反应详情

(R)-3-hydroxyoctanoyl-CoA was synthesized according to Rehm B. H. A., Kruger N., Steinbuchel A., Journal of Biological Chemistry, 273, p. 24044-24051, 1998 with slight changes as described below. Acyl-CoA synthetic enzyme (available from Sigma Co.) was dissolved in a Tris-HCl buffer (50 mM, pH 7.5) containing 2 mM ATP, 5 mM MgCl2, 2 mM coenzyme A, and 2 mM (R)-3-hydroxyoctanoate to concentration of 0.1 mU/μl. The mixture was retained in a warm bath at 37° C. and was sampled suitably to analyze progress of a reaction by HPLC. After the enzyme reaction was terminated by adding sulfuric acid to the sampled reaction solution to concentration of 0.02 N, the unreacted substrate (R)-3-hydroxyoctanoate was removed by extraction with n-heptane. The HPLC analysis employed RP18 column (nucleosil C18, 7 μm, Knauser), and elusion was conducted under a linear concentration slope of acetonitrile, using a 25 mM phosphoric acid buffer (pH 5.3) as a moving phase. A thioester compound produced through the enzymatic reaction was detected by monitoring an absorption spectrum of 200 to 500 nm using a diode array detector. (R)-3-hydroxy-5-phenylvaleryl CoA and (R)-3-hydroxy-5-(4-fluorophenyl)valeryl CoA were synthesized in a similar manner.

1. customThe mixture was retained in a warm bath at 37° C.
2. aliquotwas sampled suitably
3. customprogress of a reaction by HPLC
4. customAfter the enzyme reaction
5. customwas terminated
6. additionby adding sulfuric acid
7. customto the sampled reaction solution to concentration of 0.02 N
8. customthe unreacted substrate (R)-3-hydroxyoctanoate was removed by extraction with n-heptane
9. concentrationwas conducted under a linear concentration slope of acetonitrile