Reacción #409094

Ecuación de reacción

Pinoresinol

pinoresinol

lariciresinol

→

(+)-Pinoresinol (+)-Lariciresinol

Disolventes

MeOH

Condiciones de reacción

Tratamiento posterior

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

Procedimiento

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.

Fuente