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W tym Artykule

  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

We present a method useful for large-scale enzymatic synthesis and purification of specific enantiomers and regioisomers of epoxides of arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) with the use of a bacterial cytochrome P450 enzyme (BM3).

Streszczenie

The epoxidized metabolites of various polyunsaturated fatty acids (PUFAs), termed epoxy fatty acids, have a wide range of roles in human physiology. These metabolites are produced endogenously by the cytochrome P450 class of enzymes. Because of their diverse and potent biological effects, there is considerable interest in studying these metabolites. Determining the unique roles of these metabolites in the body is a difficult task, as the epoxy fatty acids must first be obtained in significant amounts and with high purity. Obtaining compounds from natural sources is often labor intensive, and soluble epoxide hydrolases (sEH) rapidly hydrolyze the metabolites. On the other hand, obtaining these metabolites via chemical reactions is very inefficient, due to the difficulty of obtaining pure regioisomers and enantiomers, low yields, and extensive (and expensive) purification. Here, we present an efficient enzymatic synthesis of 19(S),20(R)- and 16(S),17(R)-epoxydocosapentaenoic acids (EDPs) from DHA via epoxidation with BM3, a bacterial CYP450 enzyme isolated originally from Bacillus megaterium (that is readily expressed in Escherichia coli). Characterization and determination of purity is performed with nuclear magnetic resonance spectroscopy (NMR), high-performance liquid chromatography (HPLC), and mass spectrometry (MS). This procedure illustrates the benefits of enzymatic synthesis of PUFA epoxy metabolites, and is applicable to the epoxidation of other fatty acids, including arachidonic acid (AA) and eicosapentaenoic acid (EPA) to produce the analogous epoxyeicosatrienoic acids (EETs) and epoxyeicosatetraenoic acids (EEQs), respectively.

Wprowadzenie

As interest in the role that polyunsaturated fatty acids (particularly omega-3 and omega-6 polyunsaturated fatty acids) play in human biology has grown in recent years, researchers have taken notice of the wide range of appealing benefits that their metabolites exhibit. In particular, epoxy fatty acid metabolites produced by the cytochrome P450 class of enzymes have been a large point of focus. For example, many PUFA epoxides, including epoxyeicosatrienoic acids (EETs), epoxydocosapentaenoic acids (EDPs) and epoxyeicosatetraenoic acids (EEQs), play a critical role in regulation of blood pressure and inflammation1,2,3,4,5. Interestingly, the specific enantiomers and regioisomers of AA and EPA epoxides are known to have varying effects on vasoconstriction6,7. While the physiological effects of the enantiomers and regioisomers of EETs and EEQs have been documented, little is known about the effect of the analogous epoxydocosapentaenoic acids (EDPs) formed from DHA. Widespread use of fish oil8, which is rich in both EPA and DHA, has also stirred interest in EDPs9. The benefits of these supplements are believed to be partly due to the downstream DHA metabolites (16,17-EDP and 19,20-EDP being the most abundant) because in vivo levels of EDPs coordinate very well with the amount of DHA in the diet10,11.

Studying the mechanisms and targets of these epoxy fatty acids by metabolomics, chemical biology, and other methods has proven challenging, in part because they exist as mixtures of regio- and stereo-isomers, and a method of obtaining pure amounts of the enantiomers and regioisomers is required. Conventional means for chemically synthesizing these compounds have proved ineffective. Use of peroxyacids like meta-chloroperoxybenzoic acid for epoxidation has many drawbacks, notably the lack of epoxidation selectivity, which necessitates expensive and painstaking purification of individual regioisomers and enantiomers. Total synthesis of DHA and EPA metabolites is possible, but also suffers from drawbacks that make it impractical for large-scale synthesis such as high costs and low yields12,13. Efficient overall production can be achieved with enzymatic synthesis, as enzymatic reactions are regio- and stereoselective14. Studies show that enzymatic epoxidation of AA and EPA (with BM3) is both regioselective and enantioselective15,16,17,18, but this procedure has not been tested with DHA, or on a large scale. The overall goal of our method was to scale up and optimize this chemoenzymatic epoxidation to rapidly produce significant amounts of pure epoxy fatty acids as their individual enantiomers. Using the method presented here, researchers have access to a simple and cost-effective strategy for synthesis of EDPs and other PUFA epoxy metabolites.

