WO2012121041A1 - シアル酸含有糖鎖の製造方法 - Google Patents
シアル酸含有糖鎖の製造方法 Download PDFInfo
- Publication number
- WO2012121041A1 WO2012121041A1 PCT/JP2012/054727 JP2012054727W WO2012121041A1 WO 2012121041 A1 WO2012121041 A1 WO 2012121041A1 JP 2012054727 W JP2012054727 W JP 2012054727W WO 2012121041 A1 WO2012121041 A1 WO 2012121041A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sugar chain
- sialic acid
- derivative
- sugar
- cmp
- Prior art date
Links
- 235000000346 sugar Nutrition 0.000 title claims abstract description 319
- 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 title claims abstract description 166
- 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 title claims abstract description 149
- 238000004519 manufacturing process Methods 0.000 title claims description 27
- 150000001875 compounds Chemical class 0.000 claims abstract description 54
- 102000003838 Sialyltransferases Human genes 0.000 claims abstract description 52
- 108090000141 Sialyltransferases Proteins 0.000 claims abstract description 52
- 235000021310 complex sugar Nutrition 0.000 claims abstract description 24
- TXCIAUNLDRJGJZ-BILDWYJOSA-N CMP-N-acetyl-beta-neuraminic acid Chemical compound O1[C@@H]([C@H](O)[C@H](O)CO)[C@H](NC(=O)C)[C@@H](O)C[C@]1(C(O)=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(N=C(N)C=C2)=O)O1 TXCIAUNLDRJGJZ-BILDWYJOSA-N 0.000 claims description 45
- TXCIAUNLDRJGJZ-UHFFFAOYSA-N CMP-N-acetyl neuraminic acid Natural products O1C(C(O)C(O)CO)C(NC(=O)C)C(O)CC1(C(O)=O)OP(O)(=O)OCC1C(O)C(O)C(N2C(N=C(N)C=C2)=O)O1 TXCIAUNLDRJGJZ-UHFFFAOYSA-N 0.000 claims description 44
- 108010064886 beta-D-galactoside alpha 2-6-sialyltransferase Proteins 0.000 claims description 29
- 102100029945 Beta-galactoside alpha-2,6-sialyltransferase 1 Human genes 0.000 claims description 27
- 239000000758 substrate Substances 0.000 claims description 25
- 238000000034 method Methods 0.000 claims description 23
- 241000282414 Homo sapiens Species 0.000 claims description 17
- 102000051366 Glycosyltransferases Human genes 0.000 claims description 16
- 108700023372 Glycosyltransferases Proteins 0.000 claims description 16
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 claims description 15
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 claims description 15
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 14
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 14
- -1 UDP saccharide Chemical class 0.000 claims description 12
- 230000015572 biosynthetic process Effects 0.000 claims description 9
- 241000588724 Escherichia coli Species 0.000 claims description 8
- 102100037982 Alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase A Human genes 0.000 claims description 6
- 101000951392 Homo sapiens Alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase A Proteins 0.000 claims description 6
- HSCJRCZFDFQWRP-ABVWGUQPSA-N UDP-alpha-D-galactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-ABVWGUQPSA-N 0.000 claims description 6
- HSCJRCZFDFQWRP-UHFFFAOYSA-N Uridindiphosphoglukose Natural products OC1C(O)C(O)C(CO)OC1OP(O)(=O)OP(O)(=O)OCC1C(O)C(O)C(N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-UHFFFAOYSA-N 0.000 claims description 6
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 claims description 5
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 claims description 5
- LFTYTUAZOPRMMI-CFRASDGPSA-N UDP-N-acetyl-alpha-D-glucosamine Chemical compound O1[C@H](CO)[C@@H](O)[C@H](O)[C@@H](NC(=O)C)[C@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 LFTYTUAZOPRMMI-CFRASDGPSA-N 0.000 claims description 5
- LFTYTUAZOPRMMI-UHFFFAOYSA-N UNPD164450 Natural products O1C(CO)C(O)C(O)C(NC(=O)C)C1OP(O)(=O)OP(O)(=O)OCC1C(O)C(O)C(N2C(NC(=O)C=C2)=O)O1 LFTYTUAZOPRMMI-UHFFFAOYSA-N 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 abstract description 88
- 102000003886 Glycoproteins Human genes 0.000 abstract description 29
- 108090000288 Glycoproteins Proteins 0.000 abstract description 29
- 239000007795 chemical reaction product Substances 0.000 abstract description 17
- 230000000694 effects Effects 0.000 abstract description 17
- 238000005580 one pot reaction Methods 0.000 abstract description 16
- 239000003814 drug Substances 0.000 abstract description 8
- 238000000746 purification Methods 0.000 abstract description 3
- 230000002194 synthesizing effect Effects 0.000 abstract description 3
- 239000007858 starting material Substances 0.000 abstract description 2
- 229940079593 drug Drugs 0.000 abstract 1
- 238000006911 enzymatic reaction Methods 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 description 35
- 108090000790 Enzymes Proteins 0.000 description 35
- 239000000243 solution Substances 0.000 description 32
- IERHLVCPSMICTF-XVFCMESISA-N cytidine 5'-monophosphate Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(O)=O)O1 IERHLVCPSMICTF-XVFCMESISA-N 0.000 description 26
- IERHLVCPSMICTF-UHFFFAOYSA-N cytidine monophosphate Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(COP(O)(O)=O)O1 IERHLVCPSMICTF-UHFFFAOYSA-N 0.000 description 25
- 102000004357 Transferases Human genes 0.000 description 22
- 108090000992 Transferases Proteins 0.000 description 22
- 229930182830 galactose Natural products 0.000 description 20
- 150000004676 glycans Chemical class 0.000 description 20
- 150000001413 amino acids Chemical class 0.000 description 19
- 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 description 17
- 229940024606 amino acid Drugs 0.000 description 17
- 235000001014 amino acid Nutrition 0.000 description 17
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 16
- 229950006780 n-acetylglucosamine Drugs 0.000 description 16
- 108090000623 proteins and genes Proteins 0.000 description 16
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 15
- 125000003277 amino group Chemical group 0.000 description 15
- 238000006276 transfer reaction Methods 0.000 description 15
- 238000004128 high performance liquid chromatography Methods 0.000 description 14
- 125000005629 sialic acid group Chemical group 0.000 description 14
- 238000012546 transfer Methods 0.000 description 14
- 239000000047 product Substances 0.000 description 13
- 239000002994 raw material Substances 0.000 description 12
- 108010057005 beta-galactoside alpha-2,3-sialyltransferase Proteins 0.000 description 11
- 125000006239 protecting group Chemical group 0.000 description 11
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical group CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- 235000018102 proteins Nutrition 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 238000010438 heat treatment Methods 0.000 description 9
- 150000008163 sugars Chemical class 0.000 description 9
- 239000007993 MOPS buffer Substances 0.000 description 8
- OVRNDRQMDRJTHS-CBQIKETKSA-N N-Acetyl-D-Galactosamine Chemical compound CC(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-CBQIKETKSA-N 0.000 description 8
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 description 8
- 239000002253 acid Substances 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 8
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 7
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 7
- 229940098773 bovine serum albumin Drugs 0.000 description 7
- 238000003379 elimination reaction Methods 0.000 description 7
- 229930182470 glycoside Natural products 0.000 description 7
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 6
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 229960001230 asparagine Drugs 0.000 description 6
- 235000009582 asparagine Nutrition 0.000 description 6
- 230000015556 catabolic process Effects 0.000 description 6
- 238000006731 degradation reaction Methods 0.000 description 6
- 230000008030 elimination Effects 0.000 description 6
- 101710136188 Beta-galactoside alpha-2,6-sialyltransferase 2 Proteins 0.000 description 5
- 102100029963 Beta-galactoside alpha-2,6-sialyltransferase 2 Human genes 0.000 description 5
- 102000003951 Erythropoietin Human genes 0.000 description 5
- 108090000394 Erythropoietin Proteins 0.000 description 5
- 241000282412 Homo Species 0.000 description 5
- 241000124008 Mammalia Species 0.000 description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 5
- 239000013504 Triton X-100 Substances 0.000 description 5
- 229920004890 Triton X-100 Polymers 0.000 description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 229940105423 erythropoietin Drugs 0.000 description 5
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 238000005918 transglycosylation reaction Methods 0.000 description 5
- PXBFMLJZNCDSMP-UHFFFAOYSA-N 2-Aminobenzamide Chemical compound NC(=O)C1=CC=CC=C1N PXBFMLJZNCDSMP-UHFFFAOYSA-N 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- 241000283690 Bos taurus Species 0.000 description 4
- 102100029962 CMP-N-acetylneuraminate-beta-1,4-galactoside alpha-2,3-sialyltransferase Human genes 0.000 description 4
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 4
- KFEUJDWYNGMDBV-LODBTCKLSA-N N-acetyllactosamine Chemical group O[C@@H]1[C@@H](NC(=O)C)[C@H](O)O[C@H](CO)[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 KFEUJDWYNGMDBV-LODBTCKLSA-N 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000009833 condensation Methods 0.000 description 4
- 230000005494 condensation Effects 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 4
- 230000018044 dehydration Effects 0.000 description 4
- 238000006297 dehydration reaction Methods 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- BQINXKOTJQCISL-GRCPKETISA-N keto-neuraminic acid Chemical compound OC(=O)C(=O)C[C@H](O)[C@@H](N)[C@@H](O)[C@H](O)[C@H](O)CO BQINXKOTJQCISL-GRCPKETISA-N 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 229930014626 natural product Natural products 0.000 description 4
- CERZMXAJYMMUDR-UHFFFAOYSA-N neuraminic acid Natural products NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO CERZMXAJYMMUDR-UHFFFAOYSA-N 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 3
- 101710163570 Alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3-N-acetyl-galactosaminide alpha-2,6-sialyltransferase Proteins 0.000 description 3
- 102100031972 Alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3-N-acetyl-galactosaminide alpha-2,6-sialyltransferase Human genes 0.000 description 3
- 101710183133 Alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase 1 Proteins 0.000 description 3
- 102100031969 Alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase 1 Human genes 0.000 description 3
- 102100031970 Alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase 2 Human genes 0.000 description 3
- 102100031971 Alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase 3 Human genes 0.000 description 3
- 229930186217 Glycolipid Natural products 0.000 description 3
- 102000002068 Glycopeptides Human genes 0.000 description 3
- 108010015899 Glycopeptides Proteins 0.000 description 3
- 101000863864 Homo sapiens Beta-galactoside alpha-2,6-sialyltransferase 1 Proteins 0.000 description 3
- SQVRNKJHWKZAKO-PFQGKNLYSA-N N-acetyl-beta-neuraminic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-PFQGKNLYSA-N 0.000 description 3
- FDJKUWYYUZCUJX-UHFFFAOYSA-N N-glycolyl-beta-neuraminic acid Natural products OCC(O)C(O)C1OC(O)(C(O)=O)CC(O)C1NC(=O)CO FDJKUWYYUZCUJX-UHFFFAOYSA-N 0.000 description 3
- 108010052465 Neu5Ac N-acetylgalactosamine 2,6-sialyltransferase Proteins 0.000 description 3
- 241001489174 Ogataea minuta Species 0.000 description 3
- 102000016611 Proteoglycans Human genes 0.000 description 3
- 108010067787 Proteoglycans Proteins 0.000 description 3
- 102100027107 Type 2 lactosamine alpha-2,3-sialyltransferase Human genes 0.000 description 3
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 238000000354 decomposition reaction Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 108010082530 galactosyl-1-3-N-acetylgalactosaminyl-specific 2,6-sialyltransferase Proteins 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 241000712461 unidentified influenza virus Species 0.000 description 3
- DQJCDTNMLBYVAY-ZXXIYAEKSA-N (2S,5R,10R,13R)-16-{[(2R,3S,4R,5R)-3-{[(2S,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-5-(ethylamino)-6-hydroxy-2-(hydroxymethyl)oxan-4-yl]oxy}-5-(4-aminobutyl)-10-carbamoyl-2,13-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazaheptadecan-1-oic acid Chemical compound NCCCC[C@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)CC[C@H](C(N)=O)NC(=O)[C@@H](C)NC(=O)C(C)O[C@@H]1[C@@H](NCC)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DQJCDTNMLBYVAY-ZXXIYAEKSA-N 0.000 description 2
- HOEWKBQADMRCLO-YKNQQZBYSA-N (2r,4s,5r,6r)-2-[[(2r,3s,4r)-5-(4-amino-2-oxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-4-hydroxy-5-[(2-hydroxyacetyl)amino]-6-[(1r,2r)-1,2,3-trihydroxypropyl]oxane-2-carboxylic acid Chemical compound O=C1N=C(N)C=CN1C1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)O[C@@]2(O[C@H]([C@H](NC(=O)CO)[C@@H](O)C2)[C@H](O)[C@H](O)CO)C(O)=O)O1 HOEWKBQADMRCLO-YKNQQZBYSA-N 0.000 description 2
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 2
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 2
- DIDIOPKMWHMWQM-UHFFFAOYSA-N 9-aminopyrene-1,4,6-trisulfonic acid Chemical compound OS(=O)(=O)C1=CC=C2C(N)=CC3=C(S(O)(=O)=O)C=CC4=C(S(O)(=O)=O)C=C1C2=C34 DIDIOPKMWHMWQM-UHFFFAOYSA-N 0.000 description 2
- 102000013563 Acid Phosphatase Human genes 0.000 description 2
- 108010051457 Acid Phosphatase Proteins 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 102100027098 CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 1 Human genes 0.000 description 2
- 102100031973 CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 2 Human genes 0.000 description 2
- 108010033531 CMP-N-acetylneuraminate-poly-alpha-2,8-sialosyl sialyltransferase Proteins 0.000 description 2
- 102100021786 CMP-N-acetylneuraminate-poly-alpha-2,8-sialyltransferase Human genes 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 241000238557 Decapoda Species 0.000 description 2
- 101000616698 Homo sapiens CMP-N-acetylneuraminate-poly-alpha-2,8-sialyltransferase Proteins 0.000 description 2
- 102000004856 Lectins Human genes 0.000 description 2
- 108090001090 Lectins Proteins 0.000 description 2
- OVRNDRQMDRJTHS-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-N 0.000 description 2
- SUHQNCLNRUAGOO-UHFFFAOYSA-N N-glycoloyl-neuraminic acid Natural products OCC(O)C(O)C(O)C(NC(=O)CO)C(O)CC(=O)C(O)=O SUHQNCLNRUAGOO-UHFFFAOYSA-N 0.000 description 2
- FDJKUWYYUZCUJX-KVNVFURPSA-N N-glycolylneuraminic acid Chemical compound OC[C@H](O)[C@H](O)[C@@H]1O[C@](O)(C(O)=O)C[C@H](O)[C@H]1NC(=O)CO FDJKUWYYUZCUJX-KVNVFURPSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 108010017795 Sia(alpha2,3)Gal(beta1,4)GlcNAc alpha-2,8-sialyltransferase Proteins 0.000 description 2
- 102100029227 Sia-alpha-2,3-Gal-beta-1,4-GlcNAc-R:alpha 2,8-sialyltransferase Human genes 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000256251 Spodoptera frugiperda Species 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 2
- 230000021736 acetylation Effects 0.000 description 2
- 238000006640 acetylation reaction Methods 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 238000007259 addition reaction Methods 0.000 description 2
- 229960000643 adenine Drugs 0.000 description 2
- 229960004050 aminobenzoic acid Drugs 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000007810 chemical reaction solvent Substances 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 125000000623 heterocyclic group Chemical group 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 239000002523 lectin Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 150000002772 monosaccharides Chemical class 0.000 description 2
- 229940060155 neuac Drugs 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 210000000813 small intestine Anatomy 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 125000001981 tert-butyldimethylsilyl group Chemical group [H]C([H])([H])[Si]([H])(C([H])([H])[H])[*]C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- AVBGNFCMKJOFIN-UHFFFAOYSA-N triethylammonium acetate Chemical compound CC(O)=O.CCN(CC)CC AVBGNFCMKJOFIN-UHFFFAOYSA-N 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- WMSUFWLPZLCIHP-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 9h-fluoren-9-ylmethyl carbonate Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)ON1C(=O)CCC1=O WMSUFWLPZLCIHP-UHFFFAOYSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 1
- 125000004066 1-hydroxyethyl group Chemical group [H]OC([H])([*])C([H])([H])[H] 0.000 description 1
- 125000000979 2-amino-2-oxoethyl group Chemical group [H]C([*])([H])C(=O)N([H])[H] 0.000 description 1
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 101000628802 Bos taurus Alpha-1,3-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase A Proteins 0.000 description 1
