indicated otherwise, the reference to nucleotides is also intended to be
applicable to nucleosides.
Labeled Nucleotides
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[0100] According to an aspect of the disclosure, the described 3’-OH blocked nucleotide also
comprises a detectable label and such nucleotide is called a labeled nucleotide. The label
(e.g., a fluorescent dye) can be conjugated via an optional linker by a variety of means
including hydrophobic attraction, ionic attraction, and covalent attachment. In some aspects,
the dyes are conjugated to the substrate by covalent attachment. More particularly, the
covalent attachment is by means of a linker group. In some instances, such labeled
nucleotides are also referred to as “modified nucleotides.”
[0101] Labeled nucleosides and nucleotides are useful for labeling polynucleotides formed
by enzymatic synthesis, such as, by way of non-limiting example, in PCR amplification,
isothermal amplification, solid phase amplification, polynucleotide sequencing (e.g., solid
phase sequencing), nick translation reactions and the like.
[0102] In some embodiments, the dye may be covalently attached to oligonucleotides or
nucleotides via the nucleotide base. For example, the labeled nucleotide or oligonucleotide
may have the label attached to the C5 position of a pyrimidine base or the C7 position of a 7-
deaza purine base through a linker moiety.
[0103] Unless indicated otherwise, the reference to nucleotides is also intended to be
applicable to nucleosides. The present application will also be further described with
reference to DNA, although the description will also be applicable to RNA, PNA, and other
nucleic acids, unless otherwise indicated.
[0104] In some embodiments described herein, the purine or pyrimidine base of the
nucleotide or nucleoside molecules described herein can be linked to a detectable label as
described above. In some such embodiments, the linkers used are cleavable. The use of a
cleavable linker ensures that the label can, if required, be removed after detection, avoiding
any interfering signal with any labeled nucleotide or nucleoside incorporated subsequently. In
some embodiments, the cleavable linker comprises an azido moiety, a —O'C2'C6 alkenyl
moiety (e.g., —O-allyl), a disulfide moiety, an acetal moiety (same or similar to the 3’acetal
blocking group described herein), or a thiocarbamate moiety (same or similar to the 3’acetal
blocking group described herein).
[0105] In some other embodiments, the linkers used are non-cleavable. Since in each
instance where a labeled nucleotide of the invention is incorporated, no nucleotides need to
be subsequently incorporated and thus the label need not be removed from the nucleotide.
W0 2020/ 136170 PCT/EP2019/086926
[0106] Cleavable linkers are known in the art, and conventional chemistry can be applied to
attach a linker to a nucleotide base and a label. The linker can be cleaved by any suitable
method, including exposure to acids, bases, nucleophiles, electrophiles, radicals, metals,
reducing or oxidizing agents, light, temperature, enzymes etc. The linker as discussed herein
may also be cleaved with the same catalyst used to cleave the 3’-O-blocking group bond.
Suitable linkers can be adapted from standard chemical protecting groups, as disclosed in
Greene & Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons. Further
suitable cleavable linkers used in solid-phase synthesis are disclosed in Guillier er al. (Chem.
Rev. 10012092-2157, 2000).
[0107] Where the detectable label is attached to the base, the linker can be attached at any
position on the nucleotide base provided that Watson-Crick base pairing can still be carried
out. In the context of purine bases, it is preferred if the linker is attached via the 7-position of
the purine or the preferred deazapurine analogue, via an 8-modified purine, via an N-6
modified adenosine or an N-2 modified guanine. For pyrimidines, attachment is preferably
via the 5-position on cytosine, thymidine or uracil and the N-4 position on cytosine.
[0108] In some embodiments, the linker can comprise a spacer unit. The length of the linker
is unimportant provided that the label is held a sufficient distance from the nucleotide so as
not to interfere with any interaction between the nucleotide and an enzyme, for example, a
polymerase.
[0109] In some embodiments, the linker may consist of the similar functionality as the 3'-OH
protecting group. This will make the deprotection and deprotecting process more efficient, as
only a single treatment will be required to remove both the label and the protecting group.
[0110] Use of the term “cleavable linker” is not meant to imply that the whole linker is
required to be removed. The cleavage site can be located at a position on the linker that
ensures that part of the linker remains attached to the dye and/or substrate moiety after
cleavage. Cleavable linkers may be, by way of non-limiting example, electrophilically
cleavable linkers, nucleophilically cleavable linkers, photocleavable linkers, cleavable under
reductive conditions (for example disulfide or azide containing linkers), oxidative conditions,
cleavable via use of safety-catch linkers and cleavable by elimination mechanisms. The use
of a cleavable linker to attach the dye compound to a substrate moiety ensures that the label
can, if required, be removed after detection, avoiding any interfering signal in downstream
steps.
[0111] Useful linker groups may be found in PCT Publication No. WOZOO4/018493 (herein
incorporated by reference), examples of which include linkers that may be cleaved using
W0 2020/ 136170 PCT/EP2019/086926
water-soluble phosphines or water-soluble transition metal catalysts formed from a transition
metal and at least partially water-soluble ligands, for example, a Pd(II) complex and THP. In
aqueous solution the latter form at least partially water-soluble transition metal complexes.
Such cleavable linkers can be used to connect bases of nucleotides to labels such as the dyes
set forth herein.
[0112] Particular linkers include those disclosed in PCT Publication No. WOZOO4/018493
(herein incorporated by reference) such as those that include moieties of the formulae:
*\X}\, *1/'\/\
\»x=
*\rlwr“/\
x\* 0
(wherein X is selected from the group comprising 0, S, NH and NQ wherein Q is a C1-1o
substituted or unsubstituted alkyl group, Y is selected from the group comprising 0, S, NH
and N(allyl), T is hydrogen or a C1-C10 substituted or unsubstituted alkyl group and *
indicates where the moiety is connected to the remainder of the nucleotide or nucleoside). In
some aspects, the linkers connect the bases of nucleotides to labels such as, for example, the
dye compounds described herein.
[0113] Additional examples of linkers include those disclosed in U.S. Publication No.
2016/0040225 (herein incorporated by reference), such as those include moieties of the
formulae:
;;:‘j9<S,s\/11:7,‘ 23%/\/yillfi E 3: x\/\/“Fr
WK K X = CH2, 0, s
The linker moieties illustrated herein may comprise the whole or partial linker structure
between the nucleotides/nucleosides and the labels.
[0114] Additional examples of linkers (“L”) include moieties of the formula:
W0 2020/ 136170 PCT/EP2019/086926
B/W?“ BMWW
B/\fiJJ\/Ojz/‘o/<jfifNM’\NHI=|
O n=1,2_3,4,5
B/\/\NJ]\/Oj/‘o NM”\NH—I=I
H n=1 2 3 4 5
z o ' - ' ' ,
\/N o N /Fl
o/\/ o ‘(v)’\NH
E/\ W2/\ O n=1,2, 3, 4,5
B\/\/N\n/\O/\/OW/\O/ \'rN\(\/)/\NH/Fl
0 Z O
” =1'2'3'4'5, wherein B is a nucleobase, Z
—N3 (azido), —O-C1-C6 alkyl, —O-C2-C6 alkenyl, or -0-C2-C6 alkynyl; and F1 comprises a
fluorescent label, which may contain additional linker structure. One of ordinary skill in the
art understands that label is covalently bounded to the linker by reacting a functional group of
the label (e. g., carboxyl) with a functional group of the linker (e.g., amino).
[0115] In particular embodiments, the length of the linker between a fluorescent dye
(fluorophore) and a guanine base can be altered, for example, by introducing a polyethylene
glycol spacer group, thereby increasing the fluorescence intensity compared to the same
fluorophore attached to the guanine base through other linkages known in the art. Exemplary
linkers and their properties are set forth in PCT Publication No. WO2007020457 (herein
incorporated by reference). The design of linkers, and especially their increased length, can
allow improvements in the brightness of fluorophores attached to the guanine bases of
guanosine nucleotides when incorporated into polynucleotides such as DNA. Thus, when the
dye is for use in any method of analysis which requires detection of a fluorescent dye label
attached to a guanine-containing nucleotide, it is advantageous if the linker comprises a
spacer group of formula —((CH2)2O)n—, wherein n is an integer between 2 and 50, as
described in WO 2007/020457.
[0116] Nucleosides and nucleotides may be labeled at sites on the sugar or nucleobase. As
known in the art, a “nucleotide” consists of a nitrogenous base, a sugar, and one or more
W0 2020/ 136170 PCT/EP2019/086926
phosphate groups. In RNA, the sugar is ribose and in DNA is a deoxyribose, i.e., a sugar
lacking a hydroxy group that is present in ribose. The nitrogenous base is a derivative of
purine or pyrimidine. The purines are adenine (A) and guanine (G), and the pyrimidines are
cytosine (C) and thymine (T) or in the context of RNA, uracil (U). The C-1 atom of
deoxyribose is bonded to N-1 of a pyrimidine or N-9 of a purine. A nucleotide is also a
phosphate ester of a nucleoside, with esterification occurring on the hydroxy group attached
to the C-3 or C-5 of the sugar. Nucleotides are usually mono, di- or triphosphates.
[0117] A “nucleoside” is structurally similar to a nucleotide but is
missing the phosphate moieties. An example of a nucleoside analog would be one in which
the label is linked to the base and there is no phosphate group attached to the sugar molecule.
[0118] Although the base is usually referred to as a purine or pyrimidine, the skilled person
will appreciate that derivatives and analogues are available which do not alter the capability
of the nucleotide or nucleoside to undergo Watson-Crick base pairing. “Derivative” or
“analogue” means a compound or molecule whose core structure is the same as, or closely
resembles that of a parent compound but which has a chemical or physical modification, such
as, for example, a different or additional side group, which allows the derivative nucleotide or
nucleoside to be linked to another molecule. For example, the base may be a deazapurine. In
particular embodiments, the derivatives should be capable of undergoing Watson-Crick
pairing. “Derivative” and "analogue" also include, for example, a synthetic nucleotide or
nucleoside derivative having modified base moieties and/or modified sugar moieties. Such
derivatives and analogues are discussed in, for example, Scheit, Nucleotide analogs (John
Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90:543-584, 1990. Nucleotide
analogues can also comprise modified phosphodiester linkages including phosphorothioate,
phosphorodithioate, alkyl-phosphonate, phosphoranilidate, phosphoramidate linkages and the
like.
[0119] A dye may be attached to any position on the nucleotide base, for example, through a
linker. In particular embodiments, Watson-Crick base pairing can still be carried out for the
resulting analog. Particular nucleobase labeling sites include the C5 position of a pyrimidine
base or the C7 position of a 7-deaza purine base. As described above a linker group may be
used to covalently attach a dye to the nucleoside or nucleotide.
[0120] In particular embodiments the labeled nucleoside or nucleotide may be enzymatically
incorporable and enzymatically extendable. Accordingly, a linker moiety may be of
sufficient length to connect the nucleotide to the compound such that the compound does not
significantly interfere with the overall binding and recognition of the nucleotide by a nucleic
W0 2020/ 136170 PCT/EP2019/086926
acid replication enzyme. Thus, the linker can also comprise a spacer unit. The spacer
distances, for example, the nucleotide base from a cleavage site or label.
[0121] Nucleosides or nucleotides labeled with the dyes described herein may have the
formula:
B-L-Dye
R...
where Dye is a dye compound; B is a nucleobase, such as, for example uracil, thymine,
cytosine, adenine, guanine and the like; L is an optional linker group which may or may not
be present; R‘ can be H, monophosphate, diphosphate, triphosphate, thiophosphate, a
phosphate ester analog, -0- attached to a reactive phosphorous containing group, or —O—
protected by a blocking group; R"' can be H, OH, a phosphoramidite, or a 3’-OH blocking
group described herein, and R" is H or OH. Where R"' is phosphoramidite, R‘ is an acid-
cleavable hydroxy protecting group which allows subsequent monomer coupling under
automated synthesis conditions.
[0122] In a particular embodiment, the linker (between dye and nucleotide) and blocking
group are both present and are separate moieties. In particular embodiments, the linker and
blocking group are both cleavable under substantially similar conditions. Thus, deprotection
and deblocking processes may be more efficient because only a single treatment will be
required to remove both the dye compound and the blocking group. However, in some
embodiments a linker and blocking group need not be cleavable under similar conditions,
instead being individually cleavable under distinct conditions.
[0123] The disclosure also encompasses polynucleotides incorporating dye compounds.
Such polynucleotides may be DNA or RNA comprised respectively of deoxyribonucleotides
or ribonucleotides joined in phosphodiester linkage. Polynucleotides may comprise naturally
occurring nucleotides, non-naturally occurring (or modified) nucleotides other than the
labeled nucleotides described herein or any combination thereof, in combination with at least
one modified nucleotide (e.g., labeled with a dye compound) as set forth herein.
Polynucleotides according to the disclosure may also include non-natural backbone linkages
and/or non-nucleotide chemical modifications. Chimeric structures comprised of mixtures of
ribonucleotides and deoxyribonucleotides comprising at least one labeled nucleotide are also
contemplated.
[0124] Non-limiting exemplary labeled nucleotides as described herein include:
HZN ’N§
Dye‘
L \ \ [\/I
Dye\L NH
O H2N
Dye\L)]\N /N
H § »
\ \ N
Ve\L l \ N
Dye—L
Dye\)J\ NH2
L M §
wherein L represents a linker and R represents a sugar residue as described above, or a sugar
residue with the 5’ position substituted with one, two or three phosphates.
W0 2020/ 136170 PCT/EP2019/086926
[0125] In some embodiments, non-limiting exemplary fluorescent dye conjugates are shown
below:
N / é NJK/O N3
HO~’E>\O
Q ‘:0 ffA-LN3-D e
HO’P\ y
Hox /0
HO, \\O
N” r
O N (CH2)nDye
NH2 NJl\,o\/\O)\,o
N’ l H
0 o~FL
F0 53“
o o~ \ _ - -
PG/O K 5 O ffC LN3 Dye
HO 00
wherein PG stands for the 3' hydroxy blocking groups described herein. In any embodiments
of the labeled nucleotide described herein, the nucleotide is a nucleotide triphosphate.
[0126] The present disclosure also provides kits including one or more 3' blocked
nucleosides and/or nucleotides described herein, for example, the 3' blocked nucleotide of
Formula (I), (la), or (11). Such kits will generally include at least one 3' blocked nucleotide or
nucleoside labeled with a dye together with at least one further component. The further
component(s) may be one or more of the components identified in a method set forth herein
or in the Examples section below. Some non-limiting examples of components that can be
combined into a kit of the present disclosure are set forth below.
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[0127] In a particular embodiment, a kit can include at least one labeled 3' blocked
nucleotide or nucleoside together with labeled or unlabeled nucleotides or nucleosides. For
example, nucleotides labeled with dyes may be supplied in combination with unlabeled or
native nucleotides, and/or with fluorescently labeled nucleotides or any combination thereof.
Combinations of nucleotides may be provided as separate individual components (eg, one
nucleotide type per vessel or tube) or as nucleotide mixtures (e. g., two or more nucleotides
mixed in the same vessel or tube).
[0128] Where kits comprise a plurality, particularly two, or three, or more particularly four,
3’ blocked nucleotides labeled with a dye compound, the different nucleotides may be labeled
with different dye compounds, or one may be dark, with no dye compounds. Where the
different nucleotides are labeled with different dye compounds, it is a feature of the kits that
the dye compounds are spectrally distinguishable fluorescent dyes. As used herein, the term
"spectrally distinguishable fluorescent dyes" refers to fluorescent dyes that emit fluorescent
energy at wavelengths that can be distinguished by fluorescent detection equipment (for
example, a commercial capillary-based DNA sequencing platform) when two or more such
dyes are present in one sample. When two nucleotides labeled with fluorescent dye
compounds are supplied in kit form, it is a feature of some embodiments that the spectrally
distinguishable fluorescent dyes can be excited at the same wavelength, such as, for example
by the same laser. When four 3’ blocked nucleotides (A, C, T, and G) labeled with fluorescent
dye compounds are supplied in kit form, it is a feature of some embodiments that two of the
spectrally distinguishable fluorescent dyes can both be excited at one wavelength and the
other two spectrally distinguishable dyes can both be excited at another wavelength.
