NO166538B - MICROBIAL HYDROXYLING METHOD FOR QUININE, QUINIDINE, DIHYDROQININ AND DIHYDROQINIDINE. - Google Patents
MICROBIAL HYDROXYLING METHOD FOR QUININE, QUINIDINE, DIHYDROQININ AND DIHYDROQINIDINE. Download PDFInfo
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- NO166538B NO166538B NO871173A NO871173A NO166538B NO 166538 B NO166538 B NO 166538B NO 871173 A NO871173 A NO 871173A NO 871173 A NO871173 A NO 871173A NO 166538 B NO166538 B NO 166538B
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- quinidine
- hydroxylation
- quinine
- hydroxylated
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- LOUPRKONTZGTKE-LHHVKLHASA-N quinidine Chemical compound C([C@H]([C@H](C1)C=C)C2)C[N@@]1[C@H]2[C@@H](O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-LHHVKLHASA-N 0.000 title claims description 32
- LOUPRKONTZGTKE-UHFFFAOYSA-N cinchonine Natural products C1C(C(C2)C=C)CCN2C1C(O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-UHFFFAOYSA-N 0.000 title claims description 26
- 238000000034 method Methods 0.000 title claims description 20
- LOUPRKONTZGTKE-WZBLMQSHSA-N Quinine Chemical compound C([C@H]([C@H](C1)C=C)C2)C[N@@]1[C@@H]2[C@H](O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-WZBLMQSHSA-N 0.000 title claims description 19
- 229960001404 quinidine Drugs 0.000 title claims description 16
- 235000001258 Cinchona calisaya Nutrition 0.000 title claims description 10
- 229960000948 quinine Drugs 0.000 title claims description 10
- 230000000813 microbial effect Effects 0.000 title description 2
- 230000033444 hydroxylation Effects 0.000 claims description 24
- 238000005805 hydroxylation reaction Methods 0.000 claims description 24
- LJOQGZACKSYWCH-LHHVKLHASA-N (s)-[(2r,4s,5r)-5-ethyl-1-azabicyclo[2.2.2]octan-2-yl]-(6-methoxyquinolin-4-yl)methanol Chemical compound C1=C(OC)C=C2C([C@H](O)[C@H]3C[C@@H]4CCN3C[C@@H]4CC)=CC=NC2=C1 LJOQGZACKSYWCH-LHHVKLHASA-N 0.000 claims description 18
- LJOQGZACKSYWCH-UHFFFAOYSA-N dihydro quinine Natural products C1=C(OC)C=C2C(C(O)C3CC4CCN3CC4CC)=CC=NC2=C1 LJOQGZACKSYWCH-UHFFFAOYSA-N 0.000 claims description 18
- 150000001875 compounds Chemical class 0.000 claims description 14
- 229960000811 hydroquinidine Drugs 0.000 claims description 12
- 238000011534 incubation Methods 0.000 claims description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical class N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- 239000002609 medium Substances 0.000 claims description 10
- 241000378863 Mucor plumbeus Species 0.000 claims description 9
- 241001290628 Cunninghamella echinulata Species 0.000 claims description 7
- 244000005700 microbiome Species 0.000 claims description 7
- 229910052799 carbon Inorganic materials 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 6
- 229960004251 hydroquinine Drugs 0.000 claims description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical class [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 5
- 241000306281 Mucor ambiguus Species 0.000 claims description 5
- 241000187419 Streptomyces rimosus Species 0.000 claims description 5
- 229910052500 inorganic mineral Chemical class 0.000 claims description 5
- 239000011707 mineral Chemical class 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 150000003839 salts Chemical class 0.000 claims description 4
- 230000000707 stereoselective effect Effects 0.000 claims description 4
- 238000010564 aerobic fermentation Methods 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 2
- 241000233866 Fungi Species 0.000 claims description 2
- 239000000047 product Substances 0.000 description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 239000002207 metabolite Substances 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000002906 microbiologic effect Effects 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- BSRUJCFCZKMFMB-LGWHJFRWSA-N (2r,4s,5s)-5-ethenyl-2-[(s)-hydroxy-(6-methoxyquinolin-4-yl)methyl]-1-azabicyclo[2.2.2]octan-5-ol Chemical compound C([C@H]([C@@](C1)(O)C=C)C2)CN1[C@H]2[C@@H](O)C1=CC=NC2=CC=C(OC)C=C21 BSRUJCFCZKMFMB-LGWHJFRWSA-N 0.000 description 2
