CN117417395A - Non-natural phosphate sugar, preparation method thereof, sugar metabolism marking kit and application thereof - Google Patents
Non-natural phosphate sugar, preparation method thereof, sugar metabolism marking kit and application thereof Download PDFInfo
- Publication number
- CN117417395A CN117417395A CN202311188091.1A CN202311188091A CN117417395A CN 117417395 A CN117417395 A CN 117417395A CN 202311188091 A CN202311188091 A CN 202311188091A CN 117417395 A CN117417395 A CN 117417395A
- Authority
- CN
- China
- Prior art keywords
- sugar
- mannaz
- natural
- sate
- group
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 235000000346 sugar Nutrition 0.000 title claims abstract description 150
- 229910019142 PO4 Inorganic materials 0.000 title claims abstract description 67
- 239000010452 phosphate Substances 0.000 title claims abstract description 66
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 title claims abstract description 56
- 230000004060 metabolic process Effects 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title abstract description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 28
- 125000006239 protecting group Chemical group 0.000 claims abstract description 24
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 15
- 210000004027 cell Anatomy 0.000 claims description 82
- 238000002372 labelling Methods 0.000 claims description 38
- 238000000034 method Methods 0.000 claims description 30
- -1 amino mannose Chemical compound 0.000 claims description 21
- 125000002252 acyl group Chemical group 0.000 claims description 19
- 229910052698 phosphorus Inorganic materials 0.000 claims description 16
- 150000003215 pyranoses Chemical class 0.000 claims description 16
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 14
- 150000001720 carbohydrates Chemical group 0.000 claims description 12
- 150000002243 furanoses Chemical group 0.000 claims description 12
- 239000003550 marker Substances 0.000 claims description 10
- 150000001540 azides Chemical class 0.000 claims description 9
- 125000003837 (C1-C20) alkyl group Chemical group 0.000 claims description 8
- 125000006736 (C6-C20) aryl group Chemical group 0.000 claims description 8
- 125000000304 alkynyl group Chemical group 0.000 claims description 7
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 claims description 7
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 claims description 6
- 125000001424 substituent group Chemical group 0.000 claims description 6
- 238000005917 acylation reaction Methods 0.000 claims description 5
- 125000003342 alkenyl group Chemical group 0.000 claims description 5
- 230000004048 modification Effects 0.000 claims description 5
- 238000012986 modification Methods 0.000 claims description 5
- 125000006245 phosphate protecting group Chemical group 0.000 claims description 5
- 230000010933 acylation Effects 0.000 claims description 4
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 claims description 4
- 230000023852 carbohydrate metabolic process Effects 0.000 claims description 4
- 210000000056 organ Anatomy 0.000 claims description 4
- DPOPAJRDYZGTIR-UHFFFAOYSA-N Tetrazine Chemical compound C1=CN=NN=N1 DPOPAJRDYZGTIR-UHFFFAOYSA-N 0.000 claims description 3
- CGTWCAVZZBLHQH-UHFFFAOYSA-N [cyano-(3-phenoxyphenyl)methyl] 2,2-dichloro-1-(4-ethoxyphenyl)cyclopropane-1-carboxylate Chemical group C1=CC(OCC)=CC=C1C1(C(=O)OC(C#N)C=2C=C(OC=3C=CC=CC=3)C=CC=2)C(Cl)(Cl)C1 CGTWCAVZZBLHQH-UHFFFAOYSA-N 0.000 claims description 3
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims description 3
- ZPWOOKQUDFIEIX-UHFFFAOYSA-N cyclooctyne Chemical compound C1CCCC#CCC1 ZPWOOKQUDFIEIX-UHFFFAOYSA-N 0.000 claims description 3
- OOXWYYGXTJLWHA-UHFFFAOYSA-N cyclopropene Chemical compound C1C=C1 OOXWYYGXTJLWHA-UHFFFAOYSA-N 0.000 claims description 3
- URYYVOIYTNXXBN-OWOJBTEDSA-N trans-cyclooctene Chemical compound C1CCC\C=C\CC1 URYYVOIYTNXXBN-OWOJBTEDSA-N 0.000 claims description 3
- MSWZFWKMSRAUBD-GASJEMHNSA-N 2-amino-2-deoxy-D-galactopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O MSWZFWKMSRAUBD-GASJEMHNSA-N 0.000 claims description 2
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 claims description 2
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims description 2
- 150000001336 alkenes Chemical class 0.000 claims description 2
- 150000001345 alkine derivatives Chemical class 0.000 claims description 2
- 235000021256 carbohydrate metabolism Nutrition 0.000 claims description 2
- 229960002442 glucosamine Drugs 0.000 claims description 2
- 210000002569 neuron Anatomy 0.000 claims description 2
- 230000001225 therapeutic effect Effects 0.000 claims description 2
- 125000004432 carbon atom Chemical group C* 0.000 claims 1
- 238000003786 synthesis reaction Methods 0.000 abstract description 39
- 150000004676 glycans Chemical class 0.000 abstract description 9
- 235000018102 proteins Nutrition 0.000 abstract description 7
- 102000004169 proteins and genes Human genes 0.000 abstract description 7
- 108090000623 proteins and genes Proteins 0.000 abstract description 7
- 238000007086 side reaction Methods 0.000 abstract description 5
- 230000008901 benefit Effects 0.000 abstract description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 abstract description 3
- 235000018417 cysteine Nutrition 0.000 abstract description 3
- 125000003132 pyranosyl group Chemical group 0.000 abstract 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 81
- 238000006243 chemical reaction Methods 0.000 description 66
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 57
- 235000021317 phosphate Nutrition 0.000 description 44
- 230000015572 biosynthetic process Effects 0.000 description 38
- 239000000047 product Substances 0.000 description 37
- 150000008163 sugars Chemical group 0.000 description 34
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 33
- 150000001875 compounds Chemical class 0.000 description 27
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 26
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 21
- GVNVAWHJIKLAGL-UHFFFAOYSA-N 2-(cyclohexen-1-yl)cyclohexan-1-one Chemical compound O=C1CCCCC1C1=CCCCC1 GVNVAWHJIKLAGL-UHFFFAOYSA-N 0.000 description 20
- 101150065749 Churc1 gene Proteins 0.000 description 20
- 102100038239 Protein Churchill Human genes 0.000 description 20
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 20
- 150000008300 phosphoramidites Chemical class 0.000 description 20
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 19
- 239000000741 silica gel Substances 0.000 description 19
- 229910002027 silica gel Inorganic materials 0.000 description 19
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 18
- 210000004556 brain Anatomy 0.000 description 18
- 239000000243 solution Substances 0.000 description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 15
- 239000011574 phosphorus Substances 0.000 description 15
- 239000003480 eluent Substances 0.000 description 14
- UQEAIHBTYFGYIE-UHFFFAOYSA-N hexamethyldisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)C UQEAIHBTYFGYIE-UHFFFAOYSA-N 0.000 description 14
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- 239000002904 solvent Substances 0.000 description 13
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 13
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 12
- 238000011534 incubation Methods 0.000 description 12
- 238000001466 metabolic labeling Methods 0.000 description 12
- 230000005311 nuclear magnetism Effects 0.000 description 11
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 10
- 125000005042 acyloxymethyl group Chemical group 0.000 description 10
- 125000001280 n-hexyl group Chemical group C(CCCCC)* 0.000 description 10
- 239000012074 organic phase Substances 0.000 description 10
- 229910000029 sodium carbonate Inorganic materials 0.000 description 10
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 10
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 9
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 9
- 230000002503 metabolic effect Effects 0.000 description 9
- 229910052757 nitrogen Inorganic materials 0.000 description 9
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 9
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 238000001819 mass spectrum Methods 0.000 description 8
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 7
- 125000000217 alkyl group Chemical group 0.000 description 7
- 238000004624 confocal microscopy Methods 0.000 description 7
- 239000005457 ice water Substances 0.000 description 7
- OKKJLVBELUTLKV-VMNATFBRSA-N methanol-d1 Chemical compound [2H]OC OKKJLVBELUTLKV-VMNATFBRSA-N 0.000 description 7
- KOUKXHPPRFNWPP-UHFFFAOYSA-N pyrazine-2,5-dicarboxylic acid;hydrate Chemical compound O.OC(=O)C1=CN=C(C(O)=O)C=N1 KOUKXHPPRFNWPP-UHFFFAOYSA-N 0.000 description 7
- 102000003886 Glycoproteins Human genes 0.000 description 6
- 108090000288 Glycoproteins Proteins 0.000 description 6
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 6
- 230000001413 cellular effect Effects 0.000 description 6
- 238000003384 imaging method Methods 0.000 description 6
- 239000012046 mixed solvent Substances 0.000 description 6
- 239000003208 petroleum Substances 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 description 5
- 239000008346 aqueous phase Substances 0.000 description 5
- 125000006297 carbonyl amino group Chemical group [H]N([*:2])C([*:1])=O 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 5
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 5
- 125000003136 n-heptyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 5
- 229920001282 polysaccharide Polymers 0.000 description 5
- 239000005017 polysaccharide Substances 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 125000006853 reporter group Chemical group 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- KLDLRDSRCMJKGM-UHFFFAOYSA-N 3-[chloro-(2-oxo-1,3-oxazolidin-3-yl)phosphoryl]-1,3-oxazolidin-2-one Chemical compound C1COC(=O)N1P(=O)(Cl)N1CCOC1=O KLDLRDSRCMJKGM-UHFFFAOYSA-N 0.000 description 4
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 229960002685 biotin Drugs 0.000 description 4
- 235000020958 biotin Nutrition 0.000 description 4
- 239000011616 biotin Substances 0.000 description 4
- 230000013595 glycosylation Effects 0.000 description 4
- 238000006206 glycosylation reaction Methods 0.000 description 4
- FFUAGWLWBBFQJT-UHFFFAOYSA-N hexamethyldisilazane Chemical compound C[Si](C)(C)N[Si](C)(C)C FFUAGWLWBBFQJT-UHFFFAOYSA-N 0.000 description 4
- 238000004896 high resolution mass spectrometry Methods 0.000 description 4
- 230000000971 hippocampal effect Effects 0.000 description 4
- 210000001320 hippocampus Anatomy 0.000 description 4
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 4
- JLRMRTRHBRVQMS-UHFFFAOYSA-N hydrogen peroxide;2-methylpropan-2-ol Chemical compound OO.CC(C)(C)O JLRMRTRHBRVQMS-UHFFFAOYSA-N 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 125000001501 propionyl group Chemical group O=C([*])C([H])([H])C([H])([H])[H] 0.000 description 4
- 238000002390 rotary evaporation Methods 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 239000007858 starting material Substances 0.000 description 4
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 3
- 239000005695 Ammonium acetate Substances 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- OVRNDRQMDRJTHS-ZTVVOAFPSA-N N-acetyl-D-mannosamine Chemical compound CC(=O)N[C@@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-ZTVVOAFPSA-N 0.000 description 3
- 208000005890 Neuroma Diseases 0.000 description 3
- 108010090804 Streptavidin Proteins 0.000 description 3
- 229940043376 ammonium acetate Drugs 0.000 description 3
- 235000019257 ammonium acetate Nutrition 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- IVRMZWNICZWHMI-UHFFFAOYSA-N azide group Chemical group [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 description 3
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 3
- 210000005013 brain tissue Anatomy 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000000799 fluorescence microscopy Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000037347 glycan metabolism Effects 0.000 description 3
- 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 3
- GPRLSGONYQIRFK-UHFFFAOYSA-N hydron Chemical group [H+] GPRLSGONYQIRFK-UHFFFAOYSA-N 0.000 description 3
- 239000003456 ion exchange resin Substances 0.000 description 3
- 229920003303 ion-exchange polymer Polymers 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 210000002243 primary neuron Anatomy 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 238000001308 synthesis method Methods 0.000 description 3
- KUEFXPHXHHANKS-UHFFFAOYSA-N 5-nitro-1h-1,2,4-triazole Chemical compound [O-][N+](=O)C1=NC=NN1 KUEFXPHXHHANKS-UHFFFAOYSA-N 0.000 description 2
- 239000012114 Alexa Fluor 647 Substances 0.000 description 2
- XLYOFNOQVPJJNP-ZSJDYOACSA-N Heavy water Chemical compound [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000001311 chemical methods and process Methods 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 229940125904 compound 1 Drugs 0.000 description 2
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000010511 deprotection reaction Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000007429 general method Methods 0.000 description 2
- 150000002402 hexoses Chemical class 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 210000003016 hypothalamus Anatomy 0.000 description 2
- 239000000543 intermediate Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 210000001259 mesencephalon Anatomy 0.000 description 2
- 239000002808 molecular sieve Substances 0.000 description 2
- 150000002772 monosaccharides Chemical class 0.000 description 2
- 210000000478 neocortex Anatomy 0.000 description 2
- 210000001577 neostriatum Anatomy 0.000 description 2
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000010189 synthetic method Methods 0.000 description 2
- 150000007970 thio esters Chemical class 0.000 description 2
- NGNBDVOYPDDBFK-UHFFFAOYSA-N 2-[2,4-di(pentan-2-yl)phenoxy]acetyl chloride Chemical compound CCCC(C)C1=CC=C(OCC(Cl)=O)C(C(C)CCC)=C1 NGNBDVOYPDDBFK-UHFFFAOYSA-N 0.000 description 1
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 1
- 208000010444 Acidosis Diseases 0.000 description 1
- 125000000882 C2-C6 alkenyl group Chemical group 0.000 description 1
- 125000003601 C2-C6 alkynyl group Chemical group 0.000 description 1
- 108010051152 Carboxylesterase Proteins 0.000 description 1
- 102000013392 Carboxylesterase Human genes 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102000005348 Neuraminidase Human genes 0.000 description 1
- 108010006232 Neuraminidase Proteins 0.000 description 1
- BPQQTUXANYXVAA-UHFFFAOYSA-N Orthosilicate Chemical compound [O-][Si]([O-])([O-])[O-] BPQQTUXANYXVAA-UHFFFAOYSA-N 0.000 description 1
- KBGAYAKRZNYFFG-BOHATCBPSA-N aceneuramic acid Chemical compound OC(=O)C(=O)C[C@H](O)[C@@H](NC(=O)C)[C@@H](O)[C@H](O)[C@H](O)CO KBGAYAKRZNYFFG-BOHATCBPSA-N 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 150000002301 glucosamine derivatives Chemical class 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- CQRPUKWAZPZXTO-UHFFFAOYSA-M magnesium;2-methylpropane;chloride Chemical compound [Mg+2].[Cl-].C[C-](C)C CQRPUKWAZPZXTO-UHFFFAOYSA-M 0.000 description 1
- 150000008276 mannosamines Chemical class 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- UPPVRFOGRCBSJP-UHFFFAOYSA-N n-dichlorophosphanyl-n-propan-2-ylpropan-2-amine Chemical compound CC(C)N(C(C)C)P(Cl)Cl UPPVRFOGRCBSJP-UHFFFAOYSA-N 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 238000005580 one pot reaction Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- CHKVPAROMQMJNQ-UHFFFAOYSA-M potassium bisulfate Chemical class [K+].OS([O-])(=O)=O CHKVPAROMQMJNQ-UHFFFAOYSA-M 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- WYVAMUWZEOHJOQ-UHFFFAOYSA-N propionic anhydride Chemical compound CCC(=O)OC(=O)CC WYVAMUWZEOHJOQ-UHFFFAOYSA-N 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- ITMCEJHCFYSIIV-UHFFFAOYSA-N triflic acid Chemical compound OS(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-N 0.000 description 1
- FTVLMFQEYACZNP-UHFFFAOYSA-N trimethylsilyl trifluoromethanesulfonate Chemical compound C[Si](C)(C)OS(=O)(=O)C(F)(F)F FTVLMFQEYACZNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
- C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
- C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/02—Phosphorylation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The invention relates to the technical field of organic chemical synthesis, and particularly discloses non-natural phosphate sugar, a preparation method thereof, a sugar metabolism marking kit and application thereof, wherein the non-natural phosphate sugar is hexacarbon sugar and has a pyranose structure, the non-natural phosphate sugar contains a bio-orthogonal group, and the six-position of the non-natural phosphate sugar is modified by phosphoric acid with a protecting group. The non-natural phosphate sugar disclosed by the invention has the advantages of the existing non-natural sugar, so that the non-natural phosphate sugar can be effectively utilized by cells, and S saccharification side reaction with cysteine in protein in the metabolic process is effectively avoided. Meanwhile, the rapid metabolism marking of the glycan can be realized.
