Reaktion #52122

Reaktionsgleichung

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

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

(R,S)-3-hydroxy-7,8-epoxyoctanoyl CoA

3-chlorobenzoic acid

(R,S)-3-hydroxy-7,8-epoxyoctanoic acid

(R,S)-3-hydroxy-7,8-epoxyoctanoyl CoA

ATP

MgCl2

coenzyme A

(R)-3-hydroxyoctanoate

Sulfuric acid

3-hydroxy-7-octenoic acid

→

(R)-3-hydroxyoctanoyl-CoA

Lösungsmittel

Reaktionsbedingungen

Aufarbeitung

1. 1
   Einengenso that the concentration
2. 2
   Sonstigewas stored in a hot bat at 37° C.
3. 3
   workup.ALIQUOTwas sampled at appropriate times
4. 4
   Sonstigethe progress of the reaction by HPLC
5. 5
   Sonstigethe enzyme reaction
6. 6
   Extraktion(R)-3-hydroxyoctanoate being an unreacted substrate was extracted with n-heptane
7. 7
   Sonstigeremoved
8. 8
   Waschena RP18 column (nucleosil C18, 7 μm, Knauser), elution
9. 9
   Einengenwas conducted with the linear concentration gradient of acetonitrile using a 25 mM phosphate buffer solution (pH 5.3)
10. 10
    Sonstigeas a mobile phase, and absorption spectra of 200 to 500 nm
11. 11
    Sonstigeproduced through the enzyme reaction
12. 12
    Sonstigewas prepared
13. 13
    Sonstigesynthesized by the method

Vorschrift

(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 coenzyme A and 2 mM (R)-3-hydroxyoctanoate so that the concentration was 0.1 milliunit per microliter. The solution was stored in a hot bat 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, (R)-3-hydroxy-5-(4-fluorophenyl) valeryl CoA, (R,S)-3-hydroxy-5-phenoxyvaleryl CoA and (R,S)-3-hydroxy-7,8-epoxyoctanoyl CoA were prepared. Furthermore, (R,S)-3-hydroxy-7,8-epoxyoctanoic acid for use in preparation of (R,S)-3-hydroxy-7,8-epoxyoctanoyl CoA was prepared by epoxidizing unsaturated parts of 3-hydroxy-7-octenoic acid synthesized by the method described in Int. J. Biol. Macromol., 12, 85-91 (1990) with 3-chlorobenzoic acid.

Quelle