Synthesis of isotopically labeled arachidonic acids to probe the reaction mechanism of prostaglandin H synthase.
Prostaglandin H synthase (PGHS) catalyzes the conversion of arachidonic acid to prostaglandin G(2) in the cyclooxygenase reaction. The first step of the mechanism has been proposed to involve abstraction of the pro-S hydrogen atom from C13 to generate a pentadienyl radical spanning C11-C15. We report here the synthesis of six site-specifically deuterated arachidonic acids to investigate the structure of the radical intermediate. The preparation of these compounds was achieved using a divergent scheme that involved one advanced intermediate for all targets. The synthetic design introduced the label late in the routes and allowed the utilization of common synthetic intermediates in the preparation of various targets. Both 13(R)- and 13(S)-deuterium-labeled arachidonic acids were synthesized in high enantiomeric purity as deduced from soybean lipoxygenase assays and mass spectrometric analysis of the resulting enzymatic products. Each synthetic compound was reacted under anaerobic conditions with the wide singlet tyrosyl radical of PGHS-2 to generate a radical intermediate that was analyzed by EPR. Deuterium substitution at positions 11, 13(S), and 15 resulted in the loss of one hyperfine interaction, indicating that the protons at these positions interact with the unpaired electron. Simulation of the spectra was achieved with one set of parameters that are consistent with the assignment of a pentadienyl radical. Use of 16-[(2)H(2)]-arachidonic acid indicated that only one of the protons at C16 gives rise to a strong hyperfine interaction. The findings are discussed in the context of two proposed mechanisms for the cyclooxygenase reaction.
Peng
Sheng
S
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.
Okeley
Nicole M
NM
Tsai
Ah-Lim
AL
Wu
Gang
G
Kulmacz
Richard J
RJ
van der Donk
Wilfred A
WA
D001095:Arachidonic Acids / Q000138:chemical synthesis* / Q000737:chemistry; D003903:Deuterium / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods; D008084:Lipoxygenase / Q000737:chemistry / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013237:Stereoisomerism
D001095:Arachidonic Acids; D005609:Free Radicals; D003903:Deuterium; C101084:lipoxygenase L-1; D008084:Lipoxygenase; D011451:Prostaglandin-Endoperoxide Synthases