Name: synthesis and structure determination of &#181;-conotoxin piiia isomers with different disulfide connectivities
Uploaded: 2018-10-02T14:00:00
Description: Watch this Scientific Journal Video about Synthesis and Structure Determination of Âµ-Conotoxin PIIIA Isomers with Different Disulfide Connectivities at JoVE.com

我们要感谢 Resemann, 琼斯迈尔和 d. Suckau 从布鲁克 optics Daltonics 有限公司布里曼;d. 肋软骨炎、肋软骨炎、Schmidts 和 c. 达姆施塔特技术大学蒂勒;Ohlenschläger 从波恩大学 FLI 耶拿、Engeser m。Harzen, 和 h. Nakagami 来自最大的普朗克植物育种研究研究所, 科隆;Susanne Neupert 从动物研究所, 科隆;以及法兰克福大学的生物分子磁共振光谱学设施, 用于技术支持、培训模块和获取仪器。波恩大学向直喷的财政支助得到了感激的承认。

注: 此处使用的所有氨基酸均采用 l-构型。根据 IUB 命名委员会和 IUPAC-IUB 联合委员会关于生物化学术语的建议, 使用了氨基酸和氨基酸衍生物的缩写。
1. 固相肽合成 (许可证)
注: 采用固相多肽合成器进行合成。使用 9-fluorenylmethyloxycarbonyl (n-芴甲氧羰基) 化学的标准协议, 对常规序列 ZRLCCGFOKSCRSRQCKOHRCC-NH2的线性肽前体进行合成。应用下列受保护的氨基?...

