CN109134449A - Method for separating and purifying epothilone D based on molecular imprinting - Google Patents
Method for separating and purifying epothilone D based on molecular imprinting Download PDFInfo
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- CN109134449A CN109134449A CN201810809608.7A CN201810809608A CN109134449A CN 109134449 A CN109134449 A CN 109134449A CN 201810809608 A CN201810809608 A CN 201810809608A CN 109134449 A CN109134449 A CN 109134449A
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- Prior art keywords
- methanol
- epothilone
- epod
- isolating
- template molecule
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- XOZIUKBZLSUILX-SDMHVBBESA-N Epothilone D Natural products O=C1[C@H](C)[C@@H](O)[C@@H](C)CCC/C(/C)=C/C[C@@H](/C(=C\c2nc(C)sc2)/C)OC(=O)C[C@H](O)C1(C)C XOZIUKBZLSUILX-SDMHVBBESA-N 0.000 title claims abstract description 58
- XOZIUKBZLSUILX-UHFFFAOYSA-N desoxyepothilone B Natural products O1C(=O)CC(O)C(C)(C)C(=O)C(C)C(O)C(C)CCCC(C)=CCC1C(C)=CC1=CSC(C)=N1 XOZIUKBZLSUILX-UHFFFAOYSA-N 0.000 title claims abstract description 58
- XOZIUKBZLSUILX-GIQCAXHBSA-N epothilone D Chemical compound O1C(=O)C[C@H](O)C(C)(C)C(=O)[C@H](C)[C@@H](O)[C@@H](C)CCC\C(C)=C/C[C@H]1C(\C)=C\C1=CSC(C)=N1 XOZIUKBZLSUILX-GIQCAXHBSA-N 0.000 title claims abstract description 58
- 238000000034 method Methods 0.000 title claims abstract description 36
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 282
- 239000008367 deionised water Substances 0.000 claims abstract description 23
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 23
- 239000003480 eluent Substances 0.000 claims abstract description 16
- 229920000642 polymer Polymers 0.000 claims abstract description 16
- 239000012043 crude product Substances 0.000 claims abstract description 11
- 239000012535 impurity Substances 0.000 claims abstract description 6
- ZCHPKWUIAASXPV-UHFFFAOYSA-N acetic acid;methanol Chemical compound OC.CC(O)=O ZCHPKWUIAASXPV-UHFFFAOYSA-N 0.000 claims abstract description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 56
- 229920000344 molecularly imprinted polymer Polymers 0.000 claims description 31
- 239000000243 solution Substances 0.000 claims description 31
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical group CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 30
- HESCAJZNRMSMJG-KKQRBIROSA-N epothilone A Chemical class C/C([C@@H]1C[C@@H]2O[C@@H]2CCC[C@@H]([C@@H]([C@@H](C)C(=O)C(C)(C)[C@@H](O)CC(=O)O1)O)C)=C\C1=CSC(C)=N1 HESCAJZNRMSMJG-KKQRBIROSA-N 0.000 claims description 30
- 239000000178 monomer Substances 0.000 claims description 30
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 29
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 28
- 238000006116 polymerization reaction Methods 0.000 claims description 25
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 24
- 239000003431 cross linking reagent Substances 0.000 claims description 23
- 229930013356 epothilone Natural products 0.000 claims description 22
- 239000004088 foaming agent Substances 0.000 claims description 19
- 239000003999 initiator Substances 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 15
- 238000002360 preparation method Methods 0.000 claims description 14
- 238000002156 mixing Methods 0.000 claims description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- 239000011837 N,N-methylenebisacrylamide Substances 0.000 claims description 8
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 claims description 8
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical group N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 claims description 7
- 239000007864 aqueous solution Substances 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 7
- 239000012528 membrane Substances 0.000 claims description 7
- 229910052757 nitrogen Inorganic materials 0.000 claims description 7
- -1 2- methacrylic acid glycol ester Chemical class 0.000 claims description 6
- 239000004971 Cross linker Substances 0.000 claims description 6
- 239000007795 chemical reaction product Substances 0.000 claims description 6
- 238000011010 flushing procedure Methods 0.000 claims description 6
- 238000011068 loading method Methods 0.000 claims description 6
- 230000010355 oscillation Effects 0.000 claims description 6
- 230000003068 static effect Effects 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims description 5
- VVJKKWFAADXIJK-UHFFFAOYSA-N Allylamine Chemical compound NCC=C VVJKKWFAADXIJK-UHFFFAOYSA-N 0.000 claims description 4
- LOIYMIARKYCTBW-UHFFFAOYSA-N trans-urocanic acid Natural products OC(=O)C=CC1=CNC=N1 LOIYMIARKYCTBW-UHFFFAOYSA-N 0.000 claims description 4
- OMPJBNCRMGITSC-UHFFFAOYSA-N Benzoylperoxide Chemical compound C=1C=CC=CC=1C(=O)OOC(=O)C1=CC=CC=C1 OMPJBNCRMGITSC-UHFFFAOYSA-N 0.000 claims description 3
- 235000019400 benzoyl peroxide Nutrition 0.000 claims description 3
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 claims description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 2
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N ethylene glycol Natural products OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 2
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims description 2
- KCXFHTAICRTXLI-UHFFFAOYSA-N propane-1-sulfonic acid Chemical compound CCCS(O)(=O)=O KCXFHTAICRTXLI-UHFFFAOYSA-N 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 11
- 238000001179 sorption measurement Methods 0.000 abstract description 10
- 238000000926 separation method Methods 0.000 abstract description 7
- 238000000746 purification Methods 0.000 abstract description 4
- 238000002390 rotary evaporation Methods 0.000 abstract description 4
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 abstract description 2
- 238000002386 leaching Methods 0.000 abstract 1
- COTNUBDHGSIOTA-UHFFFAOYSA-N meoh methanol Chemical compound OC.OC COTNUBDHGSIOTA-UHFFFAOYSA-N 0.000 abstract 1
- 238000002791 soaking Methods 0.000 abstract 1
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 21
- 239000000047 product Substances 0.000 description 17
- 239000001963 growth medium Substances 0.000 description 13
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 12
- 229920001592 potato starch Polymers 0.000 description 12
- 230000001954 sterilising effect Effects 0.000 description 12
- HESCAJZNRMSMJG-HGYUPSKWSA-N epothilone A Natural products O=C1[C@H](C)[C@H](O)[C@H](C)CCC[C@H]2O[C@H]2C[C@@H](/C(=C\c2nc(C)sc2)/C)OC(=O)C[C@H](O)C1(C)C HESCAJZNRMSMJG-HGYUPSKWSA-N 0.000 description 11
- 238000004128 high performance liquid chromatography Methods 0.000 description 11
- 238000000855 fermentation Methods 0.000 description 10
- 230000004151 fermentation Effects 0.000 description 10
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 9
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical compound CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 9
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 235000019341 magnesium sulphate Nutrition 0.000 description 9
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 description 9
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 9
- 239000000843 powder Substances 0.000 description 9
- 238000011084 recovery Methods 0.000 description 9
