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 sterile LB broth in a Fernbach or Erlenmeyer flask. Shake at 37 &#730;C for 6 h at 200 rpm, then at 30 &#730;C for 18 h at 200 rpm.</li>
	<li>Collect and centrifuge the cell culture at 4 &#730;C for 10 min at 1,000 x g. Discard the supernatant and store the cell pellet at -78 &#730;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.</li>
</ol>
2. Purification of BM3.
<ol>
	<li>Cell lysis
	<ol>
		<li>Thaw the cell pellet on ice and resuspend in 40 mL of ice-cold (4 &#730;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.</li>
		<li>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 &#730;C for 30 min at 11,000 x g to pellet cell debris.</li>
	</ol>
	</li>
	<li>Affinity chromatography
	<ol>
		<li>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 &#730;C.</li>
		<li>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).</li>
		<li>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 &#730;C.</li>
	</ol>
	</li>
</ol>
3. Epoxidation of DHA by BM3
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Quench the reaction mixture by slowly adding 1 M oxalic acid, dropwise, until the pH of the solution reaches 4.</li>
</ol>
4. Extraction of EDPs
<ol>
	<li>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).</li>
	<li>Filter the MgSO4 from the solution and concentrate the dried ether layer on a rotary evaporator to yield the crude EDP residue.</li>
	<li>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)).</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>Wait 10 min and add additional TMS-diazomethane (0.050 mL) until a pale yellow color remains.</li>
	<li>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%).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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&#160;3&#160;- Figure 4 for column and conditions). Chemical purity can be assessed by C18 (achiral) HPLC (see Table of Materials and reference 14).</li>
</ol>

<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>

The authors have no conflicts of interest to disclose.

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

enzymatic-synthesis-epoxidized-metabolites-docosahexaenoic

Research

JoVE Journal

Chemistry

7.9K Views.  Michigan State University. 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).

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

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Bioengineering

Light-driven Enzymatic Decarboxylation

Oxidoreductases belong to the most-applied industrial enzymes. Nevertheless, they need external electrons whose supply is often costly and challenging. Recycling of the electron donors NADH or NADPH requires the use of additional enzymes and sacrificial substrates. Interestingly, several oxidoreductases accept hydrogen peroxide as electron donor. While being inexpensive, this reagent often reduces the stability of enzymes. A solution to this problem is the in situ generation of the cofactor. The continuous supply of the cofactor at low concentration drives the reaction without impairing enzyme stability. This paper demonstrates a method for the light-catalyzed in situ generation of hydrogen peroxide with the example of the heme-dependent fatty acid decarboxylase OleTJE. The fatty acid decarboxylase OleTJE was discovered due to its unique ability to produce long-chain 1-alkenes from fatty acids, a hitherto unknown enzymatic reaction. 1-alkenes are widely used additives for plasticizers and lubricants. OleTJE has been shown to accept electrons from hydrogen peroxide for the oxidative decarboxylation. While addition of hydrogen peroxide damages the enzyme and results in low yields, in situ generation of the cofactor circumvents this problem. The photobiocatalytic system shows clear advantages regarding enzyme activity and yield, resulting in a simple and efficient system for fatty acid decarboxylation.

We describe a protocol for the light-catalyzed generation of hydrogen peroxide &#8212; a cofactor for oxidative transformations.

Oxidoreductases belong to the most-applied industrial enzymes. Nevertheless, they need external electrons whose supply is often costly and challenging. ...

Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising approach to cancer therapy. The glycosaminoglycan placental chondroitin sulfate A (plCSA) is expressed on a wide range of cancer cells and placental trophoblasts, and malarial protein VAR2CSA can specifically bind to plCSA. A reported placental chondroitin sulfate A binding peptide (plCSA-BP), derived from malarial protein VAR2CSA, can also specifically bind to plCSA on cancer cells and placental trophoblasts. Hence, plCSA-BP-conjugated nanoparticles could be used as a tool for targeted drug delivery to human cancers and placental trophoblasts. In this protocol, we describe a method to synthesize plCSA-BP-conjugated lipid-polymer nanoparticles loaded with doxorubicin (plCSA-DNPs); the method consists of a single sonication step and bioconjugate techniques. In addition, several methods for characterizing plCSA-DNPs, including determining their physicochemical properties and cellular uptake by placental choriocarcinoma (JEG3) cells, are described.