<ol>
	<li>Fosgerau, K., Hoffmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+therapeutics:+Current+status+and+future+directions.">Peptide therapeutics: Current status and future directions.</a> Drug Discovery Today. 20 (1), 122-128 (2015).</li><li>Gongora-Ben&#237;tez, M., Tulla-Puche, J., Albericio, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifaceted+roles+of+disulfide+bonds.+peptides+as+therapeutics.">Multifaceted roles of disulfide bonds. peptides as therapeutics.</a> Chemical Reviews. 114 (2), 901-926 (2014).</li><li>Tietze, A. 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=Structurally+diverse+&#181;-conotoxin+PIIIA+isomers+block+sodium+channel+NaV1.4.">Structurally diverse &#181;-conotoxin PIIIA isomers block sodium channel NaV1.4.</a> Angewandte Chemie - International Edition. 51 (17), 4058-4061 (2012).</li><li>Carstens, B. 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=Structure-Activity+Studies+of+Cysteine-Rich+&#945;-Conotoxins+that+Inhibit+High-Voltage-Activated+Calcium+Channels+via+GABABReceptor+Activation+Reveal+a+Minimal+Functional+Motif.">Structure-Activity Studies of Cysteine-Rich &#945;-Conotoxins that Inhibit High-Voltage-Activated Calcium Channels via GABABReceptor Activation Reveal a Minimal Functional Motif.</a> Angewandte Chemie - International Edition. 55 (15), 4692-4696 (2016).</li><li>Zhang, Y., Schulten, K., Gruebele, M., Bansal, P. S., Wilson, D., Daly, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disulfide+bridges:+Bringing+together+frustrated+structure+in+a+bioactive+peptide.">Disulfide bridges: Bringing together frustrated structure in a bioactive peptide.</a> Biophysical Journal. 110 (8), 1744-1752 (2016).</li><li>Nielsen, K. J., 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=Solution+structure+of+&#181;-conotoxin+PIIIA,+a+preferential+inhibitor+of+persistent+tetrodotoxin-sensitive+sodium+channels.">Solution structure of &#181;-conotoxin PIIIA, a preferential inhibitor of persistent tetrodotoxin-sensitive sodium channels.</a> Journal of Biological Chemistry. 277 (30), 27247-27255 (2002).</li><li>Heimer, 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=Conformational+&#181;-Conotoxin+PIIIA+Isomers+Revisited:+Impact+of+Cysteine+Pairing+on+Disulfide-Bond+Assignment+and+Structure+Elucidation.">Conformational &#181;-Conotoxin PIIIA Isomers Revisited: Impact of Cysteine Pairing on Disulfide-Bond Assignment and Structure Elucidation.</a> Analytical Chemistry. 90 (5), 3321-3327 (2018).</li><li>Chang, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+pathways+of+oxidative+folding+of+disulfide+proteins:+Underlying+causes+and+folding+models.">Diverse pathways of oxidative folding of disulfide proteins: Underlying causes and folding models.</a> Biochemistry. 50 (17), 3414-3431 (2011).</li><li>B&#246;hm, M., 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=Novel+insights+into+structure+and+function+of+factor+XIIIa-inhibitor+tridegin.">Novel insights into structure and function of factor XIIIa-inhibitor tridegin.</a> Journal of Medicinal Chemistry. 57 (24), 10355-10365 (2014).</li><li>Postma, T. M., Albericio, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disulfide+Formation+Strategies+in+Peptide+Synthesis.">Disulfide Formation Strategies in Peptide Synthesis.</a> European Journal of Organic Chemistry. 2014 (17), 3519-3530 (2014).</li><li>Albericio, F., Isidro-llobet, A., Mercedes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+Acid-Protecting+Groups.">Amino Acid-Protecting Groups.</a> Chemical Reviews. 109 (6), 2455-2504 (2009).</li><li>Annis, I., Hargittai, B., Barany, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disulfide+bond+formation+in+peptides.">Disulfide bond formation in peptides.</a> Methods in Enzymology. 289 (1988), 198-221 (1997).</li><li>Kamber, 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=The+Synthesis+of+Cystine+Peptides+by+Iodine+Oxidation+of+S-Trityl-cysteine+and+S-Acetamidomethyl-cysteine+Peptides.">The Synthesis of Cystine Peptides by Iodine Oxidation of S-Trityl-cysteine and S-Acetamidomethyl-cysteine Peptides.</a> Helvetica Chimica Acta. 63 (4), 899-915 (1980).</li><li>Bosch, D. E., Zielinski, T., Lowery, R. G., Siderovski, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+Modulators+of+Regulator+of+G-Protein+Signaling+(RGS)+Proteins.">Evaluating Modulators of Regulator of G-Protein Signaling (RGS) Proteins.</a> Current Protocols in Pharmacology. 56 (2.8), 1-15 (2012).</li><li>Mochizuki, M., Tsuda, S., Tanimura, K., Nishiuchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regioselective+formation+of+multiple+disulfide+bonds+with+the+aid+of+postsynthetic+S-tritylation.">Regioselective formation of multiple disulfide bonds with the aid of postsynthetic S-tritylation.</a> Organic Letters. 17 (9), 2202-2205 (2015).</li><li>Peigneur, 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=&#948;-conotoxins+synthesized+using+an+acid-cleavable+solubility+tag+approach+reveal+key+structural+determinants+for+NaV+subtype+selectivity.">&#948;-conotoxins synthesized using an acid-cleavable solubility tag approach reveal key structural determinants for NaV subtype selectivity.</a> Journal of Biological Chemistry. 289 (51), 35341-35350 (2014).</li><li>Heimer, 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=Application+of+Room-Temperature+Aprotic+and+Protic+Ionic+Liquids+for+Oxidative+Folding+of+Cysteine-Rich+Peptides.">Application of Room-Temperature Aprotic and Protic Ionic Liquids for Oxidative Folding of Cysteine-Rich Peptides.</a> ChemBioChem. 15 (18), 2754-2765 (2014).</li><li>Kates, S. A., Albericio, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Solid-Phase Synthesis: A Practical Guide. , (2000).</li><li>W&#252;thrich, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMR+studies+of+structure+and+function+of+biological+macromolecules+(Nobel+lecture).">NMR studies of structure and function of biological macromolecules (Nobel lecture).</a> Angewandte Chemie - International Edition. 42 (29), 3340-3363 (2003).</li></ol>

本文描述的方法是合成富含半胱氨酸的多肽, 如µ-PIIIA, 是一种有选择地从同一氨基酸序列中产生二硫化物粘合异构体的可能性。因此, 建立了以 n-芴甲氧羰基为基础的固相肽合成18和确定的保护基团策略为选择性形成二硫化键的方法16。固相肽合成可以通过自动合成在聚合物支持 (树脂) 上产生氨基酸序列。这些氨基酸被保护免受不期望的副作用在序列装配期间由...

