반응 #409094

반응 방정식

Pinoresinol

pinoresinol

lariciresinol

→

(+)-Pinoresinol (+)-Lariciresinol

용매

MeOH

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후처리

1. 1
   기타the enzyme preparation (i.e., total protein extract from E. coli, 210 μl)
2. 2
   기타The enzymatic reaction
3. 3
   추출the assay mixture was extracted with EtOAc (500 μl)
4. 4
   workup.ADDITIONcontaining (±)-lariciresinols (20 μg) and (±)-secoisolariciresinols (20 μg) as radiochemical carriers
5. 5
   기타After centrifugation (13,800×g, 5 min), the EtOAc solubles were removed
6. 6
   추출the extraction procedure
7. 7
   기타removed for determination of its radioactivity
8. 8
   기타The remainder of the combined EtOAc solubles was evaporated to dryness in vacuo

실험 절차

Enzyme Assays. Pinoresinol and lariciresinol reductase activities were assayed by monitoring the formation of [3H]lariciresinol and [3H]secoisolariciresinol as set forth in Example 8, with the following modifications. Briefly, each assay for pinoresinol reductase activity consisted of (±)-pinoresinols (5 mM in MeOH, 20 μl) and the enzyme preparation (i.e., total protein extract from E. coli, 210 μl). The enzymatic reaction was initiated by addition of [4R-3H]NADPH (10 mM, 6.79 kBq/mmol in distilled H2O, 20 μl). After 3 hour incubation at 30° C. with shaking, the assay mixture was extracted with EtOAc (500 μl) containing (±)-lariciresinols (20 μg) and (±)-secoisolariciresinols (20 μg) as radiochemical carriers. After centrifugation (13,800×g, 5 min), the EtOAc solubles were removed and the extraction procedure was repeated. For each assay, the EtOAc solubles were combined with an aliquot (100 μl) removed for determination of its radioactivity using liquid scintillation counting. The remainder of the combined EtOAc solubles was evaporated to dryness in vacuo, reconstituted in MeOH/H2O (30:70, 100 μl) and subjected to reversed phase and chiral column HPLC.

출처