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

这项工作由R00 ES024806(国家卫生研究院)、DMS-1761320(国家科学基金会)和密歇根州立大学的启动基金资助。作者希望感谢杨俊博士(加州大学戴维斯分校)和拉利莎·卡查拉博士(密歇根州立大学)协助优化酶反应,以及托尼·席尔米勒博士(MSU质谱和代谢组学设施)协助HRMS数据采集。

注意:在使用所列化学品之前,请查阅所有相关的材料安全数据表 (MSDS)。
1. 野生型BM3的表达
<ol><li>接种pBS-BM3转染DH5+大肠杆菌(由F.Ann Walker博士慷慨捐赠)在5 mL无菌LB汤中加入0.5毫克氨基青霉素加入20 mL培养管。</li>
	<li>在37°C下在摇床中孵育细胞培养,在200rpm下孵育24小时。在Fernbach或Erlenmeyer烧瓶中加入隔夜开胃剂培养基(5 mL)和100毫克的阿霉素,加入1L无菌LB汤。在 37°C 下在 2...

<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>

我们在这里提出了一种操作简单且经济高效的方法,用于制备DHA的两种最丰富的环氧代谢物 - 19,20 和 16,17-EDP。这些环氧脂肪酸可以使用野生型BM3酶以高抗纯(作为S,R-等体)形式制备。下面将介绍可用于故障排除的几个关键点,以及我们制备 AA 和 EPA 的环氧树脂代谢物的方法的扩展。
BM3 存储指南
在-78°C冷冻室储存之前,将蛋白质溶液与同等?...

近年来,随着人们对多不饱和脂肪酸(特别是欧米茄-3和欧米茄-6多不饱和脂肪酸)在人类生物学中的作用的兴趣与日俱增,研究人员已经注意到其代谢物具有广泛的吸引力。展览。特别是环氧脂肪酸代谢物产生的细胞色素P450类酶一直是人们关注的焦点。例如,许多PUFA环氧乙烷,包括环氧乙烷酸(EETs)、环氧二甲酸(EDPs)和环氧乙烷四甲酸(EEQs),在调节血压和炎症方面起着关键作用1,2,3,4,5.有趣的是,已知AA和EPA环氧树脂的特定异构体对血管收缩6、7具有不同影响。虽然EET和EEQ的内窥剂和再血管异构体的生理影响已经记录在案,但人们对DHA形成的类似环氧二苯丙酸(EDPs)的影响知之甚少。鱼油8的广泛使用,富含EPA和DHA,也激起了对EDPs9的兴趣。这些补充剂的好处被认为是部分由于下游DHA代谢物(16,17-EDP和19,20-EDP是最丰富的),因为在体内的EDP水平与膳食10中的DHA量协调得很好, 11.
通过代谢组学、化学生物学和其他方法研究这些环氧脂肪酸的机制和目标已被证明具有挑战性,部分原因在于它们作为再血管和立体异构体的混合物存在,以及一种获得纯量的需要反古体和再血管等位。事实证明,用化学合成这些化合物的传统方法是无效的。使用环氧树脂等环氧树脂有许多缺点,特别是缺乏环氧化选择性,这需要昂贵和费力地纯化单个再血管体和抗氧化剂。DHA和EPA代谢物的完全合成是可能的,但也存在缺点,使得大规模合成不切实际,如高成本和低产量12,13。有效的整体生产可以通过酶合成来实现,因为酶反应是regio-和立体选择性14。研究表明,AA和EPA的酶环氧化(与BM3)同时具有反选择性和内选选择性15,16,17,18,但这个程序没有测试与DHA,或在大型规模。我们该方法的总体目标是扩大和优化这种化学环氧化,以迅速产生大量的纯环氧脂肪酸作为各自的抗氧化剂。使用这里介绍的方法,研究人员可以获得一种简单且经济高效的策略来合成EDPs和其他PUFA环氧代谢物。

<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

各种多不饱和脂肪酸(PUFAs)的环氧树脂代谢物,称为环氧脂肪酸,在人体生理学中有着广泛的作用。这些代谢物是由细胞色素P450类酶内分泌产生的。由于其多样化和强大的生物效应,人们对研究这些代谢物产生了浓厚的兴趣。确定这些代谢物在体内的独特作用是一项艰巨的任务,因为环氧脂肪酸必须首先获得大量且纯度高。从天然来源获得化合物通常是劳动密集型的,可溶性环氧乙烷(sEH)会迅速水解代谢物。另一方面,通过化学反应获得这些代谢物的效率非常低,因为很难获得纯反热敏体和抗性剂,产量低,并且广泛(和昂贵的)纯化。在这里,我们提出一个有效的酶合成19(S),20 (R) - 和16 (S),17 (R)-环氧乙烷酸 (EDPs) 从DHA通过环氧化与BM3, 一种细菌CYP450酶从杆菌分离梅盖特(在大肠杆菌中很容易表达)。使用核磁共振光谱 (NMR)、高性能液相色谱 (HPLC) 和质谱 (MS) 进行纯度的特性和测定。本程序说明了PUFA环氧代谢物酶合成的好处,并适用于其他脂肪酸的环氧化,包括甲氧乙酸(AA)和乙酸苯甲酸(EPA),以生产类似的环氧乙烷酸酸(EETs)和四烯酸(EEQs)。