An erratum was issued for: <a href="https://www.jove.com/video/58368/synthesis-structure-determination-conotoxin-piiia-isomers-with">Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities</a>. The Protocol and Materials sections were updated.
Step 1.1.3 in the Protocol section was updated from:
Weigh the individual reagents (amino acids, HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)) according to the protocol and dissolve them in dimethylformamide (DMF) to a final concentration of 2.4 M (amino acids) and 0.6 M (HBTU), respectively.
to:
Weigh the individual reagents (amino acids, HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)) according to the protocol and dissolve them in dimethylformamide (DMF) to a final concentration of 0.6 M (amino acids) and 0.6 M (HBTU), respectively.
Step 1.2 in the Protocol section was updated from:
NOTE: The protocol applies to 100 mg of resin (loading: 0.28 mmol/g) added to one reaction column for 28 &#956;mol scale.
to:
NOTE: The standardized protocol usually applies to 100 mg of resin (loading: 0.53 mmol/g) added to one reaction column for 53 &#956;mol scale leading to the following equivalents: 5 eq. HBTU, 10 eq. NMM, 5 eq. amino acid. In case of PIIIA, however, a loading of 0.28 mmol/g (28 &#181;mol scale) is used, which results in the specified higher equivalents.
Step 1.2.3.1 in the Protocol section was updated from:
HBTU (450 &#181;L; 0.6 M in DMF; 270 &#181;mol; 9.6 eq.), NMM (125 &#181;L; 50% in DMF; 568 &#181;mol; 20 eq.), Fmoc-amino acid (420 &#181;L; 2.4 M in DMF; 1.01 mmol; 36 eq.).
to:
HBTU (415 &#181;L; 0.6 M in DMF; 249 &#181;mol; 9 eq.), NMM (112 &#181;L; 50% in DMF; 510 &#181;mol; 18 eq.), Fmoc-amino acid (420 &#181;L; 0.6 M in DMF; 252 &#181;mol; 9 eq.).
The first item in the Materials table was updated from:
<table>
	<tbody>
		<tr>
			<td>Fmoc Rink amide resin</td>
			<td>Novabiochem</td>
			<td>855001</td>
			<td></td>
		</tr>
	</tbody>
</table>
to:
<table>
	<tbody>
		<tr>
			<td>PS PEG2000 Fmoc Rink-amide Resin</td>
			<td>Varian, Inc.</td>
			<td>PL3867-3764</td>
			<td>AmphiSpheres 40 RAM</td>
		</tr>
	</tbody>
</table>

An erratum was issued for: <a href="https://www.jove.com/video/58368/synthesis-structure-determination-conotoxin-piiia-isomers-with">Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities</a>. The Protocol and Materials sections were updated.

Erratum: Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

生物活性肽在药物研究和开发中的应用得到高度认可, 因为它们代表了特定生物靶1的有效和高度选择性化合物。然而, 对于它们的生物活性, 三维结构是非常重要的, 以执行结构-活动关系研究2,3,4。除影响整体构象的主要氨基酸序列外, 二硫键还能显著稳定半胱氨酸富肽5的结构。多重二硫化物桥接肽包括芋螺毒素, 如µ PIIIA圆锥 purpurascens , 其序列中含有六半胱氨酸。这种高半胱氨酸含量理论上允许形成15二硫化物异构体。正确的二硫化物连通性是非常重要的生物活动6,7。然而, 出现的问题是是否有一个以上的生物活性构象自然发生的肽, 如果是这样, 哪些异构体具有最高的生物活性？在µ-芋螺毒素的情况下, 生物靶点为电压门控钠离子通道, 特别是µ-PIIIA 对亚型 Nav1.2、nav1.4 和 nav1.73最有效。
采用多种方法可实现二硫桥多肽的合成。在肽内形成二硫化物键的最方便的方法是所谓的氧化自折叠法。在此, 首先用固相肽合成法合成所需循环肽的线性前体, 即在缓冲系统中由聚合物支持氧化后的裂解。氧化还原酶活性剂, 如还原和氧化谷胱甘肽 (GSH/GSSG) 往往增加, 以促进形成的二硫化物债券。缓冲支持自折叠的主要缺点是二硫化物债券不是以逐步的方式有选择地形成的。与原生肽相比, 通常只有一个特定的二硫化物异构体被描述, 有可能获得许多其他异构体以这个方法8。µ-PIIIA 已经被证明导致至少三种不同的折叠异构体后, 自折叠在以前的研究3。这种异构体混合物的分离是相当困难的, 由于相同的保留时间, 如果使用色谱纯化方法9。因此, 特定异构体的目标合成是有利的。为了专门生产具有定义的二硫化物连通性的异构体, 需要一个特殊的策略, 在其中二硫化物键连续关闭。因此, 在聚合物支持下合成了具有不同保护基团的线性前体在单个半胱氨酸对上。消除后, 半胱氨酸对分别和连续 deprotected 和连接在一个氧化反应, 以产生所期望的二硫化物债券10,11,12,13,14,15,16. 在反应产物的合成和纯化后, 需要用适当的分析方法确认识别和二硫化物的连通性。许多分析方法可用于阐明主要氨基酸序列, e., ms/毫秒, 而二硫化物连通性的测定仍然很少被调查。除了这种多重二硫化物粘结肽的复杂性外, 与产品相关的杂质 (例如, 从二硫化物上的), 由于样品的制备和工作, 可以进一步复杂化分析。在本文中, 我们表明, 使用不同的分析技术是必要的, 以明确澄清二硫化物键在µ-PIIIA 异构体的身份。我们将色谱方法与质谱相结合, 并提供了相同的样品到核磁共振光谱学中。在基质辅助激光解吸/电离 (MALDI) ms/质谱分析中, 我们用部分还原和 iodoacetamide 衍生的方法确定了二硫化物键, 因为这种肽的自顶向下分析是不可能的。为了获得每种异构体的三维结构, 进行了2D 核磁共振实验。因此, 通过结合不同的复杂的分析方法, 可以正确地阐明二硫化物连通性和三维结构复杂的多重二硫化物结肽7。