- 101710083563 CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 2 Proteins 0.000 description 1
- 102100031974 CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 4 Human genes 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 1
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
- 241000258955 Echinodermata Species 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101100023203 Homo sapiens MGAT5 gene Proteins 0.000 description 1
- PNIWLNAGKUGXDO-UHFFFAOYSA-N Lactosamine Natural products OC1C(N)C(O)OC(CO)C1OC1C(O)C(O)C(O)C(CO)O1 PNIWLNAGKUGXDO-UHFFFAOYSA-N 0.000 description 1
- DUKURNFHYQXCJG-UHFFFAOYSA-N Lewis A pentasaccharide Natural products OC1C(O)C(O)C(C)OC1OC1C(OC2C(C(O)C(O)C(CO)O2)O)C(NC(C)=O)C(OC2C(C(OC3C(OC(O)C(O)C3O)CO)OC(CO)C2O)O)OC1CO DUKURNFHYQXCJG-UHFFFAOYSA-N 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 108010006519 Molecular Chaperones Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- MNLRQHMNZILYPY-MDMHTWEWSA-N N-acetyl-alpha-D-muramic acid Chemical compound OC(=O)[C@@H](C)O[C@H]1[C@H](O)[C@@H](CO)O[C@H](O)[C@@H]1NC(C)=O MNLRQHMNZILYPY-MDMHTWEWSA-N 0.000 description 1
- 102100023315 N-acetyllactosaminide beta-1,6-N-acetylglucosaminyl-transferase Human genes 0.000 description 1
- 108010056664 N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase Proteins 0.000 description 1
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 1
- FDJKUWYYUZCUJX-AJKRCSPLSA-N N-glycoloyl-beta-neuraminic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@@H]1O[C@](O)(C(O)=O)C[C@H](O)[C@H]1NC(=O)CO FDJKUWYYUZCUJX-AJKRCSPLSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241001327682 Oncorhynchus mykiss irideus Species 0.000 description 1
- 241000607568 Photobacterium Species 0.000 description 1
- 241001517016 Photobacterium damselae Species 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 125000003172 aldehyde group Chemical group 0.000 description 1
- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 1
- 125000003342 alkenyl group Chemical group 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 125000000304 alkynyl group Chemical group 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 125000002837 carbocyclic group Chemical group 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 150000004649 carbonic acid derivatives Chemical class 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 210000000991 chicken egg Anatomy 0.000 description 1
- 239000013317 conjugated microporous polymer Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000009615 deamination Effects 0.000 description 1
- 238000006481 deamination reaction Methods 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000010460 detection of virus Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 125000005982 diphenylmethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])(*)C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 235000013345 egg yolk Nutrition 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 229950006191 gluconic acid Drugs 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 229960002442 glucosamine Drugs 0.000 description 1
- 229940097043 glucuronic acid Drugs 0.000 description 1
- 125000000350 glycoloyl group Chemical group O=C([*])C([H])([H])O[H] 0.000 description 1
- 102000035122 glycosylated proteins Human genes 0.000 description 1
- 108091005608 glycosylated proteins Proteins 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000008611 intercellular interaction Effects 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- DOVBXGDYENZJBJ-ONMPCKGSSA-N lactosamine Chemical compound O=C[C@H](N)[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O DOVBXGDYENZJBJ-ONMPCKGSSA-N 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 210000003643 myeloid progenitor cell Anatomy 0.000 description 1
- RBMYDHMFFAVMMM-PLQWBNBWSA-N neolactotetraose Chemical class O([C@H]1[C@H](O)[C@H]([C@@H](O[C@@H]1CO)O[C@@H]1[C@H]([C@H](O[C@H]([C@H](O)CO)[C@H](O)[C@@H](O)C=O)O[C@H](CO)[C@@H]1O)O)NC(=O)C)[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O RBMYDHMFFAVMMM-PLQWBNBWSA-N 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 125000005017 substituted alkenyl group Chemical group 0.000 description 1
- 125000000547 substituted alkyl group Chemical group 0.000 description 1
- 125000004426 substituted alkynyl group Chemical group 0.000 description 1
- 125000003107 substituted aryl group Chemical group 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid group Chemical group S(O)(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1081—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/005—Glycopeptides, glycoproteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/99—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/99—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
- C12Y204/99001—Beta-galactoside alpha-2,6-sialyltransferase (2.4.99.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
Definitions
- the present invention relates to a method for synthesizing sugar chains that can be applied to pharmaceuticals such as glycoproteins, standard products for analytical instruments, academic reagents, sugar chain arrays, and the like.
- sugar chain structure bound to a protein plays an important role in the function of the protein in biological activity.
- Sugar chains are also called “cell faces”, and it is known that sugar chains expressed on the cell surface are involved in cell-cell interactions, signal transduction, development / differentiation, fertilization, cancer metastasis, and the like.
- Asn-linked type, mucin type, proteoglycan type and the like are well known, and each has a unique sugar chain structure through different biosynthetic pathways. It is known that sugars such as fucose and sialic acid are added to the non-reducing terminal side of this sugar chain structure.
- Sialic acid is a general term for compounds in which amino group or hydroxy group is substituted for neuraminic acid, which is a special 9-carbon sugar with amino group or carboxy group bonded.
- N-acetylated 5-amino group -Acetylneuraminic acid (Neu5Ac) is believed to be most abundant in nature.
- Various structures such as N-glycolylneuraminic acid in which the amino group at the 5-position is modified with a glycolyl group and KDN which is a deamino form are known.
- sialic acid-containing sugar chains exist not only in mammals such as humans and mice, but also in vertebrates and echinoderms, and also in protozoa and some bacteria with gram-negative pathogenicity.
- the production of this sialic acid-containing sugar chain is carried out by sialyltransferase.
- Sialyltransferase uses sialic acid added to cytidine monophosphate (CMP) as a substrate donor, and via the aldehyde group present at the 2-position of sialic acid, positions 3 and 6 of galactose, N-acetylgalactosamine The sialic acid is transferred to the 6th position of the sialic acid and the 8th position of the sialic acid.
- CMP cytidine monophosphate
- an enzyme that transfers sialic acid to position 3 of galactose is ⁇ -2,3 sialyltransferase
- an enzyme that transfers sialic acid to position 6 of galactose or N-acetylgalactosamine is ⁇ -2,6-sialyltransferase
- An enzyme that further transfers sialic acid to position 8 of sialic acid is called ⁇ -2,8 polysialic acid transferase.
- ST6Gal-I and ST6Gal-II are enzymes that transfer to position 6 of galactose in humans
- ST6GalNAc-I is an enzyme that transfers to position 6 of N-acetylgalactosamine.
- ST6GalNAc-II, ST6GalNAc-III and ST6GalNAc-IV are known.
- ST6Gal-I recognizes an N-acetyllactosamine structure (Gal ⁇ 1-4GlcNAc) in which galactose is bonded to the 4-position of N-acetylglucosamine as a substrate receptor, some glycolipids and N-linked sugars Modify the non-reducing end structure of the chain.
- the specificity of the substrate receptor has been analyzed mainly by 2-branch and 3-branch N-linked sugar chains, and sialic acid can be easily transferred to lactosamine extending on ⁇ 1,3-linked mannose. It has been reported (see Non-Patent Document 1).
- erythropoietin EPO
- 4-branched N-linked sugar chain in which 4 molecules of sialic acid are added by ⁇ 2,3 bond. It has been reported that the addition of sialic acid contributes to the stability in blood (see Non-Patent Document 10). Although these are structures that exist in nature, no report has been found of actually preparing a large amount of four-branched N-linked sugar chains in which four molecules of sialic acid are added by ⁇ 2,3 bonds.
- EPO also adds a 4-branched N-linked sugar chain in which ⁇ 2,3 bond and ⁇ 2,6 bond are mixed to the glycoprotein (see Non-Patent Document 11).
- glycoproteins having ⁇ 2,6-linked sialic acid can be expected to be taken up in an organ-specific manner by binding to a specific lectin and expected to be used for drug delivery.
- Glycoproteins are known to be excreted in the urine in the kidney depending on the molecular size.
- erythropoietin In erythropoietin, it is reported that when the number of sugar chain branches is large, the apparent molecular size increases and the clearance from the blood is delayed. Therefore, a sugar chain having ⁇ 2,3-bond and / or ⁇ 2,6-linked sialic acid, in particular, a 4-branched N-type sugar chain having four molecules of ⁇ 2,3-bond and / or ⁇ 2,6-linked sialic acid is synthesized. If it is possible, it is expected that it can be applied to the production of glycoprotein pharmaceuticals with different uptake efficiency into organs.
- human influenza viruses recognize and infect ⁇ 2,6-linked sialic acid of sugar chains expressed on the cell surface
- avian-derived influenza viruses recognize and infect ⁇ 2,3-linked sialic acid.
- many viruses recognize the sugar chain structure of infected cells and start infection, so it is necessary to investigate the binding specificity with viruses using various sugar chains. Therefore, sugar chains having ⁇ 2,3 or ⁇ 2,6-linked sialic acid can be used as materials for investigating the binding specificity of viruses and can be applied to detection of viruses.
- sugar chains in which sialic acid is ⁇ 2,3-bonded or ⁇ 2,6-bonded to the non-reducing end of the sugar chain is known, and some of these sugar chain compounds exist in nature. Extraction has problems such as scarcity as natural products, difficulty in obtaining, and safety, and industrial production is required.
- sugar chains with a uniform structure are controlled by controlling the binding mode of sialic acid ( ⁇ 2,6 or ⁇ 2,3 linkage). It is essential to mass-produce.
- the activity of decomposing sialic acid on the reaction product in the presence of CMP is newly found in the present invention, and the resulting CMP is enzymatically decomposed to efficiently convert sialic acid-containing sugar chains. It was found that it can be manufactured. Furthermore, for the 4-branched N-type sugar chain in which 4 molecules of sialic acid are added through ⁇ 2,6 bonds, which has been difficult to synthesize, a sugar chain elongation reaction is carried out using a 2-branched sugar chain as a raw material. It was found that it can be prepared in a high yield by one-pot synthesis without performing purification after the reaction.
- the present invention in the presence of sialyltransferase and phosphatase, CMP-sialic acid is reacted with the first sugar chain or a derivative thereof, By transferring sialic acid to the non-reducing end of the first sugar chain or derivative thereof,
- the present invention relates to a method for producing a second sugar chain having sialic acid or a derivative thereof.
- the first sugar chain or the derivative thereof is a N-branch type or a 4-branch type N—. It is a conjugated complex sugar chain or a derivative thereof.
- the first sugar chain or a derivative thereof is Compound represented by the following formula (In the figure, Gn represents N-acetylglucosamine, Man represents mannose, and Gal represents galactose.
- Gn represents N-acetylglucosamine
- Man represents mannose
- Gal represents galactose.
- N-acetylglucosamine represents GlcNAc.
- the second sugar chain having sialic acid or a derivative thereof is a 3-branch type or a 4-branch type.
- N-linked complex type sugar chain which is a compound having sialic acid at all non-reducing ends of the sugar chain or a derivative thereof.
- the second sugar chain having sialic acid or a derivative thereof is Compound represented by the following formula (In the figure, Gn represents N-acetylglucosamine, Man represents mannose, Gal represents galactose, and Sia represents sialic acid.
- Gn represents N-acetylglucosamine
- Man represents mannose
- Gal represents galactose
- Sia represents sialic acid.
- Acetylglucosamine is sometimes expressed as GlcNAc.
- Or a derivative thereof Or a derivative thereof.
- another aspect of the present invention includes the following steps: (A) in the presence of glycosyltransferase, Sugar chain represented by the following formula Or a step of reacting a derivative thereof with a UDP saccharide serving as a substrate for the glycosyltransferase one or more times; and (B) in the presence of sialyltransferase and phosphatase, Reacting the product of step (a) with CMP-sialic acid; A sugar chain having sialic acid or a derivative thereof.
- another aspect of the present invention includes the following steps: (A) in the presence of MGAT4 and MGAT5, Reacting UDP-GlcNAc with an agaracto 2-branch complex type sugar chain or a derivative thereof; (B) reacting UDP-Gal with the product of step (a) in the presence of ⁇ 4GalT1; and (c) formation of step (b) in the presence of sialyltransferase and phosphatase. Reacting the product with CMP-sialic acid; A sugar chain having a sialic acid at a non-reducing end or a derivative thereof.
- the sialic acid transferase is ⁇ 2,6-sialyltransferase. In one embodiment of the method for producing a sugar chain having a sialic acid or a derivative thereof according to the present invention, the sialic acid transferase is a human-derived sialic acid transferase. In one embodiment of the method for producing a sugar chain having sialic acid or a derivative thereof according to the present invention, the sialic acid transferase is ST6Gal-I.
- the CMP-sialic acid is CMP-Neu5Ac.
- the phosphatase is alkaline phosphatase.
- the phosphatase is E. coli-derived alkaline phosphatase.
- Another aspect of the present invention relates to a compound having sialic acid at all of the non-reducing ends of a 4-branched N-linked complex sugar chain or a derivative thereof.
- Another aspect of the present invention relates to a compound having ⁇ 2,6-linked sialic acid at all of the non-reducing ends of a 4-branched N-linked complex sugar chain or a derivative thereof.
- Another aspect of the present invention is the following formula: It relates to a compound represented by
- a sialic acid-containing sugar chain using a sialyltransferase can be produced more efficiently than before.
- sialic acid-containing sugar chains sucgar amino acids, sugars
- sialic acid is bonded to all of the non-reducing ends of the branched chain with respect to the three-branched or four-branched complex sugar chain, which has been difficult to produce conventionally.
- Including peptides can also be efficiently produced.
- it can be easily produced in a one-pot synthesis reaction with good yield, and enables mass production of these sugar chains, which has been difficult in the past.
- FIG. 1 shows that ST6Gal-1 was added to a solution containing NA4-Fmoc and CMP-NeuAc as a 4-branched complex type sugar chain, and at 37 ° C., in order from the bottom, 0 hours, 1 hour, and 6 hours,
- the HPLC chart of the reaction product after 24 hours and after 24 hours and after adding CMP-NeuAc and ST6Gal-1 and further reacting for 24 hours is shown.
- tetrasia “trisialo”, “disialo”, and “monosialo” at the top are ( ⁇ 2,6) tetrasia-NA4-Fmoc, ( ⁇ 2,6) trisialo-NA4-Fmoc, ( ⁇ 2) in the HPLC chart, respectively. , 6) retention time (minutes) of disialo-NA4-Fmoc, ( ⁇ 2,6) monosialo-NA4-Fmoc-NA4-Fmoc.
- FIG. 2 shows that ( ⁇ 2,6) tetrasia-NA4-Fmoc was reacted at 37 ° C.
- FIG. 3 shows ( ⁇ 2,6) when sialyltransferase ST6Gal1 is reacted at 37 ° C. for 15 hours with tetrasialo-NA4-Fmoc in the presence or absence of CMP-Neu5AC or CMP.
- the abundance ratios of tetrasia-NA4-Fmoc and ( ⁇ 2,6) trisialo-NA4-Fmoc are shown.
- the right side of FIG. 3 shows ( ⁇ 2,3) tetrasiaNA4-Fmoc with sialyltransferase (ST6Gal-I or ST3Gal-III) at 37 ° C. for 15 hours in the presence or absence of CMP-Neu5AC.
- FIG. 4 shows the abundance ratio of CMP when CMP-Neu5AC is incubated at 37 ° C., 33 ° C., 30 ° C. or 25 ° C. for 2, 8, or 24 hours.
- FIG. 5 shows that NA4-Fmoc was reacted with sialyltransferase ST6Gal1 at 37 ° C., 30 ° C., 25 ° C., 20 ° C. or 10 ° C. for 2 hours, 8 hours or 24 hours in the presence of CMP-Neu5AC.