- 239000011347 resin Substances 0.000 description 9
- 229920005989 resin Polymers 0.000 description 9
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 9
- 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 8
- 239000008103 glucose Substances 0.000 description 8
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 7
- 235000019796 monopotassium phosphate Nutrition 0.000 description 7
- 229920001817 Agar Polymers 0.000 description 6
- 102100029158 Consortin Human genes 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 101000771062 Homo sapiens Consortin Proteins 0.000 description 6
- 229930012538 Paclitaxel Natural products 0.000 description 6
- 239000008272 agar Substances 0.000 description 6
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 6
- 229960001592 paclitaxel Drugs 0.000 description 6
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 6
- 239000011592 zinc chloride Substances 0.000 description 6
- 241000862997 Sorangium cellulosum Species 0.000 description 5
- QXRSDHAAWVKZLJ-PVYNADRNSA-N epothilone B Chemical compound C/C([C@@H]1C[C@@H]2O[C@]2(C)CCC[C@@H]([C@@H]([C@@H](C)C(=O)C(C)(C)[C@@H](O)CC(=O)O1)O)C)=C\C1=CSC(C)=N1 QXRSDHAAWVKZLJ-PVYNADRNSA-N 0.000 description 5
- 238000001914 filtration Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 238000004445 quantitative analysis Methods 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 238000003556 assay Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000000376 reactant Substances 0.000 description 4
- 238000001291 vacuum drying Methods 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 3
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 3
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 3
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 3
- 229910021626 Tin(II) chloride Inorganic materials 0.000 description 3
- 230000000259 anti-tumor effect Effects 0.000 description 3
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 3
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 description 3
- 239000011565 manganese chloride Substances 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- AXZWODMDQAVCJE-UHFFFAOYSA-L tin(II) chloride (anhydrous) Chemical compound [Cl-].[Cl-].[Sn+2] AXZWODMDQAVCJE-UHFFFAOYSA-L 0.000 description 3
- 235000005074 zinc chloride Nutrition 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- QXRSDHAAWVKZLJ-OXZHEXMSSA-N Epothilone B Natural products O=C1[C@H](C)[C@H](O)[C@@H](C)CCC[C@@]2(C)O[C@H]2C[C@@H](/C(=C\c2nc(C)sc2)/C)OC(=O)C[C@H](O)C1(C)C QXRSDHAAWVKZLJ-OXZHEXMSSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 229940123237 Taxane Drugs 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- FBAFATDZDUQKNH-UHFFFAOYSA-M iron chloride Chemical compound [Cl-].[Fe] FBAFATDZDUQKNH-UHFFFAOYSA-M 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 102100033350 ATP-dependent translocase ABCB1 Human genes 0.000 description 1
- WXQDFOGZIYLEGP-UHFFFAOYSA-N C(C(C)C)#N.C(C(C)C)#N.[N] Chemical compound C(C(C)C)#N.C(C(C)C)#N.[N] WXQDFOGZIYLEGP-UHFFFAOYSA-N 0.000 description 1
- 238000007445 Chromatographic isolation Methods 0.000 description 1
- 108010047230 Member 1 Subfamily B ATP Binding Cassette Transporter Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- WURBFLDFSFBTLW-UHFFFAOYSA-N benzil Chemical group C=1C=CC=CC=1C(=O)C(=O)C1=CC=CC=C1 WURBFLDFSFBTLW-UHFFFAOYSA-N 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000011097 chromatography purification Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 150000003883 epothilone derivatives Chemical class 0.000 description 1
- 125000003700 epoxy group Chemical group 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 229940041033 macrolides Drugs 0.000 description 1
- 125000005395 methacrylic acid group Chemical group 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 150000002978 peroxides Chemical group 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
- C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
- C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/52—Amides or imides
- C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
- C08F220/56—Acrylamide; Methacrylamide
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F222/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical and containing at least one other carboxyl radical in the molecule; Salts, anhydrides, esters, amides, imides, or nitriles thereof
- C08F222/36—Amides or imides
- C08F222/38—Amides
- C08F222/385—Monomers containing two or more (meth)acrylamide groups, e.g. N,N'-methylenebisacrylamide
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
- C08J9/26—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a solid phase from a macromolecular composition or article, e.g. leaching out
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
The invention discloses a method for separating and purifying epothilone D based on molecular imprinting, which comprises the steps of soaking an EpoD molecular imprinting polymer in a small amount of pure methanol, filling the soaked polymer into a separation column, leaching the column by sequentially using methanol-acetic acid, methanol and methanol-deionized water, vacuumizing and compacting the column; dissolving the EpoD crude product in 20-80% methanol water solution, and adding into a packed column; standing for 20-50min, eluting the column, removing interferents, and further reducing impurity interference; eluting the column by using 10-90% methanol PBS solution as eluent to completely dissolve EpoD in the eluent, collecting the eluent, performing rotary evaporation to dryness, and re-dissolving pure methanol to obtain a purified EpoD methanol solution. Compared with the existing separation and purification method, the method has the advantages of simple and convenient operation and low cost, and simultaneously, the method is specific adsorption in the epothilone D adsorption process, and can achieve better separation effect. Provides possibility for further separation and purification of epothilone D.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of side that Epothilone D is isolated and purified based on molecular engram
Method.
Background technique
Epothilones (epothilone) is a kind of macrolides compound, by German National biotechnology center
(GBF) G. H fle et al. was reported for the first time in 1993.It can be with from the Sorangium cellulosum strain fermentation liquid of slime bacteria suborder
Epothilones is separated, main component is Epothilone A and B.The bioactivity of ebomycin A, B and D to cancer cell
Suitable or even stronger with taxol, especially epothilone B, activity 10-100 times stronger than taxol is on the position C12
After having methyl substitution and C12-C13 double bond to change epoxy group into, the activity of Epothilones will be greatly improved.Epothilones
With antitumor properties more superior than taxol: 1. Epothilones is better than taxol soluble, and structure is simple, is conducive to chemical conjunction
At Epothilones and structure derivatization.2. Epothilones does not have taxol intracellular toxin active.3. it is different from taxol, angstrom
Rich mycin also maintains very big cytotoxicity in the multidrug resistant cells of P P-glycoprotein expression type.