<table border="0" cellpadding="0" cellspacing="0" style="width:979px;" width="979">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Fmoc Rink amide resin</td>
			<td style="width:185px;">Novabiochem</td>
			<td style="width:191px;">855001</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Pyr(Boc)</td>
			<td style="width:185px;">Bachem</td>
			<td style="width:191px;">A-3850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Arg(Pbf)</td>
			<td style="width:185px;">Iris Biotech</td>
			<td style="width:191px;">FSC1010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Asn(Trt)</td>
			<td style="width:185px;">Bachem</td>
			<td style="width:191px;">B-1785</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Asp(tBu)</td>
			<td style="width:185px;">Iris Biotech</td>
			<td style="width:191px;">FSP1020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Hyp(tBu)</td>
			<td style="width:185px;">Iris Biotech</td>
			<td style="width:191px;">FAA1627</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Lys(Boc)</td>
			<td style="width:185px;">Bachem</td>
			<td style="width:191px;">B-1080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Ser(tBu)</td>
			<td style="width:185px;">Iris Biotech</td>
			<td style="width:191px;">FSC1190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Gln(Trt)</td>
			<td style="width:185px;">Iris Biotech</td>
			<td style="width:191px;">FSC1043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Glu(tBu)</td>
			<td style="width:185px;">Iris Biotech</td>
			<td style="width:191px;">FSP1045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Trp(Boc)</td>
			<td style="width:185px;">Iris Biotech</td>
			<td style="width:191px;">FSC1225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Tyr(tBu)</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">47623</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Thr(tBu)</td>
			<td style="width:185px;">Iris Biotech</td>
			<td style="width:191px;">FSP1210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">His(Trt)</td>
			<td style="width:185px;">Iris Biotech</td>
			<td style="width:191px;">FDP1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorphosphat</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">8510060</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">DMF</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:191px;">D119</td>
			<td>Flammable, Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">DCM</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td>D37</td>
			<td>Carcinogenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Piperidine</td>
			<td style="width:185px;">Alfa Aesar</td>
			<td>A12442</td>
			<td>Flammable, Toxic, Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">N-Methyl-Morpholin</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">224286</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Cys(Acm)</td>
			<td style="width:185px;">Iris Biotech</td>
			<td style="width:191px;">FAA1506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Cys(Trt)</td>
			<td style="width:185px;">Bachem</td>
			<td style="width:191px;">E-2495</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Cys(tBu)</td>
			<td style="width:185px;">Bachem</td>
			<td style="width:191px;">B-1220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">trifluoruacetic acid</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">74564</td>
			<td>Toxic, Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">phenol</td>
			<td style="width:185px;">Merck</td>
			<td style="width:191px;">1002060</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">thioanisol</td>
			<td style="width:185px;">Alfa Aesar</td>
			<td style="width:191px;">A14846</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">ethanedithiol</td>
			<td style="width:185px;">Fluka Analytical</td>
			<td style="width:191px;">2390</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">diethyl ether</td>
			<td style="width:185px;">VWR</td>
			<td style="width:191px;">100,921</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">tert-butanol</td>
			<td style="width:185px;">Alfa Aesar</td>
			<td style="width:191px;">L12338</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">acetonitrile</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:191px;">A998</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">water</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:191px;">W5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">isopropanol</td>
			<td style="width:185px;">VWR</td>
			<td style="width:191px;">ACRO42383</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">sodium hydroxide</td>
			<td style="width:185px;">AppliChem</td>
			<td style="width:191px;">A6579,1000</td>
			<td>Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">iodoacetamide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">I6125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">iodine</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">I0385</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Hydrochloric acid</td>
			<td style="width:185px;">Merck</td>
			<td style="width:191px;">110165</td>
			<td>Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">ascorbic acid</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">A4403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">diphenylsulfoxide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">P35405</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">anisol</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">96109</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">trichloromethylsilane</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">M85301</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sample dilution buffer</td>
