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. 2021 Nov 25;10(12):1888.
doi: 10.3390/antiox10121888.

Regioselective and Stereoselective Epoxidation of n-3 and n-6 Fatty Acids by Fungal Peroxygenases

Alejandro González-Benjumea  1 , Dolores Linde  2 , Juan Carro  2 , René Ullrich  3 , Martin Hofrichter  3 , Angel T Martínez  2 , Ana Gutiérrez  1
Affiliations

Regioselective and Stereoselective Epoxidation of n-3 and n-6 Fatty Acids by Fungal Peroxygenases

Alejandro González-Benjumea et al. Antioxidants (Basel). .

Abstract

Epoxide metabolites from n-3 and n-6 polyunsaturated fatty acids arouse interest thanks to their physiological and pharmacological activities. Their chemical synthesis has significant drawbacks, and enzymes emerge as an alternative with potentially higher selectivity and greener nature. Conversion of eleven eicosanoid, docosanoid, and other n-3/n-6 fatty acids into mono-epoxides by fungal unspecific peroxygenases (UPOs) is investigated, with emphasis on the Agrocybe aegerita (AaeUPO) and Collariella virescens (rCviUPO) enzymes. GC-MS revealed the strict regioselectivity of the n-3 and n-6 reactions with AaeUPO and rCviUPO, respectively, yielding 91%-quantitative conversion into mono-epoxides at the last double bond. Then, six of these mono-epoxides were obtained at mg-scale, purified and further structurally characterized by 1H, 13C and HMBC NMR. Moreover, chiral HPLC showed that the n-3 epoxides were also formed (by AaeUPO) with total S/R enantioselectivity (ee > 99%) while the n-6 epoxides (from rCviUPO reactions) were formed in nearly racemic mixtures. The high regio- and enantioselectivity of several of these reactions unveils the synthetic utility of fungal peroxygenases in fatty acid epoxidation.

Keywords: NMR; bioactive compounds; chiral HPLC; epoxylipids; omega 3 (n-3) fatty acids; omega 6 (n-6) fatty acids; polyunsaturated fatty acids; regioselective synthesis; stereoselective synthesis; unspecific peroxygenases.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Formulae of the n-3 (1–8) and n-6 (9–11) fatty acids used in enzymatic epoxidation reactions: (1) Hexadeca-7,10,13-trienoic acid (HTA); (2) Octadeca-6,9,12,15-tetraenoic acid (SDA); (3) Eicosa-11,14,17-trienoic acid (ETE); (4) Eicosa-5,8,11,14,17-pentaenoic acid (EPA); (5) Heneicosa-6,9,12,15,18-pentaenoic acid (HPA); (6) Docosa-7,10,13,16,19-pentaenoic acid (DPA); (7) Docosa-4,7,10,13,16,19-hexaenoic acid (DHA); (8) Tetracosa-6,9,12,15,18,21-hexaenoic acid (nisinic acid); (9) Eicosa-8,11,14-trienoic acid (DGLA); (10) Eicosa-5,8,11,14-tetraenoic acid (AA); and (11) Docosa-7,10,13,16-tetraenoic acid (AdA).
Figure 2
Figure 2
Formulae of main products (12–22) from UPO epoxidation of n-3/n-6 fatty acids (Figure 1): (12) 13,14-epoxyhexadeca-7,10-dienoic acid (13,14-EpHDE); (13) 15,16-epoxyoctadeca-6,9,12-trienoic acid (15,16-EpOTE); (14) 17,18-epoxyeicosa-11,14-dienoic acid (17,18-EpEDE); (15) 17,18-epoxyeicosa-5,8,11,14-tetraenoic acid (17,18-EpETrE); (16) 18,19-epoxyheneicosa-6,9,12,15-tetraenoic acid (18,19-EpHTrE); (17) 19,20-epoxydocosa-7,10,13,16-tetraenoic (19,20-EpDTrE); (18) 19,20-epoxydocosa-4,7,10,13,16-pentaenoic acid (19,20-EpDPE); (19) 21,22-epoxytetracosa-6,9,12,15,18-pentaenoic acid (21,22-EpTPE); (20) 14,15-epoxyeicosa-8,11-dienoic acid (14,15-EpEDE); (21) 14,15-epoxyeicosa-5,8,11-trienoic acid (14,15-EpETE); and (22) 16,17-epoxydocosa-7,10,13-trienoic acid (16,17-EpDTE).
Figure 3
Figure 3
Oxyfunctionalization patterns of n-3 compound 4 (EPA) by different UPOs.
Figure 4
Figure 4
Oxyfunctionalization patterns of n-6 compound 9 (DGLA) by different UPOs.
Figure 5
Figure 5
Sections of the AaeUPO (A), CciUPO (B), MroUPO (C) and CviUPO (D) molecules, highlighting the differences in heme access channels. External surface of the protein and surface of the residues lining the channel are shown in light teal, and the buried heme is displayed as sticks in CPK coloring. From PDB entries 2YP1 (A) and 5FUJ (C) and homology models (B,D).
Figure 6
Figure 6
HMBC 2D-NMR spectra of enzymatic epoxides: (A) 19,20-EpDPE (18) from DHA (7) reaction with AaeUPO; and (B) 14,15-EpETE (21) from AA (10) reaction with rCviUPO. Several relevant correlation signals are indicated.
Scheme 1
Scheme 1
Characteristic enzymatic mono-epoxidation patterns of n-3 and n-6 polyunsaturated fatty acids by AaeUPO and rCviUPO, respectively. * Also including the rCviUPO T158F variant.
Figure 7
Figure 7
Chiral HPLC profiles (at 202 nm) and formulae of isolated n-3 and n-6 mono-epoxides: (A) 19(S),20(R)-EpDPE (18) from AaeUPO reaction with DHA (7) (top) compared with a racemic standard (bottom); and (B) 14(R),15(S)-EpETE and 14(S),15(R)-EpETE (21) from rCviUPO reaction with AA (10) (top) compared with a racemic standard (bottom).
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