Particular excitation wavelengths are 488 nm and 532 nm.
[0129] In one embodiment, a kit includes a first 3' blocked nucleotide labeled with a first dye
and a second nucleotide labeled with a second dye wherein the dyes have a difference in
absorbance maximum of at least 10 nm, particularly 20 nm to 50 nm. More particularly, the
two dye compounds have Stokes shifts of between 15-40 nm where "Stokes shift" is the
distance between the peak absorption and peak emission wavelengths.
[0130] In an alternative embodiment, the kits of the disclosure may contain 3' blocked
nucleotides where the same base is labeled with two or more different dyes. A first
nucleotide (e.g., 3' blocked T nucleotide triphosphate or 3' blocked G nucleotide
triphosphate) may be labeled with a first dye. A second nucleotide (e.g., 3’ blocked C
nucleotide triphosphate) may be labeled with a second spectrally distinct dye from the flrst
dye, for example a “green” dye absorbing at less than 600 nm, and a “blue” dye absorbs at
W0 2020/ 136170 PCT/EP2019/086926
less than 500 nm, for example 400 nm to 500, in particular 450 nm to 460 nm). A third
nucleotide (e.g., 3' blocked A nucleotide triphosphate) may be labeled as a mixture of the first
and the second dyes, or a mixture of the first, the second and a third dyes, and the fourth
nucleotide (e. g., 3' blocked G nucleotide triphosphate or 3' blocked T nucleotide
triphosphate) may be ‘dark’ and contain no label. In one example, the nucleotides 1-4 may
be labeled ‘blue’, ‘green’, ‘blue/green’, and dark. To simplify the instrumentation further,
four nucleotides can be labeled with two dyes excited with a single laser, and thus the
labeling of nucleotides 1-4 may be ‘blue 1’, ‘blue 2’, ‘blue 1/blue 2’, and dark.
[0131] In particular embodiments, the kits may contain four labeled 3’ blocked nucleotides
(e.g., A, C, T, G), where each type of nucleotide comprises the same 3' blocking group and a
fluorescent label, and wherein each fluorescent label has a distinct fluorescence maximum
and each of the fluorescent labels is distinguishable from the other three labels. The kits may
be such that two or more of the fluorescent labels have a similar absorbance maximum but
different Stokes shift. In some other embodiments, one type of the nucleotide is unlabeled.
[0132] Although kits are exemplified herein in regard to configurations having different
nucleotides that are labeled with different dye compounds, it will be understood that kits can
include 2, 3, 4 or more different nucleotides that have the same dye compound. In some
embodiments, the kit also includes an enzyme and a buffer appropriate for the action of the
enzyme. In some such embodiments, the enzyme is a polymerase, a terminal
deoxynucleotidyl transferase, or a reverse transcriptase. In particular embodiments, the
enzyme is a DNA polymerase, such as DNA polymerase 812 (Pol 812) or DNA polymerase
1901 (Pol 1901). The amino acid sequences of Pol 812 and Pol 1901 polymerases are
described, for example, in U.S. Patent Application Nos. 16/670,876, filed Oct. 31, 2019, and
16/703,569, filed Dec. 4, 2019, both of which are incorporated by reference herein.
[0133] Other components to be included in such kits may include buffers and the like. The
nucleotides of the present disclosure, and other any nucleotide components including
mixtures of different nucleotides, may be provided in the kit in a concentrated form to be
diluted prior to use. In such embodiments a suitable dilution buffer may also be included.
Again, one or more of the components identified in a method set forth herein can be included
in a kit of the present disclosure.
Methods of Seguencing
[0134] Labeled nucleotides or nucleosides according to the present disclosure may be used in
any method of analysis such as method that include detection of a fluorescent label attached
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to a nucleotide or nucleoside, whether on its own or incorporated into or associated with a
larger molecular structure or conjugate. In this context the term “incorporated into a
polynucleotide” can mean that the 5’ phosphate is joined in phosphodiester linkage to the 3'-
OH group of a second (modified or unmodified) nucleotide, which may itself form part of a
longer polynucleotide chain. The 3' end of a nucleotide set forth herein may or may not be
joined in phosphodiester linkage to the 5' phosphate of a further (modified or unmodified)
nucleotide. Thus, in one non-limiting embodiment, the disclosure provides a method of
detecting a nucleotide incorporated into a polynucleotide which comprises: (a) incorporating
at least one nucleotide of the disclosure into a polynucleotide and (b) detecting the
nucleotide(s) incorporated into the polynucleotide by detecting the fluorescent signal from
the dye compound attached to said nucleotide(s).
[0135] This method can include: a synthetic step (a) in which one or more nucleotides
according to the disclosure are incorporated into a polynucleotide and a detection step (b) in
which one or more nucleotide(s) incorporated into the polynucleotide are detected by
detecting or quantitatively measuring their fluorescence.
[0136] Some embodiments of the present application are directed to methods of sequencing
including: (a) incorporating at least one labeled nucleotide as described herein into a
polynucleotide; and (b) detecting the labeled nucleotide(s) incorporated into the
polynucleotide by detecting the fluorescent signal from the new fluorescent dye attached to
said nucleotide(s).
[0137] Some embodiments of the present disclosure relate to a method for determining the
sequence of a target single-stranded polynucleotide, comprising:
(a) incorporating a nucleotide comprising a 3'-OH blocking group and a detectable label as
described herein into a copy polynucleotide strand complementary to at least a portion of the
target polynucleotide strand,
(b) detecting the identity of the nucleotide incorporated into the copy polynucleotide strand,
(c) chemically removing the label and the 3'-OH blocking group from the nucleotide
incorporated into the copy polynucleotide strand.
[0138] In some embodiments, the sequencing method further comprises (d) washing the
chemically removed label and the 3’ blocking group away from the copy polynucleotide
strand. In some such embodiments, the 3’ blocking group and the detectable label are
removed prior to introducing the next complementary nucleotide. In some further
embodiments, the 3’ blocking group and the detectable label are removed in a single step of
W0 2020/ 136170 PCT/EP2019/086926
chemical reaction. In some embodiment, the washing step (d) also remove unincorporated
nucleotides. In some further embodiments, a palladium scavenger is also used in the washing
step after chemical cleavage of the label and the 3' blocking group.
[0139] In some embodiments, steps (a) to (d) is repeated until a sequence of the portion of
the template polynucleotide strand is determined. In some such embodiments, steps (a) to (d)
is repeated at least 50 times, at least 75 times, at least 100 times, at least 150 times, at least
200 times, at least 250 times, or at least 300 times.
[0140] In some embodiments, the label and the 3’ blocking group are removed in two
separate chemical reactions. In some such embodiments, removing the label from the
nucleotide incorporated into the copy polynucleotide strand comprises contacting the copy
strand including the incorporated nucleotide with a first cleavage solution. In some such
embodiment, the first cleavage solution contains a phosphine, such as a trialkylphosphine.
None-limiting examples of trialkylphosphines include tris(hydroxypropyl)phosphine (THP),
tris-(2-carboxyethyl)phosphine (TCEP), tris(hydroxymethyl)phosphine (THMP), or
tris(hydroxyethyl)phosphine (THEP). In one embodiment, the first cleavage solution
contains THP. In some such embodiments, removing the 3' blocking group from the
nucleotide incorporated into the copy polynucleotide strand comprises contacting the copy
strand including the incorporated nucleotide with a second cleavage solution. In some such
embodiments, the second cleavage solution contains a palladium (Pd) catalyst. In some
further embodiments, the Pd catalyst is a Pd(O) catalyst. In some such embodiments, the
Pd(O) is prepared by mixing a Pd(II) complex [(PdCl(C3H5))2] with THP in situ. The molar
ratio ofthe Pd(II) complex and the THP may be about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or
1:10. In one embodiment the molar ration of Pd: THP is 1:5. In some further embodiments,
one or more reducing agents may be added, such as ascorbic acid or a salt thereof (e.g.,
sodium ascorbate). In some embodiments, the second cleavage solution may contain one or
more buffer reagents, such as a primary amine, a secondary amine, a tertiary amine, a
carbonate salt, a phosphate salt, or a borate salt, or combinations thereof. In some further
embodiments, the buffer reagent comprises ethanolamine (EA),
tris(hydroxymethyl)aminomethane (Tris), glycine, sodium carbonate, sodium phosphate,
sodium borate, 2-dimethyalaminomethanol (DMEA), 2-diethyalaminomethanol (DEEA),
N,N,N’,N'-tetramethylethylenediamine(TEMED), or N,N,N',N'-tetraethylethylenediamine
(TEEDA), or combinations thereof. In one embodiment, the buffer reagent is DEEA. In
another embodiment, the buffer reagent contains one or more inorganic salts such as a
carbonate salt, a phosphate salt, or a borate salt, or combinations thereof. In one embodiment,
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the inorganic salt is a sodium salt. In some other embodiments, the second cleavage solution
contains NaIO4 or Oxone®. In some further embodiments, the 3’ blocked nucleotide contains
a AOM group and the second cleavage solution contains a palladium (Pd) catalyst and one or
more buffer reagents described herein (e.g., a tertiary amine such as DEEA) and have pH of
about 9.0 to about 10.0 (e.g., 9.6 or 9.8).
[0141] In some alternative embodiments, the label and the 3'-OH blocking group are
removed in a single chemical reaction. In some such embodiments, the label is attached to
the nucleotide via a cleavage linker comprising the same moiety as the 3’ blocking group, for
example, both the linker and the 3’ blocking group may comprise an acetal moiety
or a thiocarbamate moiety NR5R6 as described herein. In some such
embodiment, the single chemical reaction is carried out in a cleavage solution containing a Pd
catalyst described above.
[0142] In some further embodiments, the nucleotides used in the incorporation step (a) are
fully functionalized A, C, T and G nucleotide triphosphate each contains a 3’blocking group
described herein. In some such embodiments, the nucleotides herein provide superior stability
in solution during sequencing runs, compared to the same nucleotides protected with a
standard 3'-O-azidomethyl blocking group. For example, the acetal or thiocarbamate blocking
groups disclosed herein may confer at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%,
2000%, 2500%, or 3000% improved stability compare to an azidomethyl protected 3’-OH at
the same condition for the same period of time, thereby reducing the pre-phasing values and
resulting in longer sequencing read lengths. In some embodiments, the stability is measured
at ambient temperature or a temperature below ambient temperature (such as 4-10°C). In
other embodiments, the stability is measured at an elevated temperature, such as 40°C, 45°C,
50°C, 55°C, 60°C or 65°C. In some such embodiments, the stability is measured in solution
in a basic pH environment, eg., at pH 9.0, 9.2, 9.4, 9.6, 9.8. or 10.0. In some further
embodiments, the pre-phasing value with the 3’ blocked nucleotide described herein is less
than about 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12,
0.11, 0.10, 0.09, 0.08, 0.07, 0.06, or 0.05 after over 50, 100 or 150 cycles of SBS. In some
further embodiments, the phasing value with the 3’ blocked nucleotide is less than about
0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10,
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0.09, 0.08, 0.07, 0.06, or 0.05, after over 50, 100 or 150 cycles of SBS. In one embodiment,
each ffN contains the 3’-AOM group.
[0143] In some embodiments, the 3’ blocked nucleotides described herein provide superior
deblocking rate in solution during the chemical cleavage step of the sequencing run,
compared to the same nucleotides protected with a standard 3’-O-azidomethyl blocking
group. For example, the acetal (e.g., AOM) or thiocarbamate blocking groups disclosed
herein may confer at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,
150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, or 2000%
improved deblocking rate compare to an azidomethyl protected 3'-OH using the standard
deblocking reagent (such as tris(hydroxypropyl)phosphine), thereby reducing the overall time
for a sequencing cycle. In some embodiments, the deblocking time for each nucleotide is
reduced by about 5%, 10%, 20%, 30%, 40%, 50%, or 60%. For example, the deblocking
time for 3'-AOM and 3’-O-azidomethyl is about 4-5 seconds and about 9-10 seconds
respectively under certain chemical reaction condition. In some embodiments, the half life (t
1/2) of AOM blocking group is at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 fold faster than
azidomethyl blocking group. In some such embodiment, tug of AOM is about 1 minute while
tug of azidomethyl is about 11 minutes. In some embodiments, the deblocking rate is
measured at ambient temperature or a temperature below ambient temperature (such as 4-
°C). In other embodiments, the deblocking rate is measured at an elevated temperature,
such as 40°C, 45°C, 50°C, 55°C, 60°C or 65°C. In some such embodiments, the deblocking
rate is measured in solution in a basic pH environment, e.g., at pH 9.0, 9.2, 9.4, 9.6, 9.8. or
.0. In some such embodiments, the molar ratio of the deblocking reagent to substrate (i.e.,
3' blocked nucleoside or nucleotide) is about 10:1, about 5:1, about 2:1,about 1:1, about 1:2,
about 1:5 or about 1:10. In one embodiment, each ffN contains the 3’-AOM group.
[0144] In any embodiments of the methods described herein, the labeled nucleotide is a
nucleotide triphosphate. In any embodiments of the method described herein, the target
polynucleotide strand is attached to a solid support, such as a flow cell.
[0145] In one embodiment, at least one nucleotide is incorporated into a polynucleotide in the
synthetic step by the action of a polymerase enzyme. In some such embodiments, the
polymerase may be DNA polymerase Pol 812 or Pol 1901. However, other methods of
joining nucleotides to polynucleotides, such as, for example, chemical oligonucleotide
synthesis or ligation of labeled oligonucleotides to unlabeled oligonucleotides, can be used.
Therefore, the term "incorporating,” when used in reference to a nucleotide and
W0 2020/ 136170 PCT/EP2019/086926
polynucleotide, can encompass polynucleotide synthesis by chemical methods as well as
enzymatic methods.
[0146] In a specific embodiment, a synthetic step is carried out and may optionally comprise
incubating a template polynucleotide strand with a reaction mixture comprising labeled 3'
blocked nucleotides of the disclosure. A polymerase can also be provided under conditions
which permit formation of a phosphodiester linkage between a free 3'-OH group on a
polynucleotide strand annealed to the template polynucleotide strand and a 5' phosphate
group on the nucleotide. Thus, a synthetic step can include formation of a polynucleotide
strand as directed by complementary base-pairing of nucleotides to a template strand.
[0147] In all embodiments of the methods, the detection step may be carried out while the
polynucleotide strand into which the labeled nucleotides are incorporated is annealed to a
template strand, or after a denaturation step in which the two strands are separated. Further
steps, for example chemical or enzymatic reaction steps or purification steps, may be
included between the synthetic step and the detection step. In particular, the target strand
incorporating the labeled nucleotide(s) may be isolated or purified and then processed further
or used in a subsequent analysis. By way of example, target polynucleotides labeled with
nucleotide(s) as described herein in a synthetic step may be subsequently used as labeled
probes or primers. In other embodiments, the product of the synthetic step set forth herein
may be subject to fi,lI'lZl161” reaction steps and, if desired, the product of these subsequent steps
purified or isolated.
[0148] Suitable conditions for the synthetic step will be well known to those familiar with
standard molecular biology techniques. In one embodiment, a synthetic step may be
analogous to a standard primer extension reaction using nucleotide precursors, including
nucleotides as described herein, to form an extended target strand complementary to the
template strand in the presence of a suitable polymerase enzyme. In other embodiments, the
synthetic step may itself form part of an amplification reaction producing a labeled double
stranded amplification product comprised of annealed complementary strands derived from
copying of the target and template polynucleotide strands. Other exemplary synthetic steps
include nick translation, strand displacement polymerization, random primed DNA labeling,
etc. A particularly useful polymerase enzyme for a synthetic step is one that is capable of
catalyzing the incorporation of nucleotides as set forth herein. A Variety of naturally
occurring or modified polymerases can be used. By way of example, a thermostable
polymerase can be used for a synthetic reaction that is carried out using thermocycling
conditions, whereas a thermostable polymerase may not be desired for isothermal primer
W0 2020/ 136170 PCT/EP2019/086926
extension reactions. Suitable thermostable polymerases which are capable of incorporating
the nucleotides according to the disclosure include those described in WO 2005/024010 or
WO 06/120433, each of which is incorporated herein by reference. In synthetic reactions
which are carried out at lower temperatures such as 37 °C, polymerase enzymes need not
necessarily be thermostable polymerases, therefore the choice of polymerase will depend on a
number of factors such as reaction temperature, pH, strand-displacing activity and the like.