			<td style="width:185px;">Laborservice Onken</td>
			<td style="width:191px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">sodium dihydrogen phosphate</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">106370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">disodium hydrogen phosphate</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">795410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(2-carboxyethyl)phosphine hydrochloride</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">C4706</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">citric acid</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">251275</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">sodium citrate dihydrate</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">W302600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tris-acetate</td>
			<td style="width:185px;">Carl Roth,&#160;</td>
			<td style="width:191px;">7125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Ethylenediaminetetraacetic acid</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:191px;">E26282&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">peptide calibration standard II</td>
			<td style="width:185px;">Bruker Daltonics GmbH</td>
			<td style="width:191px;">8222570</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;"></td>
			<td style="width:185px;"></td>
			<td style="width:191px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Name of Equipment</td>
			<td style="width:185px;">Company</td>
			<td style="width:191px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">solid-phase peptide synthesizer</td>
			<td>Intavis Bioanalytical Instruments AG</td>
			<td style="width:191px;">EPS 221</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:340px;">lyophilizer&#160;</td>
			<td>Martin Christ GmbH</td>
			<td style="width:191px;">&#160;Alpha 1-2 Ldplus</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">semipreparative HPLC</td>
			<td>Jasco</td>
			<td style="width:191px;">system PV-987</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Eurospher 100 C18 column (RP, 5 &#181;m particle size, 100 &#197; pore size, 250 x 32 mm)</td>
			<td style="width:185px;">Knauer</td>
			<td style="width:191px;">25QE181E2J</td>
			<td>purification of the linear peptide</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:340px;">Vydac 218TP1022 column (RP C18, 10 &#181;m particle size, 300 &#197; pore size, 250 x 22 mm)</td>
			<td>Hichrom-VWR</td>
			<td style="width:191px;">HICH218TP1022</td>
			<td>purification of the oxidized peptide</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:340px;">analytical HPLC&#160;</td>
			<td>Shimadzu</td>
			<td style="width:191px;">system LC-20AD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Vydac 218TP54 column (C18 RP, 5 &#181;m particle size, 300 &#197; pore size, 250 x 4.6 mm)&#160;</td>
			<td>Hichrom-VWR</td>
			<td style="width:191px;">HICH218TP54</td>
			<td>analytical column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">ground steel target (MTP 384)</td>
			<td>Bruker Daltonics GmbH</td>
			<td style="width:191px;">NC0910436</td>
			<td>MALDI preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">C18-concentration filter (ZipTip)</td>
			<td style="width:185px;">Merck KGaA</td>
			<td style="width:191px;">ZTC18S096</td>
			<td>MALDI preparation&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">MALDI mass spectrometer</td>
			<td style="width:185px;">Bruker Daltonics GmbH</td>
			<td style="width:191px;">ultraflex III TOF/TOF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">amino acid analyzer</td>
			<td>Eppendorf-Biotronik GmbH</td>
			<td style="width:191px;">LC 3000 system</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">NMR spectrometer Bruker Avance III</td>
			<td>Bruker Daltonics GmbH</td>
			<td style="width:191px;">Bruker Avance III 600 MHz</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">computer program for molecular visualising</td>
			<td>YASARA Biosciences GmbH</td>
			<td style="width:191px;">Yasara structures</td>
			<td>NMR structure calculation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">computer program for MALDI data evaluation&#160;</td>
			<td>Bruker Daltonics GmbH</td>
			<td style="width:191px;">flexAnalysis, BioTools</td>
			<td>MS/MS fragmentation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">analog vortex mixer</td>
			<td>VWR</td>
			<td style="width:191px;">VM 3000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Microcentrifuge</td>
			<td>Eppendorf</td>
			<td style="width:191px;">5410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Centrifuge</td>
			<td>Hettich</td>
			<td style="width:191px;">EBA 20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Rotational vacuum concentrator</td>
			<td style="width:185px;">Christ</td>
			<td style="width:191px;">2-18 Cdplus</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Analytical Balance</td>
			<td style="width:185px;">A&#38;D Instruments</td>
			<td style="width:191px;">GR-202-EC</td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis and structure determination of &#181;-conotoxin piiia isomers with different disulfide connectivities