Epothilones has antitumor properties more superior than taxanes, in the research of substitution taxanes drug
Great potentiality are shown, and have shown its huge value in antitumor field.But in its production process
Higher cost has become people's urgent problem.In addition to it produces bacterium low output and growth cycle length, easy infection etc.
Factor, there is also very big factors in terms of the separating-purifying of Epothilones.Because of the expression product ingredient of sorangium cellulosum
Complicated multiplicity, the difficulty that the Epothilones of significant increase isolates and purifies.
Mainly have about the method that Epothilones isolates and purifies at present: sephadex combination PHPLC isolates and purifies road
Line, purity is up to 90% or more, but the rate of recovery is lower, and only 75% or so, and filler is at high cost, operates relative complex；C18
The reversed column chromatographic isolation and purification route of normal pressure, for the rate of recovery up to 80%, purity is up to 85%, filler higher cost, and recycles
Rate is lower；Ion-exchange chromatography isolates and purifies route, and the rate of recovery is up to 90%, but purity 60% is much not achieved and actually answers
It is required that.Therefore in order to reach the demand of actual production and application, in the research that Epothilones isolates and purifies also
Many problem needs overcome.
Summary of the invention
The present invention provides a kind of method that Epothilone D isolates and purifies, relative to above-mentioned isolation and purification method, operation letter
Singly, conveniently, at low cost, while being specific adsorption in Epothilone D adsorption process, better separating effect can be reached.
In order to achieve the above object, the technological means that the present invention uses is as follows:
A method of Epothilone D is isolated and purified based on molecular engram, is included the following steps:
1) preparation of epothilone D molecular imprinted polymer: taking 0.1mmol template molecule, 0.1-1.0mmol function monomer,
After the mixing of 0.5-3.0mmol crosslinking agent, the static 1-2h of 50mL pore-foaming agent is added, 30mg initiator, sonic oscillation 20min is added
Afterwards, it is passed through nitrogen 10-20min immediately；Then the polymerization reaction 10-24 h at 40-55 DEG C, then the polymerization reaction at 55-65 DEG C
10-24h ；Reaction product is crushed after the completion of polymerization reaction, be ground up, sieved after obtain the polymerization reaction that partial size is 40-60 μm
Object, then with 200mL acetic acid/methyl alcohol mixed liquor continuous flushing for several times after, the washing of pure methanol for several times, is dried in vacuo to obtain Epothilones point
Sub- imprinted polymer；
2) molecularly imprinted polymer splitter loads: a small amount of pure methanol of EpoD molecularly imprinted polymer being impregnated, 20ml is packed into
In splitter (bottom adds filter membrane), pillar successively is eluted using methanol-acetic acid, methanol, methanol-deionized water, vacuumizes compacting
Pillar；
3) it loading: takes the EpoD crude product of 2mg to be dissolved in the 20-80% methanol aqueous solution of 10-20ml, is added in the pillar installed；
4) it adsorbs, elute: after standing 20-50min, eluting pillar using or mixtures thereof deionized water, methanol, acetonitrile, acetic acid,
Chaff interferent is removed, impurity interference is further reduced；
5) it elutes: using the methanol PBS solution of 10-90% as elution pillar, so that EpoD is dissolved completely in eluent
It is interior, it collects eluent and rotation is evaporated, pure methanol redissolves, the EpoD methanol solution purified.
Template molecule described in step 1) is one or more of ebomycin A ~ D mixing.Preferably Epothilone D
The mixture mixed with ebomycin A with the ratio of 1:1.
Function monomer described in step 1) is acrylic acid, methacrylic acid, acrylamide, allylamine or 2- acrylamide
At least one of base -2- methyl propane sulfonic acid.
Crosslinking agent is 2- methacrylic acid glycol ester, 4- urocanic acid ethyl ester, N, N- di-2-ethylhexylphosphine oxide third in step 1)
At least one of acrylamide or 4- urocanic acid.
Initiator is azodiisobutyronitrile or dibenzoyl peroxide in step 1)；Pore-foaming agent be ethyl acetate, acetone or
The two mixture.
The volume ratio of methanol and acetic acid is 10:1 in methanol-acetic acid solution in step 2；First in methanol-deionized water solution
The volume ratio of alcohol and deionized water is 1:1.
Methanol aqueous solution concentration is 60% in step 3).
The methanol PBS solution (including 80mL methanol, 20mLPBS in 100mL eluent) that eluent is 80% in step 5).
It is further preferred that template molecule and crosslinker ratio are 1:20 in step 1), template molecule and function monomer
Ratio is 1:6.
Bacterial strain (Shanghai grind life) of the experiment Epothilone D Producing Strain used from purchase.Pass through EpoD molecular engram
The specific adsorption of polymer is to make the EpoD in fermentation liquid reach good separating effect.
Epothilone D crude product to be separated can be that any strain fermentation is resulting comprising Epothilone D in the present invention
Fermentation liquid, or the thick solution etc. comprising Epothilone D of any chemical method preparation.
The present invention illustrates the separation method of Epothilone D by taking the fermentation liquid that is prepared of fermenting as an example:
Sorangium cellulosum strain is inoculated on the CNST plate for being placed with sterilizing filter paper piece, cultivates 4d, be transferred to M26 culture medium
On continue cultivate 3d after seed liquor is made, and by it according to 10%(V/V) be inoculated into the fermented and cultured containing 5% XAD-16 resin
In base, resin is collected by filtration after 28 DEG C of 4 ~ 6d of culture；The resin being collected into is placed in the methanol solution of 75 % of PH 6.0,
And 2 h are parsed in 200r/min on shaking table.Desorbed solution rotary evaporation is collected to a small amount of, is dried under vacuum to dry product, it is multiple with pure methanol
Molten, 8000r/min is centrifuged 10min, filters, and it is simultaneously stored refrigerated spare by 4 DEG C of crude product that HPLC detects Epothilone D content.