Description
Technical Field
The invention relates to the technical field of organic chemical synthesis, in particular to non-natural phosphate sugar, a preparation method thereof, a sugar metabolism marking kit and application thereof.
Background
The glycan in the cell has important functions, and the labelling of the glycan is an important means for researching and regulating the function of the glycan. The synthesis of glycans does not use nucleic acid directly as a template, and therefore, conventional methods for protein and nucleic acid research are difficult to apply directly to glycans (e.g., fusion fluorescent protein technology). In recent years, the metabolic labelling of glycans with non-natural sugars bearing chemical reporter groups (such as azides or alkynyl groups) has been widely used for imaging and glycohistology analysis of sugars. Such techniques are known as non-natural glycometabolism markers and can be used for the study of living cells and living body levels. In the technique of non-natural sugar metabolism labeling, non-natural sugar is necessary. In the design of non-natural sugar, monosaccharide in glycosylation biosynthesis path is often selected as matrix, and chemical reporter group is introduced into proper position of the matrix to obtain non-natural sugar containing chemical reporter group. By utilizing the substrate tolerance of glycosylation-related enzymes in cells, these unnatural saccharides can be taken up and utilized by cells and chemical reporter groups can be integrated into the sugar chains of the cells to effect labeling of specific glycans. Fluorescence imaging and enrichment of glycans can then be achieved by derivatizing chemical reporter groups, such as modified fluorescent molecules or biotin affinity tags, using bio-orthogonal reactions (e.g., click chemistry). Therefore, the structural design and synthetic route selection of the non-natural sugar are of great importance for labeling of the polysaccharide and research on the application developed on the basis of the labeling.
In practical applications, because unnatural sugars have multiple hydroxyl groups, which are very polar and difficult to cross cell membranes, such sugars without protecting groups require high concentrations to achieve effective labeling. Thus, the hydroxyl groups of the non-natural sugars are typically peracetylated to increase the efficiency of the non-natural sugar labelling. The non-natural sugar modified by the full acetylation has better membrane penetrating property, and acetyl groups on sugar hydroxyl groups are easy to remove under the action of intracellular nonspecific esterase. Thus, peracetylated non-natural sugars are widely used. However, recent studies have found that peracetylated unnatural sugars produce non-enzymatically catalyzed side reactions of S saccharification, which produce a number of false positive signals during metabolic labeling. Recently, the literature reports that this problem can be solved using partially hydroxyalkylated modified non-natural sugars. The partially acylated sugar easily penetrates through the cell membrane and enters the cell, so that the S saccharification side reaction can be effectively avoided, and the high metabolic labeling efficiency can be maintained. The partially acylated unnatural sugar is suitable for both living cell and living body levels, and is the best choice for the current research of the metabolic markers of unnatural sugar. For sialidases, metabolic labelling is often performed using sialic acid Neu5Ac or derivatives corresponding to the precursor molecule ManNAc of its metabolism. In practical application scenarios, derivatives without protecting groups, partial acylation protection or peracetylation protection are used.
All the non-natural saccharides used for sialic acid metabolism markers at present have the problem of low marking speed no matter whether the hydroxyl groups on the saccharides are not protected, are partially protected or are fully protected. In cell experiments, cells often need to be cultured with unnatural sugars for 2 to 3 days in order to achieve a sufficiently strong metabolic labeling effect. Thus, this technique is not well suited for observing dynamic changes in glycosylation over a short period of time. In addition, acute tissue culture sections are used in large numbers in life medicine studies, where they are suitable for culture for a limited period of time (typically within 24 hours), and where conventional non-natural sugars are difficult to develop for use in these research systems. Therefore, development of unnatural saccharides that can be rapidly metabolically labeled and related research of applications have been carried out have been demanded.
Disclosure of Invention
The invention mainly aims to provide an unnatural phosphate sugar, a preparation method and application thereof, and aims to solve the problems that the labeling speed of the polysaccharide is low, the polysaccharide cannot be well used for observing dynamic change of glycosylation in a short time, and the polysaccharide is difficult to apply to scientific research in the prior art.
In a first aspect, the invention provides a non-natural phosphate sugar, which is a hexose and has a pyranose or furanose structure, wherein the non-natural phosphate sugar contains a bio-orthogonal group, and the six positions of the non-natural phosphate sugar are modified by phosphoric acid with a protecting group.
Preferably, the non-natural phosphate sugar is a hexose and is a pyranose structure.
Further, the protecting group is a protecting group that is capable of cleavage or hydrolysis, preferably comprising any one or more of SATE, AOM, proTide, AB or CycloSal groups, more preferably comprising a SATE, POM or ProTide group.
Further, the orthogonal groups on the non-natural phosphate sugar include any one or more of azide, alkyne, alkenyl, cyclopropene, trans-cyclooctene, cyclooctyne, and tetrazine.
Further, the sugar type of the pyranose is any one selected from amino mannose, glucosamine or galactosamine, preferably amino mannose;
more preferably, the mannose has a structure of either ManNAz, manNAl or mannttl.
Further, the non-natural phosphoric acid has the structure of formula I:
wherein the R is 1 Acyl groups containing C1 to C12, including but not limited to acyl groups containing C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11 or C12 carbon atoms, preferably C1 to C6 acyl groups, more preferably acetyl groups;
the R is 2 、R 5 Is a phosphate protecting group independently selected from: SATE, AOM, proTide or AB groups;
The R is 3 、R 4 Independently selected from: h or an acyl group of C1 to C12, for example, an acyl group of C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11 or C12;
x is azide, alkenyl of C2-C6 or alkynyl of C2-C6, such as alkenyl of C2, C3, C4, C5 or C6, or alkynyl of C2, C3, C4, C5 or C6;
the Y is 1 Or Y 2 Independently selected from-O-or-NH-;
the R is 8 、R 9 、R 10 、R 11 Or R is 12 Independently selected from: h or C1-C6 alkyl, for example, C1, C2, C3, C4, C5 or C6 alkyl.
Further, the R 1 Is an acyl group containing C1 to C6;
the SATE group structure is as follows:
the R is 6 Is a C1-C20 alkyl chain or a C6-C20 aryl group, including but not limited to C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 alkyl, or C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 aryl;
preferably, said R 6 Preferably a C1-C14 alkyl group, more preferably any one of methyl, ethyl, n-butyl, t-butyl or n-heptyl;
preferably, the structure of the AOM is:wherein R is 7 Is a C1-C20 alkyl chain or a C6-C20 aryl group, including but not limited to C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 alkyl, or C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 aryl; r is R 7 Preferably a C1-C14 alkyl group, more preferably any one of methyl, ethyl, n-butyl, t-butyl or n-heptyl;
in the structure of the AOM, when R 7 In the case of tert-butyl, the AOM is POM in structure.
Preferably, when the phosphate protecting group is a ProTide group, R 2 Or R is 5 Any substituent beingThe structure being simultaneously taken fromThe substituent is->Wherein said R is 13 、R 14 、R 15 、R 16 、R 17 、R 18 Or R is 19 Independently selected from: h or C1-C6 alkyl, for example, C1, C2, C3, C4, C5 or C6 alkyl;
the structure of the AB group is as follows:wherein said R is 20 、R 21 、R 22 、R 23 Or R is 24 Independently selected from: h or C1-C6 alkyl, for example, C1, C2, C3, C4, C5 or C6 alkyl;
further, the R 3 、R 4 Independently selected from: h or C1-C12 acyl, including but not limited to acyl containing C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11 or C12 carbon atoms;
further, X is azide, C2-C6 alkenyl or C2-C6 alkynyl, including but not limited to C2, C3, C4, C5 or C6 alkenyl, including but not limited to C2, C3, C4, C5 or C6 alkynyl.
Further, the non-natural phosphate sugar comprises any one of the following: 1-O-Ac-ManNAz-6-bis- (Me-SATE) -P,
1-O-Ac-ManNAz-6-bis-(Et-SATE)-P、1-O-Ac-ManNAz-6-bis-(Bu-SATE)-P、1-O-Ac-ManNAz-6-bis-(tBu-SATE)-P、1-O-Ac-ManNAz-6-bis-(He-SATE)-P、1-O-Ac-ManNAl-6-bis-(tBu-SATE)-P、1-O-Ac-ManNPtl-6-bis-(tBu-SATE)-P、Ac 3 ManNAz-6-bis- (Me-SATE) -P, 1-O-Ac-ManNAz-6-bis-POM-P, 1-O-Pr-ManNAz-6-bis-POM-P or 1-O-Pr-ManNAz-6-ProTide-P.
In a second aspect, the present invention provides a method for preparing the non-natural phosphoric acid sugar according to the first aspect, the method comprising: protecting the first position on the pyranose or furanose ring, leaving the third position and the fourth position unprotected, and selectively introducing a phosphate group with a protecting group at the sixth position to obtain non-natural phosphate sugar; or protecting the first position of the pyranose or furanose, protecting the third position and the fourth position of the pyranose or furanose, introducing a phosphate group with a protecting group at the sixth position, and finally removing the protecting groups at the third position and the fourth position of the pyranose or furanose to obtain the non-natural phosphate sugar;
preferably, the preparation method further comprises: further acylation modification of the hydroxyl group of the non-natural phosphate sugar.
Further preferably, the other hydroxyl groups on the ring (on the pyranose or furanose ring) may be in unprotected, partially protected or fully protected form.
Further preferably, the protecting the first position on the pyranose or furanose ring is to introduce an ester bond to the first position on the pyranose or furanose ring, and protect the hydroxyl group at the first position on the pyranose ring.
Further, the protecting group is a SATE or AOM group, wherein the SATE group structure is:
the R is 6 Is a C1-C20 alkyl chain or a C6-C20 aryl group including, but not limited to, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19 or C20 alkyl, or C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19 or C20 aryl, said R 6 Preferably a C1-C14 alkyl group, more preferably any one of methyl, ethyl, n-butyl, t-butyl or n-heptyl;
preferably, the structure of AOM (Acyloxymethyl) is as follows:wherein R is 7 Is a C1-C20 alkyl chain or a C6-C20 aryl group including, but not limited to, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19 or C20 alkyl, or C6, C7, C8, C9, C10C11, C12, C13, C14, C15, C16, C17, C18, C19 or C20 aryl, R 7 Preferably C1-C14 alkyl, more preferably any of methyl, ethyl, n-butyl, t-butyl or n-heptyl, when R 7 In the case of tert-butyl, the protecting group is POM (pivaloyloxymethyl).
In a third aspect, the invention provides a kit for labeling sugar metabolism, which comprises the non-natural phosphate sugar according to the first aspect of the invention or the non-natural phosphate sugar prepared by the preparation method according to the second aspect of the invention.
According to a fourth aspect of the present invention there is provided the use of a non-natural phosphate sugar according to the first aspect, or a non-natural phosphate sugar prepared by a method according to the second aspect of the present invention, or a kit according to the third aspect of the present invention, in a marker for sugar metabolism, for non-diagnostic or therapeutic purposes.
Further, the use includes labeling the sugar metabolism of any one of the following cell lines or primary cells: heLa cells, MCF-7 cells, HEK 293T cells, MDA-MB-231 cells, A549 cells, hepG2 cells, NIH 3T3 cells, CHO cells, COS-7 cells, or primary neuronal cells.
Further, the use includes a marker of carbohydrate metabolism on a mammalian organ or tissue section;
preferably, the mammalian organ or tissue slice comprises any one or more of the following: rat brain slices, human brain slices, monkey brain slices or pig brain slices, or other organ slices.
The mouse brain slice includes a mouse brain slice or a rat brain slice.
By adopting the technical scheme, the invention has at least the following beneficial effects:
the invention provides the non-natural phosphate sugar, the preparation method thereof, the sugar metabolism marking kit and the application thereof, and the non-natural phosphate sugar with the specific structure can realize the rapid metabolism marking of the polysaccharide, and effectively avoid the side reaction with cysteine in the protein in the non-natural sugar metabolism process.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1A shows the different unnatural sugars (ManNAz, siaNAz, 1, 6-Pr) observed by laser confocal microscopy 2 ManNAz, AMMS);
FIG. 1B shows different unnatural sugars (ManNAz, siaNAz, 1, 6-Pr) 2 ManNaz, AMMS) cell surface mean fluorescence intensity results;
FIG. 1C shows different unnatural sugars (ManNAz, siaNAz, 1,6-Pr 2 ManNAz, AMMS);
FIG. 2A shows the different unnatural sugars (ManNAz, siaNAz, 1, 6-Pr) observed by laser confocal microscopy 2 ManNaz, AMES);
FIG. 2B shows different unnatural sugars (ManNAz, siaNAz, 1, 6-Pr) 2 ManNAz, AMES) cell surface mean fluorescence intensity results;
FIG. 2C shows different unnatural sugars (ManNAz, siaNAz, 1,6-Pr 2 ManNaz, AMES);
FIG. 3A shows the different unnatural sugars (ManNAz, siaNAz, 1, 6-Pr) observed by laser confocal microscopy 2 ManNaz, AMBS);
FIG. 3B shows different unnatural sugars (ManNAz, siaNAz, 1, 6-Pr) 2 ManNAz, AMBS) cell surface mean fluorescence intensity results;
FIG. 3C shows different unnatural sugars (ManNAz, siaNAz, 1,6-Pr 2 ManNAz, AMBS);
FIG. 4A shows the different unnatural sugars (ManNAz, siaNAz, 1, 6-Pr) observed by laser confocal microscopy 2 ManNaz, AMtBS);
FIG. 4B shows different unnatural sugars (ManNAz, siaNAz, 1, 6-Pr) 2 ManNAz, AMtBS) cell surface mean fluorescence intensity results;
FIG. 4C shows different unnatural sugars (ManNAz, siaNAz, 1,6-Pr 2 ManNAz, AMtBS);
FIG. 5A shows the different unnatural sugars (ManNAz, siaNAz, 1, 6-Pr) observed by laser confocal microscopy 2 ManNAz, AMHS);
FIG. 5B shows different unnatural sugars (ManNAz, siaNAz, 1, 6-Pr) 2 ManNAz, AMHS) cell surface mean fluorescence intensity results;
FIG. 5C shows different unnatural sugars (ManNAz, siaNAz, 1,6-Pr 2 ManNAz, AMHS);
FIG. 6 shows the results of laser confocal microscopy of different unnatural sugars (ManNAz, siaNAz, 1,6-Pr 2 ManNAz, AMMS, AMES, AMBS, AMBtS, AMHS) metabolic labeling results;
FIG. 7 shows SATE protected and peracetylated modified non-natural phosphate sugar TAMMS and conventional non-natural sugar ManNAz, 1,6-Pr observed by laser confocal microscopy 2 Microscopic results of ManNAz;
FIG. 8 shows the results of observation of the conventional unnatural saccharide 1,6-Pr and the unnatural phosphate saccharide PMPOM by a laser confocal microscope 2 Metabolic labeling results of ManNAz;
FIG. 9 shows the results of observation of the non-natural phosphoric acid sugar PMPT and the conventional non-natural sugar 1,6-Pr by a laser confocal microscope 2 Metabolic labelling effects of ManNAz in MCF-7 cells;
FIG. 10 shows the results of achieving efficient glycoprotein labelling by the unnatural phosphoglycosylate AMMS;
FIG. 11A shows the results of the non-native phosphoglycosum AMMS to achieve efficient and rapid metabolic labeling on mouse primary neurons;
FIG. 11B shows the results of the non-native phosphoglycosum AMMS to achieve efficient and rapid metabolic labeling on the primary neurons of mice;
FIG. 12 shows that the non-natural phosphate sugar AMMS can realize rapid metabolic labeling results;
FIG. 13A is a graph showing incubation time and fluorescence intensity results for the unnatural phosphate sugars AMMS, AMES and AMtBS;
FIG. 13B is a graph showing incubation time and fluorescence intensity results for the unnatural phosphate sugars AMMS, AMES and AMtBS;
FIG. 14A shows the results of fluorescence scanning of non-native phosphoglycosylate AMMS on a mouse brain slice;
FIG. 14B shows the results of marker signal imaging of unnatural phosphoglycosylate AMMS on mouse brain sections;
FIG. 14C shows the results of signal imaging of different unnatural phosphate sugars on mouse brain sections;
FIG. 15 is an image of the striatum, hypothalamus, neocortex, hippocampus, and midbrain of a mouse, all marked with AMMS with high efficiency and rapidity;
FIG. 16A shows normal brain sections of humans using AMMS, siaNAz and 1,6-Pr 2 After 24 hours of ManNAz incubation, a map of the observed marker signal;
FIG. 16B shows a graph of marker signals in normal brain tissue and neuroma sections;
FIG. 16C shows the average intensity of marker signals in normal brain tissue and neuroma sections;
FIG. 17 shows the labeling of POM-protected ManNAz-6-P derivative PMPOM in sections of hippocampal regions of the mouse brain.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the following embodiments of the present invention will be described in further detail with reference to the accompanying drawings.