Protokół

CAUTION: Please consult all relevant material safety data sheets (MSDS) before using the listed chemicals.

1. Expression of wild-type BM3

  1. Inoculate pBS-BM3 transfected DH5α E. coli (a generous donation from Dr. F. Ann Walker) in 5 mL of sterile LB broth with 0.5 mg of ampicillin added into a 20 mL culture tube.
  2. Incubate the cell culture in a shaker at 37 ˚C for 24 h at 200 rpm. Add the overnight starter culture (5 mL) and 100 mg of ampicillin to 1 L of sterile LB broth in a Fernbach or Erlenmeyer flask. Shake at 37 ˚C for 6 h at 200 rpm, then at 30 ˚C for 18 h at 200 rpm.
  3. Collect and centrifuge the cell culture at 4 ˚C for 10 min at 1,000 x g. Discard the supernatant and store the cell pellet at -78 ˚C until enzyme purification.
    NOTE: The supernatant can be either chemically sterilized by treatment with bleach or sterilized using an autoclave and then poured down the drain.

2. Purification of BM3.

  1. Cell lysis
    1. Thaw the cell pellet on ice and resuspend in 40 mL of ice-cold (4 ˚C) solubilization buffer (10 mM Tris, 0.01 mM phenylmethylsulfonyl fluoride (PMSF;), 0.01 mM EDTA; pH 7.8).
      CAUTION: PMSF is toxic by contact.
    2. While on ice, sonicate the cells for 1 min with an ultrasonic homogenizer (output power setting 10, duty 100%), followed by a 1 min break on ice in order to lyse the cells. Repeat this procedure 6 times. Centrifuge the cell lysate at 4 ˚C for 30 min at 11,000 x g to pellet cell debris.
  2. Affinity chromatography
    1. Prepare a strong anion exchange chromatography column (see Table of Materials; diameter: 2.8 cm x 6 cm, column volume: 37 mL) by washing with 5 column volumes (CV) of buffer A (10 mM Tris, pH 7.8) at 4 ˚C.
    2. Add the cell lysates to equilibrated column and wash the column with 3 CV of cold buffer A. Elute the BM3 by washing the column with cold buffer B (10 mM Tris, 600 mM NaCl, 6 CV).
    3. Collect the reddish-brown eluent fraction. If the protein is not being used immediately, mix it with an equal volume of glycerol and flash freeze with liquid nitrogen. Store the frozen solution at -78 ˚C.

3. Epoxidation of DHA by BM3

  1. Prepare the reaction by adding 0.308 g (0.940 mmol) of DHA in 18.8 mL of dimethylsulfoxide (DMSO) to 2 L of stirring reaction buffer (0.12 M potassium phosphate, 5 mM MgCl2, pH 7.4) along with 20 nM of the thawed BM3 enzyme. The enzyme concentration can be determined by the carbon monoxide/dithionite spectral assay method19.
  2. While the solution is stirring, begin the reaction by adding 1 equivalent of NADPH (nicotinamide adenine dinucleotide phosphate reduced, tetrasodium salt, 0.808 g, 0.940 mmol) dissolved in reaction buffer. Stir the reaction for 30 min while bubbling air through the reaction mixture with an air-filled balloon attached to a syringe and needle.
  3. Using a spectrophotometer (see Table of Materials), check the absorbance of the reaction mixture at 340 nm to determine if NADPH is depleted. If there is no remaining absorbance indicating the consumption of NADPH, the reaction is complete.
    NOTE: Typically, the reaction is complete after 30 min.
  4. Quench the reaction mixture by slowly adding 1 M oxalic acid, dropwise, until the pH of the solution reaches 4.

4. Extraction of EDPs

  1. Extract the quenched buffer solution with 2 L of diethyl ether (anhydrous, peroxide-free) 3 times. Collect the ether layer (6 L) and dry with anhydrous magnesium sulfate (MgSO4).
  2. Filter the MgSO4 from the solution and concentrate the dried ether layer on a rotary evaporator to yield the crude EDP residue.
  3. Purify the residue by flash column chromatography (a 40 g silica cartridge is sufficient). Start at 10% ethyl acetate (EtOAc) in hexanes and ramp up to 60% EtOAc in hexanes over 22 min.
    NOTE: Three major peaks are obtained and collected, eluting in the order of 1. unreacted DHA; 2. mixture of EDP isomers; and 3. di-epoxide (normal over-oxidation products (See Figure 1)).
  4. Combine the fractions and concentrate them on the rotary evaporator. From this example, 0.074 g, (24%) of unreacted DHA, 0.151 g (47%) of EDP isomers, and 0.076 g, (22%) of di-epoxide were obtained.