- FIG. 6 shows that sialyltransferase ST6Gal1 is compared to ( ⁇ 2,6) tetrasia-NA4-Fmoc in the presence of control (CMP, absence of BAP), CMP, or CMP and BAP.
- FIG. 7 shows a one-pot synthesis reaction of ( ⁇ 2,6) tetrasia-NA4-Fmoc described in Example (7) as a flowchart.
- FIG. 8 schematically shows a sugar chain addition reaction in a one-pot synthesis reaction of ( ⁇ 2,6) tetrasia-NA4-Fmoc described in (7) of the Examples using a structural formula.
- Example 9 relates to a one-pot synthesis method of ( ⁇ 2,6) tetrasia-NA4-Fmoc described in Example (7).
- the upper HPLC chart indicated by “+ ST6Gal1” indicates the HPLC of the reaction product.
- sialic acid is a family name generically referring to substances in which the amino group or hydroxy group of neuraminic acid is substituted.
- “Neuraminic acid” is a special 9-carbon sugar having an amino group and a carboxyl group in the molecule, and is represented by the following formula.
- As the structure of sialic acid as the substitution of amino group with respect to the above neuraminic acid, acetylation of amino group, glycorylation and the like are known, and deamination of elimination of amino group is known. Examples of group substitution include, but are not limited to, acetylation, methylation, phosphorylation, and lactylation.
- N-acetylneuraminic acid (Neu5Ac), which is the most abundant in nature.
- N-glycolylneuraminic acid (Neu5Gc) is preferred.
- N-acetylneuraminic acid is more preferable from the viewpoint of producing a naturally occurring glycoprotein or its sugar chain as a human glycoprotein.
- CMP-sialic acid means cytidine 5′-monophosphosialic acid, and the hydroxy group at the 2-position of sialic acid and the phosphate group of Cytidine Monophosphate (CMP) are dehydrated and condensed. It has a structure.
- sialic acid more specifically identified includes CMP-N-acetylneuraminic acid (CMP-Neu5Ac), CMP-N-glycolylneuraminic acid (CMP-Neu5Gc) and the like. .
- CMP-sialic acid used in the present invention includes CMP-N-acetylneuraminic acid (CMP-Neu5Ac), CMP-N -Glycolyl neuraminic acid (CMP-Neu5Gc) is preferred, and in particular, from the viewpoint of producing a glycoprotein that naturally exists as a human glycoprotein or its sugar chain, CMP-N-acetylneuraminic acid (CMP-Neu5Ac) ) Is more preferable.
- sialyltransferase is a kind of glycosyltransferase, and is a sugar acceptor (also referred to as an acceptor substrate) from CMP-sialic acid which is a sugar donor (also referred to as a donor substrate).
- CMP-sialic acid which is a sugar donor (also referred to as a donor substrate).
- sialic acid transfer reaction an enzyme that catalyzes a reaction for transferring a sialic acid residue to a sugar chain structure
- Sialyltransferase is known to transfer sialic acid to the non-reducing end of a sugar chain.
- the sialic acid transfer reaction can be represented by the following reaction formula.
- sialic acid-sugar chain represents a compound in which sialic acid is glycosidically bonded to the non-reducing end of the sugar chain.
- sialyltransferases for example, it is known to transfer to the 3rd and 6th positions of galactose, the 6th position of N-acetylgalactosamine, and the 8th position of sialic acid, which are present at the non-reducing end of the sugar chain. Yes.
- an enzyme that transfers sialic acid to position 3 of galactose is ⁇ -2,3 sialyltransferase
- an enzyme that transfers sialic acid to position 6 of galactose or N-acetylgalactosamine is ⁇ -2,6-sialyltransferase
- An enzyme that further transfers sialic acid to position 8 of sialic acid is called ⁇ -2,8 polysialic acid transferase.
- bacteria-derived sialyltransferases those derived from rainbow trout and mammals are known, and proteins having sialyltransferase-like activity have been found in plants.
- a mammal-derived one is preferable, and a naturally occurring glycoprotein or a sugar chain thereof is produced as a human glycoprotein.
- those derived from humans are more preferable.
- those derived from humans such as ST6Gal-I (also referred to as ST6Gal1), and ST6Gal-II are enzymes that transfer to position 6 of galactose.
- ST6GalNAc-I, ST6GalNAc-II, ST6GalNAc-III and ST6GalNAc-IV are known as enzymes that transfer to the 6-position of N-acetylgalactosamine.
- ST3Gal-I to ST3Gal-VI are known as those derived from humans, for example, as enzymes that transfer to position 3 of galactose.
- Sialyltransferases are particularly ST6Gal-I, ST6Gal-II, ST3Gal-I, ST3Gal-II, ST3Gal-III, ST3Gal from the viewpoint of producing a naturally occurring glycoprotein or its sugar chain as a glycoprotein.
- ST6Gal-I, ST6Gal-II, ST3Gal-III, ST3Gal-IV, ST3Gal-VI, ST8Sia-II, ST8Sia-III, and ST8Sia-IV are preferable.
- sugar chain refers to a compound formed by connecting one or more unit sugars (monosaccharide and / or a derivative thereof). When two or more unit sugars are connected, each unit sugar is bound by dehydration condensation by a glycosidic bond.
- sugar chains include monosaccharides and polysaccharides (glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, and complexes thereof contained in the living body.
- sugar chains that are decomposed or derived from complex biomolecules such as degraded polysaccharides, glycoproteins, proteoglycans, glycosaminoglycans, glycolipids, and the like, but are not limited thereto.
- the sugar chain may be linear or branched.
- the “sugar chain” includes a compound in which a substituent of the sugar chain is modified.
- the sugar constituting the sugar chain is a sugar having a carboxyl group (for example, C-1 Oxidized aldonic acid converted to carboxylic acid (for example, D-gluconic acid oxidized D-glucose), uronic acid whose terminal C atom became carboxylic acid (D-glucose oxidized D-glucose) Glucuronic acid)), sugars having amino groups or amino group derivatives (eg acetylated amino groups) (eg N-acetyl-D-glucosamine, N-acetyl-D-galactosamine etc.), amino groups and carboxyls Sugars having both groups (eg, N-acetylneuraminic acid (sialic acid), N-acetylmuramic acid, etc.), deoxygenated sugars (eg, 2-deoxy-D-ribone) ), S
- preferred sugar chains are sugar chains that exist as complex carbohydrates (glycopeptides (or glycoproteins), proteoglycans, glycolipids, etc.) in vivo from the viewpoint of production of glycoproteins as pharmaceuticals.
- an N-linked sugar chain, an O-linked sugar chain, or the like which is a sugar chain bonded to a peptide (or protein) as a glycopeptide (or glycoprotein) in vivo.
- the N-linked sugar chain is a form of binding when a sugar chain is bound to a protein.
- the anomeric hydroxyl group in N-acetylglucosamine at the reducing end of the sugar chain is linked to the amino group (—NH 2 ) of the asparagine side chain.
- O-linked glycan is a general term for sugar chains that are bonded by dehydration condensation, and the anomeric hydroxyl group at the reducing end of the sugar chain is serine or It is a general term for sugar chains that are bonded by dehydration condensation with the hydroxyl group (—OH) of the threonine side chain.
- the N-linked sugar chain is sometimes referred to as an asparagine-linked sugar chain, an N-type sugar chain, or the like.
- the N-linked sugar chain is a group of sugar chains having Man 3 -GlcNAc-GlcNAc as a mother nucleus.
- sugar chain branch structures It is known to have specific sugar chain structures called a high mannose type, a complex (complex) type, and a hybrid (hybrid) type depending on the structure of the sugar chain that binds to the mother nucleus Man.
- multi-branched structures such as 2-branch, 3-branch, and 4-branch structures are known as sugar chain branch structures.
- the structures of these sugar chains are also described in, for example, the Biochemical Dictionary (3rd edition, published by Tokyo Chemical Co., Ltd.).
- the sugar chain or derivative thereof serving as a sugar acceptor serves as a substrate for sialyltransferase at the non-reducing end.
- a sugar chain structure it is not particularly limited.
- glycoproteins that exist in nature there are many branched-type glycans with a structure in which sialic acid is bonded to the non-reducing end in N-linked glycan complex and hybrid types. From the viewpoint of producing these sugar chains, N-linked complex sugar chains and N-linked hybrid sugar chains are preferred, and N-linked complex types that can have sialic acid at all non-reducing ends.
- a sugar chain is more preferred.
- the branched structure is preferably an N-linked tri-branched or 4-branched sugar chain, which has been difficult to produce in the past, and an N-linked tri-branched or 4-branched complex sugar chain is preferred. More preferred.
- the “first sugar chain or a derivative thereof” refers to a sugar chain or a derivative thereof that becomes a raw material compound (also referred to as a starting compound) in a sialic acid transfer reaction, and at least one of the non-reducing ends, A glycan having a glycan structure that serves as a substrate for sialyltransferase.
- the “first sugar chain or derivative thereof” is referred to as “sugar acceptor” or “acceptor substrate”, and “CMP-sialic acid” is referred to as “sugar donor” or “donor substrate”.
- first sugar chain or derivative thereof examples include, for example, a compound having a structure in which each non-reducing end is a substrate of a sialyltransferase in a branched sugar chain, in other words, “sialic acid as a target compound” It is preferable to use a compound having a structure in which all sialic acids are lacking from the “second sugar chain or derivative thereof having“.
- a sugar is sometimes referred to as an “asialo sugar chain”, “asialo body”, or “asialo”.
- as the asialo sugar chain for example, a 4-branched sugar chain represented by the following formula or a derivative thereof is preferable.
- examples of the asialo 4-branched N-linked complex sugar chain or a derivative thereof include a sugar chain represented by the following formula: Or its derivative is preferable.
- asialo 3-branched N-linked complex sugar chains or derivatives thereof include, for example, sugar chains represented by the following formula Or its derivative is preferable.
- asialo bifurcated N-linked complex sugar chains or derivatives thereof include, for example, sugar chains represented by the following formulae: Or its derivative is preferable.
- a sugar chain in which sialic acid is glycosidically bonded to one or a plurality of non-reducing ends of each sugar chain, or a derivative thereof, as a sugar receptor in the sialic acid transfer reaction of the present invention is a sugar chain represented by the following formula: Or its derivative is preferable.
- sugar chains having sialic acid at all non-reducing ends can be obtained.
- a sugar chain obtained by a conventional method a compound represented by the following formula Etc.
- the above compounds can also be produced by adjusting the reaction time in the method of the present invention.
- the first sugar chain or a derivative thereof a sugar chain or a derivative thereof in which sialic acid is glycosidically bonded to one or more of the non-reducing ends of the sugar chain, a glycoside bond of sialic acid in the compound is used.
- sialyltransferases that produce different glycosidic linkages, it is also possible to produce compounds in which the sialic acid glycoside linkage mode is not identical.
- the “sugar chain derivative” includes a compound in which another compound is bonded to the reducing end of the sugar chain by dehydration condensation or the like.
- Examples of the “derivative of sugar chain” include the following formula: It can be expressed as a compound in which R is further bonded to N-acetylglucosamine at the reducing end of the sugar chain.
- the above sugar chain derivatives are exemplifications, and in the case of derivatives for other sugar chains, they can be similarly expressed by adding -R to the reducing end.
- the sugar chain derivatives include those in which an amino acid, peptide, protein, linker, fluorescent group, lipid, low molecular weight compound, radioactive compound or the like is added as R to the reducing end of the sugar chain.
- Amino acids include non-natural amino acids such as amino acid variants and derivatives as well as natural amino acids.
- Amino acids, peptides, proteins, and the like may be those in which some or all of the functional groups such as hydroxyl group, amino group, and carboxyl group contained therein are protected with a protecting group.
- the hydroxyl protecting group include a methyl group, a benzyl group, a benzoyl group, an acetyl group, a trimethylsilyl (TMS) group, a triethylsilyl (TES) group, a tert-butyldimethylsilyl (TBS or TBDMS) group, and the like. it can.
- amino protecting group examples include, for example, carbonates such as 9-fluorenylmethoxycarbonyl (Fmoc) group, t-butyloxycarbonyl (Boc) group, benzyl group, allyloxycarbonyl group, and acetyl group as fat-soluble protecting groups. And amide-type protecting groups.
- a fat-soluble protecting group for example, when introducing an Fmoc group, it can be introduced by adding 9-fluorenylmethyl-N-succinimidyl carbonate and sodium carbonate for reaction.
- protecting group for the carboxyl group examples include a benzyl group, an allyl group, and a diphenylmethyl group.
- any adduct to the reducing end of the sugar chain can be used as long as it does not significantly affect the glycosyltransferase reaction.
- a linker is useful for binding to an amino acid, protein, or the like after production of a sugar chain.
- the fluorescent group is useful when purifying after production of a sugar chain, or when the sugar chain is used for testing or the like.
- dansylation pyridylamino (PA) formation, 2-aminobenzamide (2-AB), 2- Examples thereof include aminobenzoic acid (2-AA), 9-aminopyrene-1,4,6-trisulfonic acid (APTS).
- a linker may be further added to the sugar chain amino acid, or two or more adducts may be added in any order, such as a sugar and an amino acid being bonded via a linker.
- the sugar chain derivative is preferably a sugar chain amino acid, a glycosylated peptide, or a glycosylated protein.
- a sugar chain asparagine (sugar chain) -It may be expressed as -Asn).
- a compound in which a protecting group is bonded to the sugar chain asparagine also referred to as sugar chain-Asn-R 2 , where R 2 is a protecting group.
- R 2 is a protecting group.
- the protecting group those known to those skilled in the art can be used in addition to those exemplified above as the fat-soluble protecting group.
- sugar chain asparagine Fmoc sucgar chain-Asn-Fmoc
- sugar chain asparagine Boc sucgar chain-Asn-Boc
- Fmoc, Boc and the like, which are fat-soluble protective groups, are added are preferable.
- the “second glycan having sialic acid or a derivative thereof” refers to a glycan or a derivative thereof that is a product in a sialic acid transfer reaction, and at least one non-reducing end has sialic acid. Or a derivative thereof.
- a sugar chain having sialic acid at all non-reducing ends having a structure serving as a substrate for sialyltransferase or a derivative thereof is preferable.
- a sugar chain having sialic acid or a derivative thereof a sugar chain having sialic acid at all non-reducing ends or a derivative thereof is more preferable.
- tetrasialo sugar chain when the number of sialic acids bound to one molecule of sugar chain is specified, when four molecules of sialic acid are bound to one molecule of sugar chain Called “tetrasialo", the case where 3 molecules of sialic acid are bonded to one molecule of sugar chain is called “tricialo”, and the case where 2 molecules of sialic acid are bonded to 1 molecule of sugar chain is called “disialo" The case where one sialic acid molecule is bonded to one sugar chain molecule is called “monosialo”.
- tetrasialo sugar chain or “tetrasialo body” may be used.
- a compound in which four molecules of sialic acid are bonded to one molecule of a four-branched sugar chain is a “tetrasialo” or “four-branched” sugar chain.
- a compound in which three molecules of sialic acid are bonded is a “tricialo” or “four-branched” sugar chain
- a compound in which three molecules of sialic acid are bonded to one molecule of a three-branched sugar chain For example, it can be referred to as “tricialo” or “3-branch” sugar chain.
- tetrasialo means that if 4 molecules of sialic acid are bonded to 1 molecule of sugar chain, for example, all types of glycosidic bonds between sialic acid and sugar chain are all Examples include compounds that are ( ⁇ 2,6) bonds, compounds that are all ( ⁇ 2,3) bonds, compounds that are partly ( ⁇ 2,6) bonds, and others that are ( ⁇ 2,3) bonds.
- Tetrasialo refers to a compound in which four molecules of sialic acid are all linked to a sugar chain by ( ⁇ 2,3) linkage.
- the linkage mode of the glycosidic bond formed by the sialyltransferase between the non-reducing end of the “first sugar chain or derivative thereof” and the sialic acid is not particularly limited, but is preferably an ⁇ 2,6 bond, ⁇ 2 , 3 bonds, or ⁇ 2,8 bonds.