Synthesis and Characterization of Placental Chondroitin Sulfate A plCSA-Targeting Lipid-Polymer Nanoparticles

Here, we present a protocol for the synthesis of placental chondroitin sulfate A binding peptide (plCSA-BP)-conjugated lipid-polymer nanoparticles via single-step sonication and bioconjugate techniques. These particles constitute a novel tool for the targeted delivery of therapeutics to most human tumors and placental trophoblasts to treat cancers and placental disorders.

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising ...

Cancer Research

Enzymatic Cascade Reactions for the Synthesis of Chiral Amino Alcohols from L-lysine

Amino alcohols are versatile compounds with a wide range of applications. For instance, they have been used as chiral scaffolds in organic synthesis. Their synthesis by conventional organic chemistry often requires tedious multi-step synthesis processes, with difficult control of the stereochemical outcome. We present a protocol to enzymatically synthetize amino alcohols starting from the readily available L-lysine in 48 h. This protocol combines two chemical reactions that are very difficult to conduct by conventional organic synthesis. In the first step, the regio- and diastereoselective oxidation of an unactivated C-H bond of the lysine side-chain is catalyzed by a dioxygenase; a second regio- and diastereoselective oxidation catalyzed by a regiodivergent dioxygenase can lead to the formation of the 1,2-diols. In the last step, the carboxylic group of the alpha amino acid is cleaved by a pyridoxal-phosphate (PLP) decarboxylase (DC). This decarboxylative step only affects the alpha carbon of the amino acid, retaining the hydroxy-substituted stereogenic center in a beta/gamma position. The resulting amino alcohols are therefore optically enriched. The protocol was successfully applied to the semipreparative-scale synthesis of four amino alcohols. Monitoring of the reactions was conducted by high performance liquid chromatography (HPLC) after derivatization by 1-fluoro-2,4-dinitrobenzene. Straightforward purification by solid-phase extraction (SPE) afforded the amino alcohols with excellent yields (93% to &#62;95%).

Chiral amino alcohols are versatile molecules for use as scaffolds in organic synthesis. Starting from L-lysine, we synthesize amino alcohols by an enzymatic cascade reaction combining diastereoselective C-H oxidation catalyzed by dioxygenase followed by cleavage of the carboxylic acid moiety of the corresponding hydroxyl amino acid by a decarboxylase.

Amino alcohols are versatile compounds with a wide range of applications. For instance, they have been used as chiral scaffolds in organic synthesis. ...

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). Endocannabinoids are conserved lipid mediators that regulate multiple biological processes in a variety of organisms. In C. elegans, 2-AG has been found to possess different roles, including modulation of dauer formation and cholesterol metabolism. This report describes a method to overcome the difficulties associated with the costs and stability of deuterated standards required for 2-AG quantification. The procedure for the synthesis of the standard is simple and can be performed in any laboratory, without the need for organic synthesis expertise or special equipment. In addition, a modification of Folch&#39;s method to extract the deuterated standard from C. elegans culture is described. Finally, a quantitative and analytic method to detect 2-AG using the stable isotopically labeled analog 1-AG-d5 is described, which provides reliable results in a fast-chromatographic run. The procedure is useful for studying the multiple roles of 2-AG in C. elegans while also being applicable to other studies of metabolites in different organisms.

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work describes a robust and straightforward method to detect and quantify the endocannabinoid 2-arachidonoylglycerol (2-AG) in C. elegans. An analytical deuterated standard us prepared and used for the quantification of 2-AG by isotopic dilution and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid ...

Biochemistry

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

Biology

Quantitative Determination of De Novo Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide

Fatty acid synthesis is a complex and highly energy demanding metabolic pathway with important functional roles in the control of whole-body metabolic homeostasis and other physiological and pathological processes. Contrary to other key metabolic pathways, such as glucose disposal, fatty acid synthesis is not routinely functionally assessed, leading to incomplete interpretations of metabolic status. In addition, there is a lack of publicly available detailed protocols suitable for newcomers in the field. Here, we describe an inexpensive quantitative method utilizing deuterium oxide and gas chromatography mass spectrometry (GCMS) for the analysis of total fatty acid de novo synthesis in brown adipose tissue in vivo. This method measures the synthesis of the products of fatty acid synthase independently of a carbon source, and it is potentially useful for virtually any tissue, in any mouse model, and under any external perturbation. Details on the sample preparation for GCMS and downstream calculations are provided. We focus on the analysis of brown fat due to its high levels of de novo fatty acid synthesis and critical roles in maintaining metabolic homeostasis.

Quantitative Determination of De Novo Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide

Here, we present an inexpensive quantitative method utilizing deuterium oxide and gas chromatography mass spectrometry (GCMS) for the analysis of total fatty acid de novo lipogenesis in brown adipose tissue in vivo.

