WO1997038120A1 - PROCEDE DE PURIFICATION DES K-252a - Google Patents
PROCEDE DE PURIFICATION DES K-252a Download PDFInfo
- Publication number
- WO1997038120A1 WO1997038120A1 PCT/JP1997/001134 JP9701134W WO9738120A1 WO 1997038120 A1 WO1997038120 A1 WO 1997038120A1 JP 9701134 W JP9701134 W JP 9701134W WO 9738120 A1 WO9738120 A1 WO 9738120A1
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- WO
- WIPO (PCT)
- Prior art keywords
- microbial cells
- organic solvent
- solution
- alkali salt
- water
- Prior art date
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- KOZFSFOOLUUIGY-SOLYNIJKSA-N K-252a Chemical compound C12=C3N4C5=CC=CC=C5C3=C3CNC(=O)C3=C2C2=CC=CC=C2N1[C@H]1C[C@@](C(=O)OC)(O)[C@]4(C)O1 KOZFSFOOLUUIGY-SOLYNIJKSA-N 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 13
- 230000000813 microbial effect Effects 0.000 claims abstract description 45
- 150000001447 alkali salts Chemical class 0.000 claims abstract description 15
- 230000001035 methylating effect Effects 0.000 claims abstract description 3
- 239000012670 alkaline solution Substances 0.000 claims description 7
- 230000011987 methylation Effects 0.000 claims description 4
- 238000007069 methylation reaction Methods 0.000 claims description 4
- 159000000011 group IA salts Chemical class 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims 2
- 239000003513 alkali Substances 0.000 abstract description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 27
- 239000003960 organic solvent Substances 0.000 description 23
- 239000000243 solution Substances 0.000 description 23
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 21
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 15
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 13
- 239000013078 crystal Substances 0.000 description 12
- 239000000284 extract Substances 0.000 description 12
- 239000011347 resin Substances 0.000 description 12
- 229920005989 resin Polymers 0.000 description 12
- 239000000047 product Substances 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 150000003839 salts Chemical class 0.000 description 8
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- 238000010438 heat treatment Methods 0.000 description 6
- 238000001953 recrystallisation Methods 0.000 description 6
- 238000001179 sorption measurement Methods 0.000 description 6
- -1 7K oxidizing room Chemical compound 0.000 description 5
- 239000003456 ion exchange resin Substances 0.000 description 5
- 229920003303 ion-exchange polymer Polymers 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 4
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical class OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 2
- 241001221335 Nocardiopsis sp. Species 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 2
- 235000019270 ammonium chloride Nutrition 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 150000004649 carbonic acid derivatives Chemical class 0.000 description 2
- 239000003729 cation exchange resin Substances 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- VAYGXNSJCAHWJZ-UHFFFAOYSA-N dimethyl sulfate Chemical compound COS(=O)(=O)OC VAYGXNSJCAHWJZ-UHFFFAOYSA-N 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 150000007522 mineralic acids Chemical class 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 239000002798 polar solvent Substances 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- LBLYYCQCTBFVLH-UHFFFAOYSA-N 2-Methylbenzenesulfonic acid Chemical compound CC1=CC=CC=C1S(O)(=O)=O LBLYYCQCTBFVLH-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- DKPFZGUDAPQIHT-UHFFFAOYSA-N Butyl acetate Natural products CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 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 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- NTIZESTWPVYFNL-UHFFFAOYSA-N Methyl isobutyl ketone Chemical compound CC(C)CC(C)=O NTIZESTWPVYFNL-UHFFFAOYSA-N 0.000 description 1
- UIHCLUNTQKBZGK-UHFFFAOYSA-N Methyl isobutyl ketone Natural products CCC(C)C(C)=O UIHCLUNTQKBZGK-UHFFFAOYSA-N 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 150000001340 alkali metals Chemical class 0.000 description 1
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 1
- 150000001342 alkaline earth metals Chemical class 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- 239000000908 ammonium hydroxide Substances 0.000 description 1
- 229910052788 barium Inorganic materials 0.000 description 1
- DSAJWYNOEDNPEQ-UHFFFAOYSA-N barium atom Chemical compound [Ba] DSAJWYNOEDNPEQ-UHFFFAOYSA-N 0.000 description 1