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates.
The term "non-natural sugar" refers to a natural monosaccharide chemically modified to have bioorthogonal groups such as azides, alkynyls, etc. attached. The unnatural saccharides can be taken up by cells, integrated into the saccharide chains via natural sugar metabolic pathways, and then the glycans can be labeled, imaged, or enriched via bioorthogonal reactions.
The term "non-natural phosphate sugar", i.e., a phosphate modified non-natural sugar, also referred to as a phosphate containing non-natural sugar, refers to the covalent introduction of phosphate groups at specific positions of the non-natural sugar. They are intermediates for the metabolic conversion of unnatural sugars in cells.
The term "bioorthogonal reaction" refers to chemical reactions that can be performed in living cells or tissues without interfering with the biochemical reactions of the organism itself. Is used for researching biological macromolecules such as nucleic acid, protein, sugar or lipid.
In some embodiments of the invention, the non-natural phosphate sugar compound is a derivative of ManNAc-6-P, belonging to the ManNAc analog.
In the synthesis strategy, the first position of the ManNAc analogue is protected, then other hydroxyl groups are protected, deprotection of the sixth position of sugar is selectively carried out, and after phosphoric acid is introduced into the sixth position, the third and fourth protecting groups are removed to obtain the product. In some cases, non-natural saccharides with protected one position and unprotected three, four and six positions can be used as raw materials, and the non-natural saccharides with unprotected three, four and six positions can be directly and selectively reacted to obtain products.
In particular, in some embodiments of the invention, the compound ManNAz (or 1,6-Ac, a mono-, hexa-di-oxyacylated derivative thereof 2 ManNAz、1,6-Pr 2 ManNAz), oxygen-acyl protection is first introduced in its first position. There are two methods for introducing the acyl group in the first position, namely, the hydroxy hydrogen in the first position of ManNAz is directly utilized to have stronger acidity than other hydroxy hydrogen on sugar, and sodium carbonate and corresponding anhydride (such as acetic anhydride or propionic anhydride) are utilized to be positioned on the oxygen in the first position of ManNAzAn acyl group (such as acetyl or propionyl) is directly introduced. In particular, for acetyl protection in position one, the 1-O-Ac-ManNAz can also be synthesized using sodium carbonate, DMC (2-Chloro-1, 3-dimethylazolonium chloride) and thioacetic acid.
The second method is to use an organotin reagent [ tBu ] from the ManNAz derivative of the mono-hexadi-oxo-acylation 2 SnOH(Cl)] 2 The ester bond protection at six positions is selectively removed, and the sugar with one position being the protection of the oxo acyl is obtained. The method comprises the steps of taking DCM (dichloromethane) as a solvent, taking TMSOTf (trifluoromethanesulfonic acid trimethyl silicate) as a catalyst, taking HMDS (hexamethyldisilazane) as a reactant, carrying out total TMS (trimethyl silyl ether) protection on three, four and six hydroxyl groups on the sugar, selectively deprotecting the six TMS by using ammonium acetate to expose the hydroxyl groups, introducing phosphate groups, and finally carrying out deprotection on the three and four TMS by using hydrogen ion exchange resin to obtain a target product. In particular, when introducing SATE protected phosphates, it is also possible to use ManNAz, one position of which is an oxo-acyl group, as starting material, to react directly with an excess of SATE phosphoramidite at zero degrees, and to introduce the SATE protected ManNAz-6-P derivative directly at six positions after oxidation by a one-pot method.
In the non-natural phosphate sugar structure of the present invention, the orthogonal groups contained are not limited to azide, alkynyl and terminal alkene, and non-natural sugar containing other orthogonal groups without protecting groups can be synthesized by using the strategy. Other orthogonal groups include, but are not limited to, any of cyclopropene, trans-cyclooctene, cyclooctyne, and tetrazine. Thus, the structure of these unnatural phosphate sugars is protected by the present invention.
In the synthetic route of the present invention, the substrate to be used is not limited to only mannosamine derivatives, glucosamine derivatives and other amino group-containing sugars, and can be used as substrates for this type of synthesis. Thus, synthesis of non-natural phosphate sugars of these configurations is protected by the present invention.
In the synthetic route of the present invention, the acyl group used on the sugar hydroxyl group is not limited to the acetyl group and propionyl group, and other acyl groups may be introduced by the method of the present invention. The protecting groups on the SATE groups are not limited to methyl, ethyl, n-butyl, t-butyl, and n-heptyl, and other types of alkyl and aryl chains employ the synthetic strategies of the present invention. The protecting groups on the AOM groups are not limited to t-butyl groups and other alkyl and aryl chains may be incorporated by the methods of the present invention. The amino acid on the ProTide group can be selected in a variety of ways and is not limited to leucine in the examples of this patent. Thus, these similarly structured non-natural phosphate sugars are also protected by the present invention.
The non-natural phosphate sugar metabolism of the invention is adopted for marking, so that the non-natural phosphate sugar metabolism marking speed is high, and the non-natural phosphate sugar metabolism marking method does not have side reaction with cysteine in protein. In a preferred embodiment, the above-mentioned application comprises labeling the sugar metabolism of any one of the following cells: heLa cells, MCF-7 cells, MDA-MB-231 cells, A549 cells, hepG2 cells, NIH3T3 cells, CHO cells, COS-7 cells, and HEK 293T cells. In some embodiments of the invention, 1-O-Ac-ManNAz-6-bis- (Me-SATE) -P is used and brain slices of mice are metabolically labeled.
The advantageous effects of the present invention will be further described below with reference to specific examples.
Example 1: synthesis of 1-O-Ac-ManNAz-6-bis- (Me-SATE) -P.
1-O-Ac-ManNAz-6-bis- (Me-SATE) -P was synthesized using 1-O-Ac-ManNAz and Me-SATE phosphoramidite as starting materials. There are three synthetic methods for 1-O-Ac-ManNAz, and Me-SATE phosphoramidite uses the general synthetic method of the present invention.
1.1 1-O-Ac-ManNAz Synthesis route one
460 mg of ManNAz and 297 mg of sodium carbonate were dissolved in 40 ml of water and the ice-water bath was cooled to zero. 195.8 microliters of thioacetic acid and 297 milligrams of DMC (DMC dissolved in 630 microliters of water, DMC in aqueous solution added at the time of reaction) were added sequentially, and after 15 minutes of zero-degree reaction, 297 milligrams of sodium carbonate and 195.8 microliters of thioacetic acid were added again, then 297 mg of DMC was added and the zero-degree reaction continued for 15 minutes. The stepwise loading method was repeated three more times (5 total reactions). After the reaction was completed, the reaction system was diluted with 40 ml of water, the aqueous phase was washed once with DCM and lyophilized to give a pale yellow powder which was purified by silica gel column (dichloromethane and methanol as eluent) to give 370 mg of 1-O-Ac-ManNAz in 69% yield. 1 H NMR(600MHz,Deuterium Oxide)δ5.96(d,J＝1.7Hz,1H,H-1-α),5.88(d,J＝1.7Hz,1H,H-1-β),4.63(dd,J＝4.6,1.8Hz,1H,H-2-β),4.48(dd,J＝4.8,1.8Hz,1H,H-2-α),4.17–4.07(m,6H,H-3-α+COCH 2 N 3 -α+COCH 2 N 3 -β),3.96(dd,J＝9.5,4.5Hz,1H,H-3-β),3.91(dd,J＝12.4,2.2Hz,1H,H-6a-β),3.87–3.80(m,3H,H-6a-α+H-6b-α+H-6b-β),3.78(ddd,J＝10.0,4.1,2.7Hz,1H,H-5-α),3.69(t,J＝9.9Hz,1H,H-4-α),3.61(t,J＝9.7Hz,1H,H-4-β),3.56(ddd,J＝9.9,4.7,2.2Hz,1H,H-5-β),2.19(s,3H,COCH 3 -α),2.13(s,3H,COCH 3 -β). 13 C NMR(151MHz,D 2 O)δ172.03,171.68,171.66,171.03,92.44(C-1-α),91.69(C-1-β),77.20(C-5-β),74.39(C-5-α),71.19(C-3-β),68.67(C-3-α),66.16(C-4-β),66.05(C-4-α),60.05,60.03,52.25(C-2-β),51.66,51.60,51.55,20.22(COCH 3 ),20.19(COCH 3 ) HRMS (ESI) theory C 10 H 16 N 4 NaO 7 [M+Na] + 327.09167, detected 327.09094.
1.2 Synthesis route two of 1-O-Ac-ManNAz
620 mg of ManNAz is dissolved in 50 ml of water, the temperature is reduced to zero by ice water bath, 0.243 g of acetic anhydride is added, and 0.253 g of Na is slowly added into the reaction system 2 CO 3 (sodium carbonate was first pre-dissolved with 2 ml of water and pre-chilled to zero), and the reaction was stirred at zero for half an hour. Subsequently, 0.243 g of acetic anhydride and 0.253 g of sodium carbonate were again added and the reaction was continued with stirring at zero for half an hour. TCL shows complete reaction, and the reaction is directly lyophilized to give a white powder which is further purified to give 0.483 g of 1-O-Ac-ManNAzThe yield was 67%.
1.3 Synthesis route three of 1-O-Ac-ManNAz
478 mg of 1,6-Ac 2 ManNAz was dissolved in 60 ml of a mixed solvent of dichloromethane and methanol (equal volume ratio), and 25 mg of organotin catalyst [ tBu ] was added 2 SnOH(Cl)] 2 The reaction was carried out overnight at room temperature, TLC showed complete reaction, solvent was dried at room temperature, and the residue was purified by column chromatography on silica gel, eluting with dichloromethane and methanol to give 260 mg of product in 62% yield.
1.4 general method for synthesizing SATE phosphoramidites from mercaptoethanol
3.907 g of mercaptoethanol and 5.059 g of triethylamine are dissolved in 50 ml of anhydrous dichloromethane, the solution is cooled to-78℃and 50 mmol of acid chloride are then added dropwise by means of a syringe, the reaction is stirred at-78℃for two hours and then warmed to room temperature and reacted for one hour, TLC shows complete reaction, 40 ml of water and 40 ml of saturated sodium chloride solution are added to the reaction system, the organic phase is separated and the aqueous phase is extracted 5 times with 40 ml of dichloromethane each time. The organic phases are combined, dried by anhydrous sodium sulfate and concentrated to obtain colorless transparent liquid which is a thioester intermediate and is directly used for the next reaction. All thioesters were dissolved in 80 ml of anhydrous tetrahydrofuran and 20.49 g of triethylamine and 8 g of activated were addedMolecular sieve, stirring at room temperature for 10 min, cooling to zero deg.c in ice water bath, adding 4.546 g of dichloro-N, N-diisopropyl phosphoramidite under nitrogen protection, stirring overnight and raising temperature gradually to room temperature to form white precipitate, filtering, concentrating the filtrate, and utilizing neutral alumina to make column layer And (3) separating and purifying, namely using petroleum ether containing 2% of triethylamine and 2% of ethyl acetate as an eluent, and developing the target product SATE phosphoramidite by using ninhydrin. All the products are combined, and the solvent is distilled off to obtain SATE phosphoramidite with different substituents on acyl groups for standby.
R is H or a substituent of C1-C6.
