Stereochemistry of Oxygenation of Linoleic Acid Catalyzed by Prostaglandin–Endoperoxide H Synthase-2

Abstract

Linoleic acid was incubated with prostaglandin–endoperoxide H synthase-2 (PGHS-2) from ovine placenta. A product consisting of regio- and stereoisomeric hydroxyoctadecadienoic (HOD) acids was obtained. Analysis by straight-phase high-performance liquid chromatography followed by chiral-phase high-performance liquid chromatography demonstrated that linoleic acid was preferentially oxygenated at C-9 to produce the following mixture of HODs: 9(R)-HOD (52%), 9(S)-HOD (11%), 13(R)-HOD (2%), and 13(S)-HOD (35%). As a comparison, linoleic acid was incubated with microsomal prostaglandin–endoperoxide H synthase-1 (PGHS-1) from ovine vesicular gland. This resulted in a product having the following composition: 9(R)-HOD (73%), 9(S)-HOD (9%), 13(R)-HOD (1%), and 13(S)-HOD (17%). The stereochemistry of the hydrogen which was removed from C-11 during the conversion of linoleic acid into hydroxy acids in the presence of PGHS-1 or PGHS-2 was determined by incubation of [(11R)-2H]- and [(11S)-2H]linoleic acids followed by mass spectrometric analysis of the isotope contents of the individual hydroxy acid isomers. Both enzymes were found to catalyze oxygenations which involved stereospecific removal of the (11S) hydrogen and retention of the (11R) hydrogen. The major hydroxy acids, i.e., 9(R)-HOD and 13(S)-HOD, were formed from linoleic acid in reactions which involved antarafacial hydrogen abstraction and oxygen insertion. It is concluded that the initial steps of the PGHS-2- and PGHS-1-catalyzed oxygenations proceed with identical stereochemistry and involve stereospecific removal of the pro-Shydrogen from the ω8-methylene group of the substrate.