- the “second sugar chain having sialic acid or a derivative thereof” has a plurality of sialic acids at the non-reducing end of the sugar chain, the non-reducing end of the “first sugar chain or the derivative thereof” and the sialic acid
- the “second glycan having sialic acid or a derivative thereof” as the product of the sialic acid transfer reaction is preferably, for example, a glycan represented by the following formula or a derivative thereof.
- second glycan having sialic acid or derivative thereof a 4-branched N-linked complex glycan having sialic acid at all non-reducing ends of the second glycan (in the present specification, Examples of tetrasialo 4-branched N-linked complex sugar chains) include sugar chains represented by the following formula: Or its derivative is preferable.
- a “second glycan having sialic acid or a derivative thereof” a three-branched N-linked complex glycan having sialic acid at all non-reducing ends of the second glycan (this specification In this case, the trisialo 3-branched N-linked complex sugar chain) is, for example, a sugar chain represented by the following formula: Or its derivative is preferable.
- a bifurcated N-linked complex glycan having sialic acid at all non-reducing ends of the second glycan is, for example, a sugar chain represented by the following formula: Or its derivative is preferable.
- N-linked complex sugar chains there are known compounds in which Fuc and Gn are bound to the sugar chains, and such compounds are also included.
- Fuc is ⁇ 1,6 bonded to Gn at the reducing end
- Gn is ⁇ 1,4 bonded to the 4-position of Man bonded to Gn at the reducing end
- Fuc binds ⁇ 1,3 or ⁇ 1,4.
- a compound in which the binding mode at the branched portion of the sugar chain is Gn ( ⁇ 1,4) Man or Gn ( ⁇ 1,2) Man instead of Gn ( ⁇ 1,6) Man, Gn ( ⁇ 1,4) Man In place of Sia ( ⁇ 2,6) Gal instead of Sia ( ⁇ 2,6) Gal in the compound to which Gn ( ⁇ 1,2) Man or sialic acid is bonded, Sia ( ⁇ 2,3 A sugar chain having a different glycosidic bond, such as a compound that is Gal or a compound in which a part of Sia ( ⁇ 2,3) Gal is Sia ( ⁇ 2,3) Gal instead of Sia ( ⁇ 2,3) Gal included.
- phosphatase is an enzyme that catalyzes a reaction of hydrolyzing a phosphate ester, and has an activity of hydrolyzing a phosphate ester against CMP under the reaction conditions of glycosyltransferase.
- Known phosphatases include alkaline phosphatase having activity under alkaline conditions and acid phosphatase having activity under acidic conditions.
- Alkaline phosphatase is known to be widely distributed throughout the body including the liver, kidney, osteoblast, placenta, and small intestine. It is known that acid phosphatase is stored in lysosomes and is also present in various organs and plasma.
- phosphatases include those derived from bacteria, Escherichia coli, shrimp, and mammals.
- Escherichia coli-derived alkaline phosphatase BAP
- CIP cattle-derived alkaline phosphatase
- CAP CAP
- CIAP cattle-derived alkaline phosphatase
- SAP shrimp-derived alkaline phosphatase
- Sialyltransferases are commercially available ( ⁇ 2,3- (N) -Sialyltransferase, Rat, Recombinant, S. frugiperda, ⁇ 2,3- (O) -Sialyltransferase, Rat, Recombinant, S. frugiper6, (N) -Sialyltransferase, Human, Recombinant S.
- frugiperda Recombinant beta-galactoside-alpha-2, 3-Sialyltransferase-2, Recombinant beta-galactose PCR based on sequence Genes can be obtained by gene synthesis by width or chemical, and the obtained genes are inserted into expression vectors such as plasmids, and expression systems such as E. coli, yeast, insect cells, plant cells, and animal cells are used. Thus, the enzyme can be obtained as a recombinant.
- sialyltransferase can be purified and used from biological samples such as tissues such as bovine small intestine and cultured animal cells. In addition to the methods described in the present specification, those skilled in the art can make modifications with appropriate modifications.
- the phosphatase can also be produced as appropriate, such as commercially available products such as Bacterial Alkaline Phosphatase (E. coli), Calf intestine Alkaline Phosphatase (CIP), and Alkaline Phosphatase from Shrim (SAP).
- CMP-sialic acid is also commercially available, and can be suitably produced in addition to using cytidine-5′-monophospho-N-acetylneuraminic acid (disodium) and the like.
- the reaction solvent for the sialyltransferase reaction is not particularly limited as long as the activity of the sialyltransferase is maintained, and a stabilizer such as bovine serum albumin or a surfactant may be added.
- a stabilizer such as bovine serum albumin or a surfactant
- an aqueous solution containing 0.1 M Tris-HCl (pH 7.5), 1 mM MnCl 2 , 0.1% Triton X-100 can be used.
- the pH of the reaction solvent is not particularly limited as long as the activity of the sialyltransferase is maintained, but the optimum range of the sialyltransferase is preferably about pH 5 to 10, more preferably about pH 7 to 8. .
- the phosphatase may be prepared slightly on the alkaline side or on the acidic side.
- the reaction temperature is not particularly limited as long as the sialyltransferase activity is maintained.
- the optimum temperature of the enzyme is preferably around 37 ° C. Preferably, it is 10 ° C to 40 ° C, more preferably 20 ° C to 37 ° C, still more preferably 25 ° C to 37 ° C. From the viewpoint of preventing degradation of CMP-sialic acid by sialyltransferase, 25 ° C. to 30 ° C. is preferable.
- the reaction time is not particularly limited as long as it is sufficient for the sialic acid transfer reaction to proceed, and can be appropriately determined by those skilled in the art.
- sialic acid when sialic acid is transferred to each non-reducing end of the multi-branched sugar chain, it can be preferably 8 to 48 hours, more preferably 16 to 24 hours.
- phosphatase or sialyltransferase may be added in addition to CMP-sialic acid serving as a sugar donor, and further reacted. These may be added simultaneously or at different timings. For example, after reacting for 24 hours, CMP-sialic acid and sialyltransferase can be added, and further reacted for 24 hours.
- the first sugar chain or derivative thereof is purified from a natural product, processed, purified from a glycoprotein synthesized in an expression system, or chemically or enzymatically synthesized, etc.
- those obtained by further subjecting them to a sugar chain elongation reaction can be used.
- an enzyme that catalyzes the formation of the glycoside bond is selected in accordance with the glycoside bond mode of the desired sugar chain structure, and these are sequentially performed according to the order of binding of the sugars constituting the sugar chain. Can be manufactured.
- a multi-branched N-linked complex sugar chain used as a sugar receptor or a derivative thereof as a sugar receptor in a sialic acid transfer reaction is converted to a sugar chain represented by the following formula (hereinafter referred to as the following formula).
- a sugar chain represented by the following formula (hereinafter referred to as the following formula).
- As the sugar chain derivative for example, agaracto 2-branched sugar chain Asn-Fmoc represented by the following formula can be used.
- the agaracto 2-branched sugar chain represented by the above formula or a derivative thereof is included.
- the N-acetylglucosamine transferase may be selected according to the glycosidic bond formed between the sugar chain and the transferred sugar.
- the target glycosidic bond is a ⁇ 1-6 bond
- ⁇ 1- An enzyme that generates 6 bonds, or an enzyme that generates ⁇ 1-4 bonds when the target glycoside bond is ⁇ 1-4 bonds may be selected.
- the enzyme that generates ⁇ 1-6 bond include enzymes that generate ⁇ 1-4 bond ( ⁇ 1,4-N-acetyl, such as human MGAT5 and bovine GnT-V).
- the glucosamine transferase include human MGAT4a, human MGAT4b, bovine GnT-IVa, and the like.
- the galactose transferase is selected according to the glycoside bond generated between the sugar chain and the sugar to be transferred, and when the target glycoside bond is a ⁇ 1-4 bond, an enzyme that generates a ⁇ 1-4 bond
- an enzyme that generates a ⁇ 1-4 bond For example, ⁇ 4GalT1, ⁇ 4GalT2, Helicbacter pylori-derived ⁇ 1,4-galactose transferase, and the like.
- MGAT4a and MGAT5 as N-acetylglucosamine transferase in (a) and ⁇ 4GalT1 as galactose transferase in (b).
- this enzyme combination it can also be produced by replacing with the enzymes exemplified above.
- the first sugar chain or a derivative thereof when the first sugar chain or a derivative thereof is a 3-branched N-linked complex sugar chain, the same operation as in the case of the above-described 4-branched sugar chain can be performed.
- Tri-branched sugar chain represented by the following formula Alternatively, a derivative thereof can be produced, for example, by using MGAT4a as N-acetylglucosamine transferase in (a) and ⁇ 4GalT1 as galactose transferase in (b).
- a derivative thereof can be produced, for example, by using MGAT5 as N-acetylglucosamine transferase in (a) and ⁇ 4GalT1 as galactose transferase in (b).
- MGAT5 N-acetylglucosamine transferase
- ⁇ 4GalT1 galactose transferase in (b).
- it can also be produced by replacing with the enzymes exemplified above.
- the agaracto bibranched sugar chain represented by the above formula is (B) reacting UDP-Gal in the presence of galactose transferase; Can be manufactured.
- the galactose transferase is the same as in the case of the 4-branch type.
- the first sugar chain or a derivative thereof is a 4-branched N-linked complex sugar chain or a derivative thereof, and fucose or N-acetylglucosamine is further bound to the sugar chain.
- these sugars can be added using fucose transferase or N-acetylglucosamine transferase.
- an agaracto 2-branched sugar chain is included in addition to the case where the agaracto 2-branched sugar chain is used as a raw material. It can also be produced by performing a necessary sugar chain elongation reaction using a raw material such as a glycopeptide derived from chicken egg yolk, PA agarac bibranched sugar chain (sold by Takara Bio Inc.) and the like.
- a sugar chain or a derivative thereof as a product of each sugar chain elongation reaction is added after each sugar chain elongation reaction. After isolation and purification, it can be used for the next sugar chain elongation reaction.
- a sialic acid transfer reaction is performed following the sugar chain elongation reaction to produce a sugar chain having a sialic acid at a non-reducing end or a derivative thereof.
- one-pot synthesis refers to a method of synthesizing a target compound without isolating and purifying an intermediate product in the process leading to the synthesis of the target compound.
- One-pot synthesis reaction (A) a step of performing at least one step of reacting a raw material compound with a UDP sugar serving as a substrate of the glycosyltransferase in the presence of a glycosyltransferase; (B) reacting the product of step (a) with CMP-sialic acid in the presence of sialyltransferase and phosphatase; It can manufacture by performing.
- the reaction of (a) for example, the above-mentioned production process of a 2-4 branched N-linked complex sugar chain can be performed.
- glycosyltransferase reaction each glycosyltransferase reaction in the case of performing the glycosyltransferase reaction a plurality of times in (a)
- sialic acid transfer reaction when starting the sialic acid transfer reaction, a glycosyltransferase concentrated solution and a concentrated solution of UDP-sugar serving as a substrate thereof are prepared and added in small amounts.
- the heat treatment can be performed even after the end of the step (b).
- the conditions for the heat treatment are not particularly limited as long as the enzyme is deactivated.
- the heat treatment can be performed by keeping the temperature at 90 ° C. or higher for a certain period of time. Preferably, it can be set to 90 ° C. to 100 ° C. for about 5 to 10 minutes. Heat treatment conditions can be changed as appropriate by those skilled in the art.
- the produced sugar chain can be purified by a known method (for example, HPLC or the like).
- HPLC conditions can be appropriately changed by those skilled in the art depending on the structure of the sugar chain.
- Embodiments of the present invention may be described with reference to schematic diagrams, but in the case of schematic diagrams, they may be exaggerated for clarity of explanation.
- terms such as first, second, etc. are used to represent various elements, it is understood that these elements should not be limited by those terms. These terms are only used to distinguish one element from another, for example, the first element is referred to as the second element, and similarly, the second element is the first element. Can be made without departing from the scope of the present invention.
- the region containing this sequence was cleaved with BamHI and then introduced into the BamHI site of the expression vector pOMEA1-10H3F of the methanol-assimilating yeast Ogataea minuta to prepare pOMEA1-10H3F-ST6Gal-I.
- This plasmid pOMEA1-10H3F-ST6Gal-I was cleaved with NotI and transformed into Ogataea minuta TK-10-1-2 strain ( ⁇ och1 ⁇ pep4 ⁇ prb1 ⁇ ura3 ⁇ ade1, WO2003 / 091431). Transformation was performed using electroporation.
- the obtained YTY-2 strain was cultured and ST6Gal-I was expressed.
- 5 ml of YPAD + KCl medium (2% polypeptone, 1% yeast extract, 2% glucose, adenine (40 mg / L), 0.3 M KCl) was inoculated and pre-cultured at 30 ° C. overnight.
- 1 ml of the preculture was inoculated into 150 ml of YPAD + KCl medium and cultured at 30 ° C. for 48 hours.
- BMMY + 2% casamino acid medium 1% yeast extract, 2% polypeptone, 1.34% Yeast Nitrogen Base w / o amino acids (manufactured by Difco), 0.1M KPi (pH 6.0), 2 % Casamino acid, 0.5% methanol) and cultured at 20 ° C. for 96 hours. In addition, methanol was added so that it might become 0.5% every 12 hours. After completion of the culture, the cells were removed by centrifugation to obtain a crude enzyme solution.
- the crude enzyme solution was dialyzed against SP buffer (25 mM sodium acetate (pH 5.5), 0.1% Triton X-100) and then applied to HiTrap SP HP (5 ml) equilibrated with SP buffer. After washing with SP buffer, elution was performed with SP buffer containing 1M NaCl. A fraction showing ST6Gal-I activity was collected and dialyzed against a reaction buffer (25 mM MOPS, pH 7.3) to obtain a partially purified preparation. The enzyme activity was measured as follows.
- the reaction was started by adding 2 ⁇ l of the crude enzyme solution to 18 ⁇ l of the reaction solution (0.1 M MOPS (pH 7.3), 5 mM CMP-Neu5Ac, 50 ⁇ M PA-modified Lacto-N-neotetraose (LNnT-PA)). After reaction at 37 ° C. for 30 minutes, the reaction was stopped by boiling, and analysis by HPLC was performed.
- NA4-Fmoc a compound represented by the following formula (hereinafter referred to as NA4-Fmoc) was produced by the following method.
- a compound represented by the following formula (hereinafter referred to as NGA2-Fmoc) That is, a compound in which an agalacto-type biantennary complex type sugar chain was bonded to the side chain of an asparagine residue and the amino group of the asparagine residue was modified with Fmoc was used as a receptor substrate for the transglycosylation reaction.
- Reaction solution A (0.1 M MOPS (pH 7.3), 40 mM UDP-GlcNAc, 6.7 mM NGA2-Fmoc, 10 mM MnCl 2 , 5 mg / ml bovine serum albumin (BSA), 0.1 mM in 0.15 ml) was added to 0.15 ml. 3 mU of MGAT4a and MGAT5 were added, and the reaction was performed at 37 ° C. for 16 hours. After the reaction, heat treatment was performed at 100 ° C. for 5 minutes to inactivate the enzyme.
- reaction solution B 0.1 M MOPS (pH 7.3), 30 mM UDP-Gal, 10 mM MnCl 2 , 10 mg / ml BSA, 8 mM AMP), then 5 mU ⁇ 4GalT1 was added, The reaction was performed at 37 ° C. for 16 hours. After the reaction, heat treatment was performed at 100 ° C. for 5 minutes to inactivate the enzyme. The target sugar chain (NA4-Fmoc) was purified from the obtained reaction solution.
- the column was equilibrated with.
- Sugar chains were collected 20 minutes after sample injection. Detection was performed with a fluorescence detector (Ex: 265 nm, Em: 315 nm).
- the substrate NGA2-Fmoc was eluted as a single peak at 14 minutes and the reaction product NA4-Fmoc at 8 minutes.
- the sugar chain of the reaction product was recovered and used as NA4-Fmoc in subsequent experiments.
- FIG. 1 shows the results of HPLC analysis of the reaction solution after 0 hours, 1 hour, 6 hours, and 24 hours after the reaction. Even after 24 hours, the reaction did not proceed completely, and a sugar chain to which three molecules of sialic acid were added (hereinafter referred to as ( ⁇ 2,6) trisialo-NA4-Fmoc. In FIG. 1, it is abbreviated as “trisialo-”. .) was the main peak.