1.5 Synthesis of 1-O-Ac-ManNAz-6-bis- (Me-SATE) -P
260 mg of 1-O-Ac-ManNAz was dissolved in an appropriate volume of anhydrous tetrahydrofuran (to a concentration of 0.08 moles per liter), three equivalents of Me-SATE phosphoramidite were added to the solution under nitrogen (synthesis procedure referenced above for the general procedure for the synthesis of SATE phosphoramidite from mercaptoethanol by 1.4), four equivalents of 1-H-tetrazine (dissolved in anhydrous acetonitrile at a concentration of about 0.45 moles per liter) were then added by syringe and the reaction was completed overnight at zero degrees with TLC. At zero degree, 70% tertiary butanol hydrogen peroxide solution is added, the reaction system is stirred for 2 hours, trivalent phosphorus products are oxidized to obtain pentavalent phosphorus products, after solvent is removed by rotary evaporation, purification is carried out by using a silica gel column, dichloromethane and methanol are used as eluent, 267 mg of 1-O-Ac-ManNAz-6-bis- (Me-SATE) -P products are obtained, and the yield of 53% of 1-O-Ac-ManNAz-6-bis- (Me-SATE) -P is called AMMS for short in two continuous steps. The compound nuclear magnetism and high resolution mass spectrum are characterized as follows. 1 H NMR(600MHz,DMSO-d 6 )δ8.20(d,J＝7.9Hz,1.3H,CONH-α),7.94(d,J＝9.6Hz,1.0H,CONH-β),5.97(d,J＝1.9Hz,1.3H,H-α-1),5.85(d,J＝1.8Hz,1.0H,H-β-1),5.40(m,4.6H,OH-α-3+OH-β-3+OH-α-4+OH-β-4),4.54(ddd,J＝9.7,4.4,1.8Hz,1.0H,H-β-2),4.42(ddd,J＝11.3,5.9,1.8Hz,1.0H,H-β-6a),4.38(ddd,J＝11.4,6.2,1.8Hz,1.3H,H-α-6a),4.28–4.21(m,3.6H,H-α-6b+H-β-6b+H-α-2),4.21–4.16(d,J＝5.6Hz,9.2H,2X C(O)SCH 2 CH 2 ),4.05–3.99(m,4.6H,NHCOCH 2 N 3 ),3.96(dd,J＝9.4,4.8Hz,1.3H,H-α-3),3.84(ddd,J＝8.8,6.6,1.9Hz,1.3H,H-α-5),3.80(dd,J＝9.2,4.4Hz,1.0H,H-β-3),3.69–3.61(m,2.3H,H-α-4+H-β-5),3.49(t,J＝9.3Hz,1.0H,H-β-4),3.28(m,9.2H,2×C(O)SCH 2 CH 2 ),2.50(s,13.8H,2×CH 2 CH 2 SC(O)CH 3 ),2.24(s,3.9H,COCH 3 -α-1),2.14(s,3.0H,COCH 3 -β-1). 13 C NMR(151MHz,DMSO-d 6 )δ194.83(SC(O)C(CH 3 ) 3 ),194.81(SC(O)C(CH 3 ) 3 ),168.78(CH 3 CO-α),168.55(CH 3 CO-β),168.39(CONH-β),168.36(CONH-α),92.00(C-α-1),91.71(C-β-1),76.22(C-β-5),76.18(C-β-5),73.60(C-α-5),73.56(C-α-5),70.61(C-β-3),68.06(C-α-3),67.46(C-β-6),67.42(C-β-6),67.14(C-α-6),67.10(C-α-6),66.29(C-β-4),66.07(C-α-4),65.76(CH 2 CH 2 SC(O)CH 3 ),65.71(CH 2 CH 2 SC(O)CH 3 ),65.67(CH 2 CH 2 SC(O)CH 3 ),65.66(CH 2 CH 2 SC(O)CH 3 ),65.63(CH 2 CH 2 SC(O)CH 3 ),65.62(CH 2 CH 2 SC(O)CH 3 ),65.60(CH 2 CH 2 SC(O)CH 3 ),51.91(C-β-2),51.43(C-α-2),50.77(NHCOCH 2 N 3 ),50.56(NHCOCH 2 N 3 ),30.52(CH 2 CH 2 SC(O)CH 3 ),28.82(CH 2 CH 2 SC(O)CH 3 ),28.77(CH 2 CH 2 SC(O)CH 3 ),20.77(CH 3 CO-α-1),20.69(CH 3 CO-β-1). 31 P NMR, -1.86.HRMS (ESI) theory C 18 H 30 N 4 O 12 PS 2 [M+H] + 589.10393, detected 589.10428.
Example 2: synthesis of 1-O-Ac-ManNAz-6-bis- (Et-SATE) -P.
148 mg of 1-O-Ac-ManNAz was dissolved in an appropriate volume of anhydrous tetrahydrofuran (to a concentration of 0.08 mol/l), and three equivalents of Et-SATE phosphoramidite were added to the solution under nitrogen (synthesis method referred to above)General procedure for the synthesis of SATE phosphoramidite from mercaptoethanol) four equivalents of 1-H-tetrazine (dissolved in anhydrous acetonitrile at a concentration of about 0.45 mol/liter) were added by syringe and the reaction was allowed to proceed overnight at zero degrees, with TLC indicating completion of the reaction. At zero degree, 70% tertiary butanol hydrogen peroxide solution is added, the reaction system is stirred for 2 hours, trivalent phosphorus products are oxidized to obtain pentavalent phosphorus products, after solvent is removed by rotary evaporation, the products are purified by a silica gel column, dichloromethane and methanol are used as eluent, 172 mg of 1-O-Ac-ManNAz-6-bis- (Et-SATE) -P products are obtained, and the yield is 57% in two continuous steps (1-O-Ac-ManNAz-6-bis- (Et-SATE) -P is called AMES for short). The compound nuclear magnetism and high resolution mass spectrum are characterized as follows. 1 H NMR(600MHz,DMSO-d 6 )δ8.07(d,J＝7.9Hz,1.3H,CONH-α),7.80(d,J＝9.6Hz,1.0H,CONH-β),5.82(d,J＝1.9Hz,1.3H,H-α-1),5.70(d,J＝1.8Hz,1.0H,H-β-1),5.34(d,J＝5.8Hz,1.3H,OH-α-4),5.30(d,J＝5.5Hz,1.0H,OH-β-4),5.25(d,J＝5.0Hz,1.4H,OH-α-3),5.18(d,J＝5.4Hz,1.0H,OH-β-3),4.39(ddd,J＝9.7,4.5,1.8Hz,1.0H,H-β-2),4.28(ddd,J＝11.3,6.0,1.9Hz,1.0H,H-β-6a),4.23(ddd,J＝11.4,6.3,1.9Hz,1.3H,H-α-6a),4.14–4.07(m,3.6H,H-α-6b+H-β-6b+H-α-2),4.03(m,9.2H,2×C(O)SCH 2 CH 2 ),3.88(m,4.6H,NHCOCH 2 N 3 ),3.81(dt,J＝9.6,4.9Hz,1.3H,H-α-3),3.69(ddd,J＝8.9,6.7,1.8Hz,1.3H,H-α-5),3.65(dt,J＝9.5,4.9Hz,1.0H,H-β-3),3.56–3.45(m,2.3H,H-α-4+H-β-5),3.33(m,1.0H,H-β-4),3.14(m,9.2H,2×C(O)SCH 2 CH 2 ),2.62(m,9.2H,2×CH 3 CH 2 CO),2.10(s,3.9H,COCH 3 -α-1),1.99(s,3.0H,COCH 3 -β-1),1.08(t,J＝7.4Hz,13.8H,2×CH 3 CH 2 CO). 13 C NMR(151MHz,DMSO-d 6 )δ199.31(SC(O)CHCH),199.29(SC(O)CHCH),169.12(CHCOα-1),168.88(CHCO-β-1),168.74(CONH-β),168.72(CONH-α),92.37(C-β-1),92.05(C-α-1),76.59(C-β-5),76.55(C-β-5),73.97(C-α-5),73.93(C-α-5),70.91(C-β-3),68.38(C-α-3),67.81(C-β-6),67.77(C-α-6),67.48(C-β-4),67.45(C-α-4),66.65(CH 2 CH 2 SC(O)CH 2 CH 3 ),66.41(CH 2 CH 2 SC(O)CH 2 CH 3 ),66.15(CH 2 CH 2 SC(O)CH 2 CH 3 ),66.11(CH 2 CH 2 SC(O)CH 2 CH 3 ),66.06(CH 2 CH 2 SC(O)CH 2 CH 3 ),66.03(CH 2 CH 2 SC(O)CH 2 CH 3 ),66.02(CH 2 CH 2 SC(O)CH 2 CH 3 ),65.99(CH 2 CH 2 SC(O)CH 2 CH 3 ),52.28(C-α-2),51.77(C-β-2),51.09(NHCOCHN),50.88(NHCOCHN),37.29(CHCHCO),28.80(C(O)SCH 2 CH 2 ),28.75(C(O)SCH 2 CH 2 ),21.16(CHCO-α-1),21.07(CHCO-β-1),9.76(2×CH 3 CH 2 CO). 31 P NMR(243MHz,DMSO-d 6 ) Delta-2.36. HRMS (ESI) theoretical molecular weight C 20 H 37 N 5 O 12 PS 2 [M+NH 4 ] + 634.16177, molecular weight 634.16132.
Example 3: synthesis of 1-O-Ac-ManNAz-6-bis- (tBu-SATE) -P.
107 mg of 1-O-Ac-ManNAz was dissolved in an appropriate volume of anhydrous tetrahydrofuran (to a concentration of 0.08 moles per liter), three equivalents of tBu-SATE phosphoramidite were added to the solution under nitrogen (synthesis procedure referred to the general procedure for SATE phosphoramidite synthesis from mercaptoethanol described above), four equivalents of 1-H-tetrazine (dissolved in anhydrous acetonitrile at a concentration of about 0.45 moles per liter) were then added by syringe and the reaction was completed at zero degrees overnight with TLC. At zero degree, 70% tertiary butanol hydrogen peroxide solution is added, the reaction system is stirred for 2 hours, trivalent phosphorus products are oxidized to obtain pentavalent phosphorus products, after solvent is removed by rotary evaporation, purification is carried out by using a silica gel column, dichloromethane and methanol are used as eluent, 147 mg of 1-O-Ac-ManNAz-6-bis- (tBu-SATE) -P products are obtained, and the yield of two continuous steps is 62 percent, (1-O-Ac-ManNAz-6-bis- (tBu-SATE) -P, which is called AMtBS for short). The nuclear magnetism and mass spectrum of the compound are characterized as follows. 1 H NMR(600MHz,DMSO-d 6 )δ8.07(d,J＝7.9Hz,1.3H,CONH-α),7.80(d,J＝9.7Hz,1.0H,CONH-β),5.82(d,J＝1.9Hz,1.3H,H-α-1),5.70(d,J＝1.8Hz,1.0H,H-β-1),5.33(d,J＝5.8Hz,1.3H,OH-α-4),5.29(d,J＝5.5Hz,1.0H,OH-β-4),5.24(d,J＝5.0Hz,1.4H,OH-α-3),5.18(d,J＝5.4Hz,1.0H,OH-β-3),4.38(ddd,J＝9.7,4.5,1.7Hz,1.0H,H-β-2),4.27(ddd,J＝11.4,6.1,1.9Hz,1.0H,H-β-6a),4.23(ddd,J＝11.3,6.2,1.8Hz,1.3H,H-α-6a),4.10–4.05(m,3.6H,H-α-6b+H-β-6b+H-α-2),4.04–3.98(m,9.2H,2×C(O)SCH 2 CH 2 ),3.89–3.86(m,4.6H,NHCOCH 2 N 3 ),3.80(dt,J＝9.6,4.9Hz,1.3H,H-α-3),3.68(ddd,J＝8.9,6.7,1.8Hz,1.3H,H-α-5),3.64(dd,J＝9.6,4.5Hz,1.0H,H-β-3),3.54–3.45(m,2.3H,H-α-4+H-β-5),3.35–3.30(m,1.0H,H-β-4),3.12–3.08(m,9.2H,2×C(O)SCH 2 CH 2 ),2.09(s,3.9H,COCH-α-1),1.98(s,3.0H,COCH-β-1),1.18(s,41.4H,SC(O)C(CH 3 ) 3 ). 13 C NMR(151MHz,DMSO-d 6 )δ205.09(SC(O)C(CH 3 ) 3 ),205.07(SC(O)C(CH 3 ) 3 ),168.63(CH 3 CO-α-1),168.40(CHCO-β-1),168.29(CONH-β),168.26(CONH-α),91.95(C-β-1),91.61(C-α-1),76.15(C-β-5),76.11(C-β-5),73.54(C-α-5),73.49(C-α-5),70.46(C-β-3),67.93(C-α-3),67.35(C-β-6),67.31(C-β-6),67.03(C-α-6),66.99(C-α-6),66.20(C-β-4),65.97(C-α-4),65.67(CH 2 CH 2 SC(O)C(CH 3 ) 3 ),65.63(CH 2 CH 2 SC(O)C(CH 3 ) 3 ),65.59(CH 2 CH 2 SC(O)C(CH 3 ) 3 ),65.57(CH 2 CH 2 SC(O)C(CH 3 ) 3 ),65.54(CH 2 CH 2 SC(O)C(CH 3 ) 3 ),65.51(CH 2 CH 2 SC(O)C(CH 3 ) 3 ),51.84(C-α-2),51.32(C-β-2),50.64(NHCOCH 2 N 3 ),50.42(NHCOCH 2 N 3 ),46.02(CH 2 CH 2 SC(O)C(CH 3 ) 3 ),28.17(CH 2 CH 2 SC(O)C(CH 3 ) 3 ),28.16(CH 2 CH 2 SC(O)C(CH 3 ) 3 ),28.12(CH 2 CH 2 SC(O)C(CH 3 ) 3 ),28.10(CH 2 CH 2 SC(O)C(CH 3 ) 3 ),26.89(CH 2 CH 2 SC(O)C(CH 3 ) 3 ),20.73(CH 3 CO-α-1),20.65(CH 3 CO-β-1). 31 P NMR(243MHz,DMSO-d 6 ) Delta-2.35. HRMS (ESI) theoretical molecular weight C 24 H 45 N 5 O 12 PS 2 [M+NH 4 ] + 690.22437, molecular weight 690.22392.
Example 4: synthesis of 1-O-Ac-ManNAz-6-bis- (Bu-SATE) -P.
259 mg of 1-O-Ac-ManNAz was dissolved in an appropriate volume of anhydrous tetrahydrofuran (to a concentration of 0.08 mol/l), three equivalents of tBu-SATE phosphoramidite were added to the solution under nitrogen (synthesis method referred to the general method for SATE phosphoramidite synthesis from mercaptoethanol described above), four equivalents of 1-H-tetrazine (dissolved in anhydrous acetonitrile at a concentration of about 0.45 mol/l) were added by syringe, and the reaction was completed at zero degrees overnight by TLC. At zero degree, 70% tertiary butanol hydrogen peroxide solution is added, the reaction system is stirred for 2 hours, trivalent phosphorus products are oxidized to obtain pentavalent phosphorus products, after solvent is removed by rotary evaporation, purification is carried out by using a silica gel column, dichloromethane and methanol are used as eluent, and 350 mg of 1-O-Ac-ManNAz-6-bis- (tBu-SATE) -P products are obtained, and the yield of 61% (1-O-Ac-ManNAz-6-bis- (Bu-SATE) -P, abbreviated as AMBS) is obtained in two steps. The compound nuclear magnetism and high resolution mass spectrum are characterized as follows. 1 H NMR(500MHz,DMSO-d 6 )δ8.08(d,J＝7.9Hz,1.4H,CONH-α),7.80(d,J＝9.6Hz,1.0H,CONH-β),5.82(d,J＝1.9Hz,1.4H,H-α-1),5.70(d,J＝1.8Hz,1.0H,H-β-1),5.34(d,J＝5.8Hz,1.4H,OH-α-4),5.30(d,J＝5.5Hz,1.0H,OH-β-4),5.25(d,J＝5.0Hz,1.4H,OH-α-3),5.18(d,J＝5.3Hz,1.0H,OH-β-3),4.38(ddd,J＝9.6,4.4,1.8Hz,1.0H,H-β-2),4.27(ddd,J＝11.5,6.2,1.8Hz,1.4H,H-β-6a),4.22(ddd,J＝11.4,6.4,1.8Hz,1.4H,H-α-6a),4.14–3.97(m,13.4H,H-α-6b+H-β-6b+H-α-2+2×C(O)SCH 2 CH 2 ),3.90–3.86(m,4.8H,NHCOCH 2 N 3 ),3.80(dt,J＝9.6,4.8Hz,1.4H,H-α-3),3.68(ddd,J＝9.2,6.9,1.8Hz,1.4H,H-α-5),3.66–3.62(m,1.0H,H-β-3),3.55–3.45(m,2.4H,H-α-4+H-β-5),3.36–3.30(s,1.0H,H-β-4),3.15–3.10(m,9.6H,2×C(O)SCH 2 CH 2 ),2.60(t,J＝7.4Hz,9.6H,SC(O)CH 2 CH 2 CH 2 CH 3 ),2.10(s,4.2H,COCH 3 -α-1),1.99(s,3.0H,COCH 3 -β-1),1.58–1.52(m,9.6H,2×SC(O)CH 2 CH 2 CH 2 CH 3 ),1.34–1.26(m,9.6H,2×SC(O)CH 2 CH 2 CH 2 CH 3 ),0.87(t,J＝7.4Hz,14.4H,2×SC(O)CH 2 CH 2 CH 2 CH 3 ). 13 C NMR(126MHz,DMSO-d 6 )δ198.60(SC(O)CH 2 CH 2 ),198.58(SC(O)CH 2 CH 2 ),169.12(CH 3 CO-α-1),168.88(CH 3 CO-β-1),168.74(CONH-β),168.72(CONH-α),92.38(C-β-1),92.05(C-α-1),76.60(C-β-5),76.55(C-β-5),73.98(C-α-5),73.93(C-α-5),70.89(C-β-3),68.37(C-α-3),67.79(C-β-6),67.44(C-α-6),66.64(C-β-4),66.41(C-α-4),66.15(CH 2 CH 2 SC(O)),66.10(CH 2 CH 2 SC(O)),66.06(CH 2 CH 2 SC(O)),66.03(CH 2 CH 2 SC(O)),66.01(CH 2 CH 2 SC(O)),65.99(CH 2 CH 2 SC(O)),52.28(C-α-2),51.77(C-β-2),51.08(NHCOCH 2 N 3 ),50.87(NHCOCH 2 N 3 ),43.54(SC(O)CH 2 CH 2 CH 2 CH 3 ),28.87(C(O)SCH 2 CH 2 ),28.81(C(O)SCH 2 CH 2 CH 2 CH 3 ),27.54(SC(O)CH 2 CH 2 CH 2 CH 3 ),21.87(SC(O)CH 2 CH 2 CH 2 CH 3 ),21.18(CH 3 CO-α-1),21.09(CH 3 CO-β-1),14.02(SC(O)CH 2 CH 2 CH 2 CH 3 ). 31 PNMR(202MHz,DMSO-d 6 ) Delta-2.36. HRMS (ESI) theoretical molecular weight C 24 H 45 N 5 O 12 PS 2 [M+NH 4 ] + 690.22437, detection ofMolecular weight 690.22404.