从酶性酶氧化中纯化原油混合物时获得的闪光柱色谱图(使用下述自动闪光纯化系统执行)如图1所示。继对再血管等量体进行酯化和分离后,获得纯16(S)、17(R)-EDP和19(S)、20(R)-EDP甲基酯。通常,它们以大约 1:4 到 1:5 的比例存在,主要产品为 19(S),20 (R) -EDP。未获得其他EDP反血管体(例如,13,14-或10,11-EDP)。1 H-NMR 光?...

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

多克沙六烯、环氧体和阿拉奇酮酸的环氧代谢物酶合成

我们提出了一种有用的方法,用于使用细菌细胞色素,对阿氏酸(AA)、多沙六烯酸(DHA)和乙二苯甲酸(EPA)的特异性环氧乙烷和环氧乙烷酶的环氧乙烷和环氧乙烷的再三氧化二氮进行纯化。P450 酶 (BM3)。

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

因此，我们的协议允许我们准备环氧树脂脂肪酸，这种方法可能有助于推进研究这些代谢物在体内作用的机制。从历史上看，纯环氧脂肪酸是很难获得的。因此，我们的协议简单、快速、经济高效，它使我们能够准备大量的这些代谢物。是的，我们为DHA开发了一个协议，但它也很好地适用于EPA和气酸。快速工作，因为某些试剂对空气敏感，特别是 DHA 和 NADPH，并且确保提前配对所有内容，以便设置简单。此外，请注意危险。我们的协议在酶学和有机化学中均采用技术。可视化对于帮助不熟悉这些领域的专业技术的读者和观众至关重要。要接种，使用浸在pBS-BM3的甘油库存中的无菌循环或移液器将DH5-alpha大肠杆菌转化为5毫升的无菌LB汤，并辅以0.5毫克的安他西林。在37摄氏度的摇床中孵育细胞培养，在200转/小时24小时。然后，在 Erlenmeyer 烧瓶中加入五毫升的开胃培养剂和 100 毫克的安皮西林，加入一升无菌 LB 肉汤中。在 37 摄氏度下在 200 rpm 下摇动 6 小时，然后在 30 摄氏度下摇动 18 小时，在 200 rpm 下摇动。之后，将细胞培养收集到离心管中，在4摄氏度下将细胞培养离心10分钟，达到1000倍g。把上一液倒到另一个烧瓶里。用漂白剂或使用自洗器去污染，并在零下78摄氏度储存细胞颗粒，直到酶纯化。要进行细胞解解，首先将细胞颗粒解冻在冰上，然后重新在含有Tris、PMSF和EDTA的40毫升冰冷溶解缓冲液中。在冰上，用超声波均质器对细胞进行声波化一分钟，输出功率设置为10，工作为100，随后在冰上休息一分钟，以对细胞进行分离。重复此过程六次。将细胞在4摄氏度下离状30分钟，以11，000倍的g为单位，对细胞碎片进行颗粒。为了在4摄氏度的冷室或带滑动门的冰箱中进行亲和力色谱，首先在pH 7.8下用五列缓冲剂 A 的缓冲器 A 洗涤树脂，以四摄氏度为温度准备强的 Anion 交换色谱柱。接下来，将细胞的上清液添加到平衡柱中，用三列体积的冷缓冲液 A 洗涤柱，然后用六列体积的冷缓冲B洗涤，即10毫摩尔三酯和600毫摩尔氯化钠。收集红褐色的水线分馏。如果蛋白质没有立即使用，则与等量的甘油混合，与液氮闪冻。将冷冻溶液存放在零下78摄氏度。要对DHA进行环氧测量，首先根据一氧化碳二甲虫光谱测定法确定解冻BM3酶的浓度。然后，在含有两升反应缓冲液的烧杯中，加入0.94毫摩尔的DHA，稀释在18.8毫升的DMSO中，BM3酶稀释至20纳米摩尔。当溶液搅拌时，通过加入含有0.94毫摩尔的NADPH反应缓冲液8.7毫升开始环氧反应。搅拌反应30分钟，同时将空气冒泡到反应混合物中，将充气气球连接到注射器上，并贴在烧杯内侧。之后，绘制一个微升的反应混合物，并放入NanoDrop分光光度计，以测量340纳米的吸光度。用水作为空白。如果没有剩余的吸光度，表示 NADPH 已被消耗，则反应完成。通过缓慢地滴入一摩尔草酸，直到溶液的pH值达到大约4，使反应混合物被淬火。现在，用两升无水、无过氧化物的二乙醚提取淬火缓冲液。使用分离漏斗摇动和分离层，并将乙醚收集到 Erlenmeyer 烧瓶中。重复两次，加入约50克无水硫酸镁以干燥乙醚。在真空过滤漏斗上，倒入干乙醚以过滤硫酸镁，并将过滤过的乙醚层转移到旋转蒸发器的圆底烧瓶中。将旋转速度调整到中等，并启动真空，以浓缩液体，产生粗性EDP残留物。将残留物装入 40 克二氧化硅盒，使用自动闪存纯化机执行闪光柱色谱。从六烷中的10%乙酸乙酯开始，在22分钟内在六烷中增加至60%乙酸乙酸酯。分数自动收集在管中。然后，将每个峰中的分数组合成一个圆底烧瓶，放在旋转蒸发器上，然后开始蒸发。获得三个分数，DHA，EDP异构体和二氧化物。要将获得的0.151克环氧树脂酯化，在圆底烧瓶或小瓶中，加入两毫升无水甲醇和三毫升无水甲苯，并加入搅拌棒。通过橡胶隔膜和针头将装满砷的气球与烧瓶连接，并在搅拌时通过注射器加入 0.26 毫升的两摩尔 TMS-diazomethane。