- BSRUJCFCZKMFMB-UHFFFAOYSA-N 3-hydroxyquinine Natural products C1C(C(C2)(O)C=C)CCN2C1C(O)C1=CC=NC2=CC=C(OC)C=C21 BSRUJCFCZKMFMB-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
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- 238000000926 separation method Methods 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 150000003431 steroids Chemical class 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- MMCFRFNECGZXQH-APFZWMIBSA-N (2r,4r,5s)-5-ethyl-2-[(s)-hydroxy-(6-methoxyquinolin-4-yl)methyl]-1-azabicyclo[2.2.2]octan-5-ol;sulfuric acid Chemical compound OS(O)(=O)=O.C1=C(OC)C=C2C([C@H](O)[C@H]3C[C@H]4CCN3C[C@]4(O)CC)=CC=NC2=C1 MMCFRFNECGZXQH-APFZWMIBSA-N 0.000 description 1
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- BSRUJCFCZKMFMB-YGHPHNMRSA-N 3-hydroxyquinine Chemical compound C([C@H]([C@@](C1)(O)C=C)C2)C[N@@]1[C@@H]2[C@H](O)C1=CC=NC2=CC=C(OC)C=C21 BSRUJCFCZKMFMB-YGHPHNMRSA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 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
- 239000007836 KH2PO4 Substances 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241000364057 Peoria Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 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
- 239000003463 adsorbent Substances 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 206010003119 arrhythmia Diseases 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
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- 239000008121 dextrose Substances 0.000 description 1
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical class CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
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- 235000019319 peptone Nutrition 0.000 description 1
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- 239000008363 phosphate buffer Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 125000003410 quininyl group Chemical group 0.000 description 1
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- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
Denne oppfinnelse vedrører en fremgangsmåte og stereospesifikk hydrolysering av kinin, kinidin, dihydrokinin og dihydrokinidin i 3S-stilling. This invention relates to a method and stereospecific hydrolysis of quinine, quinidine, dihydroquinine and dihydroquinidine in the 3S position.
Hepatisk mikrosomal hydroksylering utgjør ofte et av de første metabolismetrinn av en fremmed kjemisk forbindelse som innføres i et pattedyrs hjelpe- eller blodsirkuleringssystemer. En slik hydroksylering etterfulgt av andre reaksjoner såsom 0-metylering eller konjugering foretas for å gjøre slike substanser mer vannløselige og derfor påskynde utskillelsen av dem. I visse tilfeller er imidlertid de hydroksylerte forbindelser mer aktive eller mer giftig enn det opprinnelige molekyl; dette berettiger interesse for identifisering av dem, og derfor fremstilling av dem med tanke på undersøkelse av deres aktivitet. Hepatic microsomal hydroxylation often constitutes one of the first metabolic steps of a foreign chemical compound introduced into a mammal's accessory or circulatory systems. Such hydroxylation followed by other reactions such as 0-methylation or conjugation is carried out to make such substances more water-soluble and therefore accelerate their excretion. In certain cases, however, the hydroxylated compounds are more active or more toxic than the original molecule; this warrants interest in their identification, and therefore their production with a view to investigating their activity.
Organiske syntesemetoder er åpenbart greie for metabolitter av enkle forbindelser, men den kjemiske fremstilling av mer kompliserte (og optisk aktive) molekyler kan kreve betydelig anstrengelse og tid. Organic synthesis methods are obviously suitable for metabolites of simple compounds, but the chemical preparation of more complicated (and optically active) molecules can require considerable effort and time.