具有大量半胱氨酸的多肽通常受其二硫化物连通性对三维结构的影响。因此, 在肽合成过程中避免不必要的二硫化键形成是非常重要的, 因为它可能产生完全不同的肽结构, 从而改变生物活性。但是, 如果采用常规的缓冲氧化协议等标准自折叠方法, 可以形成多硫 connectivities 的正确形成。这个协议代表了一个先进的战略需要的目标合成多重二硫桥多肽, 不能合成通过缓冲氧化的高质量和数量。本研究展示了一种独特的保护基团策略, 用于合成µ-芋螺毒素 PIIIA 的所有可能的 3-二硫结合肽异构体。用 n-芴甲氧羰基基固相肽合成方法制备多肽, 采用保护基团策略确定二硫化物键形成。各自对半胱氨酸被保护与三苯甲氧基 (Trt), acetamidomethyl (Acm) 和叔丁基 (t步) 保护小组保证在每氧化步期间仅要求的半胱氨酸是 deprotected 和连接的。除了目标合成之外, 还使用几种分析方法来澄清所需肽结构的正确折叠和生成。对不同的 3-二硫化物粘结异构体的比较表明, 准确测定和认识二硫化物连通性对三维结构的计算和生物解释的重要性肽异构体的活性。分析表征包括通过串联质谱 (ms/毫秒) 分析精确的二硫化键说明, 这是由一个适应的协议产生的完整的肽异构体的部分减少和烷基化衍生物。此外, 用2D 核磁共振 (NMR) 实验和从 ms 和 ms 分析获得的知识来确定肽的结构。

