We report the newly successful synthesis of 3-thia-glutaryl-CoA and a partial synthesis of 4-nitrobutyryl-CoA, substrate analogs to glutaryl-CoA dehydrogenase (GCD), an enzyme utilized by mammals and by bacteria in the metabolism of the amino acids L-tryptophan, L-lysine, and L-hydroxylysine. Using flavin adenine dinucleotide (FAD) as its cofactor, GCD catalyzes the oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA, thereby contributing electrons to the electron transport chain and crotonyl-CoA to b-oxidation. Earlier studies of the reductive process of GCD revealed that the electron transfer appears to be thermodynamically unfavorable when the reduction potentials of the electron transfer reactions are evaluated separately (1). This behavior has been exhibited by other acyl-CoA dehydrogenases such as the short and medium chain acyl-CoA dehydrogenases (SCAD and MCAD). Through spectroelectrochemical studies of SCAD and MCAD, where each were complexed with different substrate, product, and substrate and product analog compounds, it was revealed that the electron transfer behavior of these enzymes is regulated by the binding of certain CoA compounds in the active site (2,3,4,5). The particular substrate analog syntheses reported here were designed to supply analogs that should bind to the GCD active site, enabling GCD spectroelectrochemical studies similar in design to those for SCAD and MCAD to be performed. Glutaryl-CoA is a poor compound to use in these studies because it irreversibly loses its CO 2 end group when it binds to GCD in the presence of electron acceptors used in spectroelectrochemical analysis. The structures of the substrate and the two substrate analogs are shown in Fig. 1.
It can be seen that 3-thia-glutaryl-CoA is identical to glutaryl-CoA except for a substituted sulfur atom. Since substrate electron transfer from glutaryl-CoA to the FAD in GCD has been shown to occur via b-hydride transfer (6), this analog is expected to complex to GCD without causing the reduction of GCD, due to the absence of a b-hydride. This will allow for the evaluation of the effects of the binding process alone on GCD behavior. As an analog isoelectronic with glutaryl-CoA, 4nitrobutyryl-CoA should bind and reduce GCD; however, it should not release its 4nitro group, enabling the investigation of reversible electron transfer with GCD.
The syntheses of acyl-CoA compounds generally follow a two-step procedure that begins with the activation of a carboxylic acid via anhydride formation. This anhydride is then subjected to nucleophilic attack by the sulfide group of free CoA, forming the coupled final product.
The first step in the production of 3-thia-glutaryl-CoA is the synthesis of thiodiglycolic acid anhydride. A 2.5 g (0.017 mol) portion of thiodiglycolic acid and 7.