[0149] In specific non-limiting embodiments, the disclosure encompasses methods of nucleic
acid sequencing, re-sequencing, whole genome sequencing, single nucleotide polymorphism
scoring, any other application involving the detection of the labeled nucleotide or nucleoside
set forth herein when incorporated into a polynucleotide. Any of a variety of other
applications benefitting the use of polynucleotides labeled with the nucleotides comprising
fluorescent dyes can use labeled nucleotides or nucleosides with dyes set forth herein.
[0150] In a particular embodiment, the disclosure provides use of labeled nucleotides
according to the disclosure in a polynucleotide sequencing-by-synthesis (SBS) reaction.
Sequencing-by-synthesis generally involves sequential addition of one or more nucleotides or
oligonucleotides to a growing polynucleotide chain in the 5' to 3' direction using a
polymerase or ligase in order to form an extended polynucleotide chain complementary to the
template nucleic acid to be sequenced. The identity of the base present in one or more of the
added nucleotide(s) can be determined in a detection or "imaging" step. The identity of the
added base may be determined after each nucleotide incorporation step. The sequence of the
template may then be inferred using conventional Watson-Crick base-pairing rules. The use
of the labeled nucleotides set forth herein for determination of the identity of a single base
may be useful, for example, in the scoring of single nucleotide polymorphisms, and such
single base extension reactions are within the scope of this disclosure.
[0151] In an embodiment of the present disclosure, the sequence of a template polynucleotide
is determined by detecting the incorporation of one or more 3’ blocked nucleotides described
herein into a nascent strand complementary to the template polynucleotide to be sequenced
through the detection of fluorescent label(s) attached to the incorporated nucleotide(s).
Sequencing of the template polynucleotide can be primed with a suitable primer (or prepared
as a hairpin construct which will contain the primer as part of the hairpin), and the nascent
chain is extended in a stepwise manner by addition of nucleotides to the 3' end of the primer
in a polymerase-catalyzed reaction.
[0152] In particular embodiments, each of the different nucleotide triphosphates (A, T, G and
C) may be labeled with a unique fluorophore and also comprises a blocking group at the 3'
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position to prevent uncontrolled polymerization. Alternatively, one of the four nucleotides
may be unlabeled (dark). The polymerase enzyme incorporates a nucleotide into the nascent
chain complementary to the template polynucleotide, and the blocking group prevents further
incorporation of nucleotides. Any unincorporated nucleotides can be washed away and the
fluorescent signal from each incorporated nucleotide can be "read” optically by suitable
means, such as a charge-coupled device using laser excitation and suitable emission filters.
The 3'-blocking group and fluorescent dye compounds can then be removed (deprotected)
simultaneously or sequentially to expose the nascent chain for further nucleotide
incorporation. Typically, the identity of the incorporated nucleotide will be determined after
each incorporation step, but this is not strictly essential. Similarly, U.S. Pat. No. 5,302,509
(which is incorporated herein by reference) discloses a method to sequence polynucleotides
immobilized on a solid support.
[0153] The method, as exemplified above, utilizes the incorporation of fluorescently labeled,
3'-blocked nucleotides A, G, C, and T into a growing strand complementary to the
immobilized polynucleotide, in the presence of DNA polymerase. The polymerase
incorporates a base complementary to the target polynucleotide but is prevented from further
addition by the 3'-blocking group. The label of the incorporated nucleotide can then be
determined, and the blocking group removed by chemical cleavage to allow further
polymerization to occur. The nucleic acid template to be sequenced in a sequencing-by-
synthesis reaction may be any polynucleotide that it is desired to sequence. The nucleic acid
template for a sequencing reaction will typically comprise a double stranded region having a
free 3'-OH group that serves as a primer or initiation point for the addition of further
nucleotides in the sequencing reaction. The region of the template to be sequenced will
overhang this free 3'-OH group on the complementary strand. The overhanging region of the
template to be sequenced may be single stranded but can be double-stranded, provided that a
"nick is present" on the strand complementary to the template strand to be sequenced to
provide a free 3'-OH group for initiation of the sequencing reaction. In such embodiments,
sequencing may proceed by strand displacement. In certain embodiments, a primer bearing
the free 3'-OH group may be added as a separate component (e.g., a short oligonucleotide)
that hybridizes to a single-stranded region of the template to be sequenced. Alternatively, the
primer and the template strand to be sequenced may each form part of a partially self-
complementary nucleic acid strand capable of forming an intra-molecular duplex, such as for
example a hairpin loop structure. Hairpin polynucleotides and methods by which they may
be attached to solid supports are disclosed in PCT Publication Nos. WO O1/57248 and WO
W0 2020/ 136170 PCT/EP2019/086926
2005/047301, each of which is incorporated herein by reference. Nucleotides can be added
successively to a growing primer, resulting in synthesis of a polynucleotide chain in the 5' to
3' direction. The nature of the base which has been added may be determined, particularly
but not necessarily after each nucleotide addition, thus providing sequence information for
the nucleic acid template. Thus, a nucleotide is incorporated into a nucleic acid strand (or
polynucleotide) by joining of the nucleotide to the free 3'-OH group of the nucleic acid strand
via formation of a phosphodiester linkage with the 5’ phosphate group of the nucleotide.
[0154] The nucleic acid template to be sequenced may be DNA or RNA, or even a hybrid
molecule comprised of deoxynucleotides and ribonucleotides. The nucleic acid template may
comprise naturally occurring and/or non-naturally occurring nucleotides and natural or non-
natural backbone linkages, provided that these do not prevent copying of the template in the
sequencing reaction.
[0155] In certain embodiments, the nucleic acid template to be sequenced may be attached to
a solid support via any suitable linkage method known in the art, for example via covalent
attachment. In certain embodiments template polynucleotides may be attached directly to a
solid support (e.g., a silica-based support). However, in other embodiments of the disclosure
the surface of the solid support may be modified in some way so as to allow either direct
covalent attachment of template polynucleotides, or to immobilize the template
polynucleotides through a hydrogel or polyelectrolyte multilayer, which may itself be non-
covalently attached to the solid support.
Embodiments and Alternatives 0fSequencing-By-Synthesis
[0156] Some embodiments include pyrosequencing techniques. Pyrosequencing detects the
release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into the
nascent strand (Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P.
(1996) "Real-time DNA sequencing using detection of pyrophosphate release." Analytical
Biochemistry 242(1), 84-9, Ronaghi, M. (2001) "Pyrosequencing sheds light on DNA
sequencing." Genome Res. 11(1), 3-11; Ronaghi, M., Uhlen, M. and Nyren, P. (1998) "A
sequencing method based on real-time pyrophosphate." Science 281(5375), 363, U.S. Pat.
Nos. 6,210,891; 6,258,568 and 6,274,320, the disclosures of which are incorporated herein by
reference in their entireties). In pyrosequencing, released PPi can be detected by being
immediately converted to adenosine triphosphate (ATP) by ATP sulfurase, and the level of
ATP generated is detected via luciferase-produced photons. The nucleic acids to be
sequenced can be attached to features in an array and the array can be imaged to capture the
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chemiluminescent signals that are produced due to incorporation of a nucleotides at the
features of the array. An image can be obtained after the array is treated with a particular
nucleotide type (e.g. A, T, C or G). Images obtained after addition of each nucleotide type
will differ with regard to which features in the array are detected. These differences in the
image reflect the different sequence content of the features on the array. However, the
relative locations of each feature will remain unchanged in the images. The images can be
stored, processed and analyzed using the methods set forth herein. For example, images
obtained after treatment of the array with each different nucleotide type can be handled in the
same way as exemplified herein for images obtained from different detection channels for
reversible terminator-based sequencing methods.
[0157] In another exemplary type of SBS, cycle sequencing is accomplished by stepwise
addition of reversible terminator nucleotides containing, for example, a cleavable or
photobleachable dye label as described, for example, in W0 04/018497 and U.S. Pat. No.
7,057,026, the disclosures of which are incorporated herein by reference. This approach is
being commercialized by Solexa (now Illumina, Inc.), and is also described in WO 91/0667 8
and W0 07/123,744, each of which is incorporated herein by reference. The availability of
fluorescently-labeled terminators in which both the termination can be reversed, and the
fluorescent label cleaved facilitates efficient cyclic reversible termination (CRT) sequencing.
Polymerases can also be co-engineered to efficiently incorporate and extend from these
modified nucleotides.
[0158] Preferably in reversible terminator-based sequencing embodiments, the labels do not
substantially inhibit extension under SBS reaction conditions. However, the detection labels
can be removable, for example, by cleavage or degradation. Images can be captured
following incorporation of labels into arrayed nucleic acid features. In particular
embodiments, each cycle involves simultaneous delivery of four different nucleotide types to
the array and each nucleotide type has a spectrally distinct label. Four images can then be
obtained, each using a detection channel that is selective for one of the four different labels.
Alternatively, different nucleotide types can be added sequentially, and an image of the array
can be obtained between each addition step. In such embodiments each image will show
nucleic acid features that have incorporated nucleotides of a particular type. Different
features will be present or absent in the different images due the different sequence content of
each feature. However, the relative position of the features will remain unchanged in the
images. Images obtained from such reversible terminator-SBS methods can be stored,
processed and analyzed as set forth herein. Following the image capture step, labels can be
W0 2020/ 136170 PCT/EP2019/086926
removed, and reversible terminator moieties can be removed for subsequent cycles of
nucleotide addition and detection. Removal of the labels after they have been detected in a
particular cycle and prior to a subsequent cycle can provide the advantage of reducing
background signal and crosstalk between cycles. Examples of useful labels and removal
methods are set forth below.
[0159] Some embodiments can utilize detection of four different nucleotides using fewer
than four different labels. For example, SBS can be performed utilizing methods and systems
described in the incorporated materials of U.S. Pub. No. 2013/0079232. As a first example, a
pair of nucleotide types can be detected at the same wavelength, but distinguished based on a
difference in intensity for one member of the pair compared to the other, or based on a
change to one member of the pair (e. g. via chemical modification, photochemical
modification or physical modification) that causes apparent signal to appear or disappear
compared to the signal detected for the other member of the pair. As a second example, three
of four different nucleotide types can be detected under particular conditions while a fourth
nucleotide type lacks a label that is detectable under those conditions, or is minimally
detected under those conditions (e.g., minimal detection due to background fluorescence,
etc.). Incorporation of the first three nucleotide types into a nucleic acid can be determined
based on presence of their respective signals and incorporation of the fourth nucleotide type
into the nucleic acid can be determined based on absence or minimal detection of any signal.
As a third example, one nucleotide type can include label(s) that are detected in two different
channels, whereas other nucleotide types are detected in no more than one of the channels.
The aforementioned three exemplary configurations are not considered mutually exclusive
and can be used in various combinations. An exemplary embodiment that combines all three
examples, is a fluorescent-based SBS method that uses a first nucleotide type that is detected
in a first channel (e. g. dATP having a label that is detected in the first channel when excited
by a first excitation wavelength), a second nucleotide type that is detected in a second
channel (e. g. dCTP having a label that is detected in the second channel when excited by a
second excitation wavelength), a third nucleotide type that is detected in both the first and the
second channel (e.g. dTTP having at least one label that is detected in both channels when
excited by the first and/or second excitation wavelength) and a fourth nucleotide type that
lacks a label that is not, or minimally, detected in either channel (e. g. dGTP having no label).
[0160] Further, as described in the incorporated materials of U.S. Pub. No. 2013/0079232,
sequencing data can be obtained using a single channel. In such so-called one-dye sequencing
approaches, the first nucleotide type is labeled but the label is removed after the first image is
W0 2020/ 136170 PCT/EP2019/086926
generated, and the second nucleotide type is labeled only after a first image is generated. The
third nucleotide type retains its label in both the first and second images, and the fourth
nucleotide type remains unlabeled in both images.
[0161] Some embodiments can utilize sequencing by ligation techniques. Such techniques
utilize DNA ligase to incorporate oligonucleotides and identify the incorporation of such
oligonucleotides. The oligonucleotides typically have different labels that are correlated with
the identity of a particular nucleotide in a sequence to which the oligonucleotides hybridize.
As with other SBS methods, images can be obtained following treatment of an array of
nucleic acid features with the labeled sequencing reagents. Each image will show nucleic acid
features that have incorporated labels of a particular type. Different features will be present or
absent in the different images due the different sequence content of each feature, but the
relative position of the features will remain unchanged in the images. Images obtained from
ligation-based sequencing methods can be stored, processed and analyzed as set forth herein.
Exemplary SBS systems and methods which can be utilized with the methods and systems
described herein are described in U.S. Pat. Nos. 6,969,488, 6,172,218, and 6,306,597, the
disclosures of which are incorporated herein by reference in their entireties.
[0162] Some embodiments can utilize nanopore sequencing (Deamer, D. W. & Akeson, M.
"Nanopores and nucleic acids: prospects for ultrarapid sequencing." Trends Biotechnol. 18,
147-151 (2000); Deamer, D. and D. Branton, "Characterization of nucleic acids by nanopore
analysis", Acc. Chem. Res. 35:8l7-825 (2002); Li, J., M. Gershow, D. Stein, E. Brandin, and
J. A. Golovchenko, "DNA molecules and configurations in a solid-state nanopore
microscope" Nat. Mater. 2:611-615 (2003), the disclosures of which are incorporated herein
by reference in their entireties). In such embodiments, the target nucleic acid passes through a
nanopore. The nanopore can be a synthetic pore or biological membrane protein, such as Ot-
hemolysin. As the target nucleic acid passes through the nanopore, each base-pair can be
identified by measuring fluctuations in the electrical conductance of the pore. (U.S. Pat. No.
7,001,792, Soni, G. V. & Meller, "A. Progress toward ultrafast DNA sequencing using solid-
state nanopores." Clin. Chem. 53, 1996-2001 (2007); Healy, K. "Nanopore-based single-
molecule DNA analysis." Ncmomed. 2, 459-481 (2007); Cockroft, S. L., Chu, J ., Amorin, M.
& Ghadiri, M. R. "A single-molecule nanopore device detects DNA polymerase activity with
single-nucleotide resolution." J. Am. Chem. Soc. 130, 818-820 (2008), the disclosures of
which are incorporated herein by reference in their entireties). Data obtained from nanopore
sequencing can be stored, processed and analyzed as set forth herein. In particular, the data
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can be treated as an image in accordance with the exemplary treatment of optical images and
other images that is set forth herein.
[0163] Some other embodiments of sequencing method involves the use the 3' blocked
nucleotide described herein in nanoball sequencing technique, such as those described in U.S.
Patent No. 9,222,132, the disclosure of which is incorporated by reference. Through the
process of rolling circle amplification (RCA), a large number of discrete DNA nanoballs may
be generated. The nanoball mixture is then distributed onto a patterned slide surface
containing features that allow a single nanoball to associate with each location. In DNA
nanoball generation, DNA is fragmented and ligated to the first of four adapter sequences.
The template is amplified, circularized and cleaved with a type II endonuclease. A second set
of adapters is added, followed by amplification, circularization and cleavage. This process is
repeated for the remaining two adapters. The final product is a circular template with four
adapters, each separated by a template sequence. Library molecules undergo a rolling circle
amplification step, generating a large mass of concatemers called DNA nanoballs, which are
then deposited on a flow cell. Goodwin et al., “Coming of age: ten years of next-generation
sequencing technologies,” Nat Rev Genet. 2016;17(6):333-51.
