Synthesis of 13C-labeled γ-hydroxybutyrates for EPR studies with 4-hydroxybutyryl-CoA dehydratase

Abstract

4-Hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum catalyses the reversible dehydration of its substrate 4-hydroxybutyryl-CoA (4-HB-CoA) to crotonyl CoA. The enzyme contains one [4Fe–4S] 2+ cluster and one flavin adenine dinucleotide (FAD) molecule per homotetramer. Incubation of the enzyme with its substrate under equilibrium conditions followed by freezing at 77 K induced the EPR-spectrum of a neutral flavin semiquinone ( g = 2.005, linewidth 2.1 mT), while at 10 K additional signals were detected. In an attempt to characterize these signals, 4-HB-CoA molecules specifically labeled with 13C have been synthesized. This was achieved via 13C-labeled γ-butyrolactones, which were obtained from 13C-labeled bromoacetic acids by efficient synthetic routes. Incubation of the 13C-labeled 4-hydroxybutyrate-CoA molecules with 4-hydroxybutyryl-CoA dehydratase did not lead to marked broadening of the signals.