Molecularly imprinted polymer splitter loads in the present invention, specifically: weigh the polymerization of 400.0 mg EpoD molecular engrams
Object, a small amount of pure methanol impregnate, and are packed into 20ml syringe (bottom adds filter membrane), successively use 40ml methanol/acetic acid (10:1),
40ml methanol, 40ml methanol/deionized water (1:1) elute pillar, vacuumize compacting pillar.
Epothilone D assay in the present invention: HPLC quantitative analysis is used, testing conditions: chromatographic column, C18,10 μm,
4.6 x 250mm；UV detector；Mobile phase, methanol (chromatographically pure): deionized water=3:1；Flow velocity is 1.0mL/min；Detect wave
A length of 249nm；Sample volume is 20 μ L；Column temperature is 25 DEG C.
Pore-foaming agent is ethyl acetate, acetone or both mixture (V:V=1:1,1:3,2:3,3:2,3:1).
The culture medium prescription involved in the present invention arrived are as follows: CNST culture medium: KN030.5g/L, Na2HP04 0.25g/L,
MgSO41.0g/L, FeCl30.01g/L, TE solution 1.0ml/L, agar 16g/L, pH7.2；Wherein the group of TE solution becomes
MnCl2·4H2O 100mg/L, CoCl2 20mg/L, CuS0410mg/L, Na2Mo04·2H20 10mg/L, ZnCl2 20mg/
L, LiCl 5mg/L, SnCl2·2H20 5mg/L, H3BO3 10mg/L, KBr 20mg/L, KI 20mg/L；
M26 culture medium: epsom salt 0.05g/L, manganese sulfate monohydrate 0.05g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate
2g/L, NaCL5g/L, yeast powder 2g/L, glucose 15g/L, soy peptone 10g/L, potato starch 10g/L, agar 15g/
L, remaining is water, and pH is adjusted to 7.2,115 DEG C of sterilizing 30min；
Seed culture medium: epsom salt 0.05g/L, manganese sulfate monohydrate 0.05g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate
2g/L, NaCL5g/L, yeast powder 2g/L, glucose 15g/L, soy peptone 10g/L, potato starch 10g/L, remaining is
Water, pH are adjusted to 7.2,115 DEG C of sterilizing 30min；
Fermentation medium: epsom salt 0.05g/L, manganese sulfate monohydrate 0.05g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate
2g/L, NaCL10g/L, zinc chloride 0.01g/L, ferric trichloride 0.01g/L, cobalt chloride 0.01g/L, yeast powder 5g/L, grape
Sugared 30g/L, soy peptone 15g/L, potato starch 10g/L, potato starch 10g/L, cellulose 1g/L, remaining is water, pH
It is adjusted to 7.6,115 DEG C of sterilizing 30min.
Its principle of the separation method of Epothilones according to the present invention is: according to obtained molecularly imprinted polymer pair
The specific adsorption of Epothilones reaches separation other impurities, reaches Epothilones sterling finally by eluent.
The utility model has the advantages that
The present invention provides a kind of method that Epothilone D isolates and purifies, relative to existing isolation and purification method, operation letter
Singly, conveniently, at low cost, while being specific adsorption in Epothilone D adsorption process, better separating effect can be reached.For
Further Epothilone D isolates and purifies offer possibility.
Specific embodiment
According to following embodiments, the present invention may be better understood.
Embodiment 1
The present invention provides a kind of method that template molecule is selected, and specific step is as follows
Template molecule includes EpoA, EpoB, EpoD and its two or more 1:1 mixing, 0.1mmol template molecule is taken, by template
Molecule, function monomer and crosslinking agent arrange mixing according to the molar ratio of 1:4:20, and 50ml pore-foaming agent is added and polymerize under certain condition
Polymer is made.Polymer obtained by above-mentioned different templates molecule is weighed into 40mg in 10ml centrifuge tube, is separately added into 8ml
The EpoD that concentration is 50mg/L marks product and is adsorbed, and measures the adsorbance A of EpoD.The polymer that template molecule is not added is control group
EpoD is adsorbed, adsorbance B is measured.It is worth size to determine that (the bigger explanation of A/B value adds optimal Template molecule as reference standard using A/B
Add the resulting polymer of the template molecule better to the adsorption effect of EpoD).
It is involved in the present invention to EpoA, B, D be purchase 99% purity rubric product (Hubei prestige is got profit).Institute of the present invention
In the preparation method for the epothilone D molecular imprinted polymer being related to, function monomer is acrylamide；Crosslinking agent is N, N-
Methylene-bisacrylamide；Initiator is azodiisobutyronitrile；Its pore-foaming agent be both ethyl acetate, acetone mixture (V:V=
3:2).
This example demonstrates that A/BEpoA:EpoB=1.504、A/BEpoB:EpoD =1.537、A/BEpoD:EpoA =1.612、A/BEpoB
=1.415、A/BEpoA =1.436、A/BEpoD=1.493、A/BEpoA:EpoB:EpoD=1.562, so selection EpoD:EpoA=(1:1)
It is best as template molecule effect.
Embodiment 2
The present invention provides a kind of method that template molecule is determined with crosslinker ratio, the specific steps are as follows:
On the basis of embodiment 1, template molecule additional amount is 0.1mmol, and function monomer additional amount is 0.4mmol, template point
The molar ratio of son and crosslinking agent is 1:5,1:10,1:15,1:20,1:25 and 1:30.50ml pore-foaming agent is added in certain condition
It is lower to polymerize obtained polymer.Polymer obtained by above-mentioned different templates molecule is weighed into 40mg in 10ml centrifuge tube, is distinguished
The EpoD mark product that 8ml concentration is 50mg/L are added to be adsorbed, the adsorbance A of EpoD is measuredTemplate molecule: crosslinking agent.Template molecule is not added
Polymer be control group adsorb EpoD, measure adsorbance BTemplate molecule: crosslinking agent.It is worth size to determine most preferably as reference standard using A/B
Template molecule (A/BTemplate molecule: crosslinking agentIt is worth suction of the resulting polymer to EpoD under bigger explanation template molecule and crosslinker ratio
Attached effect is better).
It is involved in the present invention to EpoA, B, D be the homemade 99% purity rubric product in laboratory.
It is involved in the present invention to epothilone D molecular imprinted polymer preparation method in, function monomer is acryloyl
Amine；Crosslinking agent is N,N methylene bis acrylamide；Initiator is azodiisobutyronitrile；Its pore-foaming agent is ethyl acetate, acetone
The two mixture (V:V=3:2).