5. Esterification of EDPs, separation of 16(S),17(R)- and 19(S),20(R)-EDP, and saponification of esters

CAUTION: Trimethylsilyldiazomethane (TMS-diazomethane) is very toxic by both contact and inhalation. Use only in a fume hood with the proper personal protective equipment.

  1. Dilute the epoxides (0.151 g, 0.435 mmol) in a round-bottomed flask or small vial with 2 mL of anhydrous methanol (MeOH) and 3 mL of anhydrous toluene, add a stir-bar, and add TMS-diazomethane (1.2 molar equivalents, or 0.26 mL of a 2 M solution in hexanes) under argon.
  2. Wait 10 min and add additional TMS-diazomethane (0.050 mL) until a pale yellow color remains.
  3. After 30 min, carefully concentrate the mixture using the rotary evaporator and purify the residue by flash column chromatography. Elute with 4% EtOAc in hexanes (using a 40 g silica gel column or cartridge) for 22 min. In this example, 19,20-EDP methyl ester (0.116 g, 74%) and 16,17-EDP methyl ester (0.029 g, 19%), were obtained as clear oils (total yield, 93%).
  4. Collect the fractions containing the purified EDP methyl ester regioisomers. 19(S),20(R)-EDP methyl ester elutes first, followed by 16(S),17(R)-EDP methyl ester.
  5. If any mixed fractions remain (containing both isomers; can be assessed by thin-layer chromatography (TLC) in 8:1 hexane/EtOAc and stained with potassium permanganate (KMnO4)), re-chromatograph them with the same solvent system as before.
  6. Concentrate the fractions containing the individual regioisomers.
    NOTE: At this point, identity and purity may be assessed by NMR (using CDCl3 as the solvent; see the legend for Figure 2).
  7. To convert individual EDP methyl ester regioisomers to their acid forms, dilute the EDP ester in THF:water (approximately 0.7 mL/0.1 mmol of ester). Add 2 M aqueous LiOH (3 molar equivalents) and stir overnight.
    NOTE: Completeness of the reaction can be assessed by TLC, using 3:1 hexanes/EtOAc, staining with KMnO4; the product has a retention factor of ~0.3.
  8. Quench the reaction slowly with formic acid, until the pH of the mixture reaches 3-4. Add water and ethyl acetate (1-2 mL/0.100 mmol of ester) and separate the layers. Extract the water layer with EtOAc (3 x 5 mL), wash with saturated brine (NaCl) solution, and dry the EtOAc layer over anhydrous sodium sulfate (Na2SO4).
  9. Concentrate the ethyl acetate solution using a rotary evaporator, add hexanes (10 mL) and concentrate again. Repeat twice to azeotropically remove residual formic acid. Purify the residue by flash column chromatography, eluting with 10-30% EtOAc over 15 min.
  10. Concentrate the desired fractions and dry in vacuo to afford the purified acid.
    NOTE: At this stage, enantiomeric purity may be assessed (by chiral HPLC, see the legends for Figure 3 - Figure 4 for column and conditions). Chemical purity can be assessed by C18 (achiral) HPLC (see Table of Materials and reference 14).

Wyniki

The flash column chromatogram (performed using an automated flash purification system as described below) obtained upon purification of the crude mixture from enzymatic epoxidation is shown in Figure 1. Following esterification and separation of the regioisomers, pure 16(S),17(R)-EDP and 19(S),20(R)-EDP methyl esters were obtained. Typically, they are present in an approximate 1:4 to 1:5 ratio, with the major product being ...

Dyskusje

We present here an operationally simple and cost-effective method for preparing the two most abundant epoxy metabolites of DHA - 19,20 and 16,17-EDP. These epoxy fatty acids can be prepared in highly enantiopure (as their S,R-isomers) form using wild-type BM3 enzyme. Several critical points which may be used for troubleshooting, and the extension of our method to preparing enantiopure epoxy metabolites of AA and EPA, are described below.