Example 5: synthesis of 1-O-Ac-ManNAz-6-bis- (He-SATE) -P
65 mg of 1-O-Ac-ManNAz was dissolved in an appropriate volume of anhydrous tetrahydrofuran (to a concentration of 0.08 moles per liter), three equivalents of tBu-SATE phosphoramidite were added to the solution under nitrogen (synthesis procedure referred to the general procedure for SATE phosphoramidite synthesis from mercaptoethanol described above), followed by four equivalents of 1-H-tetrazine (dissolved in anhydrous acetonitrile at a concentration of about 0.45 moles per liter) using a syringe, and the reaction was completed at zero degrees overnight with TLC. At zero degree, 70% tertiary butanol hydrogen peroxide solution is added, the reaction system is stirred for 2 hours, trivalent phosphorus products are oxidized to obtain pentavalent phosphorus products, after the solvent is removed in vacuum, the pentavalent phosphorus products are purified by a silica gel column, dichloromethane and methanol are used as eluent, 104 mg of 1-O-Ac-ManNAz-6-bis- (tBu-SATE) -P products are obtained, and the yield of the 1-O-Ac-ManNAz-6-bis- (He-SATE) -P is 64 percent in two continuous steps, which is called AMHS for short. The nuclear magnetism and high resolution mass spectrum of the compounds are characterized as follows. 1 H NMR(600MHz,DMSO-d 6 )δ8.06(d,J＝7.9Hz,1.2H,CONH-α),7.80(d,J＝9.6Hz,1.0H,CONH-β),5.83(d,J＝1.9Hz,1.2H,H-α-1),5.70(d,J＝1.8Hz,1.0H,H-β-1),5.33(d,J＝5.7Hz,1.2H,OH-α-4),5.29(d,J＝5.5Hz,1.0H,OH-β-4),5.23(d,J＝5.0Hz,1.2H,OH-α-3),5.17(d,J＝5.4Hz,1.0H,OH-β-3),4.39(ddd,J＝9.7,4.5,1.8Hz,1.0H,H-β-2),4.27(ddd,J＝11.4,6.1,1.9Hz,1.0H,H-β-6a),4.23(ddd,J＝11.4,6.2,1.9Hz,1.2H,H-α-6a),4.15–4.07(m,3.4H,H-α-6b+H-β-6b+H-α-2),4.06–3.99(m,8.8H,2X C(O)SCH 2 CH 2 ),3.92–3.85(m,4.4H,NHCOCH 2 N 3 ),3.81(dt,J＝9.6,4.9Hz,1.4H,H-α-3),3.71–3.67(m,1.2H,H-α-5),3.65(dd,J＝9.4,4.7Hz,1.0H,H-β-3),3.55–3.46(m,2.2H,H-α-4+H-β-5),3.36(dd,J＝9.3,5.5Hz,1.0H,H-β-4),3.16–3.11(m,8.8H,2×C(O)SCH 2 CH 2 ),2.61–2.57(m,8.8H,2×SC(O)CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 ),2.09(s,3.6H,COCH 3 -α-1),1.99(s,3.0H,COCH 3 -β-1),1.60–1.54(t,J＝7.3Hz,1H,2×SC(O)CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 ),1.30–1.19(m,35.2H,2×SC(O)CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 ),0.86(t,J＝7.0Hz,13.2H,2×SC(O)CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 ). 13 C NMR(151MHz,DMSO-d 6 )δ198.46(SC(O)CH 2 CH 2 ),198.44(SC(O)CH 2 CH 2 ),169.04(CH 3 CO-α-1),168.82(CH 3 CO-β-1),168.71(CONH-β),168.68(CONH-α),92.39(C-β-1),92.08(C-α-1),76.60(C-β-5),76.56(C-β-5),73.97(C-α-5),73.93(C-α-5),70.94(C-β-3),68.41(C-α-3),67.80(C-β-6),67.76(C-β-6),67.48(C-α-6),67.44(C-α-6),66.63(C-β-4),66.41(C-β-4),66.14(CH 2 CH 2 SC(O)),66.10(CH 2 CH 2 SC(O)),66.06(CH 2 CH 2 SC(O)),66.04(CH 2 CH 2 SC(O)),66.01(CH 2 CH 2 SC(O)),65.98(CH 2 CH 2 SC(O)),52.28(C-α-2),51.77(C-β-2),51.11(NHCOCH 2 N 4 ),50.89(NHCOCH 2 N 3 ),43.82(SC(O)CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 ),31.54(SC(O)CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 ),28.85(C(O)SCH 2 CH 2 ),28.83(C(O)SCH 2 CH 2 ),28.79(C(O)SCH 2 CH 2 ),28.66(SC(O)CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 ),25.45(SC(O)CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 ),22.50(SC(O)CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 ),21.13(CH 3 CO-α-1),21.04(CH 3 CO-β-1),14.32(SC(O)CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 ). 31 P NMR(243MHz,DMSO-d 6 ) Delta-2.26, -2.38.HRMS (ESI) theoretical molecular weight C 30 H 54 N 4 O 12 PS 2 [M+H] + 757.29173, molecular weight 757.29021.
Example 6: synthesis of Ac3ManNAz-6-bis- (Me-SATE) -P.
137 mg of 1-O-Ac-ManNAz-6-bis- (Me-SATE) -P were dissolved in 15 ml of pyridine, 4.5 ml of acetic anhydride and 15 mg of DMAP (4-dimethylaminopyridine) were added, and the reaction was stirred overnight at room temperature, and TLC showed completion of the reaction. The solvent was distilled off in vacuo to give a pale yellow residue, which was dissolved in 50 ml of ethyl acetate, and the organic phase was washed successively with 40 ml of saturated potassium hydrogen sulfate, sodium hydrogen carbonate and sodium chloride. The aqueous phases were extracted once with 40 ml of ethyl acetate, the organic phases were combined and dried over sodium sulfate, the solvent was distilled off and the residue was purified by a silica gel column to give 152 mg of the objective compound Ac 3 ManNAz-6-bis- (Me-SATE) -P in 97% yield, (Ac) 3 Synthesis of ManNAz-6-bis- (Me-SATE) -P, abbreviated as TAMMS. The nuclear magnetism and mass spectrum characterization information of the compounds are as follows, 1 H NMR(600MHz,DMSO-d 6 )δ8.56(d,J＝9.0Hz,1H,NH-α),8.35(d,J＝9.6Hz,0.34H,NH-β),6.03(d,J＝1.9Hz,0.34H,H-1-β),5.85(d,J＝1.9Hz,1H,H-1-α),5.27–5.18(m,1.34H,H-3-β+H-4-α),5.16(dd,J＝10.0,4.5Hz,1H,H-3-α),5.07(t,J＝9.6Hz,0.34H,H-4-β),4.62(ddd,J＝9.6,4.4,1.8Hz,0.34H,H-2-β),4.45(ddd,J＝9.1,4.5,1.9Hz,1H,H-2-α),4.10–3.99(m,9.38H,H-5-α+H-5-β+H-6-α+H-6-β+2×SCH 2 CH 2 O,),3.96–3.88(m,2.68H,COCH 2 N 3 ),3.13(m,5.36H),2.36(s,8H,2×SCOCH 3 ),2.16(s,3H,COCH 3 -α),2.06(s,3H,COCH 3 -α),2.05(s,1.02H,COCH 3 -β),2.03(s,1.02H,COCH 3 -β),1.94(s,3H,COCH 3 -α),1.92(s,1.02H,COCH 3 -β). 13 C NMR(151MHz,DMSO-d 6 )δ194.74(SCOCH 3 -β),194.72(SCOCH 3 -α),169.72,169.59,169.55,169.47,168.62,168.43,168.41,168.24,91.17(C-1-α),90.63(C-1-β),72.96,72.92,70.54,70.49,70.38,68.51,66.18,66.14,65.98,65.95,65.75,65.71,65.66,65.62,65.20,65.04,50.50(COCH 2 N 3 -β),50.39(COCH 2 N 3 -α),48.83(C-2-β),48.70(C-2-α),33.37,30.47(2×SCOCH 3 ),28.70(COCH 2 N 3 -βor COCH 2 N 3 -α),28.65(COCH 2 N 3 -αor COCH 2 N 3 -β),20.64,20.58,20.53,20.50,20.47. 31 P NMR(243MHz,DMSO-d 6 ) Delta-2.07, -2.10.HRMS (ESI) theoretical molecular weight C 22 H 37 N 5 O 14 PS 2 [M+NH 4 ] + 690.15160, molecular weight 690.15180.
Example 7: synthesis of 1-O-Ac-ManNAz-6-bis-POM-P
436 mg of 1-O-Ac-ManNAz (1.43 mmol) were dispersed in 160 ml of anhydrous dichloromethane, 50. Mu.l of TMSOTF were added, the reaction was carried out at room temperature for 2 hours, TLC showed complete reaction, the solvent was distilled off under vacuum, the residue was dissolved with ethyl acetate, the organic phase was washed with saturated sodium bicarbonate and saturated sodium chloride solution in this order, and the organic phase was dried over anhydrous sodium sulfate. Concentrated in vacuo, the residue was eluted with petroleum ether and ethyl acetate and purified by silica gel column to give 745 mg of the product, total TMS protected product, quantitative yield.
1.62 g of all TMS protected 1-O-Ac-ManNAz (3.11 mmol) was dissolved in 80 ml of dichloromethane and 160 ml of methanol, 12 g of ammonium acetate (156 mmol) was added and the reaction was stirred at room temperature overnight. TLC showed complete reaction, the residue after concentration in vacuo was treated with 200 ml of ethyl acetate and the organic phase was washed with 100 ml of water, dried over anhydrous sodium sulfate and concentrated, and the residue was eluted with a mixed solvent of petroleum ether and ethyl acetate and purified on a silica gel column to give 1.06 g of the product, manNAz with TMS protected 3,4 positions and oxyacetyl protected 1 position, in 76% yield.
1.34 g of starting material (ManNAz protected at the 3, 4-position with TMS, protected at the 1-position with oxyacetyl, 2.99 mmol) were dissolved in 200 ml of anhydrous tetrahydrofuran, 1.49 g of lithium bis (neopentyloxymethyl) phosphate (1.5 eq., 4.485 mmol), 3.86 g of N, N-diisopropylethylamine (10 eq., 29.9 mmol), 1.71 g of 3-nitro-1, 2, 4-triazole and 3.81 g of BOP-Cl (bis (2-oxo-3-oxazolidinyl) phosphinic chloride) were added in succession, and the reaction was stirred overnight at room temperature, TLC showed complete reaction. After the reaction was concentrated, the residue was dissolved in a mixed solvent of 80 ml of dichloromethane and 80 ml of methanol, 6 g of hydrogen ion exchange resin was added, and stirring was continued at room temperature for 2 hours to remove TMS protection. After concentration in vacuo, the residue was purified by silica gel column (dichloromethane and methanol as eluent) to give 1.52 g of the final product 1-O-Ac-ManNAz-6-bis-POM-P, (1-O-Ac-ManNAz-6-bis-POM-P, abbreviated as AMPOM). The nuclear magnetism of the compound is characterized as follows. 1 H NMR(500MHz,DMSO-d 6 )δ8.05(d,J＝7.9Hz,1.9H),7.79(d,J＝9.6Hz,1H),5.84(d,J＝1.7Hz,1.9H),5.71(d,J＝1.6Hz,1H),5.63–5.54(m,12H),5.46–5.12(m,5.7H),4.39(ddd,J＝9.7,4.4,1.5Hz,1H),4.35–
4.24(m,2.9H),4.20–4.06(m,5H),3.88(d,J＝2.7Hz,5.7H),3.81(dd,J＝9.3,4.8Hz,1.9H),3.75–3.68(m,1.9H),3.66(dd,J＝9.2,4.4Hz,1H),3.57–3.44(m,3.8H),3.33(t,J＝9.2Hz,1H),2.09(s,5.7H),1.98(s,3H),1.18(s,52H). 13 C NMR(126MHz,DMSO-d 6 ) Delta 176.41,169.05,168.83,168.73,168.69,92.35,92.01,83.25,83.21,83.16,83.12,83.10,83.06,76.49,76.44,73.88,73.82,70.87,68.34,68.15,68.11,66.61,66.40,52.26,51.70,51.09,50.87,38.64,27.08,27.01,26.88,26.73,21.13,21.03. The high resolution mass spectrum of the compound is characterized as follows, HRMS (ESI) theoretical molecular weight C 22 H 41 N 5 O 14 P[M+NH 4 ] + 630.23876, molecular weight 630.23775.
Example 8: synthesis of 1-O-Pr-ManNAz-6-bis-POM-P
455 mg of 1-O-Pr-ManNAz (1.43 mmol) are dispersed in 160 ml of anhydrous dichloromethane, 50. Mu.l of TMSOTF are added, the reaction is carried out for 2 hours at room temperature, TLC shows complete reaction, the solvent is distilled off in vacuo, the residue is dissolved with ethyl acetate, the organic phase is washed successively with saturated sodium bicarbonate and saturated sodium chloride solution, and the organic phase is dried over anhydrous sodium sulfate. Evaporating the organic solvent under vacuum, eluting the residue with petroleum ether and ethyl acetate, and purifying with silica gel column to obtain 1-O-Pr-ManNAz protected by TMS, and quantifying yield.
1.66 g of all TMS protected 1-O-Pr-ManNAz (3.11 mmol) were dissolved in a mixed solvent of 80 ml of dichloromethane and 160 ml of methanol, 12 g of ammonium acetate (156 mmol) were added and the reaction was stirred at room temperature overnight. TLC showed complete reaction, the residue after concentration in vacuo was dissolved in 180 ml of ethyl acetate and the organic phase was washed with 100 ml of water, dried over anhydrous sodium sulfate and concentrated, the residue was eluted with a mixed solvent of petroleum ether and ethyl acetate and purified on a silica gel column to give 1.12 g of the product, manNAz with TMS protected 3,4 positions and propionyl protected 1 position, 78% yield.
