2019-05-13

DXA₃ now known as DiEpHEDE based on the synthesis, purification, and characterization provided by a team of Cayman scientists.

Andrei Kornilov, Paul D. Kennedy, Maceler Aldrovandi, Andrew J.A. Watson, Christine Hinz, Bryan Harless, Joseph Colombo, Kirk M. Maxey, Victoria J. Tyrrell, Matthew Simon, Varinder K. Aggarwal, William E. Boeglin, Alan R. Brash, Robert C. Murphy, Valerie B. O’Donnell

Highlights

• Previous studies using a lipidomic approach uncovered DXA₃, a novel eicosanoid formed by COX oxidation of arachidonic acid during platelet stimulation.
• In an attempt to isolate and verify the structure of DXA₃, non-enzymatic oxidation of arachidonic acid was performed, and a product matching DXA₃ but having different stereochemistry was isolated.
• Follow up ¹H and COSY NMR analysis was performed to verify the structure of this stereoisomer and has provided additional evidence for the structure of DXA₃.
• Structure elucidation identified this lipid as 8,9-11,12-diepoxy-13-hydroxy-eicosadienoic acid, abbreviated to 8,9-11,12-DiEp-13-HEDE or DiEpHEDE. Efforts to characterize the specific stereochemistry of both the isolated lipid and platelet-derived lipid are ongoing.

Abstract

Eicosanoids are critical mediators of fever, pain and inflammation generated by immune and tissue cells. We recently described a new bioactive eicosanoid generated by cyclooxygenase-1 (COX-1) turnover during platelet activation that can stimulate human neutrophil integrin expression. On the basis of mass spectrometry (MS/MS and MS³), stable isotope labeling and GC/MS analysis, we previously proposed a structure of 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (DXA₃). Here, we achieved enzymatic synthesis and ¹H-NMR characterization of this compound with results in conflict with the previously proposed structural assignment. Accordingly, by using LC-MS, we screened autoxidation reactions of 11-HpETE and thereby identified a candidate sharing the precise reverse phase chromatographic and MS characteristics of the platelet product. We optimized these methods to increase yield, allowing full structural analysis by ¹H-NMR. The revised assignment is presented here as 8,9-11,12-diepoxy-13-hydroxy-eicosadienoic acid, abbreviated to 8,9-11,12-DiEp-13-HEDE or DiEpHEDE, substituted for the previous name DXA₃. We found that in platelets, the lipid likely forms via dioxolane ring opening with rearrangement to the diepoxy moieties, followed by oxygen insertion at C13. We present its enzymatic biosynthetic pathway and MS/MS fragmentation pattern, and using the synthetic compound, demonstrate that it has bioactivity. For the platelet lipid, we estimate 16 isomers based on our current knowledge (and 4 isomers for the synthetic lipid). Determining the exact isomeric structure of the platelet lipid remains to be undertaken.

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