Synthesis of Acyl-CoA Derivatives

For the small scale synthesis of butyryl-CoA and succinyl-CoA with the symetric anhydride method, CoA (4 mg, 0.005 mmol, 1 eq.) was dissolved in 200 µL 0.5 M NaHCO₃. The solution was cooled down to 4 °C and 1.6 equivalents of the corresponding anhydride was added (butyryic anhydride: 1.3 µL, 0.0081 mmol, 1.6 eq.; succinic anhydride: 0.81 mg, 0.0081 mmol, 1.6 eq.). The reaction was stirred on ice for 45 min and its completion confirmed by a test for remaining free thiols with Ellman’s reagent. The reaction mixture was directly injected into the HPLC-MS for downstream analysis (see Section 4.6).
The synthesis of acetyl-CoA, propionyl-CoA and crotonyl-CoA was scaled up 50 fold using 200 mg CoA (0.25 mmol, 1 eq.) in 5 mL 0.5 M NaHCO₃ and 1.6 eq. of the corresponding anhydride (acetic anhydride: 45 µL, 0.41 mmol, 54 µL 1.6 eq. propionic anhydride: 0.41 mmol, 1.6 eq.; crotonic anhydride: 64 µL 0.41 mmol, 1.6 eq.). The reactions were stirred on ice for 45 min and then directly injected into the HPLC-MS for downstream analysis and purification (see Section 4.6).