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 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 ...

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

Qualitative Identification of Carboxylic Acids, Boronic Acids, and Amines Using Cruciform Fluorophores

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with electron-donors, and the other with electron-acceptors, cruciforms&#39; HOMO will localize along the electron-rich and LUMO along the electron-poor axis. This spatial isolation of cruciforms&#39; frontier molecular orbitals (FMOs) is essential to their use as sensors, since analyte binding to the cruciform invariably changes its HOMO-LUMO gap and the associated optical properties. Using this principle, Bunz and Miljani&#263; groups developed 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole cruciforms, respectively, which act as fluorescent sensors for metal ions, carboxylic acids, boronic acids, phenols, amines, and anions. The emission colors observed when these cruciform are mixed with analytes are highly sensitive to the details of analyte&#39;s structure and - because of cruciforms&#39; charge-separated excited states - to the solvent in which emission is observed. Structurally closely related species can be qualitatively distinguished within several analyte classes: (a) carboxylic acids; (b) boronic acids, and (c) metals. Using a hybrid sensing system composed from benzobisoxazole cruciforms and boronic acid additives, we were also able to discern among structurally similar: (d) small organic and inorganic anions, (e) amines, and (f) phenols. The method used for this qualitative distinction is exceedingly simple. Dilute solutions (typically 10-6 M) of cruciforms in several off-the-shelf solvents are placed in UV/Vis vials. Then, analytes of interest are added, either directly as solids or in concentrated solution. Fluorescence changes occur virtually instantaneously and can be recorded through standard digital photography using a semi-professional digital camera in a dark room. With minimal graphic manipulation, representative cut-outs of emission color photographs can be arranged into panels which permit quick naked-eye distinction among analytes. For quantification purposes, Red/Green/Blue values can be extracted from these photographs and the obtained numeric data can be statistically processed.

Cross-conjugated cruciform fluorophores based on 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole nuclei can be used to qualitatively identify diverse Lewis acidic and Lewis basic analytes. This method relies on the differences in emission colors of the cruciforms that are observed upon analyte addition. Structurally closely related species can be distinguished from each other.

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with ...

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g., other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2 drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.

Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges.

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

Synthesis and Reaction Chemistry of Nanosize Monosodium Titanate

This paper describes the synthesis and peroxide-modification of nanosize monosodium titanate (nMST), along with an ion-exchange reaction to load the material with Au(III) ions. The synthesis method was derived from a sol-gel process used to produce micron-sized monosodium titanate (MST), with several key modifications, including altering reagent concentrations, omitting a particle seed step, and introducing a non-ionic surfactant to facilitate control of particle formation and growth. The resultant nMST material exhibits spherical-shaped particle morphology with a monodisperse distribution of particle diameters in the range from 100 to 150&#160;nm. The nMST material was found to have a Brunauer-Emmett-Teller (BET) surface area of 285 m2g-1, which is more than an order of magnitude higher than the micron-sized MST. The isoelectric point of the nMST measured 3.34 pH units, which is a pH unit lower than that measured for the micron-size MST. The nMST material was found to serve as an effective ion exchanger under weakly acidic conditions for the preparation of an Au(III)-exchange nanotitanate. In addition, the formation of the corresponding peroxotitanate was demonstrated by reaction of the nMST with hydrogen peroxide.

The surfactant mediated sol-gel synthesis of nanosized monosodium titanate is described, along with preparation of the corresponding peroxide modified material. An ion-exchange reaction with Au(III) is also presented.

This paper describes the synthesis and peroxide-modification of nanosize monosodium titanate (nMST), along with an ion-exchange reaction to load the ...

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 Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Hydroquinone Based Synthesis of Gold Nanorods

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the synthetic methods for the preparation of gold nanorods are still not fully optimized. In this paper we describe a new, highly efficient, two-step protocol based on the use of hydroquinone as a mild reducing agent. Our approach allows the preparation of nanorods with a good control of size and aspect ratio (AR) simply by varying the amount of hexadecyl trimethylammonium bromide (CTAB) and silver ions (Ag+) present in the "growth solution". By using this method, it is possible to markedly reduce the amount of CTAB, an expensive and cytotoxic reagent, necessary to obtain the elongated shape. Gold nanorods with an aspect ratio of about 3 can be obtained in the presence of just 50 mM of CTAB (versus 100 mM used in the standard protocol based on the use of ascorbic acid), while shorter gold nanorods are obtained using a concentration as low as 10 mM.

This paper describes a protocol for the synthesis of gold nanorods, based on the use of hydroquinone as reducing agent, plus the different mechanisms for controlling their size and aspect ratio.

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the ...

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise &#945;-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial &#946;-mannoside linkage. We further attached the N-glycans to the surface of an aluminum oxide-coated glass (ACG) slide to create a covalent mixed array for the analysis of hetero-ligand interaction with an HIV antibody. In particular, the binding behavior of a newly isolated HIV-1 broadly neutralizing antibody (bNAb), PG9, to the mixture of closely spaced Man5GlcNAc2 (Man5) and 2,6-di-sialylated bi-antennary complex type N-glycan (SCT) on an ACG array, opens a new avenue to guide the effective immunogen design for HIV vaccine development. In addition, our ACG array embodies a powerful tool to study other HIV antibodies for hetero-ligand binding behavior.

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated.

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

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 Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.