Lipoxygenases (LOs) catalyze lipid peroxidation and have been implicated in a number of human diseases connected to oxidative stress and inflammation. These enzymes have also attracted considerable attention due to large kinetic isotope effects (30−80) for the rate-limiting hydrogen abstraction step with linoleic acid (LA) as substrate. Herein, we report kinetic isotope effects (KIEs) in the reactions of three human LOs (platelet 12-hLO, reticulocyte 15-hLO-1, and epithelial 15-hLO-2) with arachidonic acid (AA). Surprisingly, the observed KIEs with AA were much smaller than the previously reported values with LA. Investigation into the origins for the smaller KIEs led to the discovery of isotope sensitive branching of the reaction pathways. Product distribution analysis demonstrated an inversion in the regioselectivity of 15-hLO-1, with hydrogen abstraction from C13 being the major pathway with unlabeled AA, but abstraction from C10 predominating when the methylene group at position 13 was deuterated. Smaller but clear changes in regioselectivity were also observed for 12-hLO and 15-hLO-2. Keywordsisotope sensitive branching; lipoxygenase; arachidonic acid Lipoxygenases (LOs) are non-heme iron proteins that catalyze the hydrogen atom abstraction from a bisallylic position in polyunsaturated fatty acids and the subsequent addition of molecular oxygen to the resulting substrate radical to generate hydroperoxide products (1). Mammalian LOs catalyze key steps in the conversion of arachidonic acid (AA), 1 a C20 tetraunsaturated fatty acid (20:4, ω−6), to lipoxins and leukotrienes (2). They have been implicated in a number of pathologies including cancer (3,4), atherosclerosis (5) and Alzheimer's disease (6,7). In plants, LOs convert linoleic acid (LA), a C18 bisunsaturated fatty acid (18:2, ω−6), into 13-hydroperoxyoctadecadienoic acid (13-HPODE), a precursor for † This work was supported by the National Institutes of Health (GM44911, WAV and GM56062, TRH) and American Heart Association pre-doctoral fellowships (0615604Z, CJ and 0310006Z, CMM).vddonk@uiuc.edu. tholman@chemistry.ucsc.edu. Supporting information Synthetic procedures for 10,10,13,13-d 4 arachidonic acid and HPLC traces displaying product distributions of the human lipoxygenase reactions are available free of charge via the internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 July 8. Published in final edited form as:Biochemistry. jasmonates and aldehydes involved in signaling, germination, and senescence (8). In mammals, the isozymes are named according to the position of AA that reacts with molecular oxygen (Figure 1). Several human LOs (5-, 12-, and 15-hLOs) have thus far been identified (9), with this study focusing on the latter two groups. Two isozymes of 15-hLO have been characterized in humans and are called 15-hLO-1 (also termed 12/15-hLO) (10,11) and 15-hLO-2 (12). The former exhibits a product selectivity of 15-HPETE over 12-HPETE of about a 9:1 (13), whereas the lat...

In the presence of catalytic vitamin B(12) and a reducing agent such as Ti(III)citrate or Zn, arylalkenes are dimerized with unusual regioselectivity forming a carbon [bond] carbon bond between the benzylic carbons of each coupling partner. Dimerization products were obtained in good to excellent yields for mono- and 1,1-disubstituted alkenes. Dienes containing one aryl alkene underwent intramolecular cyclization in good yields. However, 1,2-disubstituted and trisubstituted alkenes were unreactive. Mechanistic investigations using radical traps suggest the involvement of benzylic radicals, and the lack of diastereoselectivity in the product distribution is consistent with dimerization of two such reactive intermediates. A strong reducing agent is required for the reaction and fulfills two roles. It returns the Co(II) form of the catalyst generated after the reaction to the active Co(I) state, and by removing Co(II) it also prevents the nonproductive recombination of alkyl radicals with cob(II)alamin. The mechanism of the formation of benzylic radicals from arylalkenes and cob(I)alamin poses an interesting problem. The results with a one-electron transfer probe indicate that radical generation is not likely to involve an electron transfer. Several alternative mechanisms are discussed.

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[structure: see text] Prostaglandin H synthase catalyzes the first committed step in the biosynthesis of prostaglandins and thromboxane. Herein we report the synthesis of four site-specifically labeled arachidonic acids for investigation of the radical intermediate formed during this enzymatic reaction. Two compounds were prepared using a common C9-C11 fragment, while another target was synthesized using a previously reported advanced intermediate. An alkyne coupling followed by hydrogenation and Wittig reaction was used to prepare the final labeled substrate.