- RQPZNWPYLFFXCP-UHFFFAOYSA-L barium dihydroxide Chemical compound [OH-].[OH-].[Ba+2] RQPZNWPYLFFXCP-UHFFFAOYSA-L 0.000 description 1
- 229910001863 barium hydroxide Inorganic materials 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 description 1
- 239000000920 calcium hydroxide Substances 0.000 description 1
- 229910001861 calcium hydroxide Inorganic materials 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000013256 coordination polymer Substances 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 1
- 150000004677 hydrates Chemical class 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 150000004679 hydroxides Chemical class 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- VTHJTEIRLNZDEV-UHFFFAOYSA-L magnesium dihydroxide Chemical compound [OH-].[OH-].[Mg+2] VTHJTEIRLNZDEV-UHFFFAOYSA-L 0.000 description 1
- 239000000347 magnesium hydroxide Substances 0.000 description 1
- 229910001862 magnesium hydroxide Inorganic materials 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 229940098779 methanesulfonic acid Drugs 0.000 description 1
- 239000002032 methanolic fraction Substances 0.000 description 1
- 239000012022 methylating agents Substances 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D498/22—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains four or more hetero rings
-
- 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/80—Elimination or reduction of contamination by undersired ferments, e.g. aseptic cultivation
Definitions
- the present invention relates to a method for purifying K-252a.
- K-252a is a physiologically active substance produced by a microorganism (JP-A-60-41489). Its activity is classified as protein kinase CP and exhibits various pharmacological actions.
- a culture solution is filtered to obtain microbial cells, and a water-containing or anhydrous organic solvent such as methanol or acetone is added to the microbial cells. After extraction, it is separated into microbial cells and an extract with care, and the obtained extract is concentrated and extracted with an anhydrous organic solvent with a higher yield.
- An adsorbent such as activated carbon and Diaion HP-10 And separation by column chromatography using a carrier such as silica gel, silanized silica gel, alumina oxide, dextran, etc., followed by concentration to obtain crude crystals.
- the purity and yield of crystals obtained by this method are not always sufficient, and a recrystallization step is required to increase the purity.
- the culture solution is extremely poor in filtration rate, and although a small amount of filtration is possible, it is difficult to transfer a large amount of culture solution. It is also possible to add an organic solvent directly to the culture solution, extract the solution, and obtain an extract by filtration.However, the solubility of K-252a in various organic solvents is 5 to 10; for example, acetone The solubility in water is not as high as 5 g Zl, and the amount of organic solvent to be added must be equal to or greater than that of the culture solution, and the processing capacity will increase to @ ⁇ . And so on.
- a method for purifying K-252a characterized by isolating and recovering the same, is provided.
- the microbial cells containing K-252a used in the present invention can be obtained by culturing a microorganism capable of producing K-252a in a nutrient medium.
- Nocardiopsis sp.No. 1-252 deposited with the ARS Culture Collection in the United States as NRRL 15532 (Japanese Patent Application Laid-Open No. 60-41489) ).
- the nutrient medium may be any medium that is commonly used for culturing microorganisms capable of producing 252-252a, such as a nutrient medium used for cultivation of normal actinomycetes.
- a culture method a liquid culture, particularly a submerged stirring culture method is suitable.
- the cultivation temperature is preferably 25 to 40 ⁇ and the pH is 5 to 9, preferably 6 to 8, which is desirable for culturing near neutrality.
- the culture After accumulating K-252a in the microbial cells, the culture is stopped to obtain microbial cells used for purification.
- concentration of K-252a accumulated in the microbial cells is preferably 1.0 g / liter or more, more preferably 5.0 g / liter / volume.
- Microbial cells can be harvested by centrifugation or the like. Prior to centrifugation, it is preferable to add an acid such as hydrochloric acid, sulfuric acid, nitric acid, and acetic acid, preferably sulfuric acid, to the culture solution to adjust the pH to 2 to 4, and then harvest the microbial cells.