BM3 storage guidelines

Ujawnienia

The authors have no conflicts of interest to disclose.

Podziękowania

This work is funded by R00 ES024806 (National Institutes of Health), DMS-1761320 (National Science Foundation) and startup funds from Michigan State University. The authors wish to thank Dr. Jun Yang (University of California at Davis) and Lalitha Karchalla (Michigan State University) for assistance with optimization of the enzymatic reaction, and Dr. Tony Schilmiller (MSU Mass Spectrometry and Metabolomics Facility) for assistance with HRMS data acquisition.

Materiały

NameCompanyCatalog NumberComments
Ammonium BicarbonateSigma9830NA
AmpicillinGoldBioA30125NA
Anhydrous magnesium sulfateFisher ScientificM65-3NA
Anhydrous methanolSigma-Aldrich322515NA
Anhydrous sodium sulfateFisher ScientificS421-500NA
Anhydrous tolueneSigma-Aldrich244511NA
Arachidonic Acid (AA)Nu-Chek PrepU-71AAir-sensitive. 
Diethyl EtherSigma296082NA
DMSO (molecular biology grade)Sigma-AldrichD8418NA
Docosahexaenoic Acid (DHA)Nu-Chek PrepU-84AAir-sensitive. 
EDTA (ethylenediaminetetraacetic acid)Invitrogen15576028NA
Eicosapentaenoic Acid (EPA)Nu-Chek Prep U-99AAir-sensitive. 
Ethyl acetateSigma 34858NA
Flash column cartridges 25, 40, 4, 12 g sizesFisher Scientific145170203, 145154064, 5170200Alternatively, conventional column chromatography can be used
Formic acid (HPLC Grade)J.T. Baker0128-01NA
GlycerolSigmaG7757NA
HexanesVWRBDH24575NA
LB BrothSigmaL3022NA
Lithium hydroxideSigma-Aldrich442410NA
Magnesium chlorideFisher Scientific2444-01NA
Methanol (HPLC grade)Sigma-Aldrich34860-41-RNA
NADPH Tetrasodium SaltSigma-Aldrich481973Air-sensitive. 
Oxalic acidSigma-Aldrich194131NA
pBS-BM3 transfected DH5α E. coliNANANA
PMSF (phenylmethanesulfonyl fluoride)SigmaP7626Toxic!
Potassium PermanganateSigma-Aldrich223468For TLC staining. 
Potassium phosphate dibasicSigma795496NA
Potassium phosphate monobasicSigma795488NA
Q Sepharose Fast Flow resin (GE Healthcare life sciences)Fisher Scientific17-0515-01For anion exchange purification of enzyme
Sodium ChlorideSigma71376NA
Tetrahydrofuran, anhydrousSigma-Aldrich186562NA
TMS-Diazomethane (2.0 M in hexanes)Sigma-Aldrich362832Very toxic. 
Tris-HClGoldBioT-400NA
Also necessary:
Automatic flash purification system (we used a Buchi Reveleris X2) Buchi
C18 HPLC column (Zorbax Eclipse XDB-C18)Agilent
Centrifuge capable of 10,000 x g
Chiral HPLC Column (Lux cellulose-3), 250 x 4.6 mm, 5 µM, 1000 Å)Phenomenex
General chemistry supplies: a 2 L separatory funnel, beakers and Erlenmeyer flasks with 1000-2000 L capacity, 20 mL vials, HPLC vials, small round-bottomed flasks and stir-bars.
HPLC (we use a Shimadzu Prominence LC-20AT analytical pump and SPD-20A UV-vis detectorShimadzu
Nanodrop 2000 Spectrophotometer Thermo-Fisher Scientific
NMRNMR: Agilent DD2 spectrometer (500 MHz)
Rotary evaporatorBuchi
Sonic dismembrator or ultrasonic homogenizerCole-Parmer

