Migration of deuterium during hydroxylation of aromatic substrates by liver microsomes: I. Influence of ring substituents

The hydroxylation of a variety of specifically deuterated aromatic substrates by liver microsomes has been investigated. Labeled substrates which cannot readily ionize by loss of a proton from the ring substituent display deuterium retentions varying from 40 to 64% upon hydroxylation at the ring position bearing the isotopic label. These substrates include compounds with either electron-donating or electron attracting substituents (anisole-4- 2 H, diphenyl ether-4- 2 H, biphenyl-4- 2 H, N -methyl- N -(phenyl-4- 2 H)-benzenesulfonamide, toluene-4- 2 H, fluorobenzene-4- 2 H, chlorobromobenzene-4- 2 H, benzonitrile-4- 2 H, benzamide-4- 2 H, and nitrobenzene-4- 2 H). Substrates that can, however, ionize by loss of a proton from the ring substituent exhibit lower retentions ranging from 0 to 30% at pH 8. This class of compounds includes phenols such as salicylic-5- 2 H acid, aniline-4- 2 H, N -(phenyl-4- 2 H)-benzenesulfonamide, and various N -acylanilines-4- 2 H. As the acidity of the amide hydrogen increases in the acylanilines, migration and retention of deuterium decreases. A mechanism is presented which accommodates the retention values observed for both classes of substrates. The degree of retention for acetanilide-4- 2 H is strongly dependent upon the pH of the incubating medium ranging from 21 (pH 10) to 44% (pH 6). N -(Phenyl-4- 2 H)-benzamide shows a similar pH dependence whereas nonionizable substrates (biphenyl-4- 2 H or anisole-4- 2 H) do not display this dependence. The relative reactivity of the substrates studied toward the microsomal hydroxylating system correlates well with their reactivity toward chemical reagents which cause electrophilic substitution: i.e., reactive or electron-rich rings act as the best substrates. In chemical terms, the microsomal hydroxylating system acts as though it is a weak, selective electrophile.