Hydroksyleringer ved mikrobiologiske midler har allerede blitt utviklet betydelig for mange år siden på området steroider, hvor de har gjort det mulig å oppnå i industriell skala regio-og stereoselektivt hydroksylerte produkter som stammer fra spesifikk oksydering av ikke funksjonaliserte karbonatomer. Slike molekyler ville ha krevet betydelige syntetiske anstreng-elser hvis det hadde vært nødvendig å anvende kjemiske metoder. Denne metoden er senere blitt anvendt på andre molekylgrupper Hydroxylations by microbiological agents have already been significantly developed many years ago in the area of steroids, where they have made it possible to obtain on an industrial scale regio- and stereoselectively hydroxylated products originating from specific oxidation of non-functionalized carbon atoms. Such molecules would have required considerable synthetic efforts if it had been necessary to use chemical methods. This method has later been applied to other molecular groups
og den utgjør i visse tilfeller en foretrukket måte å fremstille nye forbindelser på, som kan overføres til produksjon i stor skala ved å bruke klassiske fermenteringsteknikker og utstyr. Videre viser det seg at mikrobielle hydroksyleringer for hver type fører til et ofte snevrere område av metaboliter enn det som finnes i pattedyrets systemer, og fjerner således noen separasjonsproblemer; det er da tilstrekkelig å utnytte multiplisiteten av stammer for å rekonstituere de forskjellige muligheter for levrede metaboliter. and it constitutes, in certain cases, a preferred way of preparing new compounds, which can be transferred to large-scale production using classical fermentation techniques and equipment. Furthermore, it turns out that microbial hydroxylations for each type lead to an often narrower range of metabolites than that found in mammalian systems, thus removing some separation problems; it is then sufficient to utilize the multiplicity of strains to reconstitute the different possibilities for stored metabolites.
På alkaloideområdet er imidlertid temmelig få reaksjoner av denne type beskrevet, og de vedrører i de fleste tilfeller den ikke-nitrogenerte del av det aromatiske eller ikke-steroide molekyl. Det er derfor både ønskelig og interressant å kunne utføre under fordelaktige betingelser hydroksylering av nitrogen-holdig alkaloidekjerne hvis. produkter allerede er gjenkjent som aktive metaboliter. Spesielt beskriver fransk patent nr. 2.506.312 hydroksylerte forbindelser av kinidin såsom 3-hydroksy-10,11-dihydro-kinidin og dens 3R og 3S epimere som kan anvendes terapeutisk for behandling av kardiale arrytmier. På den annen side er man klar over at kinin hvis farmakologiske egenskaper er kjente og kinidin er to isomerer, som adskiller seg i sine konfigurasjoner med hensyn: til karbonene i stillingene 8 og 9. However, relatively few reactions of this type have been described in the alkaloid area, and in most cases they relate to the non-nitrogenated part of the aromatic or non-steroid molecule. It is therefore both desirable and interesting to be able to carry out under favorable conditions hydroxylation of nitrogen-containing alkaloid core if. products are already recognized as active metabolites. In particular, French Patent No. 2,506,312 describes hydroxylated compounds of quinidine such as 3-hydroxy-10,11-dihydro-quinidine and its 3R and 3S epimers which can be used therapeutically for the treatment of cardiac arrhythmias. On the other hand, one is aware that quinine, whose pharmacological properties are known, and quinidine are two isomers, which differ in their configurations with regard to: the carbons in positions 8 and 9.
Fremstillingen av slike aktive, optisk rene forbindelser ved en mikrobiologisk metode, som kan overføres til industriell skala „ er derfor et mål av åpenbar interresse med' hensyn til kjemiske hydroksyleringer,. hvilke fører til lave utbytter og diastereoisomere blandinger. The preparation of such active, optically pure compounds by a microbiological method, which can be transferred to an industrial scale, is therefore a measure of obvious interest with respect to chemical hydroxylations. which lead to low yields and diastereoisomeric mixtures.