1.38 starting material (ManNAz protected at the 3, 4-position with TMS, propionyl protected at the 1-position, 2.99 mmol) was dissolved in 180 ml anhydrous tetrahydrofuran and 1.49 g lithium bis (neopentyloxymethyl) phosphate (1.5 eq. 4.485 mmol), 3.86 g N, N-diisopropylethylamine (10 eq., 29.9 mmol), 1.71 g 3-nitro-1, 2, 4-triazole and 3.81 g BOP-Cl (bis (2-oxo-3-oxazolidinyl) phosphinic chloride) were added sequentially and the reaction stirred overnight at room temperature and TLC showed complete reaction. After the reaction was concentrated, the residue was dissolved in a mixed solvent of 80 ml of dichloromethane and 80 ml of methanol, 18 g of hydrogen ion exchange resin was added, and stirring was continued at room temperature for 2 hours to remove TMS protection. After concentration in vacuo, the residue was purified by column chromatography on silica gel (dichloromethane and methanol as eluent) with 1.59 g of the final product 1-O-Pr-ManNAz-6-bis-POM-P, (synthesis of 1-O-Pr-ManNAz-6-bis-POM-P, abbreviated as PMPOM). The nuclear magnetic properties of the compounds are characterized as follows, 1 H NMR(600MHz,DMSO-d 6 )δ8.06(d,J＝7.9Hz,1H,N 3 CH 2 CONH),5.91–5.86(m,1H,H-1),5.58(m,4H,tBuCOOCH 2 O),5.36(d,J＝5.8Hz,1H,OH-4),5.23(d,J＝5.0Hz,1H,OH-3),4.30(m,1H,H-6-a),4.13(m,2H,H-6-b+H-2),3.88(s,2H,N 3 CH 2 CONH),3.83(dt,J＝9.5,4.9Hz,1H,H-3),3.75–3.68(m,1H,H-5),3.50(td,J＝9.5,6.0Hz,1H,H-4),2.40(q,J＝7.4Hz,2H,COCH 2 CH 3 ),1.18(s,18H,CH 3 -POM),1.07(t,J＝7.5Hz,3H,COCH 2 CH 3 ). 13 C NMR(151MHz,DMSO-d 6 )δ176.37(tBuCOOCH 2 O),172.21(COCH2CH3),168.67(N 3 CH 2 CONH),91.92(C-1),83.13(tBuCOOCH 2 O),83.10(tBuCOOCH 2 O),83.05(tBuCOOCH 2 O),83.02(tBuCOOCH 2 O),73.88(C-5),73.83(C-5),68.43(C-3),68.15(C-6),68.11(C-6),66.43(C-4),52.28(C-2),50.89(N 3 CH 2 CONH),38.61,27.30(COCH 2 CH 3 ),26.83(CH 3 -POM),9.10(COCH 2 CH 3 ). 31 P NMR(243MHz,DMSO-d 6 ) Delta-3.99. The high resolution mass spectrum of the compound is characterized as follows, HRMS (ESI) theoretical molecular weight C 23 H 43 N 5 O 14 P[M+NH 4 ] + 644.25441, molecular weight 644.25403.
Example 9: synthesis of 1-O-Ac-ManNAl-6-bis- (tBu-SATE) -P
9.1 1-O-Ac-ManNAl synthesis:
330 mg of ManNAl (0.886 mmol, 1.0 eq) and 150 mg of sodium carbonate (1.417 mmol, 1.6 eq) were dissolved in 20 ml of water and the ice water bath was cooled to zero. 99. Mu.l of thioacetic acid and 150 mg of DMC (DMC dissolved in 315. Mu.l of water, DMC in water) were added in this order, and after 15 minutes of zero-degree reaction, 150 mg of sodium carbonate and 99. Mu.l of thioacetic acid were added again, and then 150 mg of DMC was added, and the zero-degree reaction was continued for 15 minutes. The stepwise loading method was repeated three more times (5 total reactions). After the reaction, the reaction system was diluted with 20 ml of water, the aqueous phase was washed once with DCM, and lyophilized to obtain pale yellow powder, which was purified by silica gel column Dichloromethane and methanol as eluent) to yield 179 mg of 1-O-Ac-ManNAl, 67% yield, as anomer mixture, with a 1.0/1 ratio of the a-configuration and b-configuration products. The nuclear magnetism of the compounds is characterized as follows (signals corresponding to hydrogen protons are shown in italics for assignment), 1 H NMR(500MHz,Methanol-d 4 )δ5.96(d,J＝1.7Hz,1H,H-1-α),5.77(d,J＝1.7Hz,1H,H-1-β),4.56(dd,J＝4.4,1.6Hz,1H,H-2-β),4.34(dd,J＝4.8,1.7Hz,1H,H-2-α),3.98(dd,J＝9.1,4.8Hz,1H,H-3-α),3.85(d,J＝3.3Hz,2H,H-6-β),3.81–3.79(m,2H,H-6-α),3.76(dd,J＝9.6,4.5Hz,1H,H-3-β),3.70–3.62(m,2H,H-4-α+H-5-α),3.58(t,J＝9.6Hz,1H,H-4-β),3.40–3.35(m,1H,H-5-β),2.58–2.43(m,8H,NHCOCH 2 CH 2 CCH),2.30(m,2H,NHCOCH 2 CH 2 CCH),2.13(s,3H,COCH 3 ),2.06(s,3H,COCH 3 ). 13 C NMR(126MHz,MeOD)δ173.95(NHCOCH 2 CH 2 CCH-β),173.39(NHCOCH 2 CH 2 CCH-α),169.19(CH 3 CO),169.19(CH 3 CO),92.58(C-1-α),91.96(C-1-β),82.45(NHCOCH 2 CH 2 CCH),82.26(NHCOCH 2 CH 2 CCH),77.80(C-5-β),75.01(C-5-α),72.13(C-3-β),69.05(C-3-α),68.93(NHCOCH 2 CH 2 CCH),68.83(NHCOCH 2 CH 2 CCH),66.48(C-4-α),66.40(C-4-β),60.67(C-6-α),60.39(C-6-β),52.11(C-2-β),51.91(C-2-α),34.62(NHCOCH 2 CH 2 CCH),34.37(NHCOCH 2 CH 2 CCH),19.48(COCH 3 -β),19.41(COCH 3 -α),14.35(NHCOCH 2 CH 2 CCH),14.19(NHCOCH 2 CH 2 CCH). The compound spectrum is characterized as follows, C 13 H 20 NO 7 [M+H] + The theoretical value of high-resolution mass spectrometry (ESI) was 302.12398 and the detection value was 302.12329.
9.2 Synthesis of 1-O-Ac-ManNAl-6-bis- (tBu-SATE) -P:
96 mg of 1-O-Ac-ManNAl (one equivalent) was dissolved in an appropriate volume of anhydrous tetrahydrofuran (to a concentration of 0.08 mol/liter), and three equivalents of tBu-SATE phosphoramidite were added to the solution under nitrogen (synthesis method referred to previously for SATE phosphoramidite synthesis from mercaptoethanol)General procedure for amine) then four equivalents of 1-H-tetrazine (dissolved in anhydrous acetonitrile at a concentration of about 0.45 moles per liter) were added by syringe and the reaction was left overnight at zero degrees, with TLC showing completion. At zero degree, 70% aqueous solution of tert-butanol hydrogen peroxide (five equivalents of tert-butanol hydrogen peroxide) was added, the reaction system was stirred for 2 hours, the trivalent phosphorus product was oxidized to give a pentavalent phosphorus product, after the solvent was removed in vacuo, purification was performed using a silica gel column, methylene chloride and methanol were used as eluents, and 139 mg of 1-O-Ac-ManNAl-6-bis- (tBu-SATE) -P product was obtained in a continuous two-step yield of 65% as a mixture of anomers, and the ratio of a-configuration isomer to b-configuration isomer was 1.4/1. The nuclear magnetism of the compounds is characterized as follows (signals corresponding to hydrogen protons are shown in italics for assignment), 1 H NMR(500MHz,DMSO-d 6 )δ7.85(d,J＝8.0Hz,1.4H,CONH-α),7.57(d,J＝9.7Hz,1.0H,CONH-β),5.76(d,J＝1.9Hz,1.4H,H-1-α),5.66(d,J＝1.8Hz,1.0H,H-1-β),5.29(d,J＝5.8Hz,1.4H,OH-4-α),5.25(d,J＝5.5Hz,1.0H,OH-4-β),5.11(d,J＝5.0Hz,1.4H,OH-3-α),5.05(d,J＝5.3Hz,1.0H,OH-3-β),4.38(ddd,J＝9.8,4.4,1.8Hz,1.0H,H-2-α),4.28(ddd,J＝11.2,6.2,1.9Hz,1.0H,H-6a-β),4.23(ddd,J＝11.3,6.3,1.8Hz,1.4H,H-6a-α),4.17–4.06(m,3.8H,H-6b-α+H-6b-β+H-2-α),4.06–3.98(m,9.6H,2×C(O)SCH 2 CH 2 ),3.77(dt,J＝9.6,4.9Hz,1.4H,H-3-α),3.67(ddd,J＝9.0,6.9,1.9Hz,1.4H,H-5-α),3.61(dt,J＝9.5,4.9Hz,1.0H,H-3-β),3.55–3.48(m,2.4H,H-4-α+H-5-β),3.37(td,J＝9.2,5.5Hz,1.0H,H-4-β),3.11(m,9.6H,2×C(O)SCH 2 CH 2 ),2.73(m,2.4H,NHCOCH 2 CH 2 CCH),2.46–2.33(m,9.6H,NHCOCH 2 CH 2 +NHCOCH 2 CH 2 ),2.08(s,4.2H,COCH 3 -α),1.97(s,3H,COCH 3 -β),1.19(s,43.2H,2×SC(O)C(CH 3 ) 3 ). 13 C NMR(126MHz,DMSO-d 6 )δ205.53(SC(O)C(CH 3 ) 3 ),171.73(CONH-β),171.53(CONH-α),169.11(CH 3 CO-α),168.90(CH 3 CO-β),92.57(C-1-β),92.37(C-1-α),84.32(NHCOCH 2 CH 2 CCH-β),84.20(NHCOCH2CH 2 CCH-α),76.63(C-5-β),76.58(C-5-β),74.04(C-5-α),73.99(C-5-α),71.67(NHCOCH 2 CH 2 CCH-α),71.57(NHCOCH 2 CH 2 CCH-β),70.97(C-3-β),68.49(C-3-α),67.92(C-6-β),67.87(C-6-β),67.61(C-6-α),67.56(C-6-α),66.66(C-4-β),66.45(C-4-α),66.13(C(O)SCH 2 CH 2 ),66.08(C(O)SCH 2 CH 2 ),66.03(C(O)SCH 2 CH 2 ),66.02(C(O)SCH 2 CH 2 ),65.98(C(O)SCH 2 CH 2 ),65.94(C(O)SCH 2 CH 2 ),51.97(C-2-α),51.28(C-2-β),46.47(SC(O)C(CH 3 ) 3 ),34.65(NHCOCH 2 CH 2 -β),34.39(NHCOCH 2 CH 2 -a),28.64(C(O)SCH 2 CH 2 ),28.62(C(O)SCH 2 CH 2 ),28.58(C(O)SCH 2 CH 2 ),28.56(C(O)SCH 2 CH 2 ),27.35(SC(O)C(CH 3 ) 3 ),21.20(CH 3 CO-α),21.16(CH 3 CO-β),14.83(NHCOCH 2 CH 2 CCH-β),14.61(NHCOCH 2 CH 2 CCH-α). 31 P NMR(202MHz,DMSO-d 6 ) Delta-1.81. The compound spectrum is characterized as follows, C 27 H 45 NO 12 PS 2 [M+H] + The theoretical value of high-resolution mass spectrometry (ESI) was 670.21208 and the detection value was 670.21117.
Example 10: synthesis of 1-O-Ac-ManNPtl-6-bis- (tBu-SATE) -P
10.1 1-O-Ac-ManNPtl Synthesis:
231 mg of ManNPtl (0.886 mmol, 1.0 eq) and 150 mg of sodium carbonate (1.417 mmol, 1.6 eq) were dissolved in 20 ml of water and the ice water bath was cooled to zero. 99. Mu.l of thioacetic acid and 150 mg of DMC (DMC dissolved in 315. Mu.l of water, DMC in water) were added in this order, and after 15 minutes of zero-degree reaction, 150 mg of sodium carbonate and 99. Mu.l of thioacetic acid were added again, and then 150 mg of DMC was added, and the zero-degree reaction was continued for 15 minutes. The stepwise loading method was repeated three more times (5 total reactions). After the reaction is completed, the reactionThe system was diluted with 20 ml of water, the aqueous phase was washed once with DCM and lyophilized to give a pale yellow powder which was purified by column on silica gel (dichloromethane and methanol as eluent) to give 180 mg of 1-O-Ac-mannctl in 67% yield as anomer mixture with a ratio of the products of the alpha and beta configuration of 1.9/1. The nuclear magnetism of the compounds is characterized as follows (signals corresponding to hydrogen protons are shown in italics for assignment), 1 H NMR(500MHz,Methanol-d 4 )δ5.94(d,J＝1.7Hz,1.9H,H-1-α),5.92–5.84(m,2.9H,NHCOCH 2 CH 2 CHCH 2 ),5.77(d,J＝1.7Hz,1.0H,H-1-β),5.14–5.06(m,2.9H,NHCOCH 2 CH 2 CHCH 2 ),5.03–4.98(m,2.9H,NHCOCH 2 CH 2 CHCH 2 ),4.56(dd,J＝4.4,1.6Hz,1.0H,H-2-β),4.32(dd,J＝4.8,1.7Hz,1H,H-2-α),3.97(dd,J＝9.2,4.8Hz,1.9H,H-3-α),3.85(d,J＝3.3Hz,2.0H,H-6-β),3.81–3.79(m,3.8H,H-6-α),3.76(dd,J＝9.6,4.5Hz,1.0H,H-3-β),3.70–3.61(m,3.8H,H-4-α+H-5-α),3.58(t,J＝9.6Hz,1.0H,H-4-β),3.37(dt,J＝9.7,3.3Hz,1.0H,H-5-β),2.43–2.37(m,11.6H,NHCOCH 2 CH 2 CHCH 2 ),2.13(s,5.7H,COCH 3 -α),2.05(s,3.0H,β-COCH 3 -β). 13 C NMR(126MHz,MeOD)δ175.43(NHCOCH 2 CH 2 CHCH 2 -β),174.81(NHCOCH 2 CH 2 CHCH 2 -α),169.19(COCH 3 -α),169.09(COCH 3 -β),137.10(NHCOCH 2 CH 2 CHCH 2 -β),136.97(NHCOCH 2 CH 2 CHCH 2 -α),114.37(NHCOCH 2 CH 2 CHCH 2 ),114.24(NHCOCH 2 CH 2 CHCH 2 ),92.59(C-1-α),91.98(C-1-β),77.79(C-5-β),74.99(C-5-α),72.16(C-3-β),69.02(C-3-α),66.46(C-4-α),66.37(C-4-β),60.66(C-6-α),60.36(C-6-β),52.05(C-2-β),51.86(C-2-α),34.93(NHCOCH 2 CH 2 CHCH 2 -β),34.73(NHCOCH 2 CH 2 CHCH 2 -α),29.61(NHCOCH 2 CH 2 CHCH 2 -β),29.47(NHCOCH 2 CH 2 CHCH 2 -α),19.41(COCH 3 -β),19.40(COCH 3 - α). The compound spectrum is characterized as follows, C 13 H 22 NO 7 [M+H] + The theoretical value of high-resolution mass spectrometry (ESI) was 304.13963 and the detection value was 304.13885.