对µ-芋螺毒素 PIIIA 的15种不同的二硫桥异构体进行了合成, 并进行了详细的描述 (图 1)。二硫化键通过部分还原和随后的 ms/毫秒分析来确定 (图 2)。对不同异构体进行了核磁共振分析 (图 3), 揭示了各自的肽结构。值得注意的是, 高效液相色谱、ms/毫秒碎片和核磁共振分析相结合, 是确定二硫化物连通性的必?...

Watch this Scientific Journal Video about Synthesis and Structure Determination of Âµ-Conotoxin PIIIA Isomers with Different Disulfide Connectivities at JoVE.com

Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

富含半胱氨酸的肽折叠成不同的三维结构, 这取决于它们的二硫化物连通性。当缓冲氧化不导致所需的二硫化物连接时, 需要有针对性地合成单独的二硫化物异构体。该协议涉及 3-二硫键结合肽的选择性合成及其结构分析, 采用核磁共振和 ms/质谱研究。

不同二硫 Connectivities µ-芋螺毒素 PIIIA 异构体的合成与结构测定

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus ...

该方法有助于回答肽合成领域的关键问题，如合成二硫化富肽并确定其相应的二硫化物桥模式。该技术的主要优点是，它允许选择性地合成不同的二硫化粘结肽异构体及其随后的化学和结构表征。通常，对这种方法的新鲜个体会挣扎，因为每种非硫化物丰富的肽的行为方式不同。合成和分析可能需要针对单个肽或蛋白质进行部分优化。将文本协议中列出的试剂转移到相应的容器中，并放在固体相肽合成器的适当球拍中。然后，在反应柱中加入100毫克干树脂，并放入合成器的球拍中。开始固相肽合成，详见文本协议。合成完成后，将树脂冻干过夜。现在从树脂中切出肽，对侧链进行深度保护。为此，将干树脂混合在12毫升的管中，并在冰上冷却至零摄氏度。首先，加入150微升的清除剂混合物，然后每100毫克树脂加入1毫升95%三氟乙酸。在室温下，使混合物轻轻摇动三小时。现在，通过玻璃褶皱过滤混合物。收集管中的滤气，单独填充冰冷二乙醚，每8至10毫升二乙醚的裂解混合物为一毫升。肽沉淀为白色固体。如文本协议所述，冲洗和离心管后，冷冻干燥肽。为了用半制备色谱法净化线性前体，将约70毫克的粗肽加入15毫升管中。然后将粗肽溶解在0.1%TFA和一对一乙酰三叶酸水中。在120分钟内，以每分钟10毫升的流速，使用0至50%的Eluent B梯度分离肽混合物。收集单个管中的分数，并准备选定的分数进行质谱法和 HPLC 分析，如文本协议中所述。冻结干燥分数并组合对等分数。通过执行第一次氧化，开始选择性形成二硫化键。为此，将15毫克冻干线性肽溶解在105毫升异丙醇水混合物中。使混合物在基本条件下在空气中搅拌 12 至 48 小时。逐步氧化过程非常关键。所有三个氧化步骤都采取反应控制，以确保单个二硫化物丰富的形成。第三次氧化是最关键的，因为当超过最佳反应时间时，可能发生二硫化物扰动。现在执行第二次氧化。溶解15毫克冻干肽在105毫升异丙醇水，一个摩尔盐酸混合物。在甲醇中加入 158 微升 0.1 摩尔碘溶液。在室温下搅拌反应，直到氧化完成。然后在水中加入79微升的摩尔抗坏血酸，从而停止反应。最后进行第三次氧化。溶解15毫克的冻干肽在5.5毫升TFA含有清除剂混合物。将肽沉淀到含有冷二乙醚的管中，每八到十毫升二乙醚的反应是一毫升的反应溶液。在15毫克冷冻干燥的粗制植物中对15毫升管进行肽纯化。使用 0.1%TFA 和一对一乙酰三叶酸的混合物将粗产品溶解在 HPLC 样品环的体积中。在涡流至完全溶解后，在3400倍G.下1分钟将溶液离心，将3.6毫升混合物放入5毫升注射器中，将样品注射至注射回路中，没有任何气泡。开始注入 HPLC 系统。在120分钟内，以每分钟10毫升的流速，使用0至50%的Eluent B梯度净化肽混合物。收集单个管中的分数，因为它们出现。运行完成后，准备 MS 和 HPLC 分析的选定分数，如本文中所述。冷冻干燥所有馏分，并储存在零下20摄氏度。要执行分析性 HPLC，请将肽馏分或反应对子的样品转移到 HPLC 小瓶中，将其溶解在 0.1%TFA 的混合物中，一对一乙酰三叶酸溶水。将 HPLC 小瓶放入分析反相 HPLC 设置的自动采样器中，以注入每个样品的 250 微升。在水中使用 0.1%TFA 的梯度洗脱系统，在 Acetonitrile 中用作 0.1% TFA 中的 Eluent A。在 30 分钟内以每分钟 1.0 毫升的流速使用 10%至 40%Eluent B 的梯度分析肽。为了进行氨基酸分析，将100微克的纯肽转移到1.5毫升微离心管中，将粉末溶解在6摩尔HCL的200微升中。将溶液转移到玻璃安培后，用本森燃烧器火焰加热颈部，关闭安培。然后将一个玻璃管放在一个加热块中，在110摄氏度的加热块中放置24小时，进行水解。24 小时后，打开安培并将溶液转移到两毫升微离心管中。在60摄氏度下将溶液离心6小时，在旋转真空浓缩器中离心210倍G。然后将水解产品溶解在192微升的样品稀释缓冲液中，将溶液转移到微离心过滤器中。在2300次G下离心样品1分钟后，将100微升滤液转移到氨基酸分析样品管中。将管子放入氨基酸分析仪并开始分析。在以往的还原协议中，必须采取若干反应控制，以获得两次或四次卡帕中甲基化物种，这是通过质谱法进行后续二硫化物分析所必不可缺少的。首先溶解600微克的纯肽在1.2毫升0.05摩尔柠檬酸盐缓冲液中，含有TCEP。在室温下孵育混合物，采集几个100微升反应控制样品，范围从零到30分钟。将样品混合在1.5毫升微离心管中，与300微升烷基化缓冲液，以停止反应，并执行无硫醇组碳基甲基化。五分钟后停止反应，加入100微升10%TFA和水，将样品储存在干冰上。现在，对示例执行 HPLC，如文本协议中详细说明的。将少量收集的馏分转移到1.5毫升微离心管中，用于氧化形式的MS/MS分析。将剩余的肽溶解在水中0.1%TFA中，并在100毫摩尔TCEP溶液中加入适当的体积，以获得10毫摩尔的最终TCEP浓度。此时，可以通过肽测序检查选择性半胱氨酸烷化。在中间的两个肽中，可以确定二硫化桥型。对不同的异构体进行NNR分析，以揭示单个肽结构。显示了20个能量最低的结构以及不同异构体的二硫化连接。值得注意的是，需要将反相 HPLC MS/MS 碎片和 NMR 分析相结合，以明确识别二硫化连接。根均方偏差值的比较表明，刚性肽主要导致更好的核化 NMR 结构。本视频应为您提供一个示例，了解如何选择性地合成具有所需二硫化键模式的肽。它还显示通过招标质谱法检查正确的二硫化物连接性，通过 NMR 光谱法获得三维结构的可能性。该技术开发后，为肽合成领域的研究人员探索二硫化键模式对三维结构的重要性以及由此产生的肽异构体的活性铺平了道路。按照这个程序，可以执行其他方法，如活动ASAS，以便深入了解不同肽异构体在特定生物靶点的结构活性关系。虽然这种方法可以提供对康多毒素的合成和分析的见解，它也可以应用于其他二硫化桥肽和蛋白质，如芬森、富含二硫化物的动物毒素和其他含有分子的多种半胱氨酸。

