Name: enzymatic synthesis of epoxidized metabolites of docosahexaenoic, eicosapentaenoic, and arachidonic acids
Uploaded: 2019-06-28T15:00:00
Description: Watch this Scientific Journal Video about Enzymatic Synthesis of Epoxidized Metabolites of Docosahexaenoic, Eicosapentaenoic, and Arachidonic Acids at JoVE.com

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.

CAUTION: Please consult all relevant material safety data sheets (MSDS) before using the listed chemicals.
1. Expression of wild-type BM3
<ol>
	<li>Inoculate pBS-BM3 transfected DH5&#945; 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.</li>
	<li>Incubate the cell culture in a shaker at 37 &#730;C for 24 h at 200 rpm. Add the overnight starter culture (5 mL) and 100 mg of ampicillin to 1 L of ...

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

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
<p ...

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.

<table border="0" cellpadding="0" cellspacing="0" style="width:916px;" width="916">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Ammonium Bicarbonate</td>
			<td style="width:150px;">Sigma</td>
			<td align="right" style="width:150px;">9830</td>
			<td style="width:339px;">NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Ampicillin</td>
			<td style="width:150px;">GoldBio</td>
			<td>A30125</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Anhydrous magnesium sulfate</td>
			<td style="width:150px;">Fisher Scientific</td>
			<td style="width:150px;">M65-3</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Anhydrous methanol</td>
			<td style="width:150px;">Sigma-Aldrich</td>
			<td align="right" style="width:150px;">322515</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Anhydrous sodium sulfate</td>
			<td style="width:150px;">Fisher Scientific</td>
			<td style="width:150px;">S421-500</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Anhydrous toluene</td>
			<td style="width:150px;">Sigma-Aldrich</td>
			<td align="right" style="width:150px;">244511</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Arachidonic Acid (AA)</td>
			<td style="width:150px;">Nu-Chek Prep</td>
			<td style="width:150px;">U-71A</td>
			<td>Air-sensitive.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Diethyl Ether</td>
			<td style="width:150px;">Sigma</td>
			<td align="right" style="width:150px;">296082</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DMSO (molecular biology grade)</td>
			<td style="width:150px;">Sigma-Aldrich</td>
			<td style="width:150px;">D8418</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Docosahexaenoic Acid (DHA)</td>
			<td style="width:150px;">Nu-Chek Prep</td>
			<td style="width:150px;">U-84A</td>
			<td>Air-sensitive.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">EDTA (ethylenediaminetetraacetic acid)</td>
			<td style="width:150px;">Invitrogen</td>
			<td align="right" style="width:150px;">15576028</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Eicosapentaenoic Acid (EPA)</td>
			<td style="width:150px;">Nu-Chek Prep</td>
			<td style="width:150px;">&#160;U-99A</td>
			<td>Air-sensitive.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Ethyl acetate</td>
			<td style="width:150px;">Sigma&#160;</td>
			<td align="right" style="width:150px;">34858</td>
			<td>NA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Flash column cartridges 25, 40, 4, 12 g sizes</td>
			<td style="width:150px;">Fisher Scientific</td>
			<td style="width:150px;">145170203, 145154064, 5170200</td>
			<td>Alternatively, conventional column chromatography can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Formic acid (HPLC Grade)</td>
			<td style="width:150px;">J.T. Baker</td>
			<td style="width:150px;">0128-01</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Glycerol</td>
			<td style="width:150px;">Sigma</td>
			<td>G7757</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Hexanes</td>
			<td style="width:150px;">VWR</td>
			<td style="width:150px;">BDH24575</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">LB Broth</td>
			<td style="width:150px;">Sigma</td>
			<td>L3022</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Lithium hydroxide</td>
			<td style="width:150px;">Sigma-Aldrich</td>
			<td align="right" style="width:150px;">442410</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Magnesium chloride</td>
			<td style="width:150px;">Fisher Scientific</td>
			<td style="width:150px;">2444-01</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Methanol (HPLC grade)</td>
			<td style="width:150px;">Sigma-Aldrich</td>
			<td style="width:150px;">34860-41-R</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">NADPH Tetrasodium Salt</td>
			<td style="width:150px;">Sigma-Aldrich</td>
			<td align="right" style="width:150px;">481973</td>