- any centrifuge can be used.
- a centrifuge such as Westfary or Na-Laval is preferred. These centrifuges are preferable in that they can continuously separate a large amount of culture solution and can simultaneously wash microbial cells with washing water.
- the harvested microbial cells are suspended in an alkaline solution to treat the microbial cells so as to be 10 to 80% by volume, preferably 30 to 50% by volume.
- alkalis used in the alkaline liquid include alkali metals such as lithium, sodium, and potassium; alkaline earth metals such as magnesium, calcium, and barium; monovalent to trivalent metals such as aluminum; and hydroxides such as ammonium. Products, carbonates and bicarbonates. Specifically, hydrates such as sodium hydroxide, 7K oxidizing room, magnesium hydroxide, barium hydroxide, calcium hydroxide, ammonium hydroxide, etc., carbonates such as sodium carbonate, carbonated carbonate, etc. Examples thereof include sodium hydrogen carbonate, hydrogen carbonate such as hydrogen carbonate and the like, and hydroxide of aluminum metal is preferred.
- the alkali concentration of the alkaline solution is 0.1 to 5N, preferably 1 to 2N.
- the microbial cells suspended in the Algari solution may be treated with 30 to 90, preferably 60 to 80, and 1 to 2 For example, a method of heating for 4 hours, preferably for 3 to 5 hours.
- -252a in the microbial cells is hydrolyzed to form K-252-b or K-252-b salt. And extracted outside the microbial cells.
- K-252b or an alkali salt thereof extracted with the alkaline solution can be separated by applying to a column filled with a resin such as an adsorption resin or an ion exchange resin.
- a resin such as an adsorption resin or an ion exchange resin.
- Microbial cell separation can be performed, for example, using Nutsche, Kiriyamaguchi, a filter press, a basket separator, or the like.
- the adsorption resin for example, SP-207, HP-20, HP-30, HP-40, HP-50, etc., manufactured by Mitsubishi Kasei Co., Ltd., XAD-2, XAD-4, manufactured by Rohm and Haas Co., Ltd.
- ion-exchange resins include Mitsubishi Chemical Corporation PA-304, PA-412, etc., and Rohm and Haas IRA-410, IRA-911, A-26, ES—109, A—101 D, ES—111, etc., Dow Chemical D OWE X-2, —4, etc., Bayer MP—500 A, MP—508 And a strongly basic ion exchange resin such as 0.
- water-containing organic solvent used for washing examples include polar solvents containing water such as acetone, methanol, ethanol, propanol, and butanol.
- the water content is at least 20%, preferably at least 50%.
- An organic solvent or a water-containing organic solvent is used for elution.
- the organic solvent used for elution include ketones such as acetone, methyl ethyl ketone and methyl isobutyl ketone, alcohols such as methanol, ethanol and butanol, and esters such as ethyl acetate and butyl acetate.
- the water-containing organic solvent include polar solvents containing water, such as acetate, methanol, and ethanol. The water content is preferably 20% or less, more preferably 10% or less. Hydrous organic If necessary, salts such as sodium hydroxide and sodium chloride, and ammonium chloride can be added to the solvent.
- the concentration of the added alcohol is preferably 0.1 to 1.0, and the concentration of the added salt is 0.1 to 1.0.
- the alkaline solution extract or column eluate containing K-252b or an alkali salt thereof is concentrated as it is or by adding an acid such as hydrochloric acid or sulfuric acid as needed to make the eluate acidic.
- the concentration is carried out until the concentration of K-252b or its alkali salt per lm l of the solution is 0.5 to 2 mg, more preferably 5 to 1 mg.
- the concentrated solution can also be obtained by adding an organic solvent to K-252b or an alkali salt thereof obtained by concentrating the eluate to dryness.
- the amount of the organic solvent to be added is 50 to 2000 m 1 per 1 g of K-252b or its alkali salt, more preferably 100 to 20 m 2.
- the organic solvent used for the elution is used as the organic solvent to be added.
- K-252b or its salt is converted to K-252a again by a methylation treatment.