10.2 Synthesis of 1-O-Ac-ManNPtl-6-bis- (tBu-SATE) -P.
124 mg of 1-O-Ac-ManNPtl was dissolved in an appropriate volume of anhydrous tetrahydrofuran (to a concentration of 0.08 mol/l), three equivalents of tBu-SATE phosphoramidite were added to the solution under nitrogen (synthesis procedure referred to the general procedure for SATE phosphoramidite synthesis from mercaptoethanol described above), four equivalents of 1-H-tetrazine (dissolved in anhydrous acetonitrile at a concentration of about 0.45 mol/l) were then added by syringe, and the reaction was allowed to proceed overnight at zero, with TLC showing completion. At zero degree, 70% aqueous solution of tert-butanol hydrogen peroxide (five equivalents of tert-butanol hydrogen peroxide) was added, the reaction system was stirred for 2 hours, the trivalent phosphorus product was oxidized to give a pentavalent phosphorus product, after vacuum concentration, purification was performed using a silica gel column, methylene chloride and methanol were used as eluents, and 178 mg of 1-O-Ac-ManNPtl-6-bis- (tBu-SATE) -P product was obtained, the yield was 65% in two consecutive steps, as a mixture of anomers, and the ratio of the a-configuration and b-configuration products was 2.1/1. The nuclear magnetism of the compounds is characterized as follows (signals corresponding to hydrogen protons are shown in italics for assignment), 1 H NMR(500MHz,DMSO-d 6 )δ7.77(d,J＝7.9Hz,2.1H,CONH-α),7.51(d,J＝9.7Hz,1.0H,CONH-β),5.87–5.77(m,3.1H,NHCOCH 2 CH 2 CHCH 2 ),5.76(d,J＝1.9Hz,2.1H,H-1-α),5.66(d,J＝1.8Hz,1.0H,H-1-β),5.28(d,J＝5.7Hz,2.1H,OH-4-α),5.24(d,J＝5.4Hz,1.0H,OH-4-β),5.11(d,J＝4.9Hz,2.1H,OH-3-α),5.08–5.00(m,4.2H,NHCOCH 2 CH 2 CHCH 2 +OH-3-β),4.95(dt,J＝10.0,1.7Hz,3.1H,NHCOCH 2 CH 2 CHCH 2 ),4.40(ddd,J＝9.8,4.5,1.8Hz,1.0H,H-2-β),4.28(ddd,J＝11.3,6.0,1.8Hz,1.0H,H-6a-β),4.23(ddd,J＝11.3,6.2,1.8Hz,2.1H,H-6a-α),4.18–3.99(m,5.2H,H-6b-α+
H-6b-β+H-2-α),4.04–3.99(m,12.4H,2×C(O)SCH 2 CH 2 ),3.78(dt,J＝9.5,4.8Hz,1.0H,H-3-α),3.67(ddd,J＝8.9,6.8,1.9Hz,2.1H,H-5-α),3.61(dt,J＝9.4,4.8Hz,1.0H,H-3-β),3.58–3.48(m,3.1H,H-4-α+H-5-β),3.39(td,J＝9.2,5.5Hz,1.0H,H-4-β),3.11(m,12.4H,2×C(O)SCH 2 CH 2 ),2.26(m,12.4H,NHCOCH 2 CH 2 CHCH 2 ),2.08(s,6.3H,COCH 3 -α),1.96(s,3H,COCH 3 -β),1.19(m,55.8H,2×SC(O)C(CH 3 ) 3 ). 13 C NMR(126MHz,DMSO-d 6 )δ205.49(SC(O)C(CH 3 ) 3 ),205.47(SC(O)C(CH 3 ) 3 ),172.94(CONH-β),172.73(CONH-α),169.08(CH 3 CO-a),168.82(CH 3 CO-β),138.31(NHCOCH 2 CH 2 CCH-β),138.20(NHCOCH 2 CH 2 CCH-α),115.31(NHCOCH 2 CH 2 CCH-α),115.20(NHCOCH 2 CH 2 CCH-β),92.61(C-1-β),92.39(C-1-α),76.61(C-5-β),76.56(C-5-β),73.99(C-5-α),73.94(C-5-α),71.04(C-3-β),68.51(C-3-α),67.93(C-6-β),67.89(C-6-β),67.60(C-6-α),67.56(C-6-α),66.61(C-4-β),66.41(C-4-α),66.14(C(O)SCH 2 CH 2 ),66.10(C(O)SCH 2 CH 2 ),66.07(C(O)SCH 2 CH 2 ),66.03(C(O)SCH 2 CH 2 ),66.01(C(O)SCH 2 CH 2 ),65.97(C(O)SCH 2 CH 2 ),65.93(C(O)SCH 2 CH 2 ),51.92(C-2-α),51.18(C-2-β),46.45(SC(O)C(CH 3 ) 3 ),34.90(NHCOCH 2 CH 2 -β),34.63(NHCOCH 2 CH 2 -α),29.89(NHCOCH 2 CH 2 -β),29.70(NHCOCH 2 CH 2 -a),28.63(C(O)SCH 2 CH 2 ),28.61(C(O)SCH 2 CH 2 ),28.57(C(O)SCH 2 CH 2 ),28.55(C(O)SCH 2 CH 2 ),27.34(SC(O)C(CH 3 ) 3 ),21.18(CH 3 CO-a),21.09(CH 3 CO-β). 31 P NMR(202MHz,DMSO-d 6 ) Delta-1.84, -1.86. The compound spectrum is characterized as follows, C 27 H 47 NO 12 PS 2 [M+H] + The theoretical value of high-resolution mass spectrometry (ESI) was 672.22773 and the detection value was 672.22703.
EXAMPLE 11 Synthesis of 1-O-Pr-ManNAz-6-ProTide-P
102 mg of 1-O-Pr-ManNAz (0.32 mmol) was dissolved in 1 ml of anhydrous tetrahydrofuran, and the above solution was put into a dry one-neck flask, and a proper amount of activated was addedMolecular sieves, nitrogen under protection, were stirred at room temperature for 10 minutes and the reaction was cooled to 0 ℃ with an ice-water bath. To the above solution was added 385. Mu.l of a 1 molar solution (0.38 mmol) of t-butylmagnesium chloride in tetrahydrofuran at a concentration of each liter, and then the ice-water bath was removed, and the reaction was stirred at room temperature for an additional hour. 185 mg of Compound 1 (reported compound, methods of synthesis see J.org.chem.2011,76, 8311-8319.) were dissolved in 1 ml of anhydrous tetrahydrofuran, and the solution of Compound 1 was slowly added to the one-neck flask by syringe and the mixture was stirred at room temperature for a further 24 hours. Subsequently, 1 ml of methanol was added to the reaction system, the reaction was quenched, and the target compound was purified by using a silica gel column, and petroleum ether ethyl acetate was used as an eluent to obtain 44 mg of the product 1-O-Pr-Mannaz-6-ProTide-P (abbreviated as PMPT) in 22% yield. 1 H NMR(600MHz,Methanol-D 4 )δ7.36(dt,2.0H,J＝7.8,0.5Hz OC 6 H 5 -a),7.24-7.17(m,3.0H,OC 6 H 5 -b+OC 6 H 5 -c),6.01(d,J＝1.7Hz,1.0H,H-1-α),4.38(ddd,J＝11.3,6.6,1.8Hz,1.0H，H-6a),4.32(dd,J＝4.8Hz,1.8Hz,H-2),4.26(ddd,J＝11.3,7.5,1.7Hz,1.0H，H-6b),4.13(q,J＝7.1Hz,2.0H,OCH 2 CH 3 ),4.00(dd,J＝9.6Hz,4.8H,H-3),3.94(q,J＝14.16Hz,2.0H,COCH 2 N 3 ),3.87(dt,J＝8.9,6.2Hz,1.0H,NHCH(CO)CH 2 ),3.81(ddd,J＝9.9,5.8,1.6Hz,1.0H,H-5),3.62(t,J＝9.8Hz,1.0H,H-4),2.43(dq,J＝7.6,1.9Hz,2.0H,OCOCH 2 CH 3 ),1.78-1.71(m,1.0H,CH 2 CH(CH 3 ) 2 ),1.57-1.51(m,2.0H,CH 2 CH(CH 3 ) 2 ),1.25(t,J＝7.1Hz,3.0H,OCH 2 CH 3 ),1.15(t,J＝7.5Hz,3.0H,OCOCH 2 CH 3 ),0.93(d,J＝6.7Hz,2.0H,CH 2 CH(CH 3 ) 2 ),0.89(d,J＝6.6Hz,2.0H,CH 2 CH(CH 3 ) 2 ). 13 C NMR(400MHz,Methanol-d 4 )δ175.33(CHC(O)OCH 2 CH 3 ),173.57(CONH),170.98(OCOCH 2 CH 3 ),153.32(C-Aryl),152.25(C-Aryl),130.71(C-Aryl),125.96(C-Aryl),121.30(C-Aryl),121.25(C-Aryl),93.18(C-1),74.93(C-5),74.87(C-5),70.01(C-3),67.78(C-4),67.62(C-6),67.57(C-6),62.30(OCH 2 CH 3 ),54.56(C-2),53.46(NHCOCH 2 N 3 ),52.45(NHCHCH 2 ),44.03(NHCHCH 2 ),28.20(OCOCH 2 CH 3 ),25.53(CH(CH 3 ) 2 ),23.13(CH(CH 3 ) 2 ),22.03(CH(CH 3 ) 2 ),14.46(OCH 2 CH 3 ),9.21(OCOCH 2 CH 3 ). 31 P NMR(600MHz,Methanol-D 4 )δ3.87
HRMS (ESI) theoretical molecular weight C 25 H 38 N 5 O 11 P[M+H] + 616.23837, molecular weight 616.23864.
Experimental example 1 unnatural phosphate sugar can realize high-efficiency and rapid metabolism marking at the cellular level
First, we tested the performance of SATE-protected ManNAz-6-P in markers of cellular glycan metabolism.
Using different non-natural sugars (ManNAz, siaNAz, 1,6-Pr 2 After culturing MCF-7 cells for 48 hours by ManNAz, AMMS, biotin was covalently introduced onto the azidose on the cell surface using click chemistry, and the cells were further incubated with AlexaFluor 647 (hereafter abbreviated as AF 647) coupled streptavidin (streptavidin specifically recognizes and binds biotin) and AF 647 fluorescence was labeled on the cell surface. As shown by the results of the confocal laser fluorescence microscope in FIG. 1A, the non-natural phosphate sugar AMMS can efficiently introduce azide groups on the cell surface, and the AMMS-mediated labeling intensity at the saturation labeling concentration is not lower than that of the reported non-natural sugar (2 mM ManNAz, 2 mM SiaNAz or 200 mM 1,6 Pr 2 ManNAz). Cell surface from flow cytometerAverage fluorescence intensity results (fig. 1B,MFI,Mean Fluorescence Intensity, average fluorescence intensity) and representative flow transition diagrams (fig. 1C), similar conclusions can be drawn: AMMS can introduce azide groups on the cell surface with high efficiency, which indicates that AMMS can realize high-efficiency metabolic labeling.
Subsequently, we systematically evaluated the efficiency of metabolic labeling of SATE-protected ManNAz-6-P derivatives AMES (FIGS. 2A, 2B and 2C), AMBS (FIGS. 3A, 3B and 3C), AMtBS (FIGS. 4A, 4B and 4C), and AMHS (FIGS. 5A, 5B and 5C) in MCF-7 cells using confocal laser fluorescence microscopy and flow cytometry, by and with the unnatural sugars ManNAz, siaNAz and 1,6-Pr that have been reported 2 When compared with ManNAz, the novel non-natural phosphate sugars can be used for achieving a high-efficiency labeling effect after being incubated in cells for 48 hours. Meanwhile, the ManNAz-6-P derivatives protected by SATE are simultaneously compared, and the fact that the compounds have good marking effect and have different saturation marking concentrations is confirmed by using a laser confocal fluorescence microscope (figure 6), so that the influence of the length of an alkyl chain on the SATE protecting group on the marking working concentration of the SATE protecting group is demonstrated.
The 3 and 4 hydroxyl groups of the SATE protected ManNAz-6-P derivative are subjected to acylation modification to further increase the lipophilicity of the molecule. As a representative example, we have simultaneously acetyl modified AMMS at positions 3 and 4 to give the compound TAMMS. Using different concentrations of TAMMS, 2 mmoles per liter of ManNAz or 100 micromoles per liter of 1,6-Pr 2 MCF-7 cells were cultured for 48 hours using ManNAz and AF647 was introduced on the cell surface using the click chemistry method described above, and the results with a laser confocal microscope showed that 25. Mu. Mol/L TAMMS (shown in FIG. 7) enabled 1,6-Pr per liter of 2 mM ManNAz or 100. Mu. Mol/L 2 A similar labelling effect of ManNAz indicates that ManNAz-6-P derivatives with 3 and 4 hydroxy acyl groups and phosphate groups protected by SATE can also achieve efficient glycan metabolism labelling. It is expected that the 3-and 4-hydroxyl groups of the above compounds (AMMS, AMES, AMBS, AMtBS and AMHS) are protected by other acyl groups (other than acetyl groups) and also achieve efficient labeling because the purpose of the acyl group introduction is to increaseThe lipophilicity of the molecule and the acyl groups are well removed from the cell.
POM is another phosphate protecting group in addition to SATE. As an example, we examined the efficiency of metabolic labelling of PMPOM in HeLa cells. As shown in FIG. 8, heLa cells utilized PMPOM or 1,6-Pr at various concentrations 2 After 48 hours of ManNAz (200. Mu. Mol/L) incubation, AF647 was introduced onto the cell surface using the click chemistry method described above, and the results with a laser confocal microscope showed that 10. Mu. Mol/L PMPOM was capable of achieving 200. Mu. Mol/L1, 6-Pr 2 A similar labeling effect of ManNAz suggests that POM-protected ManNAz-6-P also enables efficient metabolic labeling.
We also examined the efficiency of metabolic labelling of PMPT in MCF-7 cells. As shown in FIG. 9, MCF-7 cells utilized the unnatural sugar PMPT or 1,6-Pr 2 After 48 hours of ManNAz incubation, biotin was covalently introduced onto the cell surface azido glycans using click chemistry, and cells were incubated with Alexa Fluor 647 modified streptavidin for AF 647 fluorescence on the cell surface markers. Fluorescence from the cell surface was analyzed using a laser confocal microscope (as shown in fig. 9).
In addition to laser confocal fluorescence microscopy imaging and flow cytometry studies, we also consider AMMS as representative of unnatural phosphate sugar, the case of ManNAz-6-P derivative labeled glycoprotein was studied. After incubation of the unnatural saccharide with the various cells 48, the cells were lysed, the azide-containing glycoprotein and the alkynyl-modified Cy5 fluorescent dye were click-chemically reacted, and the proteins were separated by SDS-PAGE and the in-gel fluorescence scanned. In a variety of cell lines (as in FIG. 10), including HEK 293T, MDA-MB-231, A549, hepG2, NIH 3T3, CHO and COS-7, the passage and 200 micromoles per liter of 1,6-Pr 2 ManNAz, or 2 millimoles per liter of SiaNAz (48 hours incubation time for all unnatural sugars), found that 500 micromoles per liter of AMMS enabled similar or stronger labeling to other unnatural sugars that have been reported, indicating that ManNAz-6-P derivatives enabled efficient glycoprotein metabolism labeling.