Målet for denne oppfinnelse er derfor å frembringe en ny fremgangsmåte for mikrobiologisk hydroksylering av kinin, kinidin og deres derivater, spesielt deres hydrogenerte derivater på en regio- og stereospesifikk måte slik at man får optisk rene forbindelser. The aim of this invention is therefore to produce a new method for the microbiological hydroxylation of quinine, quinidine and their derivatives, especially their hydrogenated derivatives in a regio- and stereospecific manner so that optically pure compounds are obtained.
Et annet mål for denne oppfinnelse er å frembringe en fremgangsmåte for mikrobiologisk hydroksylering av kinin, kinidin og deres derivater, anvendelige for industrielle formål, og frembringe optisk rene forbindelser i et tilfredsstillende utbytte ved hjelp av apparatur av vanlig type. Den regio-spesifikke og stereospesifikke mikrobiologiske hydroksylerings-metode ifølge denne oppfinnelse muliggjør hydroksylering i 3S-stillingen av kinin, kinidin og deres dihydroderivater med formelen (I): hvor R betyr en etyl- eller vinylgruppe under dannelse av det tilsvarende 3S-hydroksylerte derivat med formel (II): Another aim of this invention is to produce a method for the microbiological hydroxylation of quinine, quinidine and their derivatives, applicable for industrial purposes, and to produce optically pure compounds in a satisfactory yield by means of apparatus of the usual type. The regio-specific and stereospecific microbiological hydroxylation method according to this invention enables hydroxylation in the 3S position of quinine, quinidine and their dihydroderivatives with the formula (I): where R means an ethyl or vinyl group while forming the corresponding 3S-hydroxylated derivative with formula (II):
hvor R har samme betydning som ovenfor, ved at en mikroorganisme av typen lavere sopp valgt fra gruppen bestående av Cunninghamella echinulata, Mucor circinelloides, Mucor plumbeus, eller typen bakterie så som Streptomyces rimosus, dyrkes i et dyrkningsmedium inneholdende kilder for assimilerbart karbon, nitrogen og mineralsalter under betingelser for aerob fermentering i dypkultur, og forbindelsen som skal hydroksyleres settes direkte til dyrkningsmediet når veksten av mikroorganismen er tilstrekkelig, med det forbehold at C. echinulata ikke anvendes for hydroksyleringen av kinidin. where R has the same meaning as above, in that a microorganism of the type lower fungi selected from the group consisting of Cunninghamella echinulata, Mucor circinelloides, Mucor plumbeus, or the type of bacteria such as Streptomyces rimosus, is grown in a culture medium containing sources of assimilable carbon, nitrogen and mineral salts under conditions for aerobic fermentation in deep culture, and the compound to be hydroxylated is added directly to the culture medium when the growth of the microorganism is sufficient, with the proviso that C. echinulata is not used for the hydroxylation of quinidine.
I formel (I) ovenfor, representerer R en vinylrest i tilfellet kinin og kinidin som adskilles ved karbonenes konfigurasjoner i stillingene 8 og 9, og R representerer en etylrest i tilfellet hydrogenerte derivater, dvs. dihydrokinin og dihydrokinidin. Den samme anmerkning gjelder de hydroksylerte derivater med formel (II). In formula (I) above, R represents a vinyl residue in the case of quinine and quinidine which is separated by the configurations of the carbons in positions 8 and 9, and R represents an ethyl residue in the case of hydrogenated derivatives, ie dihydroquinine and dihydroquinidine. The same remark applies to the hydroxylated derivatives of formula (II).
De heretter identifiserte stammer brukes fortrinnsvis: Cunninghamella echinulata (NRRL 3655, MMP 2203), Mucor circinelloides (CBS 108-16), Mucor plumbeus (MMP 430, CBS 111-07) og Streptomyces rimosus (NRRL 2234). The hereinafter identified strains are preferably used: Cunninghamella echinulata (NRRL 3655, MMP 2203), Mucor circinelloides (CBS 108-16), Mucor plumbeus (MMP 430, CBS 111-07) and Streptomyces rimosus (NRRL 2234).