等待 10 分钟，再添加 05 毫升的 TMS-二丁氨酯，直到出现淡黄色。30 分钟后，使用旋转蒸发器小心浓缩混合物，并使用闪光灯色谱机上的 40 克硅胶柱盒进行净化残留物。在六烷中等式浓度为4%乙酸的同位素浓度超过22分钟。首先收集含有纯化19S、20R-EDP甲酯甲酯的馏分，然后收集16S、17R-EDP甲基酯。现在，通过八对一六烷到乙酸酯混合物中通过薄层色谱评估分数。用高锰酸钾染色，以检查是否含有两种异构物的任何混合馏分。将与每个峰值对应的分数集中在旋转蒸发器上，然后权衡平衡，以确定获得的 EDP 酯量。如果混合馏分仍然存在，则使用与以前相同的溶剂系统重新色谱。将每个包含单个异构体的馏分集中在旋转蒸发器上。要将单个 EDP 甲基酯异构体转化为其酸形式，请用每 0.1 毫摩尔 0.1 毫摩尔的 EDP 酯以及 0.175 毫升的水稀释小瓶中的 EDP 酯。在冰浴中将小瓶冷却到零摄氏度，加入三个当量为氢氧化锂溶液，并在加热至室温时在搅拌板下搅拌一夜。早晨，通过缓慢添加甲酸来淬火反应，直到混合物的pH值达到三到四。然后，加入水和两毫升乙酸乙酯分离层。用10毫升乙酸酯萃取水层三次，用20毫升饱和盐水溶液清洗，并在溶液中加入几克无水硫酸钠，干燥乙酸乙酯层。使用旋转蒸发器浓缩乙酸乙酯溶液，加入 10 毫升六烷，然后再次浓缩。重复两次，以去除残留的甲酸。通过闪光柱色谱净化残留物，用 10 到 30% 乙酸乙酯在 15 分钟内进行分离。将所需分数集中在旋转蒸发器中，并在真空中干燥，以提供纯化酸。此处显示了从酶环氧气纯化粗混合物时获得的闪光柱色谱图。获得三个主要峰，第一，未反应的DHA，第二，EDP异构体的混合物，第三，二氧化二氮，这是正常的过度氧化产物。在异构体进行酯化和分离后，质子 NMR 光谱表明获得了高纯度16S、17R-EDP和19S、20R-EDP甲基酯。奇拉尔 HPLC 确认 EDP 的酸形式的异构纯度。在操作和接种时，请务必保持肉汤无菌，以避免污染。环氧树脂可用于其他脂肪酸。因此，我们也尝试了与厌食素和二甲酸，我们获得了良好的产量，这些环氧树脂，所以14，15-EET和17，18-EEQ。因此，一个允许研究人员获取环氧脂肪酸的协议可能有助于研究这些化合物的生物功能，这可能导致它们用作药物线索。危险化学品为接触危险性PMSF和接触和吸入性剧毒的TMS-二丁氨酯。因此，后者应用于烟罩和适当的个人防护设备。

Research

JoVE Journal

Chemistry

Seeded Synthesis of CdSe/CdS Rod and Tetrapod Nanocrystals

硒化镉/硫化镉棒和纳米四脚合成种子

We demonstrate a method for the synthesis of multicomponent nanostructures consisting of CdS and CdSe with rod and tetrapod morphologies. A seeded ...

科研

化学

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

使用十字型荧光基团的羧酸，硼酸，胺的定性鉴别

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

功能化的合成与表征金属 - 有机骨架

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

纳米尺寸钛酸二氢钠的合成和化学反应

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

光驱动脱羧酶

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

超分子胶体的合成与表征

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

金纳米棒基于对苯二酚合成

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

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

用于阵列发育和 HIV 抗体分析的糖的化学酶法合成

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

寡 peptoids 的合成及质谱分析

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

酶联反应合成 l-赖氨酸手性氨基醇的研究

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