- a methylating agent such as dimethyl sulfate is added to the concentrated solution in an amount of 1 to 20 equivalents, preferably 2 to 3 equivalents to K-252b or an alkali salt thereof, and
- the reaction is carried out by heating at 100, preferably 60 to 80, for 2 to 36 hours, preferably 6 to 12 hours.
- the methylation treatment is performed by adding methanol to 100 to 2
- a cation exchange resin such as 1-16, IR-120B, 200, etc., manufactured by Michi-Ha-D-Haas, and D OWE X-50W, manufactured by Dow Chemical Co., etc., and add 50 to 100 ° C, preferably 60
- the reaction can be carried out by heating at a temperature of from 80 to 2 to 36 hours, preferably from 6 to 12 hours.
- the amount of the inorganic acid, organic acid, and cation exchange resin to be added is 1 to 20 equivalents to K-252b or an alkali salt thereof, respectively, preferably 2 to 3 equivalents. Good.
- K-252a is precipitated by concentrating the methylated reaction solution under reduced pressure and cooling. Concentration is carried out at 700-76 OmmHg. Preferably 720-76 OmmHg, at a temperature of 30-80, preferably 40-50, below the saturation concentration.
- the temperature for depositing K-252a is preferably from 10 to 60.
- K-252a can be precipitated from the reaction solution by concentrating and drying the methylated reaction solution, adding water, and then adding an organic solvent.
- the amount of water to be added is preferably 0.01 to 1 times the volume of the solvent used in the reaction, and more preferably 0.1 to 0.5 times.
- the organic solvent include the above-mentioned organic solvents that dissolve water, and the volume of the added organic solvent is preferably 0.5 to 0.5 times the volume of the added water.
- the precipitated K-252a can be isolated and obtained by a usual method such as repulsion and drying.
- Nocardiopsis sp. K-252 (NRRL 15532) was used as the inoculum.
- a medium consisting of 3 gZdl (pH 7.2 to 7.4 before sterilization) was used.
- Inoculum 1 Include 14 ml of white fungus ear in the above seed medium 5
- Oml thick test tubes were inoculated, and shaking culture was performed at 30 for 3 days.
- the composition of the type 3 medium is the same as the composition of the type 2 medium.
- 90 Oml of the 3rd seed culture was inoculated into a 30 liter stainless steel jar arm mentor containing 16 liters of the main fermentation medium, and aerated at 30 for 7 days with stirring and agitation (300 rpm, 16 liters of aeration) / Min).
- the composition of the main fermentation medium is the same as that of the first type medium.
- One liter of the culture was adjusted to pH 3 with concentrated sulfuric acid, and then microbial cells were separated using a high-speed cooling centrifuge (manufactured by Hitachi, Ltd., CR-20) to collect 300 ml of microbial cells.
- a high-speed cooling centrifuge manufactured by Hitachi, Ltd., CR-20
- a total of 2 g of K-252a was contained in 300 ml of the bacterial cells.
- 600 ml of water was added to the microbial cells, and the cells were washed again by a centrifugal separator to obtain washed and concentrated microbial cells.
- a sodium hydroxide solution was added to the washed and concentrated microbial cells to adjust the pH to 12, followed by heating at 80 for 3 hours.
- the processed microbial cell suspension was adjusted to pH 7 with concentrated sulfuric acid, and then filtered with Nutsche to remove the microbial cells to obtain an extract.
- K-252a was converted to almost 100% K-252b or its salt and extracted.
- K-252a can be extracted from almost 100% bacterial cells without using an organic solvent by converting it to K-252b or an alkali salt thereof.
- a concentrated sodium hydroxide solution was added to the suspension of the microbial cells to adjust the pH to 12, followed by heating at 80 for 3 hours.
- the heat-treated microbial cell suspension was adjusted to pH 7 with concentrated sulfuric acid, followed by Nutsch II to remove the microbial cells and obtain an extract.