De ovennevnte henvisninger har sine vanlige betydninger, dvs. NRRL = Agricultural Research Culture Collection, Northern Regional Research Center, Peoria, 111.; CBS = Centraal Bureau voor Shimmelcultures, Baarn Netherlands; MMP: Mycothéque du Muséum d'histoire Naturelle, Paris, France. The above references have their usual meanings, i.e. NRRL = Agricultural Research Culture Collection, Northern Regional Research Center, Peoria, 111.; CBS = Centraal Bureau voor Shimmelcultures, Baarn Netherlands; MMP: Mycothéque du Muséum d'histoire Naturelle, Paris, France.
Dyrkningen forløper fortrinnsvis under aerobe fermenterings-betingelser og i dypkultur. Forbindelsen som skal hydroksyleres tilsettes - fortrinnsvis under sterile betingelser - til mediet, man inkuberer inntil man har fått en tilstrekkelig mengde av de hydroksylerte produkt, og ekstraksjon utføres ved vanlige teknikker. Inkuberingen kan utføres i en vanlig type fermenteringstank i et temperaturområde mellom ca. 20 og 30°C, idet mediet har pH nær 7. Om nødvendig kan man bruke et medium med en pH på mellom ca. 6 og 8 uten å nevneverdig endre resultatene. Cultivation preferably takes place under aerobic fermentation conditions and in deep culture. The compound to be hydroxylated is added - preferably under sterile conditions - to the medium, incubated until a sufficient amount of the hydroxylated product has been obtained, and extraction is carried out by usual techniques. The incubation can be carried out in a normal type of fermentation tank in a temperature range between approx. 20 and 30°C, as the medium has a pH close to 7. If necessary, you can use a medium with a pH of between approx. 6 and 8 without significantly changing the results.
Fremgangsmåten som benyttes består i å dyrke mikroorganis-mene i et egnet dyrkningsmedium slik at man får en godt utviklet biomasse; i tillegg til en kilde for assimilerbart karbon (dextrose, saccharose, stivelse, mannitol) i en 1 til 10% konsen-trasjon kan mediet inneholde en kilde for nitrogen, enten organisk (peptoner, asparagin, protein hydrolysat) eller mine-ralsk (ammoniumsalter, nitrater) og de nødvendige mineralsalter, samt som vitaminsubstanser (slike som foreligger i "maisstøp", gjærekstrakt, ...); temperaturen kan variere fra ca. 20 til 30°C avhengig av stammene, og dyrkningen utføres med røring. Når veksten anses tilfredsstillende og i alminnelighet når hydrokar-bonsubstratet er uttømt, dvs. etter ca. 2 til 5 dager, tilsettes forbindelsen som skal hydroksyleres - som et fast stoff eller oppløst i et lite volum av et vannblandbart organisk løsnings-middel (f.eks. etanol), under sterile betingelser ved en sluttkonsentrasjon som kan variere fra 0,2 til flere gram/liter, avhengig av molekylet eller stammen. Inkubering som kan følges av en svak restvekst fortsetter under kraftig røring for å belufte blandingen ved den samme temperatur i et 2 til 40 dagers tidsrom. The method used consists of growing the micro-organisms in a suitable culture medium so that a well-developed biomass is obtained; in addition to a source of assimilable carbon (dextrose, sucrose, starch, mannitol) in a 1 to 10% concentration, the medium can contain a source of nitrogen, either organic (peptones, asparagine, protein hydrolyzate) or mineral (ammonium salts) , nitrates) and the necessary mineral salts, as well as vitamin substances (such as are present in "corn cast", yeast extract, ...); the temperature can vary from approx. 20 to 30°C depending on the strains, and cultivation is carried out with stirring. When the growth is considered satisfactory and generally when the hydrocarbon substrate is exhausted, i.e. after approx. 2 to 5 days, the compound to be hydroxylated is added - as a solid or dissolved in a small volume of a water-miscible organic solvent (e.g. ethanol), under sterile conditions at a final concentration which can vary from 0.2 to several grams/litre, depending on the molecule or strain. Incubation which may be followed by a slight residual growth continues under vigorous stirring to aerate the mixture at the same temperature for a period of 2 to 40 days.
