Steric analysis of epoxyalcohol and trihydroxy derivatives of 9-hydroperoxy-linoleic acid from hematin and enzymatic synthesis

Abstract

We characterize the allylic epoxyalcohols and their trihydroxy hydrolysis products generated from 9R- and 9S-hydroperoxy-octadecenoic acid (HPODE) under non-enzymatic conditions, reaction with hematin and subsequent acid hydrolysis, and enzymatic conditions, incubation with Beta vulgaris containing a hydroperoxide isomerase and epoxide hydrolase. The products were resolved by HPLC and the regio and stereo-chemistry of the transformations were determined through a combination of 1H NMR and GC-MS analysis of dimethoxypropane derivatives. Four trihydroxy isomers were identified upon mild acid hydrolysis of 9S,10S-trans-epoxy-11E-13S-hydroxyoctadecenoate: 9S,10R,13S, 9S,12R,13S, 9S,10S,13S and 9S,12S,13S-trihydroxy-octadecenoic acids, in the ratio 40:26:22:12. We also identified a prominent -ketol rearrangement product from the hydrolysis as mainly the 9-hydroxy-10E-13-oxo isomer. Short incubation (5 min) of 9R- and 9S-HPODE with Beta vulgaris extract yielded the 9R- and 9S-hydroxy-10E-12R,13S-cis-epoxy products respectively. Longer incubation (60 min) gave one specific hydrolysis product via epoxide hydrolase, the 9R/S,12S,13S-trihydroxyoctadecenoate. These studies provide a practical approach for the isolation and characterization of allylic epoxy alcohol and trihydroxy products using a combination of HPLC, GC-MS and 1H NMR.

A fungal catalase reacts selectively with the 13S fatty acid hydroperoxide products of the adjacent lipoxygenase gene and exhibits 13S-hydroperoxide-dependent peroxidase activity

“…Linoleate hydroperoxides were prepared by vitamin E-controlled autoxidation [17] followed by separation of the 9 RS - and 13 RS -hydroperoxides by SP-HPLC and chiral column resolution of the enantiomers [18] or using soybean lipoxygenase for preparation of the 13 S -hydroperoxides [19], Anabaena 9 R -LOX for preparation of 9 R -hydroperoxides [20], and potato 9 S -LOX for preparation of 9 S -hydroperoxides [21]. [1- 14 C]Linoleic acid and [1- 14 C]arachidonic acid were from NEN (Dupont).…”

An Ancient Relative of Cyclooxygenase in Cyanobacteria Is a Linoleate 10S-Dioxygenase That Works in Tandem with a Catalase-related Protein with Specific 10S-Hydroperoxide Lyase Activity

“…The 9RS and 10RS enantiomers were resolved in a single run using a Chiralpak AD column as above and eluting in the order 10S (13.5 min), first 9 enantiomer (not designated, 18.5 min), and 10R (26 min), second 9 enantiomer (48 min). Standards of racemic conjugated diene-containing HPODEs were prepared by vitamin E-controlled autoxidation (17) and the chiral HPODEs using potato (9S) (18), Anabaena (9R) (19), or soybean (13S) lipoxygenases (20).…”

Oxidation of C18 Hydroxy‐Polyunsaturated Fatty Acids to Epoxide or Ketone by Catalase‐Related Hemoproteins Activated with Iodosylbenzene

“…When incubated with the 13 S -hydroxy derivative of linoleic acid (13-HODE) the enzyme formed four products, in order of elution from the RP-HPLC column assigned as the 9,10-epoxy-13-hydroxy derivative (product 1 in Fig. 5A), predominantly (10:1) the 9 R ,10 S ,13 S diastereomer as established in comparison to authentic standards [16], the 9,10- cis -epoxy-13-ketone (product 2 in Fig. 5A), the minor 11,12-epoxy-9-hydroxy derivative (product 3 in Fig.…”

“…The 13 S -hydroxy derivatives were synthesized from linoleic acid or α- or γ-linolenic acids using the soybean LOX [15] and reduced subsequently with sodium borohydride to the hydroxy derivatives, 13 S -HODE, and 13 S -HOTE-ω3 and 13 S -HOTE-ω6. 9 S -HODE was prepared using potato 9 S -LOX activity [16]. The 9- and 13-ketones were prepared by oxidation of the 9- and 13-hydroxy derivatives using activated manganese dioxide or hematin.…”