This example demonstrates that when template molecule and crosslinker ratio are 1:20, A/BTemplate molecule: crosslinking agent=1:20=1.642 be maximum
It is worth, A/B under remaining ratioTemplate molecule: crosslinking agent=x:y< 1.642 so select template molecule and crosslinker ratio obtained poly- for 1:20
It is best to close object function and effect.
Embodiment 3
The present invention provides the method for a kind of template molecule and function monomer ratio-dependent, the specific steps are as follows:
On the basis of embodiment 1,2, template molecule additional amount is 0.1mmol, function monomer additional amount is respectively 0.2,0.4,
0.6,0.8 and 1.0mmol, the additional amount of crosslinking agent are 2mmol.50ml pore-foaming agent is added and polymerize obtained gather under certain condition
Close object.Polymer obtained by above-mentioned different templates molecule, which is weighed 40mg in 10ml centrifuge tube, being separately added into 8ml concentration, is
The EpoD mark product of 50mg/L are adsorbed, and the adsorbance A of EpoD is measuredTemplate molecule: function monomer.The polymer that template molecule is not added is pair
According to a group absorption EpoD, adsorbance B is measuredTemplate molecule: function monomer.It is worth size to determine optimal Template molecule (A/ as reference standard using A/B
BTemplate molecule: function monomerIt is worth and bigger illustrates resulting polymer under the template molecule and function monomer ratio to the adsorption effect of EpoD more
It is good).
It is involved in the present invention to EpoA, D be the homemade 99% purity rubric product in laboratory.
It is involved in the present invention to epothilone D molecular imprinted polymer preparation method in, function monomer is acryloyl
Amine；Crosslinking agent is N,N methylene bis acrylamide；Initiator is azodiisobutyronitrile；Its pore-foaming agent is ethyl acetate, acetone
The two mixture (V:V=3:2).
This example demonstrates that when template molecule and function monomer ratio are 1:8, A/BTemplate molecule: function monomer=1:8=1.601 is most
Big value.As ratio increases A/BTemplate molecule: function monomerReduce, it may be possible to which function monomer polymerize itself, so selection template point
Son: function monomer=1:6 optimal combination.
Embodiment 4
The present invention provides a kind of method that sample solution concentration determines, specific step is as follows
1) prepared by Epothilone D mark product: the EpoD that the homemade purity in laboratory is 99% being weighed 15mg respectively and is dissolved in 15ml concentration
For 0,20,40,60,80 and 100% methanol aqueous solution.
2) preparation of epothilone D molecular imprinted polymer: taking 0.1mmol template molecule, 0.8mmol function monomer,
After the mixing of 2mmol crosslinking agent, the static 2h of 50mL pore-foaming agent is added, 30mg initiator is added.After sonic oscillation 20min, lead to immediately
Enter nitrogen 20min；Then 12 h of polymerization reaction at 45 DEG C, then the polymerization reaction 12h at 60 DEG C；It will after the completion of polymerization reaction
Reaction product is crushed, be ground up, sieved after obtain the polymerization reactant that partial size is 40-60 μm.200mL acetic acid/methanol (2:5) is used again
Mixed liquor continuous flushing for several times after, the washing of pure methanol for several times, vacuum drying both Epothilones molecularly imprinted polymer.
3) molecularly imprinted polymer splitter loads: weighing 400.0 mg EpoD molecularly imprinted polymers, a small amount of pure methanol
It impregnates, is packed into 20ml syringe (bottom adds filter membrane), successively uses 40ml methanol/acetic acid (10:1), 40ml methanol, 40ml first
Alcohol/deionized water (1:1) elutes pillar, vacuumizes compacting pillar.
4) loading: the EpoD mark product for taking 10ml step 1) to configure are dissolved in pillar.
5) Epothilone D content in efflux: HPLC quantitative analysis is measured, testing conditions: chromatographic column, C18,10 μm, 4.6
x 250mm；UV detector；Mobile phase, methanol (chromatographically pure): deionized water=3:1；Flow velocity is 1.0mL/min；Detection wavelength is
249nm；Sample volume is 20 μ L；Column temperature is 25 DEG C.
6) calculate the reserved of EpoD in column: (EpoD amount in total EpoD amount-efflux)/total EpoD 100%=EpoD of amount * is protected
Allowance.
It is involved in the present invention to EpoA, D be the homemade 99% purity rubric product in laboratory.
It is involved in the present invention to epothilone D molecular imprinted polymer preparation method in, function monomer is acryloyl
Amine；Crosslinking agent is N,N methylene bis acrylamide；Initiator is azodiisobutyronitrile；Its pore-foaming agent is ethyl acetate, acetone
The two mixture (V:V=3:2).
The experimental results showed that EpoD reserved is 89.47% when methanol concentration is 60%, it is maximum value；Methanol concentration is
When 0%, EpoD reserved is 77.25%；When methanol concentration is 20%, EpoD reserved is 80.10%；When methanol concentration is 40%,
EpoD reserved is 81.97%；When methanol concentration is 80%, EpoD reserved is 72.39%；When methanol concentration is 100%, EpoD is protected
Allowance is 49.67%.So selecting concentration is 60% methanol as sample solution.
Embodiment 5
The present invention provides a kind of method that best leacheate condition determines, the specific steps are as follows:
1) prepared by Epothilone D mark product product: the EpoD that purity is 99% being weighed 15mg and is dissolved in the methanol-water that 15ml concentration is 60%
Solution.
2) preparation of epothilone D molecular imprinted polymer: taking 0.1mmol template molecule, 0.8mmol function monomer,
After the mixing of 2mmol crosslinking agent, the static 2h of 50mL pore-foaming agent is added, 30mg initiator is added.After sonic oscillation 20min, lead to immediately
Enter nitrogen 20min；Then 12 h of polymerization reaction at 45 DEG C, then the polymerization reaction 12h at 60 DEG C；It will after the completion of polymerization reaction
Reaction product is crushed, be ground up, sieved after obtain the polymerization reactant that partial size is 40-60 μm.200mL acetic acid/methanol (2:5) is used again
Mixed liquor continuous flushing for several times after, the washing of pure methanol for several times, vacuum drying both Epothilones molecularly imprinted polymer.
3) molecularly imprinted polymer splitter loads: weighing 400.0 mg EpoD molecularly imprinted polymers, a small amount of pure methanol
It impregnates, is packed into 20ml syringe (bottom adds filter membrane), successively uses 40ml methanol/acetic acid (10:1), 40ml methanol, 40ml first
Alcohol/deionized water (1:1) elutes pillar, vacuumizes compacting pillar.
