تفاعل #82445

معادلة التفاعل

31-O-desmethylFK-506

DMSO

→

FK-506

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المعالجة

1. 1
   workup.ADDITIONmethyltransferase was introduced into TK21
2. 2
   أخرىThe resulting transformant was isolated
3. 3
   workup.ADDITIONthe solution was added to the 500 ml bioconversion culture at the time of the transfer of the seed culture
4. 4
   أخرىAt different time intervals, an aliquot of the bioconversion culture was withdrawn
5. 5
   workup.ADDITIONThe culture was mixed with an equal volume of methanol
6. 6
   استخلاصThe resulting supernatant was extracted with methylene chloride
7. 7
   أخرىthe organic phase was recovered
8. 8
   تركيزThis extract was concentrated to dryness under reduced pressure
9. 9
   غسيلThe desired product was eluted with 60-80% acetonitrile which

الإجراء التجريبي

The gene (FKMT2) encoding 3 1-O-desmethylFK-506 O:methyltransferase was introduced into TK21, a strain of Streptomyces lividaris, as described above. The resulting transformant was isolated and was grown in the seed and bioconversion media. One hundred milligrams of 31-O-desmethylFK-506 was then dissolved in 50 μl DMSO and the solution was added to the 500 ml bioconversion culture at the time of the transfer of the seed culture. At different time intervals, an aliquot of the bioconversion culture was withdrawn and examined for the formation of FK-506. The methylation of the 31-O-desmethylFK-506 started at about 18th hour of the incubation and continued until 651h hour, when there was complete conversion of the substrate. The conversion of the 31-O-desmethylFK-506 into FK-506 was also measured using established HPLC procedures. The conversion was basically quantitative with no sign of side product formation. In order to confirm the nature of the bioconversion product, 500 ml bioconversion culture was harvested after 66 hours. The culture was mixed with an equal volume of methanol and centrifuged. The resulting supernatant was extracted with methylene chloride and the organic phase was recovered. This extract was concentrated to dryness under reduced pressure and the residue was suspended in 25 ml of 25% acetonitrile. The resulting solution was applied on a semi-preparative reverse-phase column and the column was developed with a gradient of acetonitrile in water. The desired product was eluted with 60-80% acetonitrile which was worked-up to yield 65 mg of the purified FK-506 (NMR and mass spectra obtained for the isolated product were identical to those of the standard, FK-506).

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