[0164] Some embodiments can utilize methods involving the real-time monitoring of DNA
polymerase activity. Nucleotide incorporations can be detected through fluorescence
resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase
and y-phosphate-labeled nucleotides as described, for example, in U.S. Pat. Nos. 7,329,492
and 7,211,414, both of which are incorporated herein by reference, or nucleotide
incorporations can be detected with zero-mode waveguides as described, for example, in U.S.
Pat. No. 7,315,019, which is incorporated herein by reference, and using fluorescent
nucleotide analogs and engineered polymerases as described, for example, in U.S. Pat. No.
7,405,281 and U.S. Pub. No. 2008/0108082, both of which are incorporated herein by
reference. The illumination can be restricted to a zeptoliter-scale volume around a surface-
tethered polymerase such that incorporation of fluorescently labeled nucleotides can be
observed with low background (Levene, M. J. et al. "Zero-mode waveguides for single-
molecule analysis at high concentrations." Science 299, 682-686 (2003); Lundquist, P. M. er
al. "Parallel confocal detection of single molecules in real time." Opt. Lett. 33, 1026-1028
(2008), Korlach, J. et al. "Selective aluminum passivation for targeted immobilization of
single DNA polymerase molecules in zero-mode waveguide nano structures." Proc. Natl.
Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are incorporated herein by
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reference in their entireties). Images obtained from such methods can be stored, processed
and analyzed as set forth herein.
[0165] Some SBS embodiments include detection of a proton released upon incorporation of
a nucleotide into an extension product. For example, sequencing based on detection of
released protons can use an electrical detector and associated techniques that are
commercially available from Ion Torrent (Guilford, CT, a Life Technologies subsidiary) or
sequencing methods and systems described in U.S. Pub. Nos. 2009/0026082; 2009/0127589;
2010/0137143, and 2010/0282617, all of which are incorporated herein by reference.
Methods set forth herein for amplifying target nucleic acids using kinetic exclusion can be
readily applied to substrates used for detecting protons. More specifically, methods set forth
herein can be used to produce clonal populations of amplicons that are used to detect protons.
[0166] The above SBS methods can be advantageously carried out in multiplex formats such
that multiple different target nucleic acids are manipulated simultaneously. In particular
embodiments, different target nucleic acids can be treated in a common reaction vessel or on
a surface of a particular substrate. This allows convenient delivery of sequencing reagents,
removal of unreacted reagents and detection of incorporation events in a multiplex manner. In
embodiments using surface-bound target nucleic acids, the target nucleic acids can be in an
array format. In an array format, the target nucleic acids can be typically bound to a surface
in a spatially distinguishable manner. The target nucleic acids can be bound by direct
covalent attachment, attachment to a bead or other particle or binding to a polymerase or
other molecule that is attached to the surface. The array can include a single copy of a target
nucleic acid at each site (also referred to as a feature) or multiple copies having the same
sequence can be present at each site or feature. Multiple copies can be produced by
amplification methods such as, bridge amplification or emulsion PCR as described in further
detail below.
[0167] The methods set forth herein can use arrays having features at any of a variety of
densities including, for example, at least about 10 features/cmz, 100 features/cmz, 500
features/cmz, 1,000 features/cmz, 5,000 features/cmz, 10,000 features/cmz, 50,000
features/cmz, 100,000 features/cmz, 1,000,000 features/cmz, 5,000,000 features/cmz, or
higher.
[0168] An advantage of the methods set forth herein is that they provide for rapid and
efficient detection of a plurality of target nucleic acid in parallel. Accordingly the present
disclosure provides integrated systems capable of preparing and detecting nucleic acids using
techniques known in the art such as those exemplified above. Thus, an integrated system of
W0 2020/ 136170 PCT/EP2019/086926
the present disclosure can include fluidic components capable of delivering amplification
reagents and/or sequencing reagents to one or more immobilized DNA fragments, the system
comprising components such as pumps, valves, reservoirs, fluidic lines and the like. A flow
cell can be configured and/or used in an integrated system for detection of target nucleic
acids. Exemplary flow cells are described, for example, in U.S. Pub. No. 2010/0111768 and
US Ser. No. 13/273,666, each of which is incorporated herein by reference. As exemplified
for flow cells, one or more of the fluidic components of an integrated system can be used for
an amplification method and for a detection method. Taking a nucleic acid sequencing
embodiment as an example, one or more of the fluidic components of an integrated system
can be used for an amplification method set forth herein and for the delivery of sequencing
reagents in a sequencing method such as those exemplified above. Alternatively, an
integrated system can include separate fluidic systems to carry out amplification methods and
to carry out detection methods. Examples of integrated sequencing systems that are capable
of creating amplified nucleic acids and also determining the sequence of the nucleic acids
include, without limitation, the MiSeqTM platform (Illumina, Inc., San Diego, CA) and devices
described in US Ser. No. 13/273,666, which is incorporated herein by reference.
[0169] Arrays in which polynucleotides have been directly attached to silica-based supports
are those for example disclosed in W0 O0/06770 (incorporated herein by reference), wherein
polynucleotides are immobilized on a glass support by reaction between a pendant epoxide
group on the glass with an internal amino group on the polynucleotide. In addition,
polynucleotides can be attached to a solid support by reaction of a sulfur-based nucleophile
with the solid support, for example, as described in W0 2005/047301 (incorporated herein by
reference). A still further example of solid-supported template polynucleotides is where the
template polynucleotides are attached to hydrogel supported upon silica-based or other solid
supports, for example, as described in W0 00/31148, W0 Ol/01143, W0 02/12566, W0
O3/014392, U.S. Pat. No. 6,465,178 and W0 00/53812, each ofwhich is incorporated herein
by reference.
[0170] A particular surface to which template polynucleotides may be immobilized is a
polyacrylamide hydrogel. Polyacrylamide hydrogels are described in the references cited
above and in W0 2005/065814, which is incorporated herein by reference. Specific
hydrogels that may be used include those described in W0 2005/065814 and U.S. Pub. No.
2014/0079923. In one embodiment, the hydrogel is PAZAM (poly(N-(5-
azidoacetamidylpentyl) acrylamide-co-acrylamide)).
W0 2020/ 136170 PCT/EP2019/086926
[0171] DNA template molecules can be attached to beads or microparticles, for example, as
described in U.S. Pat. No. 6,172,218 (which is incorporated herein by reference). Attachment
to beads or microparticles can be useful for sequencing applications. Bead libraries can be
prepared where each bead contains different DNA sequences. Exemplary libraries and
methods for their creation are described in Nature, 437, 376-380 (2005); Science, 309, 5741,
1728-1732 (2005), each of which is incorporated herein by reference. Sequencing of arrays
of such beads using nucleotides set forth herein is within the scope of the disclosure.
[0172] Templates that are to be sequenced may form part of an "array" on a solid support, in
which case the array may take any convenient form. Thus, the method of the disclosure is
applicable to all types of high-density arrays, including single-molecule arrays, clustered
arrays, and bead arrays. Labeled nucleotides of the present disclosure may be used for
sequencing templates on essentially any type of array, including but not limited to those
formed by immobilization of nucleic acid molecules on a solid support.
[0173] However, labeled nucleotides of the disclosure are particularly advantageous in the
context of sequencing of clustered arrays. In clustered arrays, distinct regions on the array
(often referred to as sites, or features) comprise multiple polynucleotide template molecules.
Generally, the multiple polynucleotide molecules are not individually resolvable by optical
means and are instead detected as an ensemble. Depending on how the array is formed, each
site on the array may comprise multiple copies of one individual polynucleotide molecule
(e. g., the site is homogenous for a particular single- or double-stranded nucleic acid species)
or even multiple copies of a small number of different polynucleotide molecules (e.g.,
multiple copies of two different nucleic acid species). Clustered arrays of nucleic acid
molecules may be produced using techniques generally known in the art. By way of
example, WO 98/44151 and W0 00/ 18957, each of which is incorporated herein, describe
methods of amplification of nucleic acids wherein both the template and amplification
products remain immobilized on a solid support in order to form arrays comprised of clusters
or "colonies" of immobilized nucleic acid molecules. The nucleic acid molecules present on
the clustered arrays prepared according to these methods are suitable templates for
sequencing using the nucleotides labeled with dye compounds of the disclosure.
[0174] The labeled nucleotides of the present disclosure are also useful in sequencing of
templates on single molecule arrays. The term "single molecule array" or "SMA" as used
herein refers to a population of polynucleotide molecules, distributed (or arrayed) over a solid
support, wherein the spacing of any individual polynucleotide from all others of the
population is such that it is possible to individually resolve the individual polynucleotide
W0 2020/ 136170 PCT/EP2019/086926
molecules. The target nucleic acid molecules immobilized onto the surface of the solid
support can thus be capable of being resolved by optical means in some embodiments. This
means that one or more distinct signals, each representing one polynucleotide, will occur
within the resolvable area of the particular imaging device used.
[0175] Single molecule detection may be achieved wherein the spacing between adjacent
polynucleotide molecules on an array is at least 100 nm, more particularly at least 250 nm,
still more particularly at least 300 nm, even more particularly at least 350 nm. Thus, each
molecule is individually resolvable and detectable as a single molecule fluorescent point, and
fluorescence from said single molecule fluorescent point also exhibits single step
photobleaching.
[0176] The terms "individually resolved" and "individual resolution" are used herein to
specify that, when visualized, it is possible to distinguish one molecule on the array from its
neighboring molecules. Separation between individual molecules on the array will be
determined, in part, by the particular technique used to resolve the individual molecules. The
general features of single molecule arrays will be understood by reference to published
applications WO O0/06770 and W0 O1/57248, each of which is incorporated herein by
reference. Although one use of the nucleotides of the disclosure is in sequencing-by-
synthesis reactions, the utility of the nucleotides is not limited to such methods. In fact, the
nucleotides may be used advantageously in any sequencing methodology which requires
detection of fluorescent labels attached to nucleotides incorporated into a polynucleotide.
[0177] In particular, the labeled nucleotides of the disclosure may be used in automated
fluorescent sequencing protocols, particularly fluorescent dye-terminator cycle sequencing
based on the chain termination sequencing method of Sanger and co-workers. Such methods
generally use enzymes and cycle sequencing to incorporate fluorescently labeled
dideoxynucleotides in a primer extension sequencing reaction. So-called Sanger sequencing
methods, and related protocols (Sanger-type), utilize randomized chain termination with
labeled dideoxynucleotides.
[0178] Thus, the present disclosure also encompasses labeled nucleotides which are
dideoxynucleotides lacking hydroxyl groups at both of the 3' and 2' positions, such
dideoxynucleotides being suitable for use in Sanger type sequencing methods and the like.
[0179] Labeled nucleotides of the present disclosure incorporating 3' blocking groups, it will
be recognized, may also be of utility in Sanger methods and related protocols since the same
effect achieved by using dideoxy nucleotides may be achieved by using nucleotides having
3'-OH blocking groups: both prevent incorporation of subsequent nucleotides. Where
W0 2020/ 136170 PCT/EP2019/086926
nucleotides according to the present disclosure, and having a 3' blocking group are to be used
in Sanger-type sequencing methods it will be appreciated that the dye compounds or
detectable labels attached to the nucleotides need not be connected via cleavable linkers,
since in each instance where a labeled nucleotide of the disclosure is incorporated; no
nucleotides need to be subsequently incorporated and thus the label need not be removed
from the nucleotide.
[0180] In any embodiments of the methods described herein, the nucleotide used in the
sequencing application is a 3' blocked nucleotide described herein, for example, the
nucleotide of Formula (I), (la), or (II). In any embodiments, the 3' blocked nucleotide is a
nucleotide triphosphate.
examples
[0181] Additional embodiments are disclosed in further detail in the following examples,
which are not in any way intended to limit the scope of the claims.
Example 1. Preparation of 3'-Acetal Blocked Nucleosides
[0182] In this example, Various 3'-acetal protected T nucleoside were prepared according to
Scheme 2.
NHTFA
o o o
I NH KEKNH k N
HO I N,l\O TBDPSO l N,l\O TBDPSO l N,l\O
OH OH T-1
NHTFA NHTFA
NHTFA O \ O \ O
k I NH | I H0 l N
TBDPSO /go TBDPSO N N
O N o O o O
82% 69-71% 78-94%
O\/SMe O\/O\R O\/O\R
7'3 T-4 T-5
Scheme 2.
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TFA= E XCF3 TBDPS = §—Si~é
_ / /
R- ?i/\ mom) ;i// (Prowl) ?§((DPrOM)
[0183] Preparation of T1: Into an oven-dried nitrogen-purged 100 mL flask was added 5-
iodo-2’-deoxyuridine (5.0 g, 14.12 mmol). This was co-evaporated 3 times with 30 mL of
pyridine then brought under Nitrogen. Anhydrous Pyridine (25 mL) was added and the
reaction stirred at room temperature until a homogenous solution was obtained (~15
minutes). The mixture was cooled to 0°C in an ice-water bath and Iert-butyldiphenylsilyl
chloride (4.04 mL, 15.5 mmol) was added slowly, dropwise with vigorous stirring (~1 hour).
The reaction was maintained at 0°C for 8 hours until all SM consumed by TLC. Saturated
aqueous ammonium chloride solution (~15 mL) was added and the reaction allowed to warm
to room temperature. The mixture was diluted with ethyl acetate (100 mL) and washed with
saturated aqueous ammonium chloride (200 mL). The organic layer was separated, and the
aqueous layer was extracted with ethyl acetate (4 X 50 mL). The organic layers were
combined, dried (MgSO4) and concentrated in vacuo to give ~8 g clear yellow oil after
removal of residual solvent under high vacuum. The crude product T1 was purified by flash-
column chromatography on silica as a white crystalline solid. Yield is 6.94 g (83%). LC-MS
(Electrospray negative) 591.08 [M-H]
[0184] Preparation of T2: Into an oven-dried, nitrogen-purged, brown, 500 mL three-necked
flask was added T1 (6.23 g, 10.5 mmol), copper (I) iodide (200 mg, 1.05 mmol) and
bis(triphenylphosphine)palladium(II) dichloride (369 mg, 0.526 mmol) under nitrogen. The
flask was protected from light and anhydrous degassed DMF (200 mL) added. To this
solution was added 2,2,2-trifluoro-N-prop-2-ynyl-acetamide (4.74 g, 31.6 mmol), followed
by degassed triethylamine (2.92 mL, 21.0 mmol). The reaction was stirred at room
temperature under nitrogen for 6 hours when no further starting material was observed by
TLC analysis. Volatiles were removed in vacuo (~15 mins) and DMF removed under high
vacuum (~1 hour) to brown residue. This was dissolved in ethyl acetate (200 mL) and
extracted with 0.1M EDTA in water (2 x 200 mL). The aqueous layers were combined and
further extracted with ethyl acetate (200 mL). The organic phases were combined, dried
(MgSO4) and volatiles removed in vacuo (~30 min) and further dried under high vacuum (~1
hour) to give about 8 g of crude brown/yellow oil. The mixture was purified by flash-column
W0 2020/ 136170 PCT/EP2019/086926
chromatography on silica gel as an off-white solid. Yield: 6.0 g (85%). LC-MS (Electrospray
negative) 614.19 [M-H].
[0185] Preparation of T3: To an oven dried nitrogen purged 100 mL flask containing starting
nucleoside T2 (2.0 g, 3.25 mmol) under nitrogen was added anhydrous DMSO (6.9 ml, 97.5
mmol) in one portion at room temperature and stirred until a homogeneous solution was
formed. Acetic acid (11.1 mL, 195 mmol) followed by acetic anhydride (15.1 mL, 162.09
mmol) were both added dropwise (~5 minutes each). The mixture was warmed to 50°C and
stirred until complete consumption of the starting nucleoside (~5 hours) by TLC
(EtOAc/Petroleum ether 3:2). The reaction was then concentrated to half volume and cooled
down with an ice bath to approximately 0.5 °C. Work up commenced by slow addition of
cold (~0.5 °C) NaHCO3 (aq, sat.) (45 mL) and fiirther stirring allowed until no more fizzing
observed (~15 min). The solution was allowed to warm to room temperature, then the
aqueous extracted into EtOAc (3 x 100 mL). Combined organic layers were dried over
MgSO4, filtered and the volatiles evaporated under reduced pressure and further by high
vacuum. Crude product T3 was purified by flash chromatography on silica gel as an off-
white solid. Yield: 1.79 g (82%). LC-MS (Electrospray negative) 674.20 [M-H]'.