4) loading: the EpoD mark product for taking 10ml step 1) to configure are dissolved in pillar.
5) it adsorbs, elute: after standing 30min, using deionized water, methanol, acetonitrile, acetic acid/methanol 1:7 and first respectively
Alcohol/acetonitrile 5:3 is eluted pillar 3 times, is detected the content of EpoD in efflux after a certain period of time, is determined best leacheate.
6) Epothilone D assay: HPLC quantitative analysis, testing conditions: chromatographic column, C18,10 μm, 4.6 x
250mm；UV detector；Mobile phase, methanol (chromatographically pure): deionized water=3:1；Flow velocity is 1.0mL/min；Detection wavelength is
249nm；Sample volume is 20 μ L；Column temperature is 25 DEG C.
7) calculate the reserved of EpoD in column: (EpoD amount in total EpoD amount-efflux)/total EpoD 100%=EpoD of amount * is protected
Allowance.
It is involved in the present invention to EpoA, D be 99% purity rubric product.
It is involved in the present invention to epothilone D molecular imprinted polymer preparation method in, function monomer is acryloyl
Amine；Crosslinking agent is N,N methylene bis acrylamide；Initiator is azodiisobutyronitrile；Its pore-foaming agent is ethyl acetate, acetone
The two mixture (V:V=3:2).
The experimental results showed that EpoD reserved is 87.69% when leacheate is deionized water, it is maximum value；It is eluting
When liquid is methanol, EpoD reserved is 85.72%；When leacheate is acetonitrile, EpoD reserved is 48.35%；It is in leacheate
When methanol/acetonitrile 5:3, EpoD reserved is 65.61%；When leacheate is acetic acid/methanol 1:7, EpoD reserved is
41.35%.So selecting deionized water as leacheate.
Embodiment 6
The present invention provides a kind of method that best eluent condition determines, specific step is as follows
On the basis of embodiment 5, the methanol PBS solution that concentration is 10,20,30,40,50,60,70,80 and 90% is continuously added
As elution pillar 3 times, so that EpoD is dissolved completely in eluent, collects eluent and rotation is evaporated, pure methanol
It redissolves, HPLC detects the amount of EpoD.Calculate the rate of recovery.
The experimental results showed that when methanol PBS solution concentration is 80%, the rate of recovery 86.79% is maximum value；Methanol PBS
When solution concentration is 70%, the rate of recovery 76.28%；When methanol PBS solution concentration is 90%, the rate of recovery 74.51%.So selection
Methanol PBS solution concentration is 80% as best eluate concentration.
Embodiment 7
The present invention provides a kind of method that Epothilone D isolates and purifies, the specific steps are as follows:
1) prepared by Epothilone D crude product: life is ground in the Shanghai slime bacteria sorangium cellulosum ATCCC 255322() it is inoculated in and is placed with
On the CNST plate of sterilizing filter paper piece, cultivate 4d, be transferred on M26 culture medium and continue to be made seed liquor after cultivating 3d, and by its
According to 10%(V/V) it is inoculated into the fermentation medium containing 5% XAD-16 resin, resin is collected by filtration after 28 DEG C of 4 ~ 6d of culture.
The resin being collected into is placed in the methanol solution of 75 % of PH 6.0, and parses 2 h in 200r/min on shaking table.Collect solution
Liquid rotary evaporation is analysed to a small amount of, is dried under vacuum to dry product, is redissolved with pure methanol, 8000r/min is centrifuged 10min, filtering, HPLC inspection
Survey Epothilone D content is simultaneously stored refrigerated spare by 4 DEG C of crude product.
2) preparation of epothilone D molecular imprinted polymer: taking 0.1mmol template molecule, 0.8mmol function monomer,
After the mixing of 2.0mmol crosslinking agent, the static 2h of 50mL pore-foaming agent is added, 30mg initiator is added.After sonic oscillation 20min, immediately
It is passed through nitrogen 20min；Then 12 h of polymerization reaction at 55 DEG C, then the polymerization reaction 12h at 65 DEG C；After the completion of polymerization reaction
To obtain partial size be 40-60 μm of polymerization reactant after reaction product is crushed, is ground up, sieved.Again with 200mL acetic acid/methanol (2:
5) mixed liquor continuous flushing for several times after, the washing of pure methanol for several times, vacuum drying both Epothilones molecularly imprinted polymer.
3) molecularly imprinted polymer splitter loads: weighing 400.0 mg EpoD molecularly imprinted polymers, a small amount of pure methanol
It impregnates, is packed into 20ml syringe (bottom adds filter membrane), successively uses 40ml methanol/acetic acid (10:1), 40ml methanol, 40ml first
Alcohol/deionized water (1:1) elutes pillar, vacuumizes compacting pillar.
4) it loading: takes the EpoD crude product of 2mg to be dissolved in 60% methanol aqueous solution of 20ml, is added in the pillar installed.
5) it adsorbs, elute: after standing 30min, eluting column using or mixtures thereof deionized water, methanol, acetonitrile, acetic acid
Son removes chaff interferent, is further reduced impurity interference.
6) it elutes: using 80% methanol PBS solution as elution pillar, so that EpoD is dissolved completely in elution
In liquid, collects eluent and rotation is evaporated, pure methanol redissolves, and HPLC detects the amount of EpoD.
7) Epothilone D assay: HPLC quantitative analysis, testing conditions: chromatographic column, C18,10 μm, 4.6 x
250mm；UV detector；Mobile phase, methanol (chromatographically pure): deionized water=3:1；Flow velocity is 1.0mL/min；Detection wavelength is
249nm；Sample volume is 20 μ L；Column temperature is 25 DEG C.
It is involved in the present invention to epothilone D molecular imprinted polymer preparation method in, template molecule be angstrom win it is mould
Plain A and D 1:1 mixing；Function monomer is acrylamide；Crosslinking agent is N,N methylene bis acrylamide；Initiator is peroxide
Change dibenzoyl；Its pore-foaming agent is both ethyl acetate, acetone mixture (V:V=3:2).