- reaction solution was prepared by adding 50 ⁇ U of ST6Gal-I and 5 ⁇ l of CMP-Neu5Ac to a final concentration of 2 mM.
- a reaction solution was prepared by adding 50 ⁇ U of ST6Gal-I and CMP-Neu5Ac heat-treated at 100 ° C. for 5 minutes to a final concentration of 2 mM. These were similarly kept at 37 ° C. for 17 hours.
- the reaction product was heated at 100 ° C. for 5 minutes and then subjected to HPLC analysis in the same manner as shown in (2). The result is shown in FIG. When CMP-Neu5Ac as a substrate donor was not added, almost no sialic acid was eliminated.
- the degree of CMP formation was expressed as [CMP peak area] / [CMP peak area + CMP-Neu5Ac peak area] ⁇ 100 (%).
- the result is shown in FIG. After 24 hours, 45% of CMP was generated at 37 ° C., compared with 21% at 30 ° C., which was about half of that at 37 ° C. Therefore, it was suggested that CMP-dependent sialic acid decomposition activity can be suppressed by lowering the temperature and carrying out the ST6Gal-I reaction.
- reaction solution E (0.1 M MOPS (pH 7.3), 5 mM MnCl 2 , 5 mg containing 50 ⁇ M NA4-Fmoc / Ml bovine serum albumin, 5 mM CMP-Neu5Ac) was prepared.
- 100 ⁇ U of ST6Gal-I prepared in (1) was added to 10 ⁇ l of the reaction solution E and reacted at 10 ° C., 20 ° C., 25 ° C., 30 ° C., and 37 ° C. for 24 hours. As shown in FIG. 5, when the reaction was carried out at 25 ° C. and 30 ° C.
- Reaction solution E (0.1 M Tris-HCl (pH 7.5), 1 mM MnCl 2 , 0.1% Triton X-100) containing 25 pmol of ( ⁇ 2,6) tetrasia-NA4-Fmoc and 2.5 nmol of CMP was prepared. 25 ⁇ U of ST6Gal-I was added to 10 ⁇ l of this reaction solution E and incubated at 37 ° C. for 17 hours. Similarly, a reaction solution in which 25 ⁇ U of ST6Gal-I and 50 ⁇ U of Escherichia coli-derived alkaline phosphatase (BAP) (Takara Bio Inc.) were added was prepared and kept warm in the same manner. The reaction product was heated at 100 ° C.
- BAP Escherichia coli-derived alkaline phosphatase
- reaction solution F 0.1 M MOPS (pH 7.3), 40 mM UDP-GlcNAc (6 ⁇ mol), 6.7 mM NGA2-Fmoc (1 ⁇ mol), 0.3 mU MGAT4a, 0.3 mU MGAT5, 5 mg / ml BSA, 1 mM PMSF) were prepared and reacted at 37 ° C. for 16 hours. After stopping the reaction by incubating at 100 ° C.
- reaction solution G 0.1 M MOPS (pH 7.3), 24 mM UDP-Gal (6 ⁇ mol), 4.8 mU ⁇ 4GalT1, 8 mM MnCl 2 , 5 mg / ml BSA, 4 mM AMP
- reaction solution H 40 mM CMP-Neu5Ac (10 ⁇ mol), 3 mU ST6Gal-I, 15 mU BAP
- FIG. 7 shows a flow chart of the above series of one-pot synthesis reactions.
- the sugar chain addition reaction in the above series of one-pot synthesis reactions is schematically shown in FIG. 8 using a structural formula.
- the reaction raw materials and reaction products were subjected to HPLC analysis, and ( ⁇ 2,6) tetrasialo-NA4-Fmoc was quantified.
- the reaction raw material NGA2-Fmoc was eluted as a single peak at about 16 minutes, and the reaction product ( ⁇ 2,6) tetrasia-NA4-Fmoc was eluted at about 30 minutes.
- the peak area of the obtained ( ⁇ 2,6) tetrasialo-NA4-Fmoc is 90% with respect to the peak area of NGA2-Fmoc as the reaction raw material, and the target sugar chain can be synthesized in a very high yield by one-pot synthesis. Was confirmed.
- a sialic acid-containing sugar chain using a sialyltransferase can be produced more efficiently than before.
- a sialic acid-containing sugar chain in which sialic acid is bonded to all of the non-reducing ends of the branched chain or a derivative thereof is efficiently used for a three-branched or four-branched complex sugar chain, which has been difficult to produce conventionally.
- these sugar chains especially ( ⁇ 2,6) tetrasialo 4-branch complex type sugar chains) which can be easily produced with a one-pot synthesis reaction and have a high yield, have been difficult in the past. Mass production is also possible.
- These sugar chains can be used as a sugar chain having a novel function, a pharmaceutical product such as a glycoprotein, a standard product for an analytical instrument, an academic reagent, or one sugar chain of a sugar chain array.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Materials Engineering (AREA)
- Polymers & Plastics (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
Description
一方、4分岐のN-結合型糖鎖に対するα2,6シアル酸転移反応については、ウシ由来のST6Gal-Iで検討されており、4カ所のN-アセチルラクトサミン構造のうち、まずα1,3結合のマンノース上にβ1,2結合で付加したN-アセチルラクトサミン構造に転移しやすく、次にα1,3結合のマンノース上にβ1,4結合で付加したN-アセチルラクトサミン構造に、さらにα1,6結合のマンノース上に付加した2つのN-アセチルラクトサミン構造のどちらかにシアル酸が転移されるが、4分子シアル酸が入った産物は見いだされなかった(非特許文献2参照。)。またヒトのST6Gal-Iについては、ST6Gal-IIとともに基質特異性が報告されている(非特許文献3、4参照。)が、4分岐のN-結合型糖鎖を受容体基質としたシアル酸付加については検討されていない。
ST6Gal-IのCMPによるプロダクト阻害は0.25mMのCMPで49%阻害される(非特許文献5参照。)、または71%阻害される(非特許文献6参照。)という報告がある。
一方、バクテリア由来のα2,6-シアル酸転移酵素については、Photobacterium damsela JT0160(非特許文献7参照。)、Photobacterium leiognathi JT-SHIZ-145(非特許文献8参照。)などが報告されているが、いずれも4分岐のN-結合型糖鎖を受容体基質としたシアル酸付加については検討されていない。
抗体医薬やサイトカインなどの糖タンパク質医薬品のN-型糖鎖に付加するシアル酸の結合様式に関し、α2,6結合シアル酸を付加したタンパク質は、α2,3結合シアル酸を付加したタンパク質に比べ血中からの消失が早いことが報告されている。糖タンパク質の血中からのクリアランスについては、体内のレクチン分子と結合して細胞内へ取り込まれ、最終的に代謝される。従ってα2,6結合のシアル酸を有する糖タンパク質は特異的なレクチンに結合することで臓器特異的に取り込まれることが期待でき、ドラッグデリバリーへの活用が期待できる。また糖タンパク質は、分子サイズに依存し腎臓において尿中に排出されることが知られている。エリスロポエチンでは、糖鎖の分岐数が多いと見かけの分子サイズが増加し、血中からのクリアランスが遅れると報告されている。従ってα2,3結合及び/又はα2,6結合のシアル酸を有する糖鎖、とりわけ、α2,3結合及び/又はα2,6結合のシアル酸を4分子有する4分岐のN-型糖鎖を合成することが可能となれば、臓器への取り込み効率が異なる糖タンパク質医薬品の生産へ応用できることが期待される。
即ち、本発明は、シアル酸転移酵素、及び、ホスファターゼの存在下において、
第一の糖鎖またはその誘導体に対し、CMP-シアル酸を反応させ、
前記第一の糖鎖またはその誘導体の非還元末端にシアル酸を転移させることにより、
シアル酸を有する第二の糖鎖またはその誘導体を製造する方法に関する。
また、本発明のシアル酸を有する第二の糖鎖またはその誘導体を製造する方法の一実施態様においては、前記第一の糖鎖またはその誘導体が、
下記式により表わされる化合物
またはその誘導体であることを特徴とする。
また、本発明のシアル酸を有する第二の糖鎖またはその誘導体を製造する方法の一実施態様においては、前記シアル酸を有する第二の糖鎖またはその誘導体が、3分岐型または4分岐型のN-結合型複合型糖鎖であり、糖鎖の各非還元末端のすべてにシアル酸を有する化合物またはその誘導体であることを特徴とする。
また、本発明のシアル酸を有する第二の糖鎖またはその誘導体を製造する方法の一実施態様においては、前記シアル酸を有する第二の糖鎖またはその誘導体が、
下記式により表わされる化合物
またはその誘導体であることを特徴とする。
(a)糖転移酵素の存在下において、
下記式により表わされる糖鎖
(b)シアル酸転移酵素、及び、ホスファターゼの存在下において、
前記工程(a)の生成物に対し、CMP-シアル酸を反応させる工程;
を含む、シアル酸を有する糖鎖またはその誘導体を製造する方法に関する。
(a)MGAT4及びMGAT5の存在下において、
アガラクト2分岐複合型糖鎖またはその誘導体に対し、UDP-GlcNAcを反応させる工程;
(b)β4GalT1の存在下において、前記工程(a)の生成物に対し、UDP-Galを反応させる工程;及び
(c)シアル酸転移酵素及びホスファターゼの存在下において、前記工程(b)の生成物に対し、CMP-シアル酸を反応させる工程;
を含む、非還元末端にシアル酸を有する糖鎖またはその誘導体を製造する方法に関する。
また、本発明のシアル酸を有する糖鎖またはその誘導体を製造する方法の一実施態様においては、前記シアル酸転移酵素が、ヒト由来のシアル酸転移酵素であることを特徴とする。
また、本発明のシアル酸を有する糖鎖またはその誘導体を製造する方法の一実施態様においては、前記シアル酸転移酵素が、ST6Gal-Iであることを特徴とする。
また、本発明のシアル酸を有する糖鎖またはその誘導体を製造する方法の一実施態様においては、前記CMP-シアル酸が、CMP-Neu5Acであることを特徴とする。
また、本発明のシアル酸を有する糖鎖またはその誘導体を製造する方法の一実施態様においては、前記ホスファターゼが、アルカリホスファターゼであることを特徴とする。
また、本発明のシアル酸を有する糖鎖またはその誘導体を製造する方法の一実施態様においては、前記ホスファターゼが、大腸菌由来アルカリホスファターゼであることを特徴とする。
シアル酸の構造として、上記ノイラミン酸に対する、アミノ基の置換としては、アミノ基のアセチル化、グライコリル化などが知られている他、アミノ基が脱離したデアミノ化などが知られており、ヒドロキシ基の置換としては、アセチル化、メチル化、リン酸化、ラクチル化などが知られているが、これらに限定されない。
本明細書において、天然に存在する糖タンパク質またはその糖鎖を製造するという観点から、転移させるシアル酸としては、天然に最も多く存在するN-アセチルノイラミン酸(Neu5Ac)、次に多く存在するN-グライコリルノイラミン酸(Neu5Gc)が好ましい。特に、ヒトの糖タンパク質として天然に存在する糖タンパク質またはその糖鎖を製造するという観点から、N-アセチルノイラミン酸がより好ましい。
[式中、シアル酸-糖鎖は、糖鎖の非還元末端にシアル酸がグリコシド結合した化合物を示す。]
シアル酸転移酵素としては、例えば、糖鎖の非還元末端に存在する、ガラクトースの3位や6位、N-アセチルガラクトサミンの6位、また、シアル酸の8位に転移することが知られている。例えば、ガラクトースの3位にシアル酸を転移する酵素はα-2,3シアル酸転移酵素、ガラクトースまたはN-アセチルガラクトサミンの6位にシアル酸を転移する酵素はα-2,6シアル酸転移酵素、シアル酸の8位にさらにシアル酸を転移する酵素はα-2,8ポリシアル酸転移酵素と呼ばれている。
シアル酸転移酵素は、細菌由来のもののほか、ニジマス由来、哺乳類由来のものなどが知られており、また植物からはシアル酸転移酵素様の活性を有するタンパク質が見いだされている。特に、哺乳類の糖タンパク質として天然に存在する糖タンパク質またはその糖鎖を製造するという観点から、哺乳類由来のものが好ましく、ヒトの糖タンパク質として天然に存在する糖タンパク質またはその糖鎖を製造するという観点から、ヒト由来のものがより好ましい。
α-2,6シアル酸転移酵素については、ヒト由来のものとして、例えば、ガラクトースの6位に転移する酵素としてST6Gal-I(ST6Gal1と表記することもある、以下同様。)及びST6Gal-IIが、またN-アセチルガラクトサミンの6位に転移する酵素としてST6GalNAc-I、ST6GalNAc-II、ST6GalNAc-III及びST6GalNAc-IVが知られている。
α-2,3シアル酸転移酵素については、ヒト由来のものとして、例えば、ガラクトースの3位に転移する酵素としてST3Gal-I~ST3Gal-VIが知られている。
シアル酸転移酵素は、特に、糖タンパク質として天然に存在する糖タンパク質またはその糖鎖を製造するという観点からは、ST6Gal-I、ST6Gal-II、ST3Gal-I、ST3Gal-II、ST3Gal-III、ST3Gal-IV、ST3Gal-VI、ST6GalNAc-I、ST6GalNAc-II、ST6GalNAc-III、ST6GalNAc-IV、ST8Sia-II、ST8Sia-III、ST8Sia-IVが好ましい。また、N-結合型糖鎖を製造するという観点からは、ST6Gal-I、ST6Gal-II、ST3Gal-III、ST3Gal-IV、ST3Gal-VI、ST8Sia-II、ST8Sia-III、ST8Sia-IVが好ましい。
N-結合型糖鎖は、アスパラギン結合型糖鎖、N-型糖鎖等と呼ばれることもある。N-結合型糖鎖は、Man3-GlcNAc-GlcNAcを母核とする糖鎖群である。母核のManに結合する糖鎖の構造により、高マンノース(ハイマンノース)型、複合(コンプレックス)型、混成(ハイブリッド)型と呼ばれる特定の糖鎖構造を有することが知られている。また、糖鎖の分岐構造としても、2分岐型、3分岐型、4分岐型などの多分岐型構造が知られている。これらの糖鎖の構造は、例えば、生化学辞典(第3版、株式会社東京化学同人発行)等にも記載されている。
本明細書において、アシアロ糖鎖としては、例えば、下記式で表される4分岐型の糖鎖またはその誘導体が好ましい。
本明細書において、アシアロ4分岐型N-結合型複合型糖鎖またはその誘導体としては、例えば、下記式で表される糖鎖
本明細書において、アシアロ3分岐型N-結合型複合型糖鎖またはその誘導体としては、例えば、下記式で表される糖鎖
本明細書において、アシアロ2分岐型N-結合型複合型糖鎖またはその誘導体としては、例えば、下記式で表される糖鎖
また、上記糖鎖に対し、各糖鎖の非還元末端のうちの1つないし複数の箇所にシアル酸がグリコシド結合した糖鎖またはその誘導体を、本発明のシアル酸転移反応における糖受容体として用いることもできる。従来の方法では、多分岐型の糖鎖の場合にすべての非還元末端にシアル酸を有する糖鎖を製造することが困難であったところ、これらの従来方法により得られる糖鎖も本発明のシアル酸転移反応によって、すべての非還元末端にシアル酸を有する糖鎖にすることができる。例えば、従来方法により得られる糖鎖としては、下記式で表される化合物
上記化合物は、従来方法により得られる他、本発明の方法において、反応時間を調節することにより製造することもできる。
第一の糖鎖またはその誘導体として、糖鎖の非還元末端のうちの1つないし複数の箇所にシアル酸がグリコシド結合した糖鎖またはその誘導体を用い、当該化合物中のシアル酸のグリコシド結合とは異なるグリコシド結合を生じさせるシアル酸転移酵素を用いることにより、シアル酸のグリコシド結合様式が同一ではない化合物を製造することもできる。