Fatty acid synthesis is a complex and highly energy demanding metabolic pathway with important functional roles in the control of whole-body metabolic ...

Preparation of Epoxides

<h4>Overview</h4>
Epoxides result from&#160;alkene oxidation, which can be achieved by a) air, b) peroxy acids, c) hypochlorous acids, and d) halohydrin cyclization.
<h4>Epoxidation with Peroxy Acids</h4>
Epoxidation of alkenes via oxidation with peroxy acids involves the conversion of a carbon&#8211;carbon double bond to an epoxide using the oxidizing agent&#160;meta-chloroperoxybenzoic acid, commonly known as MCPBA. Since the O&#8211;O bond of peroxy acids is very weak, the addition of electrophilic oxygen of peroxy acids to alkenes occurs with ease, thereby following syn addition. Hence, the epoxides are produced with the retention of the alkene configuration.
<h4>Epoxidation via Air Oxidation</h4>
Although peroxy-mediated epoxidation is the most common method for alkene oxidation, ethylene oxide is synthesized at the industrial scale via air oxidation by treating a mixture of ethylene and air in the presence of a silver catalyst.
<h4>Cyclization of Halohydrins</h4>
Cyclization of halohydrins of alkenes in the presence of a base also yields epoxides, and the reaction follows the SN2 substitution mechanism. Hence, the nucleophile&#8212;the oxygen anion&#8212;and the leaving group&#8212;the chloride ion&#8212;must orient anti to each other in the transition state to make the halohydrins cyclization feasible.
<img alt="Figure1" src="/files/ftp_upload/12094/12094Figure 1.svg" style="text-align: center; height: 260x;" />
In noncyclic halohydrins, this anti-relationship is achieved by an internal rotation. For instance, in 1-chloro-2-methyl-2-propanol, shown in Figure 1, the hydroxyl and the chloro group are not oriented anti to each other. To achieve the anti-relationship, the carbon-bearing chloro group undergoes an internal rotation, thereby making the nucleophile attack&#8212;from the backside of the C&#8211;X bond&#8212;and the ejection of the leaving group feasible. Thus, the epoxides formed via halohydrin cyclization also retain the alkene configuration.
Similarly, the cyclic halohydrins must undergo conformational changes to achieve the anti-relationship. For example, the halohydrin of cyclohexane, shown in Figure 2, undergoes a conformational change from diequatorial to diaxial to successfully form&#160;an epoxide.
<img alt="Figure2" src="/files/ftp_upload/12094/12094Figure 2.svg" style="text-align: center; height: 120px;" />

Preparation of Epoxides via Alkene Oxidation

Overview
Epoxides result from&#160;alkene oxidation, which can be achieved by a) air, b) peroxy acids, c) hypochlorous acids, and d) halohydrin ...

Education

JoVE Core

Organic Chemistry

Unit 1

Ethers, Epoxides, Sulfides

KEY TERMS AND CONCEPTS

Sharpless Epoxidation

The conversion of allylic alcohols into epoxides using the chiral catalyst was discovered by K. Barry Sharpless and is known as Sharpless epoxidation. The use of a chiral catalyst enables the formation of one enantiomer of the product in excess. This chiral catalyst is mainly a chiral complex of titanium tetraisopropoxide and tartrate ester (specific stereoisomer). The stereoisomer used in the chiral catalyst dictates the formation of the enantiomer of the product. In other words, the use of L-(+)-diethyl tartrate leads to enantiomers having the epoxide ring below the plane, while with D-(&#8722;)-diethyl tartrate, to enantiomers with the epoxide ring above the&#160;plane. The high enantioselectivity of the reaction can be explained by considering the activation energies required for the reaction to proceed in the forward direction in the presence of the chiral catalyst. As shown in Figure 1, compared to the uncatalyzed reaction (blue curve), the activation energy of the reaction decreases dramatically with the addition of the chiral catalyst (red and green curves). Moreover, the activation energy for the formation of one enantiomer (red curve) is lowered more than that of another enantiomer (green curve), leading to the formation of one enantiomer in excess. Hence, Sharpless epoxidation reaction can be utilized for the synthesis of desired enantiomers of the product.
<img alt="Figure1" src="/files/ftp_upload/12108/12108Figure 1.png" style="text-align: center; height: 300px;" />
The stereochemistry of the product formed when any allylic alcohol is subjected to Sharpless epoxidation can be predicted by simply orienting the allylic alcohol molecule in a plane with the hydroxyl groups pointing towards the lower right corner, as shown in Figure 2. On this planar structure, D-(&#8722;)-diethyl tartrate delivers the oxygen from the top face of the alkene, making the epoxide formation feasible from above the plane, while L-(+)-diethyl tartrate delivers the oxygen from the bottom face of the alkene, thereby installing the epoxide ring from below the plane.
<img alt="Figure2" src="/files/ftp_upload/12108/12108Figure 2.svg" style="text-align: center; height: 300px;" />

