Synthesis and evaluation of (+) and (−)‐2,2‐difluorocitrate as inhibitors of rat‐liver ATP‐citrate lyase and porcine‐heart aconitase

The enantiomers (+) and (−)‐2,2‐difluorocitrate have been synthesized. Both are good inhibitors of ATP‐citrate lyase, showing competitive inhibition against citrate, with K_is= 0.7 μM for (+)‐2,2‐difluorocitrate and 3.2 μM for (−)‐2,2‐difluorocitrate. The inhibition patterns with either ATP or CoA as the varied substrate were uncompetitive and mixed, respectively, but with much weaker inhibition constants. Neither isomer undergoes carbon‐carbon bond cleavage as a substrate and there is no evidence of irreversible time‐dependent inactivation. When ATP‐citrate lyase is incubated with CoA and difluorocitrate, the maximal intrinsic ATPase rate is 10% of the citrate‐induced rate for the (+)‐enantiomer and 2% for the (−)‐enantiomer. ¹⁹F‐NMR studies confirm that only the (+)‐enantiomer is chemically processed. The effects of the difluorocitrate enantiomers on the reaction catalysed by aconitase were examined. (−)‐2,2‐Difluorocitrate is a competitive inhibitor against citrate (K_is= 1.5 μM), whereas the (+)‐enantiomer is a relatively poor mixed inhibitor (K_i > 300 μM). The (−)‐enantiomer irreversibly inactivates aconitase at 1.1 min^‐1· mM^‐1 at 25°C and pH 7.4, whereas no irreversible inhibition is seen with the (+)‐enantiomer. Therefore, it would be expected that the (+)‐enantiomer would slow the rate of acetyl‐CoA synthesis in vivo, without inhibiting the citric acid cycle.