The culture medium prescription involved in the present invention arrived are as follows: CNST culture medium: KN030.5g/L, Na2HP04 0.25g/L,
MgSO41.0g/L, FeCl30.01g/L, TE solution 1.0ml/L, agar 16g/L, pH7.2；Wherein the group of TE solution becomes
MnCl2·4H2O 100mg/L, CoCl2 20mg/L, CuS0410mg/L, Na2Mo04·2H20 10mg/L, ZnCl2 20mg/
L, LiCl 5mg/L, SnCl2·2H20 5mg/L, H3BO3 10mg/L, KBr 20mg/L, KI 20mg/L；M26 culture
Base: epsom salt 0.05g/L, manganese sulfate monohydrate 0.05g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 2g/L, NaCL5
G/L, yeast powder 2g/L, glucose 15g/L, soy peptone 10g/L, potato starch 10g/L, agar 15g/L, remaining is water,
PH is adjusted to 7.2,115 DEG C of sterilizing 30min；Seed culture medium: epsom salt 0.05g/L, manganese sulfate monohydrate 0.05g/L, phosphoric acid
Potassium dihydrogen 1g/L, dipotassium hydrogen phosphate 2g/L, NaCL5g/L, yeast powder 2g/L, glucose 15g/L, soy peptone 10g/L,
Potato starch 10g/L, remaining is water, and pH is adjusted to 7.2,115 DEG C of sterilizing 30min；Fermentation medium: epsom salt 0.05g/L,
Manganese sulfate monohydrate 0.05g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 2g/L, NaCL10g/L, zinc chloride 0.01g/L, three
Iron chloride 0.01g/L, cobalt chloride 0.01g/L, yeast powder 5g/L, glucose 30g/L, soy peptone 15g/L, potato starch
10g/L, potato starch 10g/L, cellulose 1g/L, remaining is water, and pH is adjusted to 7.6,115 DEG C of sterilizing 30min.
The present embodiment result is that the rate of recovery of EpoD is 89.75%, purity 95.99%.The result shows that the present invention can be
Further Epothilone D isolates and purifies offer accurately scientific basis.
Embodiment 8
The present invention provides a kind of method that Epothilone D isolates and purifies, and specific step is as follows
1) prepared by Epothilone D crude product: life is ground in the Shanghai slime bacteria sorangium cellulosum ATCCC 255322() it is inoculated in and is placed with
On the CNST plate of sterilizing filter paper piece, cultivate 4d, be transferred on M26 culture medium and continue to be made seed liquor after cultivating 3d, and by its
According to 10%(V/V) it is inoculated into the fermentation medium containing 5% XAD-16 resin, resin is collected by filtration after 28 DEG C of 4 ~ 6d of culture.
The resin being collected into is placed in the methanol solution of 75 % of PH 6.0, and parses 2 h in 200r/min on shaking table.Collect solution
Liquid rotary evaporation is analysed to a small amount of, is dried under vacuum to dry product, is redissolved with pure methanol, 8000r/min is centrifuged 10min, filtering, HPLC inspection
Survey Epothilone D content is simultaneously stored refrigerated spare by 4 DEG C of crude product.
2) preparation of epothilone D molecular imprinted polymer: taking 0.1mmol template molecule, 0.8mmol function monomer,
After the mixing of 2.0mmol crosslinking agent, the static 2h of 50mL pore-foaming agent is added, 30mg initiator is added.After sonic oscillation 20min, immediately
It is passed through nitrogen 20min；Then 12 h of polymerization reaction at 45 DEG C, then the polymerization reaction 12h at 60 DEG C；After the completion of polymerization reaction
To obtain partial size be 40-60 μm of polymerization reactant after reaction product is crushed, is ground up, sieved.Again with 200mL acetic acid/methanol (2:
5) mixed liquor continuous flushing for several times after, the washing of pure methanol for several times, vacuum drying both Epothilones molecularly imprinted polymer.
3) molecularly imprinted polymer splitter loads: weighing 400.0 mg EpoD molecularly imprinted polymers, a small amount of pure methanol
It impregnates, is packed into 20ml syringe (bottom adds filter membrane), successively uses 40ml methanol/acetic acid (10:1), 40ml methanol, 40ml first
Alcohol/deionized water (1:1) elutes pillar, vacuumizes compacting pillar.
4) it loading: takes the EpoD crude product of 2mg to be dissolved in 60% methanol aqueous solution of 20ml, is added in the pillar installed.
5) it adsorbs, elute: after standing 30min, eluting column using or mixtures thereof deionized water, methanol, acetonitrile, acetic acid
Son removes chaff interferent, is further reduced impurity interference.
6) it elutes: using 80% methanol PBS solution as elution pillar, so that EpoD is dissolved completely in elution
In liquid, collects eluent and rotation is evaporated, pure methanol redissolves, and HPLC detects the amount of EpoD.
7) Epothilone D assay: HPLC quantitative analysis, testing conditions: chromatographic column, C18,10 μm, 4.6 x
250mm；UV detector；Mobile phase, methanol (chromatographically pure): deionized water=3:1；Flow velocity is 1.0mL/min；Detection wavelength is
249nm；Sample volume is 20 μ L；Column temperature is 25 DEG C.
It is involved in the present invention to epothilone D molecular imprinted polymer preparation method in, template molecule be angstrom win it is mould
Plain A and D 1:1 mixing；Function monomer is methacrylic acid；Crosslinking agent is N,N methylene bis acrylamide；Initiator is even
Nitrogen bis-isobutyronitrile or dibenzoyl peroxide；Its pore-foaming agent is ethyl acetate, acetone or both mixture (V:V=2:3).
The culture medium prescription involved in the present invention arrived are as follows: CNST culture medium: KN030.5g/L, Na2HP04 0.25g/L,
MgSO41.0g/L, FeCl30.01g/L, TE solution 1.0ml/L, agar 16g/L, pH7.2；Wherein the group of TE solution becomes
MnCl2·4H2O 100mg/L, CoCl2 20mg/L, CuS0410mg/L, Na2Mo04·2H20 10mg/L, ZnCl2 20mg/
L, LiCl 5mg/L, SnCl2·2H20 5mg/L, H3BO3 10mg/L, KBr 20mg/L, KI 20mg/L；M26 culture
Base: epsom salt 0.05g/L, manganese sulfate monohydrate 0.05g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 2g/L, NaCL5
G/L, yeast powder 2g/L, glucose 15g/L, soy peptone 10g/L, potato starch 10g/L, agar 15g/L, remaining is water,
PH is adjusted to 7.2,115 DEG C of sterilizing 30min；Seed culture medium: epsom salt 0.05g/L, manganese sulfate monohydrate 0.05g/L, phosphoric acid
Potassium dihydrogen 1g/L, dipotassium hydrogen phosphate 2g/L, NaCL5g/L, yeast powder 2g/L, glucose 15g/L, soy peptone 10g/L,
Potato starch 10g/L, remaining is water, and pH is adjusted to 7.2,115 DEG C of sterilizing 30min；Fermentation medium: epsom salt 0.05g/L,
Manganese sulfate monohydrate 0.05g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 2g/L, NaCL10g/L, zinc chloride 0.01g/L, three
Iron chloride 0.01g/L, cobalt chloride 0.01g/L, yeast powder 5g/L, glucose 30g/L, soy peptone 15g/L, potato starch
10g/L, potato starch 10g/L, cellulose 1g/L, remaining is water, and pH is adjusted to 7.6,115 DEG C of sterilizing 30min.