糖鎖の誘導体としては、糖鎖の還元末端に、Rとして、アミノ酸、ペプチド、タンパク質、リンカー、蛍光基、脂質、低分子化合物、放射性化合物などが付加されたものも含む。アミノ酸は、天然のアミノ酸のみならずアミノ酸変異体及び誘導体といったような非天然アミノ酸を含む。アミノ酸、ペプチド、タンパク質等は、これらに含まれる水酸基、アミノ基、カルボキシル基のような官能基の一部ないし全部を保護基で保護したものであってもよい。水酸基の保護基としては、例えば、メチル基、ベンジル基、ベンゾイル基、アセチル基、トリメチルシリル(TMS)基、トリエチルシリル(TES)基、tert-ブチルジメチルシリル(TBSまたはTBDMS)基などを挙げることができる。アミノ保護基としては、例えば、脂溶性保護基として、9-フルオレニルメトキシカルボニル(Fmoc)基、t-ブチルオキシカルボニル(Boc)基、ベンジル基、アリルオキシカルボニル基、アセチル基などの、カーボネート系またはアミド系の保護基等を挙げることができる。脂溶性保護基を導入する場合には、例えば、Fmoc基を導入する場合には、9-フルオレニルメチル-N-スクシニミジルカーボネートと炭酸ナトリウムを加えて反応を行うことにより導入できる。
カルボキシル基の保護基としては、例えば、ベンジル基、アリル基、ジフェニルメチル基などを挙げることができる。上記は例示であって、これらに限定されない。シアル酸転移酵素は、糖鎖の非還元末端に作用するものであるから、糖鎖の還元末端に対する付加物は、糖転移反応に大きな影響を及ぼすものでない限り、いかなるものでも用いることができる。リンカーは、糖鎖製造後に、アミノ酸やタンパク質等に結合させる際に有用であり、例えば、-NH-(CO)-(CH2)a-CH2-
(式中、aは整数であり、目的とするリンカー機能を阻害しない限り限定されるものではないが、好ましくは0~4の整数を示す。)、
C1-10ポリメチレン、-CH2-R1-(ここで、R1は、アルキル、置換されたアルキル、アルケニル、置換されたアルケニル、アルキニル、置換されたアルキニル、アリール、置換されたアリール、炭素環基、置換された炭素環基、複素環基及び置換された複素環基からなる群より選択される基から水素原子が1つ脱離して生ずる基である)等を挙げることができるが、これらに限定されない。また、蛍光基は、糖鎖製造後の精製や、糖鎖を検査等に用いる際に有用であり、例えば、ダンシル化、ピリジルアミノ(PA)化、2-アミノベンズアミド(2-AB)、2-アミノ安息香酸(2-AA)、9-アミノピレン-1,4,6-トリスルホン酸(APTS)化等を挙げることができる。また、糖鎖アミノ酸にさらにリンカーを付加する、あるいは、糖とアミノ酸がリンカーを介して結合するなど、2つ以上の付加物を任意の順序で付加したものであってもよい。
本明細書において、糖鎖の誘導体は、好ましくは、糖鎖アミノ酸、糖鎖付加ペプチド、糖鎖付加タンパク質であり、天然の糖タンパク質の糖鎖を製造する観点からは、糖鎖アスパラギン(糖鎖-Asnと表記することもある)がより好ましい。また、製造した糖鎖アスパラギンを固相合成に用いる観点からは、糖鎖アスパラギンに保護基が結合した化合物(糖鎖-Asn-R2と表記することもある。ここで、R2は保護基を示す。)が好ましい。保護基としては、脂溶性保護基として、上記に例示したものの他、当業者に公知のものを用いることができる。例えば、脂溶性保護基である、Fmoc、Boc等が付加した、糖鎖アスパラギンFmoc(糖鎖-Asn-Fmoc)、糖鎖アスパラギンBoc(糖鎖-Asn-Boc)等が好ましい。
本明細書において、シアル酸を有する糖鎖またはその誘導体について、糖鎖1分子に対して結合したシアル酸の数を特定する場合、糖鎖1分子に対してシアル酸4分子が結合した場合を「テトラシアロ」と呼び、糖鎖1分子に対してシアル酸3分子が結合した場合を「トリシアロ」と呼び、糖鎖1分子に対してシアル酸2分子が結合した場合を「ジシアロ」と呼び、糖鎖1分子に対してシアル酸1分子が結合した場合を「モノシアロ」と呼ぶ。また、「テトラシアロ糖鎖」や「テトラシアロ体」等の呼び方をすることもある。例えば、4分岐型の糖鎖1分子に対して、シアル酸が4分子結合した化合物であれば、「テトラシアロ」「4分岐型」の糖鎖であり、4分岐型の糖鎖1分子に対して、シアル酸が3分子結合した化合物であれば、「トリシアロ」「4分岐型」の糖鎖であり、3分岐型の糖鎖1分子に対して、シアル酸が3分子結合した化合物であれば、「トリシアロ」「3分岐型」の糖鎖ということができる。
本明細書において、「テトラシアロ」という場合には、糖鎖1分子に対してシアル酸4分子が結合していれば、シアル酸と糖鎖の間のグリコシド結合の種類を問わず、例えば、全て(α2,6)結合である化合物、全て(α2,3)結合である化合物、一部が(α2,6)結合であり、その他が(α2,3)結合である化合物等を含む。ただし、本明細書において、単に「(α2,6)テトラシアロ」と記載した場合には、4分子のシアル酸がすべて(α2,6)結合により糖鎖に結合した化合物をいい、単に「(α2,3)テトラシアロ」と記載した場合には、4分子のシアル酸がすべて(α2,3)結合により糖鎖に結合した化合物をいう。 シアル酸転移酵素により、「第一の糖鎖またはその誘導体」の非還元末端とシアル酸との間に形成されるグリコシド結合の結合様式は特に限定されないが、好ましくは、α2,6結合、α2,3結合、または、α2,8結合である。「シアル酸を有する第二の糖鎖またはその誘導体」が、糖鎖の非還元末端に複数のシアル酸を有する場合には、「第一の糖鎖またはその誘導体」の非還元末端とシアル酸との間に形成されるグリコシド結合の結合様式は同一であっても異なってもよい。
本明細書において、シアル酸転移反応の生成物としての、「シアル酸を有する第二の糖鎖またはその誘導体」は、例えば、下記式により表される糖鎖またはその誘導体が好ましい。
「シアル酸を有する第二の糖鎖またはその誘導体」として、第二の糖鎖の全ての非還元末端にシアル酸を有する4分岐型のN-結合型複合型糖鎖(本明細書において、テトラシアロ4分岐型N-結合型複合型糖鎖ともいう)としては、例えば、下記式により表わされる糖鎖
また、「シアル酸を有する第二の糖鎖またはその誘導体」として、第二の糖鎖の全ての非還元末端にシアル酸を有する3分岐型のN-結合型複合型糖鎖(本明細書において、トリシアロ3分岐型N-結合型複合型糖鎖ともいう)としては、例えば、下記式により表わされる糖鎖
また、「シアル酸を有する第二の糖鎖またはその誘導体」として、第二の糖鎖の全ての非還元末端にシアル酸を有する2分岐型のN-結合型複合型糖鎖(本明細書において、ジシアロ2分岐型N-結合型複合型糖鎖ともいう)としては、例えば、下記式により表わされる糖鎖
なお、N-結合型複合型糖鎖としては、上記糖鎖にFucやGnが結合した化合物も存在することが知られており、そのような化合物も含まれる。より具体的には、還元末端のGnに対して、Fucがα1,6結合することや、還元末端のGnに結合したManの4位にGnがβ1,4結合すること、分岐部分のGnにFucがα1,3またはα1,4結合することが知られている。また、上記糖鎖の分岐部分における結合様式が、Gn(β1,6)Manに代えて、Gn(β1,4)ManまたはGn(β1,2)Manである化合物、Gn(β1,4)Manに代えて、Gn(β1,2)Manである化合物や、シアル酸が結合する部分における、Sia(α2,6)Galの一部がSia(α2,6)Galに代えてSia(α2,3)Galである化合物や、Sia(α2,3)Galの一部がSia(α2,3)Galに代えてSia(α2,6)Galである化合物などのグリコシド結合の結合様式が異なる糖鎖も含まれる。
シアル酸転移酵素は、市販のもの(α2,3-(N)-Sialyltransferase, Rat, Recombinant, S. frugiperda、α2,3-(O)-Sialyltransferase, Rat, Recombinant, S. frugiperda、α2,6-(N)-Sialyltransferase, Human, Recombinant S. frugiperda、Recombinant beta-galactoside-alpha-2,3-sialyltransferase、Recombinant beta-galactoside-alpha-2,6-sialyltransferaseなど)を用いる他、公知の遺伝子配列やアミノ酸配列に基づいて、PCR増幅や化学的に遺伝子合成を行い遺伝子を取得することが出来、この得られた遺伝子をプラスミド等の発現ベクターに挿入し、大腸菌、酵母、昆虫細胞、植物細胞、動物細胞などの発現系を用いて組換え体として酵素を得ることができる。またウシ小腸などの組織や培養した動物細胞などの生体試料からシアル酸転移酵素を精製し利用することが出来る。本明細書に記載した方法の他、当業者であれば、適宜変更等を加えて、製造することが可能である。
ホスファターゼも市販のもの、例えばBacterial Alkaline Phosphatase(E.coli)、Calf intestine Alkaline Phosphatase(CIP)、Alkaline Phosphatase from Shrimp(SAP)を用いる他、適宜製造することが可能である。
CMP-シアル酸も市販のものとして、シチヂン-5'-モノホスホ-N-アセチルノイラミン酸(二ナトリウム)等を用いる他、適宜製造することが可能である。
シアル酸転移酵素反応時の反応溶媒としては、シアル酸転移酵素の活性が保たれる条件であれば特に限定されず、ウシ血清アルブミン等の安定化剤や界面活性剤などを入れてもよい。例えば、0.1M Tris-HCl(pH7.5),1mM MnCl2,0.1% Triton X-100を含む水溶液を用いることができる。当業者であれば、適宜変更等を加えて用いることができる。
反応溶媒のpHは、シアル酸転移酵素の活性が保たれる範囲であれば、特に限定されないが、シアル酸転移酵素の至適範囲として、pH5~10程度が好ましく、pH7~8程度がより好ましい。また、用いるホスファターゼの活性が保たれる範囲を考慮し、中性よりも少しアルカリ側、又は、酸性側に調製してもよい。
反応温度は、シアル酸転移酵素の活性が保たれる条件であれば特に限定されない。酵素の至適温度としては、37℃前後が好ましい。好ましくは、10℃~40℃、より好ましくは、20℃~37℃、さらに好ましくは、25℃~37℃である。シアル酸転移酵素によるCMP-シアル酸の分解を防ぐ観点からは、25℃~30℃が好ましい。
反応時間は、シアル酸転移反応が進行するのに十分な時間であれば特に制限されず、当業者であれば、適宜決定することができる。特に、多分岐型糖鎖の各非還元末端にシアル酸を転移する場合には、好ましくは、8時間~48時間、より好ましくは、16~24時間とすることができる。
また、反応に際し、一定時間反応させた後、糖供与体となるCMP-シアル酸の他、ホスファターゼやシアル酸転移酵素を追加し、さらに反応させてもよい。これらは同時に追加してもよく、また、異なるタイミングで追加してもよい。例えば、24時間反応させた後、CMP-シアル酸及びシアル酸転移酵素を追加し、さらに24時間反応させることもできる。
(a)N-アセチルグルコサミン転移酵素の存在下において、UDP-GlcNAcを反応させる工程;
(b)(a)の生成物に対し、ガラクトース転移酵素の存在下において、UDP-Galを反応させる工程;
を行うことによって、製造することができる。
N-アセチルグルコサミン転移酵素としては、糖鎖と転移する糖との間に生じさせるグリコシド結合に応じて選択すればよく、例えば、目的とするグリコシド結合がβ1-6結合である場合にはβ1-6結合を生じさせる酵素、目的とするグリコシド結合がβ1-4結合である場合にはβ1-4結合を生じさせる酵素を選択すればよい。β1-6結合を生じさせる酵素(β1,6-N-アセチルグルコサミン転移酵素)としては、例えば、ヒトMGAT5、ウシGnT-V等、β1-4結合を生じさせる酵素(β1,4-N-アセチルグルコサミン転移酵素)としては、例えば、ヒトMGAT4a、ヒトMGAT4b、ウシGnT-IVaなどを挙げることができる。
ガラクトース転移酵素としては、糖鎖と転移する糖との間に生じさせるグリコシド結合に応じて選択し、目的とするグリコシド結合がβ1-4結合である場合には、β1-4結合を生じさせる酵素を選択すればよく、例えば、β4GalT1、β4GalT2、Helicbacter pylori由来β1,4-ガラクトース転移酵素等を挙げることができる。
下記式で表される4分岐型の糖鎖
下記式で表される3分岐型の糖鎖
また、下記式で表される3分岐型の糖鎖
(b)ガラクトース転移酵素の存在下において、UDP-Galを反応させる工程;
を行うことによって、製造することができる。
ガラクトース転移酵素については、4分岐型の場合と同様である。
本明細書において、ワンポット合成とは、目的化合物の合成に至る過程で、中間生成物の単離・精製を行わずに目的化合物を合成する方法をいう。ワンポット合成反応は、
(a)糖転移酵素の存在下において、原料化合物に対し、前記糖転移酵素の基質となるUDP糖を反応させる工程を1回以上行う工程;
(b)シアル酸転移酵素、及び、ホスファターゼの存在下において、工程(a)の生成物に対し、CMP-シアル酸を反応させる工程;
を行うことにより製造することができる。
この際に、(a)の反応としては、例えば、上記の、2~4分岐型のN-結合型複合型糖鎖の製造工程を行うことができる。
ワンポット合成反応においては、例えば、(a)の糖転移反応((a)において糖転移反応を複数回行う場合には、各糖転移反応)、(b)のシアル酸転移反応を開始する際に、(a)であれば、糖転移酵素濃縮液、及び、その基質となるUDP-糖の濃縮液を調製しておき、これらを少量添加することにより、行うことができる。
ワンポット合成反応においては、(a)の糖転移反応の後((a)において糖転移反応を複数回行う場合には、各糖転移反応の後、次の糖転移反応開始の前に、)、加熱処理を行うことにより、反応系内の糖転移酵素による糖転移反応を停止させることができる。これにより、さらに、反応生成物の収率を上げることができる。また、(b)工程終了後にも、加熱処理を行うことができる。
加熱処理の条件は、酵素が失活する程度のものであれば、特に限定されないが、例えば、90℃以上の温度において一定時間保温することにより行うことができる。好ましくは、90℃から100℃、5~10分程度とすることができる。加熱処理条件は当業者が適宜変更することができる。
また、本明細書において用いられる「含む」との用語は、文脈上明らかに異なる理解をすべき場合を除き、記述された事項(部材、ステップ、要素、数字など)が存在することを意図するものであり、それ以外の事項(部材、ステップ、要素、数字など)が存在することを排除しない。
異なる定義が無い限り、ここに用いられるすべての用語(技術用語及び科学用語を含む。)は、本発明が属する技術の当業者によって広く理解されるのと同じ意味を有する。ここに用いられる用語は、異なる定義が明示されていない限り、本明細書及び関連技術分野における意味と整合的な意味を有するものとして解釈されるべきであり、理想化され、又は、過度に形式的な意味において解釈されるべきではない。
本発明の実施態様は模式図を参照しつつ説明される場合があるが、模式図である場合、説明を明確にするために、誇張されて表現されている場合がある。
第一の、第二のなどの用語が種々の要素を表現するために用いられるが、これらの要素はそれらの用語によって限定されるべきではないことが理解される。これらの用語は一つの要素を他の要素と区別するためのみに用いられているのであり、例えば、第一の要素を第二の要素と記し、同様に、第二の要素は第一の要素と記すことは、本発明の範囲を逸脱することなく可能である。
ヒトST6Gal-IのmRNA配列は公共のデータベースであるGenBankにAccession No.X62822として、またアミノ酸配列はAccession No.P15907として登録されている。このアミノ酸配列を基に、Ogataea minutaのコドン使用に合わせた形で遺伝子全合成を行なった。なお合成した遺伝子はヒトST6Gal-Iの細胞質ドメインと膜貫通領域を含むN-末端側48アミノ酸を除いて発現させ、その外側には、発現ベクターへの導入を容易にすべく、制限酵素BamHI部位を導入した。その配列を配列番号1に示す。この配列を含む領域をBamHIで切断後、メタノール資化性酵母Ogataea minutaの発現ベクターpOMEA1-10H3FのBamHI部位に導入し、pOMEA1-10H3F-ST6Gal-Iを作製した。このプラスミドpOMEA1-10H3F-ST6Gal-IをNotIで切断後、Ogataea minuta TK-10-1-2株(Δoch1Δpep4Δprb1Δura3Δade1、WO2003/091431)を形質転換した。形質転換はエレクトロポレーション法を用いて行なった。形質転換後、SD-Ade(2%グルコース、0.17%Yeast Nitrogen Base w/o amino acids (Difco社製)、アデニンを除く核酸塩基及びアミノ酸混合物(20-400mg/L))培地にまいて、30℃にて2日間培養し形質転換体を得た。形質転換体をプレートから掻きとり、PCR反応液に懸濁する簡易法PCRにて染色体上への組み込みを確認し、YTY-1株とした。
次に、より発現量を向上させるため、シャペロン遺伝子の導入を行なった。特願2009-539162に記載された、OmPDI1、OmERO1、OmKAR2を恒常的に発現する遺伝子を含むベクターOnaP11007をNotIで切断し、YTY-1の形質転換を行なった。形質転換はエレクトロポレーション法を用いて行なった。形質転換後、SD-Ura(2%グルコース、0.17%Yeast Nitrogen Base w/o amino acids(Difco社製)、ウラシルを除く核酸塩基及びアミノ酸混合物(20-400mg/L))培地にまいて、30℃にて2日間培養し形質転換体を得た。形質転換体をプレートから掻きとり、PCR反応液に懸濁する簡易法PCRにて染色体上への組み込みを確認し、YTY-2株とした。
得られたYTY-2株を培養し、ST6Gal-Iの発現を行なった。5mlのYPAD+KCl培地(2%ポリペプトン、1%酵母エキス、2%グルコース、アデニン(40 mg/L)、0.3 M KCl)に植菌し、30℃、一晩前培養した。次に150mlのYPAD+KCl培地に1mlの前培養液を植菌し、30℃、48時間培養した。集菌後、100mlのBMMY+2%カザミノ酸培地(1%酵母エキス,2%ポリペプトン,1.34% Yeast Nitrogen Base w/o amino acids (Difco社製),0.1M KPi(pH6.0),2%カザミノ酸,0.5%メタノール)に再懸濁し、20℃、96時間培養した。なお、12時間ごとに0.5%になるようにメタノールを添加した。培養終了後遠心して菌体を除き、粗酵素液とした。
粗酵素液はSPバッファー(25mM酢酸ナトリウム(pH5.5),0.1%Triton X-100)に透析後、SPバッファーで平衡化したHiTrap SP HP(5ml)に供した。SPバッファーで洗浄後、1MのNaClを含むSPバッファーで溶出を行なった。ST6Gal-I活性を示す画分を回収し、反応バッファー(25mM MOPS,pH7.3)に透析し、部分精製標品とした。
酵素活性測定は以下のように行った。反応液(0.1M MOPS(pH7.3),5mM CMP-Neu5Ac,50μM PA化Lacto-N-neotetraose(LNnT-PA))18μlに対し、粗酵素液を2μl加え反応を開始した。37℃,30分反応後、煮沸することで反応を停止し、HPLCによる解析を行った。カラムはAsahipak NH2P-50(4.6×250mm:Shodex社)を使用し、移動相は0.2Mトリエチルアミン-酢酸(pH7.0)(A液)とアセトニトリル(B液)を用い、A液:B液=30:70でカラムを平衡化した。試料注入後20分かけてA液:B液の割合を50:50まで直線的に変化させ、グラジェント溶出を行った。検出は蛍光検出器(Ex:315nm,Em:380nm)にて行った。基質であるLNnT-PAは9分に、反応産物である6’-Sialyl-LNnT-PAは18.5分に溶出される。ピーク面積から得られた反応産物を定量し、活性(U)とした。なお1Uは、1分間に1μmolの反応産物を生成する酵素量と定義した。
アシアロ4分岐複合型糖鎖誘導体の1種として、下記式で表わされる化合物(以下、NA4-Fmocという。)を以下の方法により製造した。