Ifølge en variant av den oppfunnede fremgangsmåte utføres inkuberingen av forbindelsen som skal hydroksyleres med celler som er forut vasket og gjenoppslemmet i et bufret vandig medium som ikke inneholder noe karbon eller nitrogen. Hydroksyleringen utføres så etter at mikroorganismen har vokst og etter at mycelet er samlet uten vekst. According to a variant of the invented method, the incubation of the compound to be hydroxylated is carried out with cells that have previously been washed and resuspended in a buffered aqueous medium containing no carbon or nitrogen. The hydroxylation is then carried out after the microorganism has grown and after the mycelium has been collected without growth.
Ekstraksjon av inkuberingsproduktene samt for resten av substratet utføres ved hjelp av et organisk løsningsmiddel som ikke er blandbart med vann (metylenklorid, kloroform, etylacetat osv.) etterat mycelet er fjernet ved filtrering og løsningsmidlet mettet med salter (NaCl, Na2S04). Extraction of the incubation products as well as of the rest of the substrate is carried out using an organic solvent that is not miscible with water (methylene chloride, chloroform, ethyl acetate, etc.) after the mycelium has been removed by filtration and the solvent saturated with salts (NaCl, Na2S04).
Påvisningen og identifisering av hydroksyleringsproduktene kan utføres ved tynnsjikt kromatografi på kiselgel eller ved revers-fase-høytrykksvæske-kromatografi. Separasjon av de hydroksylerte produkt fra det gjenværende opprinnelige substrat, om sådant foreligger - og dets rensning utføres ved klassiske teknikker for krystallisering eller kromatografi på mineralske adsorbenter. The detection and identification of the hydroxylation products can be carried out by thin-layer chromatography on silica gel or by reverse-phase high-pressure liquid chromatography. Separation of the hydroxylated product from the remaining original substrate, if any - and its purification is carried out by classical techniques for crystallization or chromatography on mineral adsorbents.
De rensede hydroksyleringsprodukter er blitt identifisert ved sammenligning med de autentiske (3S)-hydroksy produkter The purified hydroxylation products have been identified by comparison with the authentic (3S)-hydroxy products
(fremstilt ved kjemiske metoder eller beskrevet i litteraturen) (produced by chemical methods or described in the literature)
i forskjellige kromatografiske systemer, samt ved vanlig spektro-skopiske (UV, ^-H- eller <13>C-NMR) og polarometriske teknikker. in different chromatographic systems, as well as by usual spectroscopic (UV, ^-H- or <13>C-NMR) and polarometric techniques.
De oppnådde resultater er gjengitt i de følgende eksempler, hvori flere stammer av mikroorganismer som er i stand til å utføre disse hydroksyleringer regio- og stereoselektivt ut fra flere av de aktuelle substrater med en variabel omdannelses-hastighet er blitt karakterisert. I tillegg til den vesentlige fordelen med hydroksyleringsselektivitet, er en annen fordel at det ikke dannes andre metaboliter i nevneverdige mengder, hvilket forenkler rensningen og muliggjør resirkulering av substratet som eventuelt ikke er blitt metabolisert. The results obtained are reproduced in the following examples, in which several strains of microorganisms capable of carrying out these hydroxylations regio- and stereoselectively from several of the relevant substrates with a variable conversion rate have been characterized. In addition to the significant advantage of hydroxylation selectivity, another advantage is that no other metabolites are formed in appreciable quantities, which simplifies the purification and enables the recycling of the substrate that may not have been metabolized.
De følgende eksempler illustrerer oppfinnelsen uten å begrense dens omfang. The following examples illustrate the invention without limiting its scope.