[0186] Preparation of T4: To a solution of the starting nucleoside T3 (179 g, 2.649 mmol) in
anhydrous CH2Cl2 (50 mL) under N2 was added cyclohexene (1.34 mL, 13.2 mmol). The
mixture was cooled with an ice bath to 0°C and distilled sulfuryl chloride (322 DL, 3.97
mmol) was slowly added dropwise (~20 min) under N2, After stirring for 20 min at that
temperature TLC (EtOAc:petroleum ether = 3:2 v/v) indicated the firll consumption of the
starting nucleoside. The chloride intermediate was then quenched by direct, dropwise
addition of freshly distilled the corresponding unsaturated alcohol (5 eq.) as shown in Scheme
3. The resulting solution was stirred at room temperature for 2 hours followed by evaporation
of the volatiles under reduced pressure. The oily residue was partitioned between
EtOAc:brine (3:2) (125 mL). The organic layer was separated and the aqueous was further
extracted into EtOAc (2 x 50 mL). Combined organic extracts were dried over MgSO4,
filtered and the volatiles evaporated under reduced pressure. The oily residue was partitioned
between EtOAc:brine (3:2) (125 mL). The organic layer was separated and the aqueous was
further extracted into EtOAc (2 x 50 mL). Combined organic extracts were dried over
MgSO4, filtered and the volatiles evaporated under reduced pressure. The crude products T4
was purified by flash chromatography on silica gel to yield the final products as a yellow oil.
Yield: 1.20 g (69%) for AOM; 1.29 g (71%) for PrOM; 1.34 g (71%) for DPrOM.
[0187] 3'-AOM: Yellow oil. LC-MS (Electrospray negative) [M-H] 684.24.
W0 2020/ 136170 PCT/EP2019/086926
[0188] 3'-PrOM: Yellow oil. LC-MS (Electrospray negative) [M-H] 682.22.
[0189] 3'-DPrOM: Yellow oil. LC-MS (Electrospray negative) [M-H] 710.25.
é NHTFA é
HN | HN
NHTFA
TBDPSO 00 N \ TBDPSO N R = M
O SMe 0 O\
Scheme 3.
[0190] Preparation of T5: The starting material T4 (1.04 g, 1.516 mmol) in a 50 mL round
bottom flask under nitrogen was added anhydrous THF (9 mL) at room temperature. Then
TBAF (1.0 M in THF, 1.7 mL, 1.70 mmol) was added dropwise and the solution stirred until
all SM consumed by TLC (~2 hours). The solution turned orange over the course of the
reaction. Volatiles were removed in vacuo to give an orange residue which was dissolved in
EtOAc (100 mL) and separated with NaHCO3 (sat. aq) (60 mL). The two layers were
separated, and the aqueous layer was extracted with EtOAc (60 mL). The organic layers
were combined, dried (MgSO4), filtered, and evaporated to give the crude product as a yellow
oil. The crude product was purified by flash chromatography on silica gel to yield a clear
yellow oil. Yield: 637 mg (94%) for AOM, 526 mg (78%) for PrOM, 617 mg (86%) for
DPrOM.
[0191] 3'-AOM: clear yellow oil. LC-MS (Electrospray negative) [M-H] 446.12.
[0192] 3'-PrOM: Clear yellow oil (526 mg 78%). LC-MS (Electrospray negative): [M-H]
444.10.
[0193] 3'-DPrOM: Clear yellow oil (617 mg 86%). LC-MS (Electrospray negative): [M-H]
472.13.
[0194] In addition, two additional 3’ blocked T nucleosides (3’-eAOM T and 3’-iAOM T)
were prepared following the similar fashion as described above. 3’-iAOM T: LC-MS (ES):
(negative ion) m/z 325.5 (M-H+), (positive ion) 327.3 (M+H+). 3’-eAOM T: LC-MS (ES):
(positive ion) m/z 341.3 (M+lH+).
W0 2020/ 136170 PCT/EP2019/086926
ft 1%
H0 N/KO HO l N/KO
o\/O\/\/\ O\/Ox
3'_eAQM T 3'-iAOM T
Example 2. 3’-OH Blocking Group Stabilitv Testing
[0195] In this example, the stability tests for 5'-mP 3'-AOM T nucleotide was performed side
by side in an incorporation buffer solution with standard 5'-mP 3'-O-azidomethyl T
nucleotide.
O O O
YKNH TKNH \fikNH
9 NAG 9 NAG 9 A0 |N/go
HO-F|>—O O HO—F|>—O O HO—F|’-O ‘R
OH OH OH H0‘F,"O 0
OH R J
2 in T V3
'mP 3'-AOM T 5'mP 3'-PrOM T 5'mP 3'-DPrOM T 5'mP 3'-O-azidomethyl T
Formulation ofthe buffer solution
[0196] 1 mL of 0.1 mM of each 5’-monophosphate 3’-protected T nucleotide in a solution of
100 mM ethanolamine buffer (pH 9.8), 100 mM NaCl, and 2.5 mM EDTA, was incubated at
65°C in a heating block for 2 weeks. At set time points, 40 [LL aliquots were taken and
analyzed by HPLC to determine the percentage of blocked nucleotide remaining and the
eventual formation of unblocked nucleotide.
[0197] The stability testing results relating to the 5'-monophosphate 3’-protected nucleotides
with AOM, PrOM, DPrOM acetal protecting groups and the standard azidomethyl blocking
group are illustrated in It was observed that 3' blocked nucleotide monophosphate
with AOM, PrOM and DPrOM blocking groups offered over 30-50 fold improvement in the
reduction of the deblocking rate in the solution. This experiment mimics how the
corresponding fully fiinctionalized nucleotides (ffNs) would behave when stored in an
incorporation mix on the cartridge of a sequencing device. The stability improvement offered
W0 2020/ 136170 PCT/EP2019/086926
by these acetal protecting groups would also lead to a lower pre-phasing rate in sequencing
runs. Finally, it improves the shelf-life of the incorporation mix reagent.
Example 3. 3’-AOM Deblocking Testing
[0198] In this example, deblocking tests for 5’-mP 3’-AOM T and the standard 5’-mP 3’-O-
azidomethyl T nucleotide were performed individually in a solution unique to each blocking
group. Conditions were formulated to mimic Illumina’s standard deblock reagent as closely
as possible, and follow the same methodology. Concentrations of active deblock reagent,
buffer, and nucleoside are kept the same across all tests, but the identity of each component
was unique. In this way, the observed difference in rate between the individual deblocking
chemistries cannot due to the differences in concentration of formulation.
| NH
O N/g0 i ii
I] O N O
HO—F|’-O O ,,
OH HO—P-O O
Deblock reagent: (‘DH T( Deblock reagent:
O\/O Pd(||/O) + THP I THP
/ O\/N3
.mP 3._AOM T 5'mP 3'—O—azidomethy| T
Standard Azidomethyl Deblocking Condition
[0199] Nucleotide: 5'-monophosphate 3'-O-azidomethyl T. Active Deblock reagent:
tris(hydroxypropyl)phosphine (THP) (lM in 18 m9 water). (Optional) Additive: Sodium
ascorbate (0.1 mM in 18 m9 water) final conc. = 1 mM. Buffer: Ethanolamine pH 9.8 (2M in
18 m9 water). Quenching reagent: H202.
AOM Deblocking Condition
[0200] Nucleotide: 5’-monophosphate 3'-O-azidomethyl T. A stock solution of 3'-AOM T
was diluted to 0.1 mM in a 100 mM ethanolamine buffer (pH 9.8), in a glass vial under
nitrogen. A stock solution of sodium ascorbate additive was added to a final concentration of
0.1 mM and the solution stirred 5 minutes. To commence the assay, the deblock reagents
(Pd/THP =1/5; sodium ascorbate, ethanolamine) were added, to a final concentration of 1
mM THP, to the stirring solution at room temperature. At specified time points, 40uL
aliquots were taken and quenched with 6 [LL of a 1:3 mixture of EDTA/H202 (0.025:0.075
M). I-IPLC analysis was performed by measuring the area of the starting nucleoside peak, the
W0 2020/ 136170 PCT/EP2019/086926
3'-OH peak, and any other nucleotide peaks that appear in the HPLC chromatogram. No other
nucleotide-based side products were observed.
[0201] The comparative result is shown in . It was observed that AOM offered a 10-
fold speed improvement in term of deblocking rate in solution compared to the standard
azidomethyl blocking group. This experiment serves the purpose of mimicking how the
corresponding ffN s would behave in sequencing during the deblocking step. The substantial
improvement in the deblocking speed would allow for a flush-through deblocking step
instead of the 10 to 20 second incubation time typically used in certain Illumina sequencing
platforms. As a result, the deblocking rate will have a significant impact on sequencing by
synthesis (SB S) cycle time.
[0202] Similar experimental conditions were used for the deblocking assay for 3’-eAOM T
and 3'-iAOM T. As a single alteration, Pd catalyst to substrate ratio was reduced to 5:1 in
order to observe less distinct differences in the deblocking rate. 3'-AOM T was used as a
reference and the results are illustrated in . These results showed that the deblocking
rate of eAOM and iAOM are 2 to 3 times slower than AOM at this specific concentration of
the Pd catalyst deblocking reagent. It can be expected that the difference in deblocking rates
among the substituted version and unsubstituted version of the AOM blocking groups would
be smaller when Pd catalyst to substrate ratio is higher.
Example 4. Optimization of Palladium Cleavage Mix for Sequencing
[0203] The Pd/THP catalyst used in the deblocking reaction described in Example 2 is very
air sensitive. When exposed to air, it showed a substantial loss of activity. In this example,
an oxidation stress assay was developed to assess air sensitivity of different formulations of
the palladium cleavage mix.
[0204] 0.5 mL of Pd cleave mix were aliquoted in a 5 mL glass vial and left open to air for 3
hours at room temperature. The residual activity of the oxidized cleavage mix was assessed
by measuring the cleavage of 3'-AOM T as follows. A stock solution of 3'-AOM T was
diluted to 0.1 mM in 100 mM cleavage mix buffer. A stock solution of sodium ascorbate was
added to a final concentration of 1 mM, followed by the oxidized cleavage mix to a final l/20
dilution. After 1 hour, 40 [LL of the solution were immediately quenched with 10 [LL of a 1:1
mixture of EDTA/H202 (O.25:O.25 M) and analyzed by HPLC. In this experiment, various
buffer reagents were screened, including: primary amines (such as ethanolamine, Tris and
glycine), tertiary amines (such as 2-dimethyalaminomethanol ((DMEA), 2-
diethyalaminomethanol (DEEA), N,N,N',N‘-tetramethylethylenediamine (TEMED) or
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N,N,N’,N'-tetraethylethylenediamine (TEEDA)); and various inorganic salts (such as a borate
salt, an carbonate salt, a phosphate salt). It was observed that inorganic buffer reagents (such
as sodium borate, sodium carbonate, sodium phosphate) offered the best air stability and the
palladium complex retained high % activities. In addition, tertiary amines also substantially
improved the stability of the Pd cleavage mix as compared to primary amines.
[0205] Based on these findings, two palladium cleavage mix were prepared. In a first
example, a stock solution of 250 mM borate buffer aq. (pH 9.6, 20 mL) was diluted with
water (14 mL) before addition of a stock solution of THP (1 M in 100 mM Tris, pH 9, 5 mL,
.0 mmol) and of allylpalladium (II) chloride dimer (183 mg, 0.5 mmol). The mixture was
vigorously stirred for a few minutes at room temperature before addition of 1 M sodium
ascorbate aq. (0.5 mL, 0.5 mmol), 5 M NaCl aq. (10 mL) and 10% v/v Tween20 (0.5 mL). In
a second example, a stock solution of 2 M DEEA buffer aq. (pH 9.6, 0.6 mL) was diluted
with water (7.6 mL) before addition of a stock solution of THP (1 M in 100 mM Tris, pH 9,
1.2 mL, 1.2 mmol) and of solid allylpalladium (II) chloride dimer (43.9 mg, 0.12 mmol). The
mixture was vigorously stirred for a few minutes at room temperature before addition of 1 M
sodium ascorbate aq. (0.12 mL, 0.12 mmol), 5 M NaCl aq. (2.4 mL) and 10% v/v Tween20
(0.12 mL).
Example 5. Preparation of Fullv Functionalized Nucleotides and Uses for Sequencing
Application
[0206] In this example, the preparation of various fully functionalized nucleotides (ffN s) with
3'-AOM blocking group are described in details. These ffN s were also used in the sequencing
by synthesis application on Illumina MiniSeq® platform.
Scheme 4. Synthesis of 3'-AOM-ffC-LN3-SO7181
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NH-n:A O NHTFA O NHTFA O
H~ \ do \ do
\db \ vi
Tanpso N’i§o TBDPSO N O HO N O
W O I 52% j 0 66% j 0
O\/S\ O O\/O\/\ O\/O\/\
C1 TFA = §)J\CF3 AOM C2 AOM C3
% NH2
Ho,°:+o,9,Ho5>,Ho NAG
II II I] O
O\/O\/\
AOM C4
N3 O/WFNH
3'-AOM-ffC-LN3-SO7181 \ \ N
OH OH oH I /k
HO\|l_f|,,O\|_1,,O\P,O O N o
o as 5 Q
oVo\/\
[0207] Synthesis of intermediate AOM C2: Nucleoside C1 (0.5 g, 0.64 mmol) was
dissolved in anhydrous DCM (12 mL) under N2, and the mixture was cooled to 0 °C.
Cyclohexene (0.32 mL, 321 mmol) was added, followed by dropwise SO2Cl2 (1.0 M in
DCM, 1.27 mL, 1.27 mmol). Additional cyclohexene (0.32 mL, 3.21 mmol) was added
before quickly transferring the reaction to a rotary evaporator to remove all the volatiles
under reduced pressure. The solid residue was additionally dried under high vacuum for 10
min before being dissolved in anhydrous DCM (5 mL) under N2. The mixture was cooled to
0°C and ice-cold allyl alcohol (5 mL) was added dropwise. The reaction was stirred at 0°C
for 2h, before being quenched by addition of sat. NaHCO3 aq. (50 mL) and DCM (30 mL).
The two phases were separated, and the aqueous layer was extracted with EtOAc (2 X 50
mL). The organic layers were combined, dried over MgSO4, filtered and the volatiles were
evaporated under reduced pressure. The crude product was purified by flash chromatography
on silica gel using a EtOAc/petroleum ether to give AOM C2 as a white solid (264 mg, 52%
yield). LC-MS (Electrospray negative): [M-H] 787, [M+Cl] 823.
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[0208] Synthesis of intermediate AOM C3: AOM C2 (246 mg, 0.31 mmol) was dissolved
in anhydrous THF (9.5 mL) under N2 and the mixture was cooled to 0°C. Acetic acid (0.054
mL, 094 mmol) was added, followed by dropwise TBAF (1.0 M in THF, 5 wt.% water, 099
mL, 0.94 mmol). The reaction was stirred at 0°C for 5 h, before being diluted with EtOAc (20
mL) and then poured into 0.05 M HCl aq. (20 mL). The two layers were separated, and the
aqueous layer was extracted with EtOAc (2 x 20 mL). The organic layers were combined,
dried over MgSO4, filtered and the volatiles were evaporated under reduced pressure. The
crude product was purified by flash chromatography on silica gel using a DCM/EtOAc to
give AOM C3 as a yellowish solid (114 mg, 66% yield). LC-MS (Electrospray negative):
[M-H] 549, [M+H2O-H] 567, [M+Cl] 585, (Electrospray positive): [M+H] 551, [M+H2O+H]
569.
[0209] Synthesis of intermediate AOM C4: AOM C3 (0.114 g, 0.21 mmol), freshly
activated 4A molecular sieves, proton sponge (0.066 g, 0.31 mmol) and a magnetic stirrer
were placed under N2 and anhydrous trimethyl phosphate (1.0 mL) was added. The reaction
was cooled at -10°C and freshly distilled POCI3 (23 uL, 0.25 mmol) was added dropwise.