Research

Education

JoVE Journal

JoVE Core

Organic Chemistry

Chemistry

Unit 1

Stereoisomerism

SCIENTISTS IN ACTION

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

生产二硫键稳定的跨膜肽复合物的结构研究

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

科研

化学

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

From Constructs to Crystals &#8211; Towards Structure Determination of &#946;-barrel Outer Membrane Proteins

从构造到晶体 - 迈向β桶外膜蛋白的结构解析

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% ...

X-ray Powder Diffraction in Conservation Science: Towards Routine Crystal Structure Determination of Corrosion Products on Heritage Art Objects

X射线粉末衍射在保护科学：迈向文物艺术品腐蚀产物的常规晶体结构测定

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder ...

Synthesis and Characterization of Fe-doped Aluminosilicate Nanotubes with Enhanced Electron Conductive Properties

以增强电子导电性能的合成与表征Fe掺杂碳纳米管硅铝的

The goal of the protocol is to synthesize Fe-doped aluminosilicate nanotubes of the imogolite type with the formula (OH)3Al2-xFexO3SiOH. Doping with Fe ...

Probing the Structure and Dynamics of Interfacial Water with Scanning Tunneling Microscopy and Spectroscopy

用扫描隧穿显微镜和光谱法探讨界面水的结构和动力学

Water/solid interfaces are ubiquitous and play a key role in many environmental, biophysical, and technological processes. Resolving the internal ...

Fabricating High-viscosity Droplets using Microfluidic Capillary Device with Phase-inversion Co-flow Structure

反相共流微流控毛细管装置制备高粘度水滴的方法

The generation of monodisperse droplets with high viscosity has always been a challenge in droplet microfluidics. Here, we demonstrate a phase-inversion ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

通过活性表面生长机制合成基片束缚金纳米线

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a ...

Synthesis of Graphene Nanofluids with Controllable Flake Size Distributions

具有可控片状分布的石墨烯纳米流体合成

A method for synthesizing graphene nanofluids with controllable flake size distributions is presented. Graphene nanoflakes can be obtained by the ...

Organic Structure-directing Agent-free Synthesis for *BEA-type Zeolite Membrane

有机结构定向无代理合成，用于_BEA型沸石膜

Membrane separation has drawn attention as a novel-energy saving separation process. Zeolite membranes have great potential for hydrocarbon separation in ...