Enzymatic synthesis and chemical inversion provide both enantiomers of bioactive epoxydocosapentaenoic acids

Abstract

Epoxy PUFAs are endogenous cytochrome P450 (P450) metabolites of dietary PUFAs. Although these metabolites exert numerous biological effects, attempts to study their complex biology have been hampered by difficulty in obtaining the epoxides as pure regioisomers and enantiomers. To remedy this, we synthesized 19,20- and 16,17-epoxydocosapentaenoic acids (EDPs) (the two most abundant EDPs in vivo) by epoxidation of DHA with WT and the mutant (F87V) P450 enzyme BM3 from Bacillus megaterium WT epoxidation yielded a 4:1 mixture of 19,20:16,17-EDP exclusively as (S,R) enantiomers. Epoxidation with the mutant (F87V) yielded a 1.6:1 mixture of 19,20:16,17-EDP; the 19,20-EDP fraction was ∼9:1 (S,R):(R,S), but the 16,17-EDP was exclusively the (S,R) enantiomer. To access the (R,S) enantiomers of these EDPs, we used a short (four-step) chemical inversion sequence, which utilizes 2-(phenylthio)ethanol as the epoxide-opening nucleophile, followed by mesylation of the resulting alcohol, oxidation of the thioether moiety, and base-catalyzed elimination. This short synthesis cleanly converts the (S,R)-epoxide to the (R,S)-epoxide without loss of enantiopurity. This method, also applicable to eicosapentaenoic acid and arachidonic acid, provides a simple, cost-effective procedure for accessing larger amounts of these metabolites.

Keywords: chemoenzymatic synthesis; cytochrome P450; docosahexaenoic acid; epoxide inversion; epoxy fatty acids; lipids/chemistry; polyunsaturated fatty acids.

Fig. 1.

LC/MS/MS analysis reveals that WT-BM3 produces 16,17-EDP (12%) and 19,20-EDP (88%) from DHA (A). No other regioisomers or diols (DHDPs) are obtained (assessed by comparison to standards) (B).

Fig. 2.

Chiral LC/MS/MS analysis reveals that WT-BM3 stereospecifically produces 19(S),20(R)-EDP [19(S),20(R)-2b, ≥99.5% ee, >99.5:0.05 er] (A) and 16(S),17(R)-EDP [16(S),17(R)-3b, ≥97.6% ee, >98:2 er] (B). Racemic standards are shown on the bottom. The enantiopurities remain consistent upon scaleup.

Fig. 3.

Representative chiral HPLC traces (see Materials and Methods) showing inversion of 16(S),17(R)-2b. Enantiopure 16(S),17(R)-2b produced by BM3 (F87V) (A); artificial mixture of 2b enantiomers before and after inversion (B); and 16(R),17(S)-2b produced by chemical inversion (C). See Materials and Methods. The two peaks at ca. 3 min in each trace correspond to the void time of the column.

Fig. 4.

Representative chiral HPLC traces (see Materials and Methods) showing inversion of 19(S),20(R)-2a. Enantiopure 19(S),20(R)-2a produced by WT-BM3 (A); artificial mixture of 2a before and after inversion (B); and 19(R),20(S)-2a produced by chemical inversion (C). See Materials and Methods. The two peaks at ca. 3 min correspond to the void time of the column.