The reaction was stirred at -10°C for 1 hour. A solution of pyrophosphate as bis-tri-n-
butylammonium salt (0.5 M in DMF, 1.7 mL, 0.85 mmol) and anhydrous tri-n-butyl amine
(0.41 mL, 1.74 mmol) were premixed and added to the ice-cold activated nucleoside solution
in one portion. The mixture was vigorously stirred for 5 minutes at room temperature. The
reaction mixture was poured into a separate flask containing a vigorously stirred solution of 2
M TEAB aq. (~10 mL). The reaction flask was rinsed with a small amount of H20 and the
washings added into the 2 M TEAB solution. The combined mixture was then stirred at room
temperature for 4 hours, after which the solvent was evaporated under reduced pressure. The
residue was dissolved in NH3 aq. (35%, ~10 mL) and stirred at room temperature overnight.
The reaction was concentrated under vacuum and purified by flash chromatography on
DEAE-Sephadex. The product was further purified by preparative HPLC to give pure AOM
C4 (62 Dmol, 30% yield, determined by UV-Vis spectrometry, Xmax = 294 nm, 5 = 8600 M'1
cm-1). LC-MS (Electrospray negative): [M-H] 589.
[0210] Synthesis of 3'-AOM-ffC-LN3-SO7181: LN3-SO7l8l (0.0205 mmol) was dissolved
in anhydrous DMA (4 mL) under N2. N,N-diisopropylethylamine (28.6 uL, 0.164 mmol) was
added, followed by TSTU (0.1 M in DMA, 234 uL, 0.0234 mmol). The reaction was stirred
under N2 at room temperature for 1 hour. In the meantime, an aqueous solution of AOM C4
(0.0101 mmol) was evaporated to dryness under reduced pressure, resuspended in 0.1 M
TEAB aq. (400 uL) and added to the LN3-SO7181 solution. The reaction was stirred at RT
for 17.5 hours and then quenched with 0.1M TEAB aq. (4 mL). The crude product was
purified by flash chromatography on DEAE-Sephadex. The product was further purified by
preparative HPLC to give pure 3'-AOM-ffC-LN3-SO7181 (6.81 Dmol, 67% yield,
determined by UV-Vis spectrometry, kmx = 644 nm, 8 = 200000 M'1 cm-1). LC-MS
(Electrospray negative): [M-H] 1561, [M-2H] 781, [M-3H] 520.
Scheme 5. Synthesis of 3'-AOM-ffA
TFAHN \N/ TFAHN \N/ TFAHN \N/
\\ N/J \\ N/J \\ N/J
/ l \jJ / | \jJ / l \N
TBDPSO N N’ TBDPSO N N’ HO N N?
:0: 1 T, :0: | T» O
O\/S\ O\/O\/\ O\/O\/\
A1 AOM A2 AOM A3
\\ NH2 \\ NH2
\N \
I / N
Ho}; ,;3,Ho,§3t‘o O N N4 HO\CI)|:|o\C1)|:|o\E|;iQ N ‘Na
as as as g j + as 5 as $0)!
O\/O\/\ O\/O\/\
3'-AOM-ffA AOM A4
silk, 0 BL H O
\/\O 3 Y0 \<<—,LDye(NR650C5)
R: O O HN n
C7/«Wyn
3'-AOM-ffA-LN3-BL-NR650C5
Zglk/O\/\O/K/O H Dye (NR7180A)
N/\/N\n)g\ n
3'-AOM-ffA-LN3-NR7180A
W0 2020/ 136170 PCT/EP2019/086926
o N O
/Elk/O\/\ /Q/o O H HN/[L0/R o
O \(D/U\N/\/N7,/K/\/\ /[Le/).Dye(NR55OS0)
3'-AOM-ffA-LN3-BL-N R550S0
[0211] Synthesis of intermediate AOM A2: Nucleoside A1 (716 mg, 0.95 mmol) was
dissolved in 10 mL of anhydrous dichloromethane under N2 atmosphere, cyclohexene (481
uL, 4.75 mmol) was added and the solution was cooled to approximately -15 °C. Sulfuryl
chloride (distilled, 92 uL, 1.14 mmol) was added dropwise and the reaction was stirred for 20
minutes. After all the starting material had been consumed, an extra portion of cyclohexene
was added (481 uL, 4.75 mmol) and the reaction was evaporated to dryness under reduced
pressure. The residue was quickly purged with nitrogen, then allyl alcohol (5 mL, ~100
mmol) was added under stirring at 0°C. The reaction was stirred at 0°C for 1 hour, then
quenched with 50 mL of saturated aq. NaHCO3. The mixture was extracted with 2x 100 mL
of ethyl acetate. The pooled organic phases were washed with 100 mL of water and 100 mL
of brine, then dried over MgSO4, filtered and evaporated to dryness. The residue was purified
by flash chromatography on silica gel using Petroleum ether/EtOAc. 60% yield (435 mg, 0.57
mmol). LC-MS (ES and CI): (positive ion) m/z 763 (M+H+), (negative ion) m/z 761 (M-H+).
[0212] Synthesis of intermediate AOM A3: Nucleoside AOM A2 (476 mg, 0.62 mmol)
was dissolved in dry THF (5 mL) under N2 atmosphere, then a solution of 1.0 M TBAF in
THF (750 uL, 0.75 mmol) was added. The solution was stirred at room temperature for 1.5
hours. The solution was diluted with 50 mL of EtOAc, then washed with 100 mL of
NaH2PO4 sat. (pH=3), and with 100 mL of brine. The organic phase was dried over MgSO4,
filtered and evaporated to dryness. The residue was purified by fiash chromatography on
silica gel using EtOAc/MeOH. 90% yield (292 mg, 0.55 mmol). LC-MS (ES and CI):
(positive ion) m/z 525 (M+H+), (negative ion) m/z 523 (M-H+).
[0213] Synthesis of intermediate AOM A4: Nucleoside AOM A3 (285 mg, 0.544 mmol,)
was dried under reduced pressure over P205 for 18 hrs. Anhydrous triethyl phosphate (2 mL)
and some freshly activated 4 A molecular sieves were added to it under nitrogen, then the
reaction flask was cooled to 0 °C in an ice-bath. Freshly distilled POCI3 (61 uL, 0.65 mmol)
was added drop-wise followed by Proton Sponge® (175 mg, 0.816 mmol). After the addition,
the reaction was further stirred at 0°C for 15 minutes. Then, a 0.5 M solution of
W0 2020/ 136170 PCT/EP2019/086926
pyrophosphate as bis-tri-n-butylammonium salt (5.4 mL, 2.72 mmol) in anhydrous DMF was
quickly added, followed immediately by tri-n-butyl amine (540 uL, 2.3 mmol). The reaction
was kept in the ice-water bath for another 10 minutes, then quenched by pouring it into 1 M
aqueous triethylammonium bicarbonate (TEAB, 20 mL) and stirred at room temperature for 4
hours. All the solvents were evaporated under reduced pressure. A 35% aqueous solution of
ammonia (20 mL) was added to the above residue and the mixture was stirred at room
temperature for at least 5 hours. The solvents were then evaporated under reduced pressure.
The crude product was purified firstly by ion-exchange chromatography on DEAE-Sephadex
A25 (100g). The column was eluted with a gradient of aqueous triethylammonium
bicarbonate. The fractions containing the triphosphate were pooled and the solvent was
evaporated to dryness under reduced pressure. The crude material was further purified by
preparative scale HPLC using a YTVIC-Pack-Pro C18 column, eluting with 0.1 M TEAB and
acetonitrile. Compound AOM A4 was obtained as triethylammonium salt. 56% yield (306
umol). LC-MS (ES and CI): (negative ion) m/z 612 (M-H1), (positive ion) m/z 614 (M+H+),
715 (M+Et3NH+).
[0214] General procedure for ffA synthesis: The dye-linker (0.020 mmol) was dissolved in
2 mL of anhydrous N,N’-dimethylacetamide (DMA). N,N-diisopropylethylamine (28.4 11L,
0.163 mmol) was added, followed by N,N,N',N‘-tetramethvl-O-(N-succinimidvl)uronium
tetrafluoroborate as 0.1 M solution in anhydrous DMA (TSTU, 232 uL, 0.023 mmol). The
reaction was stirred under nitrogen at room temperature for 1 hour. In the meantime, an
aqueous solution of the triphosphate AOM A4 (0.01 mmol) was evaporated to dryness under
reduced pressure and resuspended in 200 11L of 0.1 M triethylammonium bicarbonate
(TEAB) solution in water. The activated dye-linker solution was added to the triphosphate
and the reaction was stirred at room temperature for 18 hours. The crude product was purified
firstly by ion-exchange chromatography on DEAE-Sephadex A25 (25 g). The fractions
containing the triphosphate were pooled and the solvent was evaporated to dryness under
reduced pressure. The crude material was further purified by preparative scale RP-HPLC
using a YMC-Pack-Pro C18 column. 3'-AOM—ffA-LN3-NR7180A: 38% yield (3.8 pmol).
LC-MS (ES): (negative ion) m/z 1459 (M-H1“), 729 (M-2H+), 486 (M-3H+). 3’-AOM-ffA-
LN3-BL-NR550S0: 37% yield (3.7 pmol). LC-MS (ES): (negative ion) m/z 1771 (M-H1),
885 (M-2H+), 589 (M-3H+). 3'-AOM ffA-LN3-BL-NR65OC5: 51% yield (51 umol). LC-MS
(ES): (negative ion) m/z 1917 (M-H1), 958 (M-2H+), 645 (M-3H+).
Scheme 6. Synthesis of 3'-AOM-pppG
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<, | NH O <, | NH O
N N/J\”)g N N/A”
>~S|-O—| O O $~Si-O—| O O\®
O\/Sx O\/O\/\
dG3 AOM G4
Mr <’”l1“°
000 N’ ‘j N/)5
II II II N NH2 N N
Ho".°‘o".°‘o"?‘o O HO O H 0
OH OH OH \®
O\/O\/\ O\/O\/\
3'-AOM-pppG AOM GS
[0215] Synthesis of intermediate AOM G4: Known nucleoside dG3 (100 mg, 0.143 mmol)
was dissolved in 10 mL of anhydrous dichloromethane under N2 atmosphere, Cyclohexene
(72 uL, 0.714 mmol) was added and the solution cooled to -12°C. Sulfuryl chloride (distilled,
(lM in DCM), 171 uL, 0.171 mmol) was added dropwise and the reaction was stirred for 10
min. An extra portion of cyclohexene (72 uL, 0.714 mmol) was added and the reaction stirred
for 30 min at -12°C. The reaction was evaporated to dryness under reduced pressure, the
residue was purged with nitrogen, and ice cold, neat allyl alcohol (distilled, 0.8 mL, 12
mmol) was added under stirring at -12°C. The reaction was stirred at -12°C for 60 mins, then
quenched with 2 mL of saturated aq. NaHCO3. The mixture was separated with ethyl acetate
(2mL), the aqueous layer extracted with ethyl acetate. Combined organic phases were washed
with 4 mL of water and 4 mL of brine, dried over MgSO4, filtered and evaporated to crude
oil. The residue was purified by flash chromatography on silica gel to give AOM G4 as a
clear oil. 36% yield (50.9 mg, 0.072 mmol). LC-MS (ES and CI): (positive ion) m/z 710
[M-I-H]+; (negative ion) m/z 708 [M-H]‘.
[0216] Synthesis of intermediate AOM G5: Nucleoside AOM-G4 (111 mg, 0.156 mmol)
was dissolved in dry THF (5 mL) under N2 atmosphere. Acetic acid (27 uL, 0.468 mmol) was
added, followed by a solution of 1.0 M TBAF in THF (296 uL, 0.296 mmol). The solution
was stirred at room temperature for 5 hours. The solution was diluted with 10 mL of EtOAc,
washed with 10 mL of 0.05 M aq. HCl and organic separated. The aqueous phase was
extracted with ethyl acetate. The combined organic phases were dried over MgSO4, filtered
W0 2020/ 136170 PCT/EP2019/086926
and evaporated to dryness. The residue was purified by flash chromatography on silica gel to
give AOM G5 as a white solid. 44% yield (32.4 mg, 0.068 mmol). LC-MS (ES and CI):
(positive ion) m/z 472 [M+H]+; (negative ion) m/z 470 [M-H]'.
[0217] Synthesis of 3'-AOM-pppG: Nucleoside AOM-G5 (79 mg, 0.168 mmol,) with
freshly activated 4 A molecular sieves were dried under reduced pressure over P205 for 18
hrs. Proton Sponge® (175 mg, 0.816 mmol) and anhydrous triethyl phosphate (0.8 mL) was
added under nitrogen and stirred at room temperature for 1 hour. The reaction flask was
cooled to 0°C in an ice-bath, freshly distilled POCI3 (19 uL, 0.202 mmol) was added drop-
wise and the reaction was stirred at 0°C for 15 minutes. Then, a 0.5 M solution of
pyrophosphate as bis-tri-n-butylammonium salt (1.68 mL, 0.84 mmol) in anhydrous DMF
was quickly added, followed immediately by tri-n-butyl amine (168 uL, 0.705 mmol). The
reaction was removed from the ice/water bath and stirred vigorously for 5 minutes, then
quenched by pouring it into 1 M aqueous triethylammonium bicarbonate (TEAB, 6 mL) and
stirred at room temperature for 18 hours. All the solvents were evaporated under reduced
pressure. The residue was dissolved in 35% aqueous ammonia solution (10 mL) and was
stirred at room temperature for at least 5 hours. The solvents were then evaporated under
reduced pressure and further co-evaporated with water. The crude product was purified firstly
by ion-exchange chromatography on DEAE-Sephadex A25 (50 g). The column was eluted
with a linear gradient of aqueous triethylammonium. The fractions containing the
triphosphate were collected and the solvent was evaporated to dryness under reduced
pressure. The crude material was further purified by preparative scale HPLC using a YMC-
Pack-Pro C18 column. 3'-AOM-pppG was obtained as triethylammonium salt. 24% yield
(39.7 umol). LC-MS (ES and CI): (negative ion) m/z 576 [M-H]', (positive ion) m/z 578
[M+H]+.
[0218] 3'-AOM-ffT-LN3-NR550S0 was synthesized in a similar fashion as described in the
preparation of the 3’-AOM ffA and ffC.
Scheme 7. Synthesis of 3'-AOM-ffT-LN3'-NR550S0
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o o O o
HN | I HN | \ NHTFA Ifij/\ANHTFA |-BLJE/\/\NHTFA
Ho O¢J\N Ho OéJ\N TBDPSO O N Tgppso o N
:0: _. :0: —» ‘:0: 4» F0:
OH OH OH Vs\
T1 T2 ‘rs
0 o o
IDLJ]/\ANH2 |-Ljjlj/\/\NHTFA HNWNHTFA
OH OH 0
HO\ ',O\ ' ,O\ "/0 O N Ho 0 N TBDPSQ OAN
P P P 0
II II II 0 O
o o o <— i 9
Ox/O\/\ O\/O\/\ O\/O\/\
T6 T5 T4
i O O \/\O)3\/D
HN \ )\\/O NH
A ' N 0 “
Ho\9'jo\9'jo\9Eo 0 N NH
E’ .F.’ E’ o °‘<+
o o o ( Dye (NR550S0)
3'-AOM-fI‘T-LN3'-NR550S0
[0219] Synthesis of intermediate T1: 5-Iodo-2’-deoxyuridine (3 g, 8.4 mmol) and
palladium (II) acetate (1.6 g, 7.14 mmol) were dissolved in dry degassed DMF, then N-
allyltrifluoroacetamide (6.4 mL, 42 mmol) was added. The solution was placed under
vacuum, then purged with nitrogen for 3 times, then degassed triethylamine (2.3 mL, 16.8
mmol) was added. The solution was heated to 80°C for 2 hours. The black mixture was
cooled down to room temperature then diluted with 50 mL of methanol. Approximately 0.5 g
of activated charcoal was added, and the solution was filtered on Celite, then evaporated
under reduced pressure to afford a brown thick oil. This crude was purified by
chromatography on silica gel using a EtOAc/MeOH. Yield: (2.27 g, 5.99 mmol). LC-MS (ES
and CI): (negative ion) m/z 378 (M-H+).