得られた反応液から目的糖鎖(NA4-Fmoc)の精製を行なった。カラムはKromasil 100-5C18(4.6×250mm:EKA-CHEMICAL社)を使用し、移動相は25mM酢酸アンモニウム(A液)とアセトニトリル(B液)を用い、A液:B液=82:18でカラムを平衡化した。試料注入後20分で糖鎖を回収した。検出は蛍光検出器(Ex:265nm,Em:315nm)にて行った。基質であるNGA2-Fmocは14分に、反応産物であるNA4-Fmocは8分にシングルピークとして溶出された。反応産物の糖鎖を回収し、NA4-Fmocとして以後の実験に用いた。
α2,6シアル酸が付加した4分岐複合型糖鎖誘導体の1種として、下記式で表わされる化合物(以下、(α2,6)tetrasialo-NA4-Fmocという。)
シアル酸が4分子付加した糖鎖に対応するピークを回収し、(α2,6)tetrasialo-NA4-Fmocとして以後の実験に用いた。
酵素量の増加に伴う反応産物の増加が見られなかったため、反応産物が分解されている可能性が示唆された。そこで反応産物の分解が起きているかどうかを評価するため以下の実験を行なった。
50pmol分の(α2,6)tetrasialo-NA4-Fmoc型糖鎖を含む反応液D(0.1M Tris-HCl(pH7.5),1mM MnCl2,0.1% Triton X-100)を調製した。反応液D5μlに50μUのST6Gal-I5μlを添加し、37℃で17時間保温した。同様に50μUのST6Gal-Iと終濃度として2mMとなるようにCMP-Neu5Ac 5μlを加えた反応液を調製した。また同様に、50μUのST6Gal-Iと100℃において5分間加熱処理を行なったCMP-Neu5Acを終濃度として2mMとなるように加えた反応液を調製した。これらも同様に37℃で17時間保温した。反応産物は100℃において5分間加熱後、(2)に示した方法と同様にHPLC解析を行なった。その結果を図2に示す。基質供与体であるCMP-Neu5Acを加えなかった場合にはシアル酸の脱離はほとんど見られなかった。一方、CMP-Neu5Acを加えた場合には、(α2,6)tetrasialo-NA4-Fmocのうち約30%が(α2,6)trisialo-NA4-Fmocとなり、シアル酸の脱離が確認できた。さらに、加熱したCMP-Neu5Acを加えた場合では、(α2,6)trisialo-NA4-Fmocが58%みられた。
一方、図3に示す通り、(α2,3)tetrasialo-NA4-Fmocに対してST6Gal-Iを添加しても分解活性は全く見られなかった。また、(α2,3)tetrasialo-NA4-Fmocに対し、α2,3シアル酸転移酵素であるST3Gal-IIIを添加しても分解は見られなかった。このことからST6Gal-Iによるシアル酸の脱離はα2,6結合のシアル酸に特異的であることが示唆された。
基質供与体であるCMP-Neu5Acの分解によってCMPが生成しているかどうかを確認するため、5mMのCMP-Neu5Ac/0.1M MOPS(pH7.3)を25℃、30℃、33℃、37℃で保温し、CMPの生成量を測定した。測定はHPLCで行ない、カラムはTSKgel SuperQ-5PW(7.5×75mm:東ソー社)を用い、溶媒は50mM KPi(pH8.0)を用いた。検出はUV検出器(検出波長254nm)にて行なった。CMPの生成度は[CMPのピーク面積]/[CMPのピーク面積+CMP-Neu5Acのピーク面積]×100(%)として表わした。その結果を図4に示す。24時間後では、37℃で45%のCMPが生成しているのに対し、30℃では21%であり、37℃の場合の約半分であった。従って温度を下げてST6Gal-Iの反応を行なうことにより、CMP依存的なシアル酸分解活性を抑制できることが示唆された。
次に、α2,6シアル酸が付加した4分岐複合型糖鎖の生成率を評価するため、50μM NA4-Fmocを含む反応液E(0.1M MOPS(pH7.3),5mM MnCl2,5mg/mlウシ血清アルブミン,5mM CMP-Neu5Ac)を調製した。反応液E10μlに、(1)で調製したST6Gal-Iを100μU添加し、10℃、20℃、25℃、30℃、37℃で24時間反応を行なった。図5に示すように、反応24時間後において、25℃と30℃で反応させた場合に、目的の糖鎖である(α2,6)tetrasialo-NA4-Fmocの収率が高かった。従って、(α2,6)tetrasialo-NA4-Fmocの収率を上げるためには、シアル酸転移酵素の至適温度から外れた25℃~30℃で反応させた方がよいことが示唆された。
基質供与体であるCMP-Neu5Acの分解の他にも、合成反応の副産物としてCMPが生成するため、これらのCMPを分解することによりシアル酸転移酵素によるシアル酸脱離反応を抑制することを試みた。25pmol分の(α2,6)tetrasialo-NA4-Fmocと2.5nmolのCMPを含む反応液E(0.1M Tris-HCl(pH7.5),1mM MnCl2,0.1% Triton X-100)を調製した。この反応液E10μlに25μUのST6Gal-Iを添加し、37℃で17時間保温した。同様に25μUのST6Gal-Iと50μUの大腸菌由来アルカリホスファターゼ(BAP)(タカラバイオ社)を加えた反応液を調製し、同様に保温した。反応産物は100℃において5分間加熱後、(3)に示した方法で分析を行なった。その結果を図6に示す。CMPを加えた場合には、24時間後には(α2,6)tetrasialo-NA4-Fmocのうち約70%が(α2,6)trisialo-NA4-Fmocとなり、シアル酸の脱離が確認された。一方、大腸菌由来アルカリホスファターゼ(BAP)を添加した場合には、(α2,6)tetrasialo-NA4-Fmocの分解はほとんど見られなかった。このことから、CMPをホスファターゼで分解し5’-シチジン酸とすることにより、ST6Gal-Iのテトラシアロ糖鎖に対するシアル酸脱離活性を抑制できることを確認した。
150μlの反応液F(0.1M MOPS(pH7.3),40mM UDP-GlcNAc(6μmol),6.7mM NGA2-Fmoc(1μmol),0.3mU MGAT4a,0.3mU MGAT5,5mg/ml BSA,1mM PMSF)を調製し、37℃において16時間反応を行なった。100℃において5分間保温し反応を停止後、反応液Fに対して250μlの反応液G(0.1M MOPS(pH7.3),24mM UDP-Gal(6μmol),4.8mU β4GalT1,8mM MnCl2,5mg/ml BSA,4mM AMP)を添加し、37℃、16時間反応を行なった。100℃において5分間保温し反応を停止後、減圧乾固を行なった。このチューブに250μlの反応液H(40mM CMP-Neu5Ac(10μmol),3mU ST6Gal-I,15mU BAP)を添加し、30℃において16時間反応を行なった。さらにCMP-Neu5Ac(20mM CMP-Neu5Ac)5μlを添加し、30℃において16時間反応を行なった。反応後、100℃において5分間の加熱処理を行ない、反応を停止させた。上記の一連のワンポット合成反応をフローチャートとして、図7に示す。また、上記の一連のワンポット合成反応における糖鎖付加反応を、構造式を用いて模式的に図8に示す。
反応原料及び反応生成物について、HPLC解析を行ない、(α2,6)tetrasialo-NA4-Fmocの定量を行なった。HPLC解析条件として、カラムはAmido-80(3μm、4.6×150mm:TOSOH社)を使用し、移動相はアセトニトリル(A液)と0.2M TEAA(pH7.0)(B液)を用い、A液:B液=75:25でカラムを平衡化した。試料注入後35分で糖鎖を回収した。検出は蛍光検出器(Ex:265nm,Em:315nm)にて行った。その結果を図9に示す。反応原料であるNGA2-Fmocは約16分に、反応生成物である(α2,6)tetrasialo-NA4-Fmocは約30分にシングルピークとして溶出された。反応原料のNGA2-Fmocのピーク面積に対し、得られた(α2,6)tetrasialo-NA4-Fmocのピーク面積は90%であり、ワンポット合成で非常に収率よく目的糖鎖の合成が出来ることが確認された。
Claims (16)
- シアル酸転移酵素、及び、ホスファターゼの存在下において、
第一の糖鎖またはその誘導体に対し、CMP-シアル酸を反応させ、
前記第一の糖鎖またはその誘導体の非還元末端にシアル酸を転移させることにより、
シアル酸を有する第二の糖鎖またはその誘導体を製造する方法。 - 請求項1に記載の前記第一の糖鎖またはその誘導体が、3分岐型または4分岐型のN-結合型複合型糖鎖またはその誘導体である、方法。
- 請求項1に記載の前記シアル酸を有する第二の糖鎖またはその誘導体が、3分岐型または4分岐型のN-結合型複合型糖鎖であり、糖鎖の各非還元末端のすべてにシアル酸を有する化合物またはその誘導体である、方法。
- 以下の工程;
(a)MGAT4及びMGAT5の存在下において、
アガラクト2分岐複合型糖鎖またはその誘導体に対し、UDP-GlcNAcを反応させる工程;
(b)β4GalT1の存在下において、前記工程(a)の生成物に対し、UDP-Galを反応させる工程;及び
(c)シアル酸転移酵素及びホスファターゼの存在下において、前記工程(b)の生成物に対し、CMP-シアル酸を反応させる工程;
を含む、非還元末端にシアル酸を有する糖鎖またはその誘導体を製造する方法。 - 請求項1~7に記載の方法であって、前記シアル酸転移酵素が、α2,6シアル酸転移酵素である方法。
- 請求項1~7に記載の方法であって、前記シアル酸転移酵素が、ヒト由来のシアル酸転移酵素である方法。
- 請求項1~7に記載の方法であって、前記シアル酸転移酵素が、ST6Gal-Iである方法。
- 請求項1~10のいずれか1項に記載の方法であって、前記CMP-シアル酸が、CMP-Neu5Acである方法。
- 請求項1~10のいずれか1項に記載の方法であって、前記ホスファターゼが、アルカリホスファターゼである方法。
- 請求項1~10のいずれか1項に記載の方法であって、前記ホスファターゼが、大腸菌由来アルカリホスファターゼである方法。
- 4分岐型のN-結合型複合型糖鎖またはその誘導体の各非還元末端のすべてにシアル酸を有する化合物。
- 4分岐のN-結合型複合型糖鎖またはその誘導体の各非還元末端のすべてにα2,6結合型のシアル酸を有する化合物。
Priority Applications (11)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SG2013066527A SG193021A1 (en) | 2011-03-04 | 2012-02-27 | Method for producing sialic-acid-containing sugar chain |
BR112013022277-8A BR112013022277B1 (pt) | 2011-03-04 | 2012-02-27 | Método para produção de cadeia de açúcar contendo ácido siálico |
JP2013503458A JP5975577B2 (ja) | 2011-03-04 | 2012-02-27 | シアル酸含有糖鎖の製造方法 |
RU2013143650/10A RU2597975C2 (ru) | 2011-03-04 | 2012-02-27 | Способы получения сахарной цепи, содержащей сиаловую кислоту |
AU2012225900A AU2012225900B2 (en) | 2011-03-04 | 2012-02-27 | Method for producing sialic-acid-containing sugar chain |
KR1020137024893A KR101906623B1 (ko) | 2011-03-04 | 2012-02-27 | 시알산 함유 당쇄의 제조방법 |
EP12754383.3A EP2682473B1 (en) | 2011-03-04 | 2012-02-27 | Method for producing sialic-acid-containing sugar chain |
US14/002,645 US9376506B2 (en) | 2011-03-04 | 2012-02-27 | Method for producing sialic-acid-containing sugar chain |
CA2828905A CA2828905C (en) | 2011-03-04 | 2012-02-27 | Method for producing sialic-acid-containing sugar chain |
CN201280011234.5A CN103502462B (zh) | 2011-03-04 | 2012-02-27 | 含有唾液酸的糖链的制造方法 |
US14/702,535 US20170240655A1 (en) | 2011-03-04 | 2015-05-01 | Method for producing sialic-acid-containing sugar chain |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2011-047378 | 2011-03-04 | ||
JP2011047378 | 2011-03-04 |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/002,645 A-371-Of-International US9376506B2 (en) | 2011-03-04 | 2012-02-27 | Method for producing sialic-acid-containing sugar chain |
US14/702,535 Division US20170240655A1 (en) | 2011-03-04 | 2015-05-01 | Method for producing sialic-acid-containing sugar chain |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2012121041A1 true WO2012121041A1 (ja) | 2012-09-13 |
Family
ID=46798009
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2012/054727 WO2012121041A1 (ja) | 2011-03-04 | 2012-02-27 | シアル酸含有糖鎖の製造方法 |
Country Status (12)
Country | Link |
---|---|
US (2) | US9376506B2 (ja) |
EP (1) | EP2682473B1 (ja) |
JP (1) | JP5975577B2 (ja) |
KR (1) | KR101906623B1 (ja) |
CN (1) | CN103502462B (ja) |
AU (1) | AU2012225900B2 (ja) |
BR (1) | BR112013022277B1 (ja) |
CA (1) | CA2828905C (ja) |
RU (1) | RU2597975C2 (ja) |
SG (1) | SG193021A1 (ja) |
TW (1) | TWI546385B (ja) |
WO (1) | WO2012121041A1 (ja) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2013147639A (ja) * | 2011-12-19 | 2013-08-01 | Kagawa Univ | 糖ペプチドの製造方法、糖アミノ酸の製造方法および糖タンパク質の製造方法 |
CN107429237A (zh) * | 2014-12-22 | 2017-12-01 | 豪夫迈·罗氏有限公司 | Cmp依赖性的唾液酸酶活性 |
US10610537B2 (en) | 2016-08-25 | 2020-04-07 | Masayoshi Ltd. | Methods of producing sialooligosaccharides and uses thereof |
WO2020096057A1 (ja) * | 2018-11-08 | 2020-05-14 | 日立化成テクノサービス株式会社 | メタボロームの分離方法 |
WO2020096056A1 (ja) * | 2018-11-08 | 2020-05-14 | 日立化成テクノサービス株式会社 | メタボローム分析用分離材及びメタボローム分析用カラム |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20160021075A (ko) * | 2013-03-14 | 2016-02-24 | 글리코비아, 인크. | 올리고사카라이드 조성물, 당단백질, 및 원핵생물에서의 이들의 제조 방법 |
US10450361B2 (en) | 2013-03-15 | 2019-10-22 | Momenta Pharmaceuticals, Inc. | Methods related to CTLA4-Fc fusion proteins |
ES2802274T3 (es) * | 2013-05-02 | 2021-01-18 | Momenta Pharmaceuticals Inc | Glicoproteínas sialiladas |
WO2014186310A1 (en) | 2013-05-13 | 2014-11-20 | Momenta Pharmaceuticals, Inc. | Methods for the treatment of neurodegeneration |
WO2015057622A1 (en) | 2013-10-16 | 2015-04-23 | Momenta Pharmaceuticals, Inc. | Sialylated glycoproteins |
KR101755430B1 (ko) * | 2014-09-25 | 2017-07-27 | 한국생명공학연구원 | 개선된 효율을 갖는 시알산전달효소를 이용한 당단백질의 당사슬에 시알산을 부가하는 방법 |
TWI757238B (zh) * | 2015-06-30 | 2022-03-11 | 日商糖鎖工學研究所股份有限公司 | 白蛋白-糖鏈複合物 |
CN110041443A (zh) * | 2019-04-24 | 2019-07-23 | 广东药科大学附属第一医院 | 多分支聚唾液酸衍生物及其制备方法 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003091431A1 (fr) | 2002-04-26 | 2003-11-06 | Kirin Beer Kabushiki Kaisha | Methylotrophe produisant une chaine de sucre de type mammifere |
WO2004058824A1 (ja) * | 2002-12-26 | 2004-07-15 | Otsuka Chemical Co., Ltd. | 3分岐型糖鎖アスパラギン誘導体、該糖鎖アスパラギン、該糖鎖およびそれらの製造方法 |
JP2009539162A (ja) | 2006-05-29 | 2009-11-12 | マイクロソフト コーポレーション | 複製時のシャドーコピーデータの保持 |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
RU2125092C1 (ru) * | 1991-10-15 | 1999-01-20 | Дзе Скриппс Рисерч Инститьют | Способ получения фукозилированного углевода, способ получения фукозилированной сиалилированной углеводной молекулы, реакционная система in vitro |
US6458574B1 (en) | 1996-09-12 | 2002-10-01 | Transkaryotic Therapies, Inc. | Treatment of a α-galactosidase a deficiency |