EKSEMPEL 1 EXAMPLE 1
Hydroksylering av kinidin med M. plumbeus Hydroxylation of quinidine by M. plumbeus
Noen få dråper av en suspensjon av M.plumbeus MMP-430 sporer settes til 100 ml medium A beskrevet nedenunder justert til pH A few drops of a suspension of M.plumbeus MMP-430 spores are added to 100 ml of medium A described below adjusted to pH
7 i en 250 ml Erlenmeyerkolbe. Inkuberingen fortsetter i 72 timer ved 37°C på en roterende rister (327 OPM). En løsning av 50 mg kinidin i 1 ml etanol tilsettes så under sterile betingelser til kulturen, og ristingen fortsetter i 14 dager under de samme betingelser. Kulturen filtreres på celit, filtratet mettes med NaCl, bringes til pH 10-12 med 2N NaOH, ekstraheres så seks ganger med metylenklorid. De tørkede og deretter inndampede organiske fraksjoner kromatograferes på et preparativt sjikt av 7 in a 250 ml Erlenmeyer flask. Incubation continues for 72 hours at 37°C on a rotary shaker (327 OPM). A solution of 50 mg of quinidine in 1 ml of ethanol is then added under sterile conditions to the culture, and shaking is continued for 14 days under the same conditions. The culture is filtered on celite, the filtrate is saturated with NaCl, brought to pH 10-12 with 2N NaOH, then extracted six times with methylene chloride. The dried and then evaporated organic fractions are chromatographed on a preparative layer of
kiselgel ved bruk av løsningsmidlet CHCl2-MeOH-NH4OH (85:14:1). (3S)-3-hydroksy-kinidinbåndet som identifiseres ved sin fluores-cens ved 254 nm og sin vandring som er mindre enn kinedinets, skrapes av og elueres med metanol. Man får 1 mg (3S)-3-hydroksy-kinidin, identisk med en autentisk prøve. Ikke-omsatt kinidin gjenvinnes på samme måte og nesten kvantitativt (42 mg). silica gel using the solvent CHCl2-MeOH-NH4OH (85:14:1). The (3S)-3-hydroxyquinidine band identified by its fluorescence at 254 nm and its migration which is less than that of quinidine is scraped off and eluted with methanol. You get 1 mg of (3S)-3-hydroxyquinidine, identical to an authentic sample. Unreacted quinidine is recovered in the same way and almost quantitatively (42 mg).
EKSEMPEL 2 EXAMPLE 2
Hydroksylering av dihydrokinidin ved M. plumbeus Hydroxylation of dihydroquinidine by M. plumbeus
Den samme inkubering utføres på en lignende kultur av The same incubation is carried out on a similar culture of
M. plumbeus CBS 111-07 med 1 g/liter sluttkonsentrasjon dihydrokinidin, utbytter etter 20 dager 5 mg (3S)-3-hydroksy dihydrokinidin, identisk med en autentisk prøve og 80 mg gjenvunnet dihydrokinidin. M. plumbeus CBS 111-07 with 1 g/liter final concentration of dihydroquinidine, yields after 20 days 5 mg of (3S)-3-hydroxy dihydroquinidine, identical to an authentic sample and 80 mg of recovered dihydroquinidine.
EKSEMPEL 3 EXAMPLE 3
Hydroksylering av dihydrokinidin med S. rimosus Hydroxylation of dihydroquinidine by S. rimosus
Noen få ml av en forkultur av S. rimosus NRRL 2334 forkultur settes til 100 ml medium B (beskrevet nedenunder), justert til pH 7. Dyrking utføres ved 72 timer ved 27°C; 100 mg dihydrokinidin oppløst i 1 ml etanol tilsettes og risting fortsettes i 25 dager under de samme betingelser. Man får på samme måten 4 mg (3S)-3-hydroksy dihydrokinidin og 85 mg gjenvunnet dihydrokinidin. A few ml of a pre-culture of S. rimosus NRRL 2334 pre-culture is added to 100 ml of medium B (described below), adjusted to pH 7. Cultivation is carried out at 72 hours at 27°C; 100 mg of dihydroquinidine dissolved in 1 ml of ethanol is added and shaking is continued for 25 days under the same conditions. 4 mg of (3S)-3-hydroxy dihydroquinidine and 85 mg of recovered dihydroquinidine are obtained in the same way.