[0220] Synthesis of intermediate T2: 5-[3-(2,2,2-trifluoroacetamido)-allyl]-2'-deoxyuridine
(T1) (2.55g, 6.72 mmol) was dissolved in dry DMF. Imidazole (1.37 g, 20.1 mmol) was
added followed by 4-(dimethylamino)pyridine (410 mg, 3.36 mmol). The reaction was cooled
W0 2020/ 136170 PCT/EP2019/086926
to 0°C, then iert-butyl(chloro)diphenylsilane (1.92 mL, 7.39 mmol) was added slowly in 3
portions, 30 minutes apart. The reaction was stirred at 0°C for 6 hours. The solvent was then
evaporated, and the residue resuspended in 200 mL of EtOAc and washed with 2x 200 mL
aq. saturated NaHCO3 and 200 mL of water, then 100 mL of brine. The organic phase was
dried over MgSO4, filtered and evaporated to dryness. The crude was purified by flash
chromatography on silica using a DCM/EtOAc. 68% yield (2.806 g, 4.54 mmol). LC-MS (ES
and CI): (positive ion) m/z 618 (M+H+); (negative ion) m/z 616 (M-H1).
[0221] Synthesis of intermediate T3: 5'-O-(tert-butyldiphenylsilyl)-5-[3-(2,2,2-
trifluoroacetamido)-allyl]-2'-deoxyuridine (T2) (2.8 g, 4.53 mmol) was dissolved in 10 mL of
anhydrous DMSO (136 mmol), then glacial acetic acid (16 mL, 272 mmol) and acetic
anhydride (16 mL, 158 mmol) were added. The reaction was heated to 50 °C for 6 hours then
quenched with 200 mL of aq. saturated NaHCO3. After the solution stopped bubbling, it was
extracted with 2x 150 mL of EtOAc. The organic phases were pooled and washed with 2
x200 mL of aq. saturated NaHCO3, 200 mL of water and 100 mL of brine. The organic phase
was dried over MgSO4, filtered and evaporated to dryness. The crude was purified by flash
chromatography on silica using a DCM/EtOAc. 77% yield (2.375g, 3.51 mmol). LC-MS (ES
and CI): (positive ion) m/z 678 (M+H+); (negative ion) n1/z 676 (M-H1).
[0222] Synthesis of intermediate T4: 5'-O-(tert-butyldiphenylsilyl)-3'-O-methylthiomethyl-
-[3-(2,2,2-trifluoroacetamido)-allyl]-2'-deoxyuridine (T3) (310 mg, 0.45 mmol) was
dissolved in 5 mL of anhydrous dichloromethane under N2 atmosphere, cyclohexene (228
uL, 2.25 mmol) was added and the solution was cooled to approximately -15°C. Sulfuryl
chloride (distilled, 55 uL, 0.675 mmol) was added dropwise and the reaction was stirred for
minutes. After all the starting material had been consumed, an extra portion of
cyclohexene was added (228 uL, 2.25 mmol) and the reaction was evaporated to dryness
under reduced pressure. The residue was quickly purged with nitrogen, then ice-cold allyl
alcohol (2.5 mL) was added under stirring at 0°C. The reaction was stirred at 0°C for 35
minutes, then quenched with 25 mL of saturated aq. NaHCO3, then diluted further with 100
mL of saturated aq. NaHCO3. The mixture was extracted with 2x 50 mL of ethyl acetate. The
pooled organic phases were dried over MgSO4, filtered and evaporated to dryness. The
residue was purified by flash chromatography on silica gel using a DCM/EtOAc. 69% yield
(214 mg, 0.311 mmol). LC-MS (ES and CI): (positive ion) m/z 688 (M+H+); (negative ion)
m/z 686 (M-H+).
[0223] Synthesis of intermediate T5: 5'-O-(tert-butyldiphenylsilyl)-3'-O-allyloxymethyl-5-
[3-(2,2,2-trifluoroacetamido)-allyl]-2’-deoxyuridine (T4) (210 mg, 0.305 mmol) was
W0 2020/ 136170 PCT/EP2019/086926
dissolved in dry THF (3 mL) under N2 atmosphere. A solution of 1.0 M TBAF in THF (367
uL, 0.367 mmol) was added. The solution was stirred at room temperature for 3 hours. The
solution was diluted with 50 mL of EtOAc, then washed with 50 mL of NaH2PO4 sat.
(pH=3), and with 50 mL of water. The organic phase was dried over MgSO4, filtered and
evaporated to dryness. The residue was purified by flash chromatography on silica gel using
DCM/EtOAc. 95% yield (130 mg, 0.289 mmol). LC-MS (ES and CI): (negative ion) m/z 448
(M-Hi“), 484 (M+Cl').
[0224] Synthesis of intermediate T6: 3'-O-allyloxymethyl-5-[3-(2,2,2-trifluoroacetamido)-
allyl]-2'-deoxyuridine (T5) (120 mg, 0.267 mmol,) was dried under reduced pressure over
P205 for 18 hrs. Anhydrous triethyl phosphate (1 mL) and some freshly activated 4 A
molecular sieves were added to it under nitrogen, then the reaction flask was cooled to 0°C.
Freshly distilled POCI3 (30 uL, 0.32 mmoles) was added drop-wise followed by Proton
Sponge® (85 mg, 0.40 mmol). After the addition, the reaction was further stirred at 0°C for
minutes. Then, a 0.5 M solution of pyrophosphate as bis-tri-n-butylammonium salt (2.7
mL, 133 mmol) in anhydrous DMF was quickly added, followed immediately by tri-n-butyl
amine (270 uL, 1.2 mmol). The reaction was kept in the ice-water bath for another 10
minutes, then quenched by pouring it into 1 M aqueous triethylammonium bicarbonate
(TEAB, 10 mL) and stirred at room temperature for 4 hours. All the solvents were evaporated
under reduced pressure. A 35% aqueous solution of ammonia (10 mL) was added to the
above residue and the mixture was stirred at room temperature for 18 hours. The solvents
were then evaporated under reduced pressure, the residue resuspended in 10 mL of 0.1 M
TEAB and filtered. The filtrate was purified firstly by ion-exchange chromatography on
DEAE-Sephadex A25 (100 g). The column was eluted with aqueous triethylammonium
bicarbonate (TEAB). The fractions containing the triphosphate were pooled and the solvent
was evaporated to dryness under reduced pressure. The crude material was further purified by
preparative scale HPLC using a YMC-Pack-Pro C18 column. Compound T6 was obtained as
triethylammonium salt. 33% yield (89 umol). LC-MS (ES and CI): (negative ion) m/z 592
(M-H+), 295 (M-2H*).
[0225] Synthesis of 3'-AOM-flT-LN3'-NR550S0: The dried known compound LN3-
NR550S0 (0.015 mmol) was dissolved in anhydrous DMA (2 mL) under N2. N,N-
diisopropylethylamine (17 uL, 0.1 mmol) was added, followed by TSTU (0.1 M in DMA,
180 uL, 0.018 mmol). The reaction was stirred under N2 at room temperature for 1 hour. In
the meantime, an aqueous solution of T6 (0.01 mmol) was evaporated to dryness under
W0 2020/ 136170 PCT/EP2019/086926
reduced pressure, resuspended in 0.1 M TEAB aq. (200 11L) and added to the LN3-NR550S0
solution. The reaction was stirred at RT for 18 hours and then quenched with 0.1M TEAB aq.
(4 mL). The crude product was purified by flash chromatography on DEAE-Sephadex. The
product was further purified by preparative HPLC to give pure 3'-AOM-ffT-LN3'-NR550S0.
67% yield (41 Dmol, determined by UV-Vis spectrometry, max = 550 nm, 5 = 125000 M'1
cm-1). LC-MS (ES): (negative ion) m/z 1521 (M-H+), 761 (M-2H+), 507 (M-3H+).
Sequencing-by-Synthesis Experiments
[0226] The ffN s were subsequently tested in sequencing using an Illumina MiniSeq®
instrument. With the exception of a new incorporation mix including these ffi\Is, all standard
commercial reagents were used. A standard 2x150 recipe was used. In addition to the
standard sequencing-by-synthesis (SBS) protocols, a 5 seconds incubation in a solution of
palladium cleavage mix (Pd:THP = 1/5 in DEEA as described in Example 4) were added to
unblock 3'-AOM.
[0227] In a first experiment, the following ffN s were used in the incorporation mix: 3'-AOM-
ffT-LN3-NRSSOSO, 3'-AOM-ffA-LN3—BL-NRSSOSO, 3'-AOM-ffA-LN3-BL-NR65OC5, 3'-
AOM-ffA-LN3-NR7180A, 3'-AOM-ffC-LN3-SO7181, and 3'-AOM-pppG (dark G). and the
sequencing result for Read 1 is summarized below.
Read %PF Phasing Pre-phasing ER
1 90.2% 0.798 0.159 3.12
%PF: Percentage of cluster passing filter after 26 cycles
[0228] In a second experiment, unlabeled 3'-AOM-pppT was synthesized similarly to the
preparation of 3'-AOM-pppG described above (LC-MS (ES): (negative ion) m/z 551 (M-
H+)). It was used in presence of commercial green ffG-LN3-PEG12-ATTO532 (used on
Illumina 4-channel systems) in sequencing and the same ffAs and ffC were used as those
described in the first experiment above. The results are summarized below. There were
significant improvements on phasing and pre-phasing values and no signal decay was
observed (). In addition, error rates for both Read 1 and Read 2 were also reduced.
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Dark 3'—AOM—pppT
Read Phasing Pre-phasing ER
1 0.173 0.046 1.21
2 0.175 0.057 1.99
[0229] In another experiment, an incorporation mix containing 3’-AOM-ffT-LN3’-NRSSOSO,
3 '-AOM-ffA-LN3 -BL-NR5 5 0S0, 3 '—AOM-ffA-LN3 —BL-NR65 0C5 , 3 '-AOM-ffA-LN3 -
NR7180A, 3’-AOM-ffC-LN3-SO7181, and 3’-AOM-pppG (dark G) was used. Similarly to
previous runs, a 5 second incubation with a cleavage mixture containing a palladium catalyst
(Pd/THP = 1:10, 100mM DEEA as described in Example 4) was added to the standard SBS
cycle. Standard MiniSeq® DNA polymerase was used but at 2x incorporation time. No signal
decay phenotype was observed (). In addition, these sequencing results were
compared to commercial MiniSeq® runs (average of 3; N = 3) using ffN s with the standard
azidomethyl blocking group. It was observed that the error rates were nearly identical (). The sequencing results are summarized below.
Read Phasing Pre-phasing ER
1 0.121 0.063 0.40
2 0.129 0.062 0.57
[0230] In addition, the primary sequencing metrics for the ffNs with 3'-AOM blocking
groups were compared to those produced by the standard MiniSeq® commercial kit including
DNA polymerase Pol 812 and the comparative results are demonstrated in . Very
low pre-phasing was observed due to the improved stability of the 3'-AOM-ffN s. However,
phasing was still elevated even if 2x incorporation time was used.
[0231] In yet another experiment, a different DNA polymerase (Pol 1901) was used instead
of the DNA polymerase in the commercial MiniSeq® kits (P01 812). Pol 1901 allowed for
W0 2020/ 136170 PCT/EP2019/086926
standard lx incorporation time in sequencing instead of the 2x incorporation time described
above. In addition, incubation in the Pd cleavage mixture was reduced by half compared to
standard run. This allowed for a 10% time saving on the complete SBS chemistry cycle. The
sequencing metrics were significantly improved and exceeded the values obtained from the
standard commercial kits containing 3’-O-azidomethyl blocking group ().
3 ’Bl0ckin2' Group Stability Test in Sequencing
[0232] To demonstrate stability improvement of the ffN s with 3'-AOM, they were compared
side by side with standard MiniSeq® ffN s with 3’-O-azidomethyl group. The two sets of ffN s
were incubated at 45°C for several days in standard incorporation mix formulations
excluding only the DNA polymerase. For each time point, fresh polymerase was added to
complete the incorporation mix directly prior loading on MiniSeq®. Sequencing conditions
described previously were used. Pre-phasing% is a direct indicator of the percentage of
3’OH-ffNs present in the mix therefore directly correlates to the stability of the 3’ block
group. Pre-phasing values for both sets of ffNs were recorded and plotted (. At 45°C,
it was observed that 3'-AOM containing ffNs appeared to be 6x more stable than standard
ffNs with 3’-O-azidomethyl group. Sequencing metrics also confirmed the trend observed
during the stability assay in solution — 3'-AOM block was significantly more stable than 3’-O-
azidomethyl group.
Example 6. Preparation of 3'-O-Thiocarbamate Blocked Nucleosides
[0233] In this example, various 3'-O-thiocarbamate protected T nucleoside were prepared
according to Scheme 8.
Scheme 8. Synthesis of 3'-O-Dimethvlthiocarbamate T Nucleoside
W0 2020/ 136170 PCT/EP2019/086926
o \fib 0
1 J: NH
| NH DMTO N o l
DMTO O N/KO —> ‘W DMTO O N/KO
W OYFQN )4
OH 3 Y T-8
T-7 /N\
HO ON 0 lg
r 0
[0234] Preparation of T-7 : Into an oven-dried nitrogen-purged 100 mL flask was added 5’-O-
(4,4’-Dimethoxytrityl)thymidine (1.0 g, 1.836 mmol). This was co-evaporated with
anhydrous Dl\/[F (3 X 20 mL) and brought under nitrogen. Anhydrous DCM (9.2 mL) and 4-
dimethylaminopyridine (224 mg, 0.184 mmol) were added and stirred at room temperature
until a homogeneous solution was formed. Then 1,1’-thiocarbonyldiimidazole (360 mg, 2.02
mmol) was added quickly over a stream of nitrogen, the reaction resealed and stirred at room
temperature for 2 hours until all starting material was consumed. The reaction mixture is
filtered through a pad of silica gel and the filter cake washed with EtOAc (10 mL). Volatiles
were removed in vacuo and the crude residue used without further purification.
[0235] Preparation of T-8: Compound T-7 from the previous step was used immediately after
drying in vacuo. The residue was brought under nitrogen in a 25 mL round bottomed flash
and dimethylamine (2 M in THF, 7.3 mL, 14.6 mmol) was added and the reaction stirred for
2 hours until all starting material was consumed according to TLC. All volatiles were
removed in vacuo to a form a clear crude residue, which was purified by flash-column
chromatography on silica gel to afford T-8 as a white solid. Yield: l.l5g (99%). LC-MS
(Electrospray negative) 630.23 [M-H].
[0236] Preparation of T-9: Starting nucleoside T-8 (320 mg, 0.504 mmol) was dissolved in
minimal acetonitrile in a 50 mL round bottomed flask in air. A solution of AcOH/H20 5:1
(12.5 mL:2.5 mL) was added in one go and the reaction stirred at room temperature until all
starting material was consumed (2-4 hours). Evaporation of all volatiles under vacuum and
co-evaporating the residue in toluene (2 X 60 mL) provide crude product as an off white solid.
W0 2020/ 136170 PCT/EP2019/086926
The crude product was purified by flash column chromatography to afford T—9 as a white
solid. Yield: 123mg (74%). LC-MS (Electrospray negative) [M-H] 328.10.