			<td>Air-sensitive.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Oxalic acid</td>
			<td style="width:150px;">Sigma-Aldrich</td>
			<td align="right" style="width:150px;">194131</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">pBS-BM3 transfected DH5&#945; E. coli</td>
			<td style="width:150px;">NA</td>
			<td style="width:150px;">NA</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">PMSF (phenylmethanesulfonyl fluoride)</td>
			<td style="width:150px;">Sigma</td>
			<td style="width:150px;">P7626</td>
			<td>Toxic!</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Potassium Permanganate</td>
			<td style="width:150px;">Sigma-Aldrich</td>
			<td align="right" style="width:150px;">223468</td>
			<td>For TLC staining.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Potassium phosphate dibasic</td>
			<td style="width:150px;">Sigma</td>
			<td align="right" style="width:150px;">795496</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Potassium phosphate monobasic</td>
			<td style="width:150px;">Sigma</td>
			<td align="right" style="width:150px;">795488</td>
			<td>NA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Q Sepharose Fast Flow resin (GE Healthcare life sciences)</td>
			<td style="width:150px;">Fisher Scientific</td>
			<td style="width:150px;">17-0515-01</td>
			<td>For anion exchange purification of enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Sodium Chloride</td>
			<td style="width:150px;">Sigma</td>
			<td align="right" style="width:150px;">71376</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Tetrahydrofuran, anhydrous</td>
			<td style="width:150px;">Sigma-Aldrich</td>
			<td align="right" style="width:150px;">186562</td>
			<td>NA</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:279px;">TMS-Diazomethane (2.0 M in hexanes)</td>
			<td style="width:150px;">Sigma-Aldrich</td>
			<td align="right" style="width:150px;">362832</td>
			<td>Very toxic.&#160;</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:279px;">Tris-HCl</td>
			<td style="width:150px;">GoldBio</td>
			<td style="width:150px;">T-400</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Also necessary:</td>
			<td style="width:150px;"></td>
			<td style="width:150px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Automatic flash purification system (we used a Buchi Reveleris X2)&#160;</td>
			<td style="width:150px;">Buchi</td>
			<td style="width:150px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">C18 HPLC column (Zorbax Eclipse XDB-C18)</td>
			<td style="width:150px;">Agilent</td>
			<td style="width:150px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Centrifuge capable of 10,000 x g</td>
			<td style="width:150px;"></td>
			<td style="width:150px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Chiral HPLC Column (Lux cellulose-3), 250 x 4.6 mm, 5 &#181;M, 1000 &#197;)</td>
			<td style="width:150px;">Phenomenex</td>
			<td style="width:150px;"></td>
			<td></td>
		</tr>
		<tr height="104">
			<td height="104" style="height:104px;width:279px;">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.</td>
			<td style="width:150px;"></td>
			<td style="width:150px;"></td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:279px;">HPLC (we use a Shimadzu Prominence LC-20AT analytical pump and SPD-20A UV-vis detector</td>
			<td style="width:150px;">Shimadzu</td>
			<td style="width:150px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Nanodrop 2000 Spectrophotometer&#160;</td>
			<td style="width:150px;">Thermo-Fisher Scientific</td>
			<td style="width:150px;"></td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:279px;">NMR</td>
			<td style="width:150px;">NMR: Agilent DD2 spectrometer (500 MHz)</td>
			<td style="width:150px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Rotary evaporator</td>
			<td style="width:150px;">Buchi</td>
			<td style="width:150px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Sonic dismembrator or ultrasonic homogenizer</td>
			<td style="width:150px;">Cole-Parmer</td>
			<td style="width:150px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

enzymatic synthesis of epoxidized metabolites of docosahexaenoic, eicosapentaenoic, and arachidonic acids

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.

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 ...

Watch this Scientific Journal Video about Enzymatic Synthesis of Epoxidized Metabolites of Docosahexaenoic, Eicosapentaenoic, and Arachidonic Acids at JoVE.com

Enzymatic Synthesis of Epoxidized Metabolites of Docosahexaenoic, Eicosapentaenoic, and Arachidonic Acids

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).

The epoxidized metabolites of various polyunsaturated fatty acids (PUFAs), termed epoxy fatty acids, have a wide range of roles in human physiology. These ...

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