The present embodiment result is that the rate of recovery of EpoD is 87.49%, purity 95.49%.The result shows that the present invention can reach
To a possibility that testing is repeated, is isolated and purified for further Epothilone D and accurately scientific basis is provided.
Claims (10)
1. a kind of method for isolating and purifying Epothilone D based on molecular engram, which comprises the steps of:
1) preparation of epothilone D molecular imprinted polymer: taking 0.1mmol template molecule, 0.1-1.0mmol function monomer,
After the mixing of 0.5-3.0mmol crosslinking agent, the static 1-2h of 50mL pore-foaming agent is added, 30mg initiator, sonic oscillation 20min is added
Afterwards, it is passed through nitrogen 10-20min immediately；Then the polymerization reaction 10-24 h at 40-55 DEG C, then the polymerization reaction at 55-65 DEG C
10-24h ；Reaction product is crushed after the completion of polymerization reaction, be ground up, sieved after obtain the polymerization reaction that partial size is 40-60 μm
Object, then with 200mL acetic acid/methyl alcohol mixed liquor continuous flushing for several times after, the washing of pure methanol for several times, is dried in vacuo to obtain Epothilones point
Sub- imprinted polymer；
2) molecularly imprinted polymer splitter loads: a small amount of pure methanol of EpoD molecularly imprinted polymer being impregnated, bottom is packed into
Add in the splitter of filter membrane, successively elutes pillar using methanol-acetic acid, methanol, methanol-deionized water, vacuumize compacting pillar；
3) loading: EpoD crude product is dissolved in 20-80% methanol aqueous solution, is added in the pillar installed；
4) it adsorbs, elute: after standing 20-50min, eluting pillar using or mixtures thereof deionized water, methanol, acetonitrile, acetic acid,
Chaff interferent is removed, impurity interference is further reduced；
5) it elutes: using the methanol PBS solution (methanol and PBS are mixed by certain volume) of 10-90% as elution column
Son collects eluent and rotation is evaporated, pure methanol redissolves, the EpoD purified so that EpoD is dissolved completely in eluent
Methanol solution.
2. a kind of method for isolating and purifying Epothilone D based on molecular engram according to claim 1, which is characterized in that
Template molecule described in step 1) is one or more of ebomycin A ~ D mixing.
3. a kind of method for isolating and purifying Epothilone D based on molecular engram according to claim 1, which is characterized in that
Function monomer described in step 1) is acrylic acid, methacrylic acid, acrylamide, allylamine or 2- acrylamido -2- methyl
At least one of propane sulfonic acid.
4. a kind of method for isolating and purifying Epothilone D based on molecular engram according to claim 1, which is characterized in that
In step 1) crosslinking agent be 2- methacrylic acid glycol ester, 4- urocanic acid ethyl ester, N,N methylene bis acrylamide or
At least one of 4- urocanic acid.
5. a kind of method for isolating and purifying Epothilone D based on molecular engram according to claim 1, which is characterized in that
Initiator is azodiisobutyronitrile or dibenzoyl peroxide in step 1)；Pore-foaming agent is ethyl acetate, acetone or both mixing
Object.
6. a kind of method for isolating and purifying Epothilone D based on molecular engram according to claim 1, which is characterized in that
The volume ratio of methanol and acetic acid is 10:1 in methanol-acetic acid solution in step 2；In methanol-deionized water solution methanol and go from
The volume ratio of sub- water is 1:1.
7. a kind of method for isolating and purifying Epothilone D based on molecular engram according to claim 1, which is characterized in that
Methanol aqueous solution concentration is 60% in step 3).
8. a kind of method for isolating and purifying Epothilone D based on molecular engram according to claim 1, which is characterized in that
The methanol PBS solution that eluent is 80% in step 5).
9. a kind of method for isolating and purifying Epothilone D based on molecular engram according to claim 2, which is characterized in that
Template molecule described in step 1) is the mixture that Epothilone D and ebomycin A are mixed with the ratio of 1:1.
10. a kind of method for isolating and purifying Epothilone D based on molecular engram according to claim 2, which is characterized in that
Template molecule and crosslinker ratio are 1:20 in step 1), and the ratio of template molecule and function monomer is 1:6.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102373251A (en) * | 2011-11-04 | 2012-03-14 | 陕西科技大学 | Fermentation production method of Epothilone D provided with molecular imprinting polymer |
| CN102504100A (en) * | 2011-11-04 | 2012-06-20 | 陕西科技大学 | Epothilone D molecular imprinted polymer and preparation method thereof |
| CN103724656A (en) * | 2013-12-18 | 2014-04-16 | 陕西科技大学 | Method for preparing epothilone molecularly imprinted polymer by adopting mixed template |
| CN103937852A (en) * | 2014-04-23 | 2014-07-23 | 陕西科技大学 | Method for preparing epothilone B based on coupling of separation and fermentation of molecularly imprinted membrane |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102373251A (en) * | 2011-11-04 | 2012-03-14 | 陕西科技大学 | Fermentation production method of Epothilone D provided with molecular imprinting polymer |
| CN102504100A (en) * | 2011-11-04 | 2012-06-20 | 陕西科技大学 | Epothilone D molecular imprinted polymer and preparation method thereof |
| CN103724656A (en) * | 2013-12-18 | 2014-04-16 | 陕西科技大学 | Method for preparing epothilone molecularly imprinted polymer by adopting mixed template |
| CN103937852A (en) * | 2014-04-23 | 2014-07-23 | 陕西科技大学 | Method for preparing epothilone B based on coupling of separation and fermentation of molecularly imprinted membrane |
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| 孙瑞成: "分子印迹技术在埃博霉素精制纯化中的研究与应用", 《齐鲁工业大学硕士学位论文》 * |
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