title: Synthesis of chloromethyl ketone derivatives of fatty acids. Their use as specific inhibitors of acetoacetyl-coenzyme A thiolase, cholesterol biosynthesis and fatty acid synthesis.
abstract: A general route for the synthesis of chloromethyl ketone derivatives of fatty acids is described. 5-Chloro-4-oxopentanoic acid, 7-chloro-6-oxoheptanoic acid, 9-chloro-8-oxononanoic acid and 11-chloro-10-oxoundecanoic acid were synthesized by this method and tested as covalent inhibitors of pig heart acetoacetyl-CoA thiolase. The K1 decreased by approx. 20-fold for each pair of methylenes added to the chain length, showing that the initial stage in inhibitor binding occurs at a non-polar region of the protein. This region is probably located at the enzyme active site, since inhibition was prevented by acetoacetyl-CoA or acetyl-CoA but not by CoA. The site of modification by chloromethyl ketone derivatives of fatty acids is restricted to a thiol group, since inactivation of the enzyme was prevented by reversible thiomethylation of the active-site thiol. In contrast, an amino-directed reagent, citraconic anhydride, still inactivated the enzyme, even when the active-site thiol was protected. Evidence that the enzyme thiol was particularly reactive came from studies on the pH-dependence of the alkylation reaction and thiol-competition experiments. Inhibition of the enzyme proceeded suprisingly well at acidic pH values and a 10(5) molar excess of external thiol over active-site thiol was required to prevent inhibition by 0.3 mM-9-chloro-8-oxononanoic acid. In addition to inhibiting isolated acetoacetyl-CoA thiolase, in hepatocytes the chloromethyl ketone derivatives of fatty acids also inhibited chloresterol synthesis, which uses this enzyme as an early step in the biosynthetic pathway. In isolated cells, the chloromethyl ketone derivatives of fatty acids were considerably less specific in their inhibitory action compared with 3-acetylenic derivatives of fatty acids, which act as suicide inhibitors of acetoacetyl-CoA thiolase. However, 9-chloro-8-oxononanoic acid was also an effective inhibitor of both hepatic cholesterol and fatty acid synthesis in mice in vivo, whereas the acetylenic fatty acid derivative, dec-3-ynoic acid, was completely ineffective. The effective inhibitory  dose of 9-chloro-8-oxononanoic acid (2.5-5 mg/kg) was substantially lower than the estimated LD50 for the inhibitor (100 mg/kg).
mesh_terms: D000101:Acetyl-CoA C-Acetyltransferase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D000123:Acetyltransferases / Q000037:antagonists & inhibitors*; D000812:Anhydrides; D000818:Animals; D002784:Cholesterol / Q000096:biosynthesis*; D005227:Fatty Acids / Q000096:biosynthesis / Q000138:chemical synthesis / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007659:Ketones / Q000138:chemical synthesis / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D051381:Rats; D013438:Sulfhydryl Compounds
chemical_list: D000812:Anhydrides; D005227:Fatty Acids; D007659:Ketones; D013438:Sulfhydryl Compounds; D002784:Cholesterol; D000123:Acetyltransferases; D000101:Acetyl-CoA C-Acetyltransferase