Sharpless Epoxidation: Allylic Alcohols to Epoxides

The conversion of allylic alcohols into epoxides using the chiral catalyst was discovered by K. Barry Sharpless and is known as Sharpless epoxidation. The ...

Oxidation of Alkenes: Syn Dihydroxylation with Osmium Tetraoxide

Alkenes are converted to 1,2-diols or glycols through a process called dihydroxylation. It involves the addition of two hydroxyl groups across the double bond with two different stereochemical approaches, namely anti and syn. Dihydroxylation using osmium tetroxide progresses with syn stereochemistry.
<img alt="Cyclohexene dihydroxylation reaction mechanism, chemical equation, reagent diagram." src="/files/ftp_upload/11781/11781_PT_Figure_1.svg" style="text-align: center; height: 60px;" data-altupdatedat="1747908711000" data-alt="Figure1">
<h4>Syn Dihydroxylation Mechanism</h4>
The reaction comprises a two-step mechanism. It begins with the addition of osmium tetroxide across the alkene double bond in a concerted manner forming a five-membered cyclic osmate ester as an intermediate, which can be isolated and characterized. Osmium tetroxide is electrophilic in nature, serving as a strong oxidizing agent. It accepts an electron pair from the alkene π bond undergoing a reduction from +VIII to +VI.
In the next step,  the cyclic osmate ester reacts with a reducing agent like sodium bisulfite that cleaves the Os–O bond producing a cis-glycol with retention of the syn stereochemistry of the two newly formed C–O bonds.
<img alt="Organic reaction mechanism diagram; stepwise cycloaddition with reagent NaHSO3." src="/files/ftp_upload/11781/11781_PT_Figure_2.svg" style="text-align: center; height: 200px;" data-altupdatedat="1747908711000" data-alt="Figure2">
A major drawback of the method is the use of toxic and expensive osmium tetroxide. To overcome this, osmium tetroxide is often used as a catalyst along with the co-oxidants like N-methylmorpholine N-oxide (NMO) or tert-butyl hydroperoxide (TBHP). The co-oxidants reoxidize the osmium +VI species to +VIII, thereby regenerating osmium tetroxide for further oxidation of the remaining alkenes.
<h4>Stereochemical Outcome</h4>
As oxidation of alkenes using osmium tetroxide is a stereospecific syn addition process, the two oxygens of osmium tetroxide are simultaneously added to the same face of the alkene π bond. Based on this, dihydroxylation of (E)-hex-3-ene produces a pair of enantiomers, while (Z)-hex-3-ene gives a meso compound.
<img alt="Syn dihydroxylation process; chemical reaction diagram; showing reagent conversion; organic chemistry." src="/files/ftp_upload/11781/11781_PT_Figure_3.svg" style="text-align: center; height: 130px;" data-altupdatedat="1747908711000" data-alt="Figure3">
<h4>Sharpless Asymmetric Dihydroxylation</h4>
Interestingly, Karl Barry Sharpless developed an enantioselective method for syn dihydroxylation of alkenes, for which he was awarded the Nobel prize. This method is known as Sharpless asymmetric dihydroxylation that is carried out using osmium tetroxide, a stoichiometric amount of the co-oxidant, and a chiral amine ligand. 
<img alt="Organic reaction diagram; enantiomer synthesis using AD-mix; chemical equations; 0°C condition." src="/files/ftp_upload/11781/11781_PT_Figure_4.svg" style="text-align: center; height: 60px;" data-altupdatedat="1747908711000" data-alt="Figure4">

Alkenes are converted to 1,2-diols or glycols through a process called dihydroxylation. It involves the addition of two hydroxyl groups across the double ...

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

Summary

Explore More Videos

Chapters in this video

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

Light-driven Enzymatic Decarboxylation

Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles

Enzymatic Cascade Reactions for the Synthesis of Chiral Amino Alcohols from L-lysine

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Quantitative Determination of De Novo Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide

Preparation of Epoxides

Sharpless Epoxidation

Oxidation of Alkenes: Syn Dihydroxylation with Osmium Tetraoxide