At the cellular level, apart from the cell line, we found that ManNAz-6-P derivatives were alsoCan perform high-efficiency metabolic labeling on primary cultured cells. In isolated mouse primary neurons, we take AMMS as an example, and after 48 hours of incubation, we found that 50. Mu. Moles per liter of AMMS can achieve a ratio of 1,6-Pr per liter of 200. Mu. Moles 2 ManNAz or 2 millimoles per liter of SiaNAz. AMMS implementation ratio 1,6-Pr in cell surface fluorescence imaging experiments 2 ManNAz, higher efficiency labeling (as shown in FIGS. 11A and 11B).
At the cellular level, we have confirmed that non-natural phosphate sugars can achieve high efficiency metabolic markers. We further examined whether the unnatural phosphate sugar has an advantage over the already reported unnatural sugars in terms of cell labelling rate. We selected SATE protected phosphoglycose as representative of ManNAz-6-P derivatives, comparing four unnatural sugars 1,6-Pr 2 The label intensities of ManNAz (200 micromole per liter), AMMS (200 micromole per liter), manNAz (2 millimoles per liter) and SiaNAz (2 millimoles per liter) after different incubation times, as can be seen from the laser confocal fluorescence imaging diagram of fig. 8, AMMS have the strongest cell surface fluorescence signal at each time point (2, 4, 6, 8 hours of incubation, respectively). After a short incubation time of 4 hours, a significant marker signal was introduced on the surface of MCF-7 cells (FIG. 12).
Further, as shown in FIGS. 13A and 13B, we quantitatively studied the average fluorescence intensities of the cell surfaces after MCF-7 cells were cultured with AMMS (250. Mu. Mol/liter), AMES (250. Mu. Mol/liter) or AMtBS (250. Mu. Mol/liter) for various times by using a flow cytometer, found that the labeling intensities of the cell surfaces gradually increased and tended to be smooth in the range from 2 hours to 48 hours, and that the labeling intensities of three unnatural phosphate sugars were close at the time nodes of the respective tests described above. The above results demonstrate that ManNAz-6-P can achieve rapid labeling of glycan metabolism at the cellular level.
Experimental example 2 labelling of unnatural Phosphorose on various cultured living tissue sections
After confirming that the non-natural phosphoglycoses can realize high-efficiency and rapid metabolic markers at the cellular level, we further studied the living groups of the non-natural phosphoglycoses in various cultures Labeling on the woven section. As shown in fig. 14A to 14C, after the hippocampal section of the mouse and the different unnatural saccharides were cultured for 24 hours, the sections were lysed, and the azide-containing glycoprotein and the alkynyl-modified Cy5 fluorescent dye were subjected to click chemistry, and proteins were separated by SDS-PAGE, and the intra-gel fluorescence was scanned (fig. 14B). It was found that the strength of the glycoprotein label gradually increased with increasing AMMS concentration and reached saturation at a concentration of 400. Mu. Mol per liter, and that AMMS was marked much more strongly than 1,6-Pr 2 ManNAz (as shown in FIGS. 14A-14C). The results of tissue slice imaging of the hippocampus of the mouse brain also showed (FIG. 14C), AMMS-mediated markers were also much stronger than ManNAz, 1,6-Pr 2 ManNAz and SiaNAz. In time gradient experiments, mouse brain hippocampal sections were incubated with AMMS for different times to label cell surface glycans in tissues, and the intensity of the labels was found to increase gradually with time. Imaging results show that obvious labeling signals can be introduced on the surface of the sectioned cells after one hour of culture. In addition to the hippocampal region, other brain regions such as striatum, hypothalamus, neocortex, hippocampus, and midbrain can be labeled with AMMS with high efficiency and rapidity (shown in FIG. 15).
In addition to tissue sections of mouse origin, tissues from humans can also be labeled efficiently and rapidly using the unnatural sugar phosphate sugar AMMS. Normal brain sections of humans using AMMS, siaNAz and 1,6-Pr 2 After 24 hours of ManNAz incubation, a clear label signal was observed only in AMMS treated groups. By simultaneously culturing normal brain tissue and neuroma sections using AMMS, a stronger marker signal was observed in tumor tissue (shown in fig. 16A to 16C).
In addition to SATE protected ManNAz-6-P, we also tested the labeling of AOM protected ManNAz-6-P derivative PMPOM in sections. The hippocampus of the brain of the mice was incubated with 25. Mu. Moles per liter of PPOMM for 24 hours, and obvious markers were introduced on the cell surface (FIG. 17).
Compounds such as AMMS, AMES, AMtBS, AMBS, TAMMS, AMPOM, PMPOM, PMPT in the experimental examples of the present invention were prepared from the above examples.
It should be noted that, each component or step in each embodiment may be intersected, replaced, added, and deleted, and therefore, the combination formed by these reasonable permutation and combination transformations shall also belong to the protection scope of the present invention, and shall not limit the protection scope of the present invention to the embodiments.
The foregoing is an exemplary embodiment of the present disclosure, and the order in which the embodiments of the present disclosure are disclosed is merely for the purpose of description and does not represent the advantages or disadvantages of the embodiments. It should be noted that the above discussion of any of the embodiments is merely exemplary and is not intended to suggest that the scope of the disclosure of embodiments of the invention (including the claims) is limited to these examples and that various changes and modifications may be made without departing from the scope of the invention as defined in the claims. The functions, steps and/or actions of the method claims in accordance with the disclosed embodiments described herein need not be performed in any particular order. Furthermore, although elements of the disclosed embodiments may be described or claimed in the singular, the plural is contemplated unless limitation to the singular is explicitly stated.
Those of ordinary skill in the art will appreciate that: the above discussion of any embodiment is merely exemplary and is not intended to imply that the scope of the disclosure of embodiments of the invention, including the claims, is limited to such examples; combinations of features of the above embodiments or in different embodiments are also possible within the idea of an embodiment of the invention, and there are many other variations of the different aspects of the embodiments of the invention as described above, which are not provided in detail for the sake of brevity. Therefore, any omissions, modifications, equivalent substitutions, improvements, and the like, which are made within the spirit and principles of the embodiments of the invention, are included within the scope of the embodiments of the invention.
Claims (15)
1. A non-natural phosphate sugar, which is characterized in that the non-natural phosphate sugar is hexacarbon sugar and has a pyranose or furanose structure, the non-natural phosphate sugar contains a bio-orthogonal group, and six positions of the non-natural phosphate sugar are modified by phosphoric acid with a protecting group.
2. The non-natural phosphate sugar of claim 1, wherein the protecting group is a protecting group that is cleavable or hydrolyzable, including any one or more of SATE, AOM, proTide, AB or CycloSal groups.
3. The non-natural phosphate sugar of claim 1, wherein the orthogonal groups on the non-natural phosphate sugar comprise any one or more of azide, alkyne, alkene, cyclopropene, trans-cyclooctene, cyclooctyne, and tetrazine.
4. The non-natural phosphoric acid sugar according to claim 1, wherein the sugar type of pyranose is any one selected from the group consisting of amino mannose, glucosamine and galactosamine.
5. The non-natural phosphate sugar of claim 4, wherein the mannose has a structure of either ManNAz, manNAl or mannttl.
6. The non-natural phosphoric acid saccharide according to any one of claims 1 to 5, wherein the non-natural phosphoric acid has the structure of formula I:
Wherein the R is 1 An acyl group having 1 to 12 carbon atoms;
the R is 2 、R 5 Is a phosphate protecting group independently selected from: SATE, AOM, proTide, AB or a CycloSal group;
the R is 3 、R 4 Independently selected from: h or acyl of C1-C12;
x is azide, alkenyl of C2-C6 or alkynyl of C2-C6;
the Y is 1 Or Y 2 Independently selected from-O-or-NH-;
the R is 8 、R 9 、R 10 、R 11 Or R is 12 Independently selected from: h or C1-C6 alkyl.
7. The non-natural phosphate sugar of claim 6, wherein R 1 Is an acyl group containing C1 to C6;
the SATE group structure is as follows;
the R is 6 Is a C1-C20 alkyl chain or a C6-C20 aryl group;
the AOM group has the structure:wherein R is 7 Is a C1-C20 alkyl chain or a C6-C20 aryl group;
when the phosphate protecting group is a ProTide group, R 2 Or R is 5 Any substituent beingStructure, at the same time, the other substituent is +.>Wherein said R is 13 、R 14 、R 15 、R 16 、R 17 、R 18 Or R is 19 Independently selected from: h or C1-C6 alkyl;
the structure of the AB group is as follows:wherein said R is 20 、R 21 、R 22 、R 23 Or R is 24 Independently selected from: h or C1-C6 alkyl.
8. The non-natural phosphate sugar of claim 6, wherein the non-natural phosphate sugar isThe natural phosphoric acid sugar comprises any one of the following components: 1-O-Ac-ManNAz-6-bis- (Me-SATE) -P, 1-O-Ac-ManNAz-6-bis- (Et-SATE) -P, 1-O-Ac-ManNAz-6-bis- (Bu-SATE) -P, 1-O-Ac-ManNAz-6-bis- (tBu-SATE) -P, 1-O-Ac-ManNAz-6-bis- (He-SATE) -P, 1-O-Ac-ManNAl-6-bis- (tBu-SATE) -P, 1-O-Ac-ManNPtl-6-bis- (tBu-SATE) -P, ac 3 ManNAz-6-bis- (Me-SATE) -P, 1-O-Ac-ManNAz-6-bis-POM-P, 1-O-Pr-ManNAz-6-bis-POM-P or 1-O-Pr-ManNAz-6-ProTide-P.
9. A method of preparing a non-natural phosphoric acid saccharide according to any one of claims 1 to 8, comprising: protecting the first position on the pyranose or furanose ring, leaving the third position and the fourth position unprotected, and selectively introducing a phosphate group with a protecting group at the sixth position to obtain non-natural phosphate sugar;
or protecting the first position of the pyranose or furanose, protecting the third position and the fourth position of the pyranose or furanose, introducing a phosphate group with a protecting group at the sixth position, and finally removing the protecting groups at the third position and the fourth position of the saccharide to obtain the non-natural phosphate sugar.
10. The method for preparing non-natural phosphoric acid sugar according to claim 9, further comprising: further acylation modification of the hydroxyl group of the non-natural phosphate sugar.
11. The process according to claim 9, wherein the protecting group is a SATE or AOM group,
wherein the SATE group structure is as follows;
the R is 6 Is a C1-C20 alkyl chain or a C6-C20 aryl group;
the AOM has the structure that:wherein R is 7 Is a C1-C20 alkyl chain or a C6-C20 aryl group.
12. A kit for labeling sugar metabolism, characterized in that it comprises the non-natural phosphoric acid sugar according to any one of claims 1 to 8 or the non-natural phosphoric acid sugar prepared by the method according to any one of claims 9 to 11.
13. Use of the non-natural phosphate sugar according to any one of claims 1 to 8 or the non-natural phosphate sugar prepared by the method according to any one of claims 9 to 11, or the kit according to claim 12, in a marker for sugar metabolism, said use being for non-diagnostic or therapeutic purposes.
14. The use according to claim 13, characterized in that it comprises the labelling of the glycometabolism of any of the following cell lines or primary cells: heLa cells, MCF-7 cells, HEK 293T cells, MDA-MB-231 cells, A549 cells, hepG2 cells, NIH 3T3 cells, CHO cells, COS-7 cells, or primary neuronal cells.
15. The use of claim 14, wherein the use comprises a marker of carbohydrate metabolism on a mammalian organ or tissue section.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311188091.1A CN117417395A (en) | 2023-09-15 | 2023-09-15 | Non-natural phosphate sugar, preparation method thereof, sugar metabolism marking kit and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311188091.1A CN117417395A (en) | 2023-09-15 | 2023-09-15 | Non-natural phosphate sugar, preparation method thereof, sugar metabolism marking kit and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117417395A true CN117417395A (en) | 2024-01-19 |
Family
ID=89525413
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311188091.1A Pending CN117417395A (en) | 2023-09-15 | 2023-09-15 | Non-natural phosphate sugar, preparation method thereof, sugar metabolism marking kit and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117417395A (en) |
-
2023
- 2023-09-15 CN CN202311188091.1A patent/CN117417395A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Imoto et al. | Total synthesis of Escherichia coli lipid A, the endotoxically active principle of cell-surface lipopolysaccharide. | |
| Kondo et al. | Glycosyl phosphites as glycosylation reagents: scope and mechanism | |
| Codée et al. | Automated solid‐phase synthesis of protected oligosaccharides containing β‐mannosidic linkages | |
| JP2518739B2 (en) | Tumor suppressive saccharide conjugate | |
| Liu et al. | A divergent synthesis of lipid A and its chemically stable unnatural analogues. | |
| Yano et al. | Total synthesis of Myc-IV (C16: 0, S) via automated electrochemical assembly | |
| Ando et al. | Proceedings in synthetic chemistry of sialo-glycoside | |
| Nikrad et al. | Use of human-milk fucosyltransferase in the chemoenzymic synthesis of analogues of the sialyl Lewis and sialyl Lewis tetrasaccharides modified at the C-2 position of the reducing unit | |
| CN117417395A (en) | Non-natural phosphate sugar, preparation method thereof, sugar metabolism marking kit and application thereof | |
| Dinev et al. | A convenient gram-scale synthesis of uridine diphospho (13C6) glucose | |
| CN112375108B (en) | Method for selectively synthesizing 1, 2-cis-glycoside compound | |
| Vermeer et al. | Synthesis and conjugation of a sulfated disaccharide involved in the aggregation process of the marine sponge Microciona prolifera | |
| Khaled et al. | Exploring specificity of glycosyltransferases: synthesis of new sugar nucleotide related molecules as putative donor substrates | |
| JP4599295B2 (en) | α-selective glycosylation reaction method | |
| Sherman et al. | Preparative route to N-glycolylneuraminic acid phenyl 2-thioglycoside donor and synthesis of Neu5Gc-α-(2→ 3′)-lactosamine 3-aminopropyl glycoside | |
| Kurimoto et al. | Chemical approach for the syntheses of GM4 isomers with sialic acid to non-natural linkage positions on galactose | |
| van Dorst et al. | Synthesis of Hexp-(1→ 4)-β-d-GlcpNac-(1→ 2)-α-d-Manp-(1→ O)(CH2) 7CH3 probes for exploration of the substrate specificity of glycosyltransferases: Part II, Hex= 3-O-methyl-β-d-Gal, 3-deoxy-β-d-Gal, 3-deoxy-3-fluoro-β-d-Gal, 3-amino-3-deoxy-β-d-Gal, β-d-Gul, α-l-Alt, or β-l-Gal | |
| Arif et al. | Synthesis of N-acetylmannosamine-6-phosphate derivatives to investigate the mechanism of N-acetylmannosamine-6-phosphate 2-epimerase | |
| WO2024087806A1 (en) | Non-natural sugar, and synthesis method therefor and use thereof | |
| Cheng et al. | Synthesis of a fluorescence-labeled K30 antigen repeating unit using click chemistry | |
| CN111208284A (en) | Sugar metabolism labeling probe, kit containing sugar metabolism labeling probe and application of sugar metabolism labeling probe | |
| KR102164196B1 (en) | Novel steroid -based amphiphiles and uses thereof | |
| EP3057975B1 (en) | N-glycosylation of peptides and proteins | |
| JP4890805B2 (en) | O-linked sugar amino acid derivative having core 3 type structure and method for producing the same | |
| EP3170832A1 (en) | Method of preparation of 6-azido-2,4-diacetamido-2,4,6-trideoxy-d-mannose |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |