反応 #543551

反応方程式

Sulfuric acid

ATP

MgCl2

CoA

(R)-3-hydroxyoctanoate

(R)-3-hydroxy-5-phenylvaleryl CoA

(R)-3-hydroxy-5-(4-fluorophenyl)valeryl CoA

→

(R)-3-hydroxyoctanoyl-CoA

溶媒

反応条件

後処理

1. 1
   濃縮so that the concentration
2. 2
   その他The solution was stored in a warm bath at 37° C.
3. 3
   workup.ALIQUOTwas sampled at appropriate times
4. 4
   その他the progress of the reaction by HPLC
5. 5
   その他the enzyme reaction
6. 6
   抽出(R)-3-hydroxyoctanoate being an unreacted substrate was extracted with n-heptane
7. 7
   その他removed
8. 8
   洗浄a RP18 column (nucleosil C18, 7 μm, Knauser), elution
9. 9
   濃縮was conducted with the linear concentration gradient of acetonitrile using a 25 mM phosphate buffer solution (pH 5.3)
10. 10
    その他as a mobile phase, and absorption spectra of 200 to 500 nm
11. 11
    その他produced through the enzyme reaction

実験手順

(R)-3-hydroxyoctanoyl-CoA was synthesized in accordance with the following procedure, based on the method of Rehm BHA, Kruger N, Steinbuchel A (1998) Journal of Biological Chemistry 273 pp 24044–24051, with the method slightly modified. Acyl-CoA synthetase (manufactured by Sigma Co., Ltd.) was dissolved in a tris hydrochloric buffer solution (50 mM, pH 7.5) containing 2 mM ATP, 5 mM MgCl2, 2 mM CoA and 2 mM (R)-3-hydroxyoctanoate so that the concentration was 0.1 milliunit per microliter. The solution was stored in a warm bath at 37° C., and was sampled at appropriate times to analyze the progress of the reaction by HPLC. Sulfuric acid was added in the sampled reaction solution to make a concentration 0.02 N to stop the enzyme reaction, and thereafter (R)-3-hydroxyoctanoate being an unreacted substrate was extracted with n-heptane and removed. For the analysis by HPLC, using a RP18 column (nucleosil C18, 7 μm, Knauser), elution was conducted with the linear concentration gradient of acetonitrile using a 25 mM phosphate buffer solution (pH 5.3) as a mobile phase, and absorption spectra of 200 to 500 nm were monitored by a diode array detector, thereby detecting a thioester compound produced through the enzyme reaction. In a similar way, (R)-3-hydroxy-5-phenylvaleryl CoA, and (R)-3-hydroxy-5-(4-fluorophenyl)valeryl CoA were prepared.

出典