- the obtained extract was passed through an adsorption resin (200 ml, SP-207, manufactured by Mitsubishi Chemical Corporation) at a flow rate of SV2, and the resin was passed through 200 ml of water and 400 ml of water. Washed with 50% methanol. Next, elution was performed with 100% methanol 2000 Om1.
- the methanol fraction containing K-252b or K-252b alkaline salt was concentrated to 1000 ml.
- the concentrated solution was added with concentrated sulfuric acid (lm1) and heated at 60 T: for 3 hours to convert to K-252a.
- the obtained reaction solution was cooled and then filtered to obtain crude K-252a crystals having a purity of 75% in a yield of 70% (1.4 g in terms of pure product).
- the crude crystal of -252a can be easily improved by 5% by ordinary recrystallization, and 1 ⁇ -252a with a purity of 95% was obtained with a yield of 80% (1% in terms of pure product). 1 g).
- Microbial cells were treated with sodium hydroxide in the same manner as in Example 1, the filtrate was passed through an adsorption resin, and then the resin was washed. Next, the K-252b or the alkali salt thereof adsorbed on the resin was eluted with methanol: sodium hydroxide (9: 1), and the eluate was dried and dried to give 100 mL of acetone. And 2 equivalents of dimethylsulfuric acid was added and converted to K-252a by heating at 60 for 3 hours. The resulting reaction solution was cooled and then filtered to obtain 75% of S1 ⁇ 2S 75% K-252a crude crystals in a 70% yield.
- Example 2 Microbial cells were treated with sodium hydroxide in the same manner as in Example 1. Then, the cells It was directly applied to the adsorption resin without separation, and then K-252a was obtained in the same manner as in Example 2. As a result, crude crystals of 5% K—252a were obtained in a yield of 70% (1.4 g in terms of pure product). The purity of the crude crystals of —252a can be easily improved by ordinary recrystallization, and 13 ⁇ 4-252a with 5% of J3 ⁇ 4S95 was obtained with a yield of 80% (by pure product conversion). 1.1 g).
- Example 5 The procedure was performed in the same manner as in Example 2 except that the column was made of a strongly basic ion exchange resin (PA-412) and the eluate was changed to methanol: 0.1 N ammonium chloride (9: 1). As a result, crude K-252a crystals with a purity of 75% were obtained in a yield of 70% (1.4 g in terms of pure product). This crude crystal of K-252a can easily improve ⁇ by ordinary recrystallization, and ⁇ 95% of -252a was obtained at a yield of 80% (pure product equivalent). At 1.1 g).
- Example 5 Example 5
- K-252a can be purified on an industrial scale with simple, inexpensive and high purification efficiency without using a large amount of organic solvent.
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- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
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Description
Claims
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU21774/97A AU2177497A (en) | 1996-04-09 | 1997-04-02 | Method of purifying k-252a |
CA002228471A CA2228471C (en) | 1996-04-09 | 1997-04-02 | A process for purification of k-252a |
JP53604497A JP3865781B2 (ja) | 1996-04-09 | 1997-04-02 | K−252aの精製方法 |
US08/973,127 US6004801A (en) | 1996-04-09 | 1997-04-02 | Method of purifying K-252a |
AT97914579T ATE207127T1 (de) | 1996-04-09 | 1997-04-02 | Methode zur reinigung von k-252a |
DE69707389T DE69707389T2 (de) | 1996-04-09 | 1997-04-02 | METHODE ZUR REINIGUNG VON K-252a |
EP97914579A EP0834574B1 (en) | 1996-04-09 | 1997-04-02 | METHOD OF PURIFYING K-252a |