Odniesienia

  1. Campbell, W. B., Gebremedhin, D., Pratt, P. F., Harder, D. R. Identification of epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factors. Circulation Research. 78, 415-423 (1996).
  2. Ulu, A., et al. An omega-3 epoxide of docosahexaenoic acid lowers blood pressure in angiotensin-II-dependent hypertension. Journal of Cardiovascular Pharmacology. 64, 87-99 (2014).
  3. Ye, D., et al. Cytochrome p-450 epoxygenase metabolites of docosahexaenoate potently dilate coronary arterioles by activating large-conductance calcium-activated potassium channels. Journal of Pharmacology and Experimental Therapeutics. 303, 768-776 (2002).
  4. Imig, J. D. Epoxyeicosatrienoic acids, hypertension, and kidney injury. Hypertension. 65, 476-682 (2015).
  5. Capozzi, M. E., Hammer, S. S., McCollum, G. W., Penn, J. S. Epoxygenated fatty acids inhibit retinal vascular inflammation. Scientific Reports. 6, 39211 (2016).
  6. Zou, A. P., et al. Stereospecific effects of epoxyeicosatrienoic acids on renal vascular tone and K(+)-channel activity. American Journal of Physiology. 270, F822-F832 (1996).
  7. Lauterbach, B., et al. Cytochrome P450-dependent eicosapentaenoic acid metabolites are novel BK channel activators. Hypertension. 39, 609-613 (2002).
  8. Clarke, T. C., Black, T. I., Stussman, B. J., Barnes, P. M., Nahin, R. L. . Trends in the use of complementary health approaches among adults: United States, 2002–2012. , (2015).
  9. Mozaffarian, D., Wu, J. H. Y. Omega-3 fatty acids and cardiovascular disease. Journal of the American College of Cardiology. 58, 2047-2067 (2011).
  10. Shearer, G., Harris, W., Pederson, T., Newman, J. Detection of omega-3 oxylipins in human plasma in response to treatment with omega-3 acid ethyl esters. Journal of Lipid Research. 51, 2074-2081 (2010).
  11. Ostermann, A. I., Schebb, N. H. Effects of omega-3 fatty acid supplementation on the pattern of oxylipins: a short review about the modulation of hydroxy-, dihydroxy-, and epoxy-fatty acids. Food & Function. 8, 2355-2367 (2017).
  12. Khan, M. A., Wood, P. L. . Method for the synthesis of DHA. , (2012).
  13. Nanba, Y., Shinohara, R., Morita, M., Kobayashi, Y. Stereoselective synthesis of 17,18-epoxy derivative of EPA and stereoisomers of isoleukotoxin diol by ring-opening of TMS-substituted epoxide with dimsyl sodium. Organic and Biomolecular Chemistry. 15, 8614-8626 (2017).
  14. Cinelli, M. A., et al. Enzymatic synthesis and chemical inversion provide both enantiomers of bioactive epoxydocosapentaenoic acids. Journal of Lipid Research. 59, 2237-2252 (2018).
  15. Falck, J. R., et al. Practical, enantiospecific syntheses of 14,15-EET and leukotoxin B (vernolic acid). Tetrahedron Letters. 41, 4131-4133 (2001).
  16. Celik, A., Sperandio, D., Speight, R. E., Turner, N. Enantioselective epoxidation of linolenic acid catalyzed by cytochrome P450BM3 from Bacillus megaterium. Organic and Biomolecular Chemistry. 3, 1688-2690 (2005).
  17. Capdevila, J. H., et al. The highly stereoselective oxidation of polyunsaturated fatty acids by cytochrome P450BM-3. Journal of Biological Chemistry. 271, 22663-22671 (1996).
  18. Lucas, D., et al. Stereoselective epoxidation of the last double bond of polyunsaturated fatty acids by human cytochromes P450. Journal of Lipid Research. 51, 1125-1133 (2010).
  19. Guengerich, F. P., Martin, M. V., Sohl, C. D., Cheng, Q. Measurement of cytochrome P450 and NADPH-cytochrome P450 reductase. Nature Protocols. 4, 1245-1251 (2009).
  20. . Cayman Chemical, 19,20-EpDPA Available from: https://www.caymanchem.com/product/10175 (2019)
  21. Graham-Lorence, S., et al. An active site substitution, F87V, converts cytochrome P450 BM-3 into a regio- and stereoselective (14S, 15R)-arachidonic acid epoxygenase. Journal of Biological Chemistry. 272, 1127-1135 (1996).

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Enzymatic SynthesisEpoxy Fatty AcidsDocosahexaenoic Acid DHAEicosapentaenoic Acid EPAArachidonic AcidEnantiopure MetabolitesCell CultureCentrifugationCell LysisAffinity ChromatographyOrganic Chemistry TechniquesTris BufferPMSFEDTA

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