EKSEMPEL 4 EXAMPLE 4
Hydroksylering av kinidin med C. echinulata Hydroxylation of quinidine by C. echinulata
Den samme inkubering utført på lignende kultur (medium A) The same incubation performed on similar culture (medium A)
av C. echinulata NRRL 3655 med kinin i en 1 g/liter sluttkonsentrasjon gir etter 30 dager 9,5 mg (3S)-3-hydroksy kinin identisk med en autentisk prøve og 75 mg gjenvunnet kinin. Også dannelsen av små mengder av to andre produkter (som fluorescerer ved 2 54 nm) påvises. of C. echinulata NRRL 3655 with quinine in a 1 g/liter final concentration gives after 30 days 9.5 mg of (3S)-3-hydroxy quinine identical to an authentic sample and 75 mg of recovered quinine. The formation of small amounts of two other products (which fluoresce at 2 54 nm) is also detected.
EKSEMPEL 5 EXAMPLE 5
Hydroksylering av dihydrokinin med M. circinelloides Hydroxylation of dihydroquinine by M. circinelloides
Den samme inkubering utført på en lignende kultur (medium The same incubation performed on a similar culture (medium
A) av M. circinelloides CBS 108-16, med dihydrokinin i en sluttkonsentrasjon på 1 gram/liter, gir etter 36 dager 17 mg (3S)-3-hydroksy dihydroksykinin identisk med en autentisk prøve og 70 mg gjenvunnet dihydrokinin. A) of M. circinelloides CBS 108-16, with dihydroquinine in a final concentration of 1 gram/liter, gives after 36 days 17 mg of (3S)-3-hydroxy dihydroxyquinine identical to an authentic sample and 70 mg of recovered dihydroquinine.
EKSEMPEL 6 EXAMPLE 6
Hydroksylering av dihydrokinidin med M. plumbeus Hydroxylation of dihydroquinidine by M. plumbeus
To hundre ml kultur av M. plumbeus MMP 430 i det samme medium A som i eksempel 1 blandes, sentrifugeres og mycelet, vasket under sterile betingelser med en fosfatbuffer inneholdende 2 gram K2HP04 og 1 gram KH2P04 pr. liter destillert vann, plasseres i 100 ml av den samme buffer og inkuberes med dihydrokinidin i en sluttkonsentrasjon på 0,5 gram/liter. Two hundred ml culture of M. plumbeus MMP 430 in the same medium A as in example 1 is mixed, centrifuged and myceled, washed under sterile conditions with a phosphate buffer containing 2 grams of K2HP04 and 1 gram of KH2PO4 per liter of distilled water, placed in 100 ml of the same buffer and incubated with dihydroquinidine in a final concentration of 0.5 gram/litre.
Etter 5 dagers inkubering ved 27°C oppnås 6,5 mg (3S)-3-hydroksy dihydrokinidin og 40 mg gjenvunnet dihydrokinidin ved vanlige teknikker. After 5 days of incubation at 27°C, 6.5 mg of (3S)-3-hydroxy dihydroquinidine and 40 mg of recovered dihydroquinidine are obtained by usual techniques.
Claims (4)
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR8511221A FR2585366B1 (en) | 1985-07-23 | 1985-07-23 | PROCESS FOR THE MICROBIOLOGICAL HYDROXYLATION OF QUININE, QUINIDINE, AND DERIVATIVES |
PCT/FR1986/000260 WO1987000552A1 (en) | 1985-07-23 | 1986-07-22 | Hydroxylation method by microbiological process of quinine, quinidine and derivatives |
Publications (4)
Publication Number | Publication Date |
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NO871173L NO871173L (en) | 1987-03-20 |
NO871173D0 NO871173D0 (en) | 1987-03-20 |
NO166538B true NO166538B (en) | 1991-04-29 |
NO166538C NO166538C (en) | 1991-08-07 |
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Application Number | Title | Priority Date | Filing Date |
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NO871173A NO166538C (en) | 1985-07-23 | 1987-03-20 | MICROBIAL HYDROXYLING METHOD FOR QUININE, QUINIDINE, DIHYDROQININ AND DIHYDROQINIDINE. |
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NO871173D0 (en) | 1987-03-20 |
NO166538C (en) | 1991-08-07 |
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