[0237] Following similar synthetic procedure using the corresponding MeNH2 or NH3,
nucleosides with two other thiocarbamate protecting groups were also prepared. The general
reaction scheme is demonstrated below:
i) /,\N}KN/\\ ,DMAP,DCM, rt, N2,2—10h DMTO T
DMTO T N\/, \\/N 0
PW ’ ‘
l .. O S
OH u) H2NMe (2M THF), N2, rt, 2-4 h ‘[7
99% (2Steps)
DMTO
DMTO T N//\N N/\\N, DMAP, DCM, rt, N2, 2-10 n O T
:0: / \9’ \§/
OH ii) NH3 (0.4M THF), N2, :1, 2-4 h
91% (2 Steps)
3 '-O-T//iiocarbamaie Blocking Groups Stability T esiing
[0238] The stability tests for 5'—mP 3’-DMTC T nucleotide was performed side by side in an
incorporation buffer solution with standard 5'—mP 3'-O-azidomethyl T nucleotide.
HO 3 o O N O
— — ll
6H 0 HO-Fl’-O O
W? Ox/Ne
'—mP 3'—O—azidomethy| T
'—mP 3'-DMTC T
[0239] For both 5'—mP 3'-DMTC T and 5'—mP 3'-O-azidomethyl T, the final solution Volume
was 1 mL and the final concentrations of the corresponding nucleotides were both 0.1 mM.
Other components of the aqueous buffer solution include ethanolamine (EA), ethanolamine
HCl, NaCl (100 mM), and EDTA (2.5 mM). The buffer solution has the following
concentration: 0.5 M EA Buffer, 0.5 M NaCl, 0.01 M EDTA.
W0 2020/ 136170 PCT/EP2019/086926
Stability Test Methodology
[0240] 200 uL of 10x Buffer solution was added to a 1.7 mL polypropylene snap lock
microtube and diluted with the correct volume of 18 ms) water. The corresponding
nucleoside was then added, the vial was sealed and mixed via inversion, gentle stirring or
pumping with a micropipette. A 40uL aliquot was taken and analyzed by HPLC to act as a
starting (or t=0) value. The vials are then placed in a pre-heated heating mantle set to 65°C,
covered with a thick layer of aluminum foil and left to heat for one month. 40 uL aliquots
was taken periodically (week 1: once daily. Weeks 2-4: 1 every 2 days) and analyzed by
HPLC to determine the percentage starting material and the percentage deblocked (3'-OH)
nucleotide in the samples. HPLC analysis was performed by measuring the area of the stating
nucleotide peak and the 3’OH peak. These values were used to calculate a percentage of
deblocked nucleotide which was presented graphically and used to compare stability in an
incorporation buffer between samples. illustrates the comparative results of stability
of three different thiocarbamate 3’ blocking nucleotides to the nucleotide blocked with 3’-O-
azidomethyl blocking group at 65 °C. It was observed that while the nucleotide with 3’-O-
C(=S)NH2 or 3’—O-C(=S)NHCH3 was less stable than the nucleotide protected with the
standard 3'-O-azidomethyl group, the nucleotide with 3'-DMTC conferred improved stability
during the 9-day testing period. As such, DMTC demonstrated superior stability over the
standard azidomethyl blocking group.
Example 7. 3’-O-Thiocarbamate Blocking Group Deblocking Testing
[0241] In this example, deblocking or deblock tests for 5'-mP 3’-DMTC T and the standard
'-mP 3'-O-azidomethyl T nucleotide were performed individually in a solution unique to
each blocking group. Conditions were formulated to mimic Illumina’s standard deblock
reagent as closely as possible, and follow the same methodology. Concentrations of active
deblock reagent, buffer, and nucleoside are kept the same across all tests, but the identity of
each component was unique. In this way, the observed difference in rate between the
individual deblocking chemistries cannot due to the differences in concentration of
formulation.
W0 2020/ 136170 PCT/EP2019/086926
\fkNH
O N’i§o l /]§
" N O
HO—F|>—O O 9
D bl k t: HO-P-O
OH N:|Oocorr::%::® 6H 0 Deblock reagent:
o\f3 4 THP
O\/N3
'mP 3'-DMTC T 5'mP 3'-O-azidomethyl T
Deblock Test General Methodology
[0242] Each reaction component was formulated individually as a concentrated stock in 18
m9 water, stored appropriately, and aliquots combined in a specific order given below.
Reaction was commenced by addition of the preformulated deblock reagent. Final
Concentrations: nucleoside (0.1 mM), active deblock reagent (1 mM), additive (specific to
deblock reagent), buffer (100 mM). Final Volume: 2000 [LL
[0243] In a 3 mL glass vial was added the preformulated buffer solution followed by
preformulated additive solution. This was diluted with the correct volume of 18 m9 water
and stirred for 10 minutes. An aliquot of nucleotide solution was then added and stirred for 5
minutes. A 40 uL aliquot was then taken, quenching reagent added and analyzed by HPLC as
a reference (or t=0 min) peak. The deblock reagent was then added in one go to the stirring
solution and timing was commenced. At specified time points, 40uL aliquots were taken and
quenched immediately with an appropriate quenching reagent, then analyzed by HPLC to
determine the amount of deblocked nucleotide that has occurred at these specified time
points. Results were plotted graphically and are used to compare deblock efficiency and
efficacy.
DMT C Deblocking
[0244] Nucleotide: 5’-mP 3’-DMTC T. Active deblock reagent: NaIO4 (0.1M in 18 m9
water) or Oxone® (0.1M in 18 m9 water). Additive: none. Buffer for NaIO4: pH 6.75
phosphate buffer (1M in 18 m9 water). Buffer for OXone®: pH 8.65 phosphate buffer (IM in
18 ms) water). Quenching reagent: sodium thiosulfate. The 3’-O-azidomethyl deblocking
condition is the same as those described in Example 3.
[0245] HPLC analysis was performed by measuring the area of the stating nucleoside peak,
the 3’-OH peak, and any other nucleotide peaks that appear in the HPLC chromatogram.
W0 2020/ 136170 PCT/EP2019/086926
These values were used to calculate a percentage of starting nucleotide and deblocked
nucleotide which was presented graphically and used to compare deblock rate, efficiency and
efficacy between samples. The comparative result is shown in It was observed that
deblocking of DMTC with NaIO4 was not efficient. However, the % of starting material
remaining was significantly less for the nucleotide with the DMTC blocking group when
DMTC was cleaved with OXone®. In summary, DMTC has demonstrated a superior
deblocking rate (with Oxone®) over the deblocking rate of the standard azidomethyl
blocking group.
W0 2020/ 136170 PCT/EP2019/086926
Claims (9)
- WHAT IS CLAIMED IS:1. A nucleoside or nucleotide comprising a ribose or deoxyribose having aR18 R28removable 3’-OH blocking group forming a structure R covalentlyattached to the 3'-carbon atom, wherein:each R13‘ and R13 is independently H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6alkoxy, C1-C6 haloalkoxy, cyano, halogen, optionally substituted phenyl, or optionallysubstituted aralkyl,each R23 and R23 is independently H, C1-C6 alkyl, C1-C6 haloalkyl, cyano, orhalogen;alternatively R” and R23 together with the atoms to which they are attachedform an optionally substituted five to eight membered heterocyclyl group;R3 is H, optionally substituted C2-C5 alkenyl, optionally substituted C3-C7cycloalkenyl, optionally substituted C2-C6 alkynyl, or optionally substituted (C1-C6alkylene)Si(R4)3; andeach R4 is independently H, C1-C6 alkyl, or optionally substituted C6-C10 aryl,provided that when each R13, R11’, R23‘ and R21’ is H, then R3 is not H.
- 2. The nucleoside or nucleotide of claim 1, wherein at least one of R13 and R13 is H.
- 3. The nucleoside or nucleotide of claim 2, wherein each R13‘ and R“’ is H.
- 4. The nucleoside or nucleotide of any one of claims 1 to 3, wherein each of R23 andR21’ is independently H, halogen or C1-C6 alkyl.
- 5. The nucleoside or nucleotide of claim 4, wherein each R23 and R23 is H.
- 6. The nucleoside or nucleotide of claim 4, wherein each R2‘ and R2” isindependently Cl-C6 alkyl or halogen.
- 7. The nucleoside or nucleotide of claim 6, wherein each R23 and R23 is methyl.
- 8. The nucleoside or nucleotide of claim 4, wherein R23 is H, and R2” is halogen orC1-C6 alkyl.
- 9. The nucleoside or nucleotide of any one of claims 1 to 7, wherein R3 is C2-C6alkynyl optionally substituted with one or more substituents independently selected from thegroup consisting of halogen, C1-C6 alkyl, C1-C6 haloalkyl and combinations thereof.291%,10. The nucleoside or nucleotide of claim 9, wherein R3 isW0 2020/ 136170 PCT/EP2019/08692611. The nucleoside or nucleotide of any one of claims 1 to 7, wherein R3 is C2-C6alkenyl optionally substituted with one or more substituents independently selected from thegroup consisting of halogen, C1-C6 alkyl, C1-C6 haloalkyl and combinations thereof.31/ ;e‘\/\712. The nucleoside or nucleotide of claim 11, wherein R3 is\ F CI ,I“:/\/, :53‘/\F7 Pi‘/\C|7 3“¢\|= ,}3\2\C|7 F CI F13. The nucleoside or nucleotide of any one of claims 1 to 7, wherein R3 is optionallysubstituted (C1.C6 alkylene)Si(R4)3 and wherein each R4 is C1-C6 alkyl.14. The nucleoside or nucleotide of claim 13, wherein R3 is —(CH2)-SiMe3.15. The nucleoside or nucleotide of claim 1, wherein R” and R3‘‘ together with theatoms to which they are attached form a six membered heterocyclyl.16. The nucleoside or nucleotide of claim 15, wherein the six membered heterocyclylgroup has the structureR1b O Rzb.17. The nucleoside or nucleotide of claim 15 or 16, wherein each Rlb, R3” and R3 is H.18. The nucleoside or nucleotide of claim 1, wherein the 3’-OH blocking groupcomprises the structure selected from the group consisting of:‘fix /\ / Ex /\ / E /\ / 9&0/\OO O/\/ O O/\/\ O O/\/\/$0/\O/\//7 \O/\Oj\% ~50/Eoj’ andgio/\O/\/Si(Me)3covalentlyattached to the 3’-carbon of the ribose or deoxyribose.19. A nucleoside or nucleotide comprising a ribose or deoxyribose having aJ'V\IIV\oremovable 3’-OH blocking group forming a structure NR5R6 covalently attached to the 3'-carbon atom, wherein:each of R3 and R6 is independently H, C1-C6 alkyl, C2.Cs alkenyl, C2-Csalkynyl, C1-C6 haloalkyl, C2.Cs alkoxyalkyl, optionally substituted —(CH2)m—phenyl,optionally substituted —(CH2)n—(5 or 6 membered heteroaryl), optionally substituted —W0 2020/ 136170 PCT/EP2019/086926(CH2)k~C3.C7 carbocyclyl, or optionally substituted —(CH2)p—(3 to 7 memberedheterocyclyl),each of —(CH2)m—, —(CH2)n—, —(CH2)k—, and —(CH2)p— is optionally substituted;each of m, n, k, and p is independently 0, 1, 2, 3, or 4.20. The nucleoside or nucleotide of claim 19, wherein at least one of R5 and R6 is H orC1-C6 alkyl.21. The nucleoside or nucleotide of claim 20, wherein each R5 and R6 is H.22. The nucleoside or nucleotide of claim 20, wherein R5 is H, and R6 is C1-C6 alkyl,C2-C6 alkenyl, C2-C6 alkynyl, optionally substituted —(CH2)m—phenyl or optionally substituted—(CH2)n—6 membered heteroaryl, and wherein each of m and n is O or 1.23. The nucleoside or nucleotide of claim 20, wherein each R5 and R6 is C1-C6 alkyl.24. The nucleoside or nucleotide of claim 23, wherein each R5 and R6 is methyl.25. The nucleoside or nucleotide of claim 19, wherein the 3'-OH blocking groupj g JL /comprises the structure selected from the group consisting of 350 NH2 , O N ,;gOJL /and I26. The nucleoside or nucleotide of any one of claims 1 to 25, wherein the nucleosideor nucleotide is covalently attached to a detectable label, optionally via a cleavable linker.27. The nucleoside or nucleotide of claim 26, wherein the detectable label iscovalently attached to a nucleobase of the nucleoside or nucleotide via a cleavable linker.28. The nucleoside or nucleotide of claim 26, wherein the detectable label iscovalently attached to 3’-oxygen of the nucleoside or nucleotide via a cleavable linker.29. The nucleoside or nucleotide of claim 27 or 28, wherein the linker is a cleavablelinker comprising an azido moiety, a —O-allyl moiety, a disulfide moiety, an acetal moiety, ora thiocarbamate moiety.30. The nucleoside or nucleotide of any one of claims 27 to 29, wherein the 3’-OHblocking group and the cleavable linker may be removed under the same chemical reactionconditions.31. The nucleoside or nucleotide of any one of claims 1 to 30, comprising a 2’deoxyribose.W0 2020/ 136170 PCT/EP2019/08692632. The nucleoside or nucleotide of claim 31, wherein the nucleotide is a nucleotidetriphosphate.33. An oligonucleotide comprising a nucleotide of any one of claims 1 to 32.34. A method of preparing a growing polynucleotide complementary to a targetsingle-stranded polynucleotide in a sequencing reaction, comprising incorporating anucleotide of any one of claims 1 to 32 into a growing complementary polynucleotide,wherein the incorporation of the nucleotide prevents the introduction of any subsequentnucleotide into the growing complementary polynucleotide.35. The method of claim 34, wherein the incorporation of the nucleotide isaccomplished by a polymerase, a terminal deoxynucleotidyl transferase, or a reversetranscriptase.36. A method of determining the sequence of a target single-stranded polynucleotide,comprising:(a) incorporating a nucleotide of any one of claims 26 to 32 into a copypolynucleotide strand complementary to at least a portion of the target polynucleotidestrand;(b) detecting the identity of the nucleotide incorporated into the copypolynucleotide strand; and(c) chemically removing the label and the 3’ blocking group from thenucleotide incorporated into the copy polynucleotide strand.37. The method of claim 36, further comprising ((1) washing the chemically removedlabel and the 3' blocking group away from the copy polynucleotide strand.38. The method of claim 37, further comprising repeating steps (a) to ((1) until asequence of the portion of the template polynucleotide strand is determined.39. The method of claim 37, wherein the steps (a) to (d) is repeated at least 50 times.40. The method of any one of claims 36 to 39, wherein the label and the 3' blockinggroup from the nucleotide incorporated into the copy polynucleotide strand are removed in asingle chemical reaction.41. The method of claim 40, wherein step (c) comprises contacting the incorporatednucleotide with a cleavage solution comprising a palladium catalyst.42. The method of any one of claims 36 to 39, wherein the label and the 3' blockinggroup from the nucleotide incorporated into the copy polynucleotide strand are removed intwo separate chemical reactions.W0 2020/ 136170 PCT/EP2019/08692643. The method of claim 41, wherein step (c) comprises contacting the incorporatednucleotide with a cleavage solution comprising a phosphine, and a cleavage solutioncomprising a palladium catalyst.44. The method of claim 41 or 43, wherein the phosphine istris(hydroxymethyl)phosphine, tris(hydroxyethyl)phosphine ortris(hydroXypropyl)phosphine.45. The method of any one of claims 41, 43 or 44, wherein the cleavage solutioncomprising the palladium catalyst further comprises one or more buffer reagents selectedfrom the group consisting of a primary amine, a secondary amine, a tertiary amine, acarbonate salt, a phosphate salt, and a borate salt, and combinations thereof.46. The method of claim 45, wherein the buffer reagents are selected from the groupconsisting of ethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine, acarbonate salt, a phosphate salt, a borate salt, 2-dimethyalaminomethanol (DMEA), 2-diethyalaminomethanol (DEEA), N,N,N’,N’-tetramethylethylenediamine (TEMED), andN,N,N’,N'-tetraethylethylenediamine (TEEDA), and combinations thereof.47. A kit comprising one or more nucleosides or nucleotides of any one of claims 1 to48. The kit of claim 47, further comprising an enzyme and a buffer appropriate for theaction of the enzyme.49. The kit of claim 48, wherein the enzyme is a polymerase, a terminaldeoxynucleotidyl transferase, or a reverse transcriptase.50. The kit of claim 49, wherein the polymerase is a DNA polymerase.
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