DK97914579T DK0834574T3 (da) | 1996-04-09 | 1997-04-02 | Fremgangsmåde til oprensning af K-252a |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP8646996 | 1996-04-09 | ||
JP8/86469 | 1996-04-09 |
Publications (1)
Publication Number | Publication Date |
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WO1997038120A1 true WO1997038120A1 (fr) | 1997-10-16 |
Family
ID=13887827
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP1997/001134 WO1997038120A1 (fr) | 1996-04-09 | 1997-04-02 | PROCEDE DE PURIFICATION DES K-252a |
Country Status (11)
Country | Link |
---|---|
US (1) | US6004801A (ja) |
EP (1) | EP0834574B1 (ja) |
JP (1) | JP3865781B2 (ja) |
AT (1) | ATE207127T1 (ja) |
AU (1) | AU2177497A (ja) |
CA (1) | CA2228471C (ja) |
DE (1) | DE69707389T2 (ja) |
DK (1) | DK0834574T3 (ja) |
ES (1) | ES2166071T3 (ja) |
PT (1) | PT834574E (ja) |
WO (1) | WO1997038120A1 (ja) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2006519237A (ja) * | 2003-02-27 | 2006-08-24 | アボット・ラボラトリーズ | K−252aの調製 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS6041489A (ja) * | 1983-08-12 | 1985-03-05 | Kyowa Hakko Kogyo Co Ltd | 新規生理活性物質k―252 |
-
1997
- 1997-04-02 AU AU21774/97A patent/AU2177497A/en not_active Abandoned
- 1997-04-02 JP JP53604497A patent/JP3865781B2/ja not_active Expired - Fee Related
- 1997-04-02 US US08/973,127 patent/US6004801A/en not_active Expired - Lifetime
- 1997-04-02 PT PT97914579T patent/PT834574E/pt unknown
- 1997-04-02 DK DK97914579T patent/DK0834574T3/da active
- 1997-04-02 AT AT97914579T patent/ATE207127T1/de active
- 1997-04-02 EP EP97914579A patent/EP0834574B1/en not_active Expired - Lifetime
- 1997-04-02 ES ES97914579T patent/ES2166071T3/es not_active Expired - Lifetime
- 1997-04-02 CA CA002228471A patent/CA2228471C/en not_active Expired - Fee Related
- 1997-04-02 DE DE69707389T patent/DE69707389T2/de not_active Expired - Lifetime
- 1997-04-02 WO PCT/JP1997/001134 patent/WO1997038120A1/ja active IP Right Grant
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS6041489A (ja) * | 1983-08-12 | 1985-03-05 | Kyowa Hakko Kogyo Co Ltd | 新規生理活性物質k―252 |
Non-Patent Citations (4)
Title |
---|
J. NEUROCHEM., (1994), Vol. 62, No. 4, BIN CHENG et al., "Staurosporine, K-252a and K-252b Stabilize Calcium Homeostasis and Promote Survival of CNS Neurons in the Absence of Glucose", p. 1319-1329. * |
J. NEUROCHEM., (1995), Vol. 65, No. 6, ALONZO H. ROSS et al., "Differential Biological Effects of K252 Kinase Inhibitors Are Related to Membrane Solubility but not to Permiability", p. 2748-2756. * |
NOZOMI OTAKE et al., "Isolation and Purification of Substances (in Japanese)", (15 November 1981), ZAIDAN HOJIN TOKYO DAIGAKU SHUPPAN KAI, p. 251-253. * |
TOSHINOBU ASAI, "Microbiological Industry (in Japanese)", (30 June 1956), K.K. ASAKURA SHOTEN, p. 700-709. * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2006519237A (ja) * | 2003-02-27 | 2006-08-24 | アボット・ラボラトリーズ | K−252aの調製 |
Also Published As
Publication number | Publication date |
---|---|
PT834574E (pt) | 2002-04-29 |
DK0834574T3 (da) | 2001-12-27 |
JP3865781B2 (ja) | 2007-01-10 |
AU2177497A (en) | 1997-10-29 |
EP0834574A1 (en) | 1998-04-08 |
EP0834574A4 (ja) | 1998-05-20 |
ATE207127T1 (de) | 2001-11-15 |
CA2228471C (en) | 2005-05-24 |
ES2166071T3 (es) | 2002-04-01 |
DE69707389T2 (de) | 2002-06-27 |
CA2228471A1 (en) | 1997-10-16 |
US6004801A (en) | 1999-12-21 |
EP0834574B1 (en) | 2001-10-17 |
DE69707389D1 (de) | 2001-11-22 |
MX9709802A (es) | 1998-07-31 |
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