NZ522320A (en) * | 2000-05-12 | 2007-03-30 | Neose Technologies Inc | In vitro modification of glycosylation patterns of recombinant glycopeptides |
WO2006088017A1 (ja) * | 2005-02-16 | 2006-08-24 | National University Corporation Hokkaido University | 4位ハロゲン化ガラクトース含有糖鎖及びその応用 |
US20120100609A1 (en) * | 2009-03-27 | 2012-04-26 | Crawford Brett E | N-linked glycan biosynthesis modulators |
KR102175017B1 (ko) * | 2011-09-04 | 2020-11-05 | 가부시키가이샤 도우사 고가쿠 겐큐쇼 | 당쇄 부가 폴리펩티드 및 당해 폴리펩티드를 포함하는 의약 조성물 |
-
2012
- 2012-02-27 WO PCT/JP2012/054727 patent/WO2012121041A1/ja active Application Filing
- 2012-02-27 JP JP2013503458A patent/JP5975577B2/ja active Active
- 2012-02-27 AU AU2012225900A patent/AU2012225900B2/en not_active Ceased
- 2012-02-27 KR KR1020137024893A patent/KR101906623B1/ko active IP Right Grant
- 2012-02-27 SG SG2013066527A patent/SG193021A1/en unknown
- 2012-02-27 RU RU2013143650/10A patent/RU2597975C2/ru active
- 2012-02-27 CA CA2828905A patent/CA2828905C/en active Active
- 2012-02-27 US US14/002,645 patent/US9376506B2/en active Active
- 2012-02-27 CN CN201280011234.5A patent/CN103502462B/zh not_active Expired - Fee Related
- 2012-02-27 BR BR112013022277-8A patent/BR112013022277B1/pt not_active IP Right Cessation
- 2012-02-27 EP EP12754383.3A patent/EP2682473B1/en active Active
- 2012-03-03 TW TW101107162A patent/TWI546385B/zh active
-
2015
- 2015-05-01 US US14/702,535 patent/US20170240655A1/en not_active Abandoned
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003091431A1 (fr) | 2002-04-26 | 2003-11-06 | Kirin Beer Kabushiki Kaisha | Methylotrophe produisant une chaine de sucre de type mammifere |
WO2004058824A1 (ja) * | 2002-12-26 | 2004-07-15 | Otsuka Chemical Co., Ltd. | 3分岐型糖鎖アスパラギン誘導体、該糖鎖アスパラギン、該糖鎖およびそれらの製造方法 |
JP2009539162A (ja) | 2006-05-29 | 2009-11-12 | マイクロソフト コーポレーション | 複製時のシャドーコピーデータの保持 |
Non-Patent Citations (14)
Title |
---|
"Seikagaku Jiten", TOKYO KAGAKU DOJIN CO., LTD. |
JOZIASSE ET AL., JBC, vol. 262, 1987, pages 2025 - 2033 |
JOZIASSE, D.H. ET AL.: "Branch specificity of bovine colostrum CMP-sialic acid: Galbeta1? 4GlcNAc-R a2-6-sialyltransferase. Sialylation of bi-, tri-, and tetraantennary oligosaccharides and glycopeptides of the N-acetyllactosamine type", J.BIOL.CHEM., vol. 262, no. 5, 15 February 1987 (1987-02-15), pages 2025 - 2033, XP055126875 * |
KLEINEIDAM ET AL., GLYCOCONJ. J., vol. 14, 1997, pages 57 - 66 |
KRZEWINSKI-RECCHI ET AL., EJB, vol. 270, 2003, pages 950 - 961 |
LIU, Y.C. ET AL.: "Chemo-enzymatic synthesis of trimeric sialyl Lewisx pentadecasaccharide", CAN.J.CHEM., vol. 80, no. 6, 2002, pages 540 - 545, XP008173830 * |
MIYAZAKI T ET AL., GLYCOBIOLOGY, vol. 18, 2008, pages 187 - 194 |
TAKASHIMA ET AL., JBC, vol. 277, 2002, pages 45719 - 45728 |
TAKEUCHI ET AL., J. BIOL. CHEM., vol. 263, no. 8, 1988, pages 3657 - 63 |
THOMAS, V.H. ET AL.: "Enzymatic synthesis of N- linked oligosaccharides terminating in multiple sialyl-Lewisx and GalNAc-Lewisx determinants: clustered glycosides for studying selectin interactions", CARBOHYDR.RES., vol. 306, no. 3, 1998, pages 387 - 400, XP004156133 * |
TSUDA ET AL., EUR J BIOCHEM., vol. 188, no. 2, 1990, pages 405 - 11 |
VAN DEN EIJNDEN DH ET AL., BIOCHEM BIOPHYS RES COMMUN., vol. 92, no. 3, 1980, pages 839 - 45 |
YAMAMOTO T ET AL., BBB, vol. 62, 1998, pages 210 - 214 |
YAMAMOTO T ET AL., GLYCOBIOLOGY, vol. 17, 2007, pages 1167 - 1174 |
Cited By (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2013147639A (ja) * | 2011-12-19 | 2013-08-01 | Kagawa Univ | 糖ペプチドの製造方法、糖アミノ酸の製造方法および糖タンパク質の製造方法 |
CN107429237B (zh) * | 2014-12-22 | 2021-09-28 | 豪夫迈·罗氏有限公司 | Cmp依赖性的唾液酸酶活性 |
CN107429237A (zh) * | 2014-12-22 | 2017-12-01 | 豪夫迈·罗氏有限公司 | Cmp依赖性的唾液酸酶活性 |
JP2018500934A (ja) * | 2014-12-22 | 2018-01-18 | エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft | Cmp依存性シアリダーゼ活性 |
JP2019088306A (ja) * | 2014-12-22 | 2019-06-13 | エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft | Cmp依存性シアリダーゼ活性 |
US11788108B2 (en) | 2014-12-22 | 2023-10-17 | Roche Diagnostics Operations, Inc. | CMP-dependent sialidase activity |
US11078511B2 (en) | 2014-12-22 | 2021-08-03 | Roche Diagnostics Operations, Inc. | Aqueous composition |
US10610537B2 (en) | 2016-08-25 | 2020-04-07 | Masayoshi Ltd. | Methods of producing sialooligosaccharides and uses thereof |
WO2020096056A1 (ja) * | 2018-11-08 | 2020-05-14 | 日立化成テクノサービス株式会社 | メタボローム分析用分離材及びメタボローム分析用カラム |
JPWO2020096056A1 (ja) * | 2018-11-08 | 2021-09-30 | 昭和電工マテリアルズ・テクノサービス株式会社 | メタボローム分析用分離材及びメタボローム分析用カラム |
JPWO2020096057A1 (ja) * | 2018-11-08 | 2021-10-07 | 昭和電工マテリアルズ・テクノサービス株式会社 | メタボロームの分離方法 |
JP7341434B2 (ja) | 2018-11-08 | 2023-09-11 | 株式会社レゾナック・テクノサービス | メタボローム分析用分離材及びメタボローム分析用カラム |
JP7341435B2 (ja) | 2018-11-08 | 2023-09-11 | 株式会社レゾナック・テクノサービス | メタボロームの分離方法 |
WO2020096057A1 (ja) * | 2018-11-08 | 2020-05-14 | 日立化成テクノサービス株式会社 | メタボロームの分離方法 |
Also Published As
Publication number | Publication date |
---|---|
RU2013143650A (ru) | 2015-04-10 |
SG193021A1 (en) | 2013-09-30 |
AU2012225900A1 (en) | 2013-09-19 |
RU2597975C2 (ru) | 2016-09-20 |
EP2682473B1 (en) | 2019-09-25 |
CN103502462A (zh) | 2014-01-08 |
EP2682473A4 (en) | 2015-06-03 |
JP5975577B2 (ja) | 2016-08-23 |
BR112013022277A2 (pt) | 2018-08-21 |
AU2012225900B2 (en) | 2015-09-17 |
TW201247873A (en) | 2012-12-01 |
EP2682473A1 (en) | 2014-01-08 |
CA2828905A1 (en) | 2012-09-13 |
KR20140114279A (ko) | 2014-09-26 |
KR101906623B1 (ko) | 2018-10-10 |
BR112013022277B1 (pt) | 2021-08-03 |
JPWO2012121041A1 (ja) | 2014-07-17 |
US9376506B2 (en) | 2016-06-28 |
CN103502462B (zh) | 2016-08-10 |
CA2828905C (en) | 2021-05-04 |
US20170240655A1 (en) | 2017-08-24 |
US20140066617A1 (en) | 2014-03-06 |
TWI546385B (zh) | 2016-08-21 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP5975577B2 (ja) | シアル酸含有糖鎖の製造方法 | |
Li et al. | Strategies for chemoenzymatic synthesis of carbohydrates | |
Chao et al. | Recent progress in chemo-enzymatic methods for the synthesis of N-glycans | |
Weijers et al. | Glycosyltransferase-catalyzed synthesis of bioactive oligosaccharides | |
Palcic | Biocatalytic synthesis of oligosaccharides | |
Brockhausen | Crossroads between bacterial and mammalian glycosyltransferases | |
Blixt et al. | Efficient Preparation of Natural and Synthetic Galactosides with a Recombinant β-1, 4-Galactosyltransferase-/UDP-4 ‘-Gal Epimerase Fusion Protein | |
Yu et al. | Recent developments in enzymatic synthesis of modified sialic acid derivatives | |
Chien et al. | Sequential one-pot enzymatic synthesis of oligo-N-acetyllactosamine and its multi-sialylated extensions | |
Council et al. | Enzymatic glycosylation involving fluorinated carbohydrates | |
KOTANI et al. | Knockout of mouse β1, 4-galactosyltransferase-1 gene results in a dramatic shift of outer chain moieties of N-glycans from type 2 to type 1 chains in hepatic membrane and plasma glycoproteins | |
Nilsson | Enzymatic synthesis of complex carbohydrates and their glycosides | |
Cao et al. | General consideration on sialic acid chemistry | |
Ramakrishnan et al. | Role of a single amino acid in the evolution of glycans of invertebrates and vertebrates | |
Schoenhofen et al. | Biosynthesis of legionaminic acid and its incorporation into glycoconjugates | |
JP4910091B2 (ja) | 4位ハロゲン化ガラクトース含有糖鎖及びその応用 | |
JP5464578B2 (ja) | Ssea系新規糖鎖化合物 | |
US20170121361A1 (en) | Hydrolysis Resistant Sialic Acid Derivatives and Methods for Their Use | |
Huang | Chemoenzymatic Synthesis of Carbohydrates and Glycoconjugates | |
Xiao | Enzymatic Synthesis of Common Sugar Nucleotide and Therapeutic Oligosaccharides | |
Liu et al. | Efficient Coupling of Complex Fluorooligosaccharides to Phenolic Peptide Mediated by Calcium Iodide | |
Schachter | Biosynthesis 1. Introduction | |
Flitsch et al. | Enzymes in carbohydrate chemistry: formation of glycosidic linkages | |
Thon | Bacterial Glycosyltransferases, a Disaccharide Phosphorylase, and a Uridylyltransferase for Chemoenzymatic Synthesis of Carbohydrates | |
Martini et al. | Synthesis of oligosaccharides for glycosyltransferase characterization |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 12754383 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2828905 Country of ref document: CA |
|
ENP | Entry into the national phase |
Ref document number: 2013503458 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2012225900 Country of ref document: AU Date of ref document: 20120227 Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 20137024893 Country of ref document: KR Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2013143650 Country of ref document: RU Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 14002645 Country of ref document: US |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112013022277 Country of ref document: BR |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01E Ref document number: 112013022277 Country of ref document: BR |
|
ENP | Entry into the national phase |
Ref document number: 112013022277 Country of ref document: BR Kind code of ref document: A2 Effective date: 20130830 |