作者承认中国自然科学基金 (21372023、21778009和81701818号) 的财政支持;中华人民共和国科学技术部 (2015DFA31590 号);深圳市科技创新委员会 (no。JCYJ20170412150719814、JCYJ20170412150609690、JCYJ20150403101146313、JCYJ20160301111338144、JCYJ20160331115853521、JSGG20160301095829250 和 GJHS20170310093122365);和中国博士后科学基金会 (2017M610704)。

1. 设备准备
<ol><li>对于手动多肽合成装置, 放置一个真空流形 (材料表) 在一个有效的油烟机罩。接下来, 将三路 stopcocks 到真空流形上, 并将其连接到氮气或氩气线。使用橡胶隔膜盖所有未使用的入口。</li>
	<li>使用三路 stopcocks 连接树脂填充柱 (0.8 x 4 厘米, 10 毫升储层, 见材料表) 到歧管 (图 1)。使用与真空系统连接的泵作为真空过滤器或橡胶吸管灯泡, 通过挤出来去除列中的溶剂。</li>
	<li>使用 photoreactor (图 2), 配有十 350 nm 灯 (材料表) 用于紫外线照射。通过 photoreactor 进气口将这些连接到氩气罐, 以确保 photoreactor 在 photoreactions 之前和期间充满氩气。</li>
	<li>在打开 photoreactor 的 uv 灯之前, 确保 photoreactor 门关闭, 以防紫外线照射。</li>
</ol>2. 树脂制备
注: 一般情况下, 采用 n-芴甲氧羰基基固相肽合成协议进行多肽基质的构建。这些是使用溜冰场酰胺树脂, 它留下一个 C 末端酰胺剩余的肽分裂。此协议在整个文件中使用。
注意: n, n-甲基酰胺 (DMF), 二氯甲烷 (DCM), 4-甲基啉) (NMM), 和n, n-diisoproylethylamine (DIPEA) 是有毒和有害的吸入, 摄取, 或皮肤接触。乙醚是高度易燃的。三氟乙酸酸 (TFA) 具有腐蚀性。12-ethanedithiol (EDT) 是高度恶臭。因此, 所有有机溶剂和化学品应使用适当的个人防护设备 (腈手套, 实验室大衣和防护眼镜) 处理, 并在化学油烟机内处理。
<ol><li>使用以下公式计算所需的树脂量: 鳞片 (毫摩尔)/(树脂负载能力 (毫摩尔/克) 1000 (毫克/克)) = 树脂质量 (毫克) 例如, 溜冰场酰胺 MBHA 树脂的数量 (0.5 毫摩尔/克) 为25µmol = 0.025 毫摩尔/(0.5 毫摩尔/克1000毫克/克) = 50 毫克。接下来, 在一列中重50毫克树脂, 用三路 stopcocks 在真空歧管上设置。</li>
	<li>在柱体 (0.8 x 4 厘米, 10 毫升储层) 中加入 1-2 毫升的 DCM。要膨胀树脂, 用氮气或氩气流轻轻搅动10分钟。接下来, 用真空过滤法去除溶剂。</li>
</ol>3. n-末端 n-芴甲氧羰基脱和洗涤
<ol><li>准备 n-末端 n-甲基芴甲氧羰基 deprotecting 溶液: 在玻璃瓶中准备一个适当体积 (200 毫升) 的 50% (v/v) 啉在 DMF 中。</li>
	<li>在树脂上加入 1-2 毫升的脱溶液, 轻轻搅动10分钟, 然后用真空排出溶液。按该顺序使用 DMF (1-2 毫升) 和 DCM (1-2 毫升), 彻底清洗树脂和反应容器, 共3x。接下来, 重复脱和洗涤程序1x。</li>
</ol>4. n-芴甲氧羰基保护氨基酸偶联
<ol><li>以丙氨酸渣的偶联为例, 在25µmol 的人工合成情况下, 称 n-芴甲氧羰基-5 equiv, 41.4 毫克, 2-(6-氯-1 h 苯三唑-1-基)-11, 33-tetramethylaminium 六氟磷酸盐 (HCTU; 4.9 equiv, 50.5 毫克) 在聚丙烯容器并将其溶解于 DMF (0.5 毫升)。</li>
	<li>添加 DIPEA (10 equiv, 43.5 µl), 以产生0.25 米活化氨基酸溶液 (表 1)。经过大约1分钟的预活化, 加入溶液的树脂, 然后气泡与 N2约 1-2 小时。</li>
	<li>从这一步开始, 将肽链中的每一种氨基酸作为一系列步骤进行整合: 首先是 n-端芴甲氧羰基基团的脱, 然后是洗涤, 然后用 HCTU 活化氨基酸的偶联。 注: 较长的耦合时间 (e.2 h) 建议, 如果耦合后, sterically 受阻氨基酸残留 [e., n-芴甲氧羰基-胱氨酸 (trt)-哦, n-芴甲氧羰基-他 (trt)-哦, n-芴甲氧羰基-Arg (Pbf)-哦]。含非天然氨基酸的烯烃/炔烃用于3当量而不是 5, 剩下的反应为 3 h。</li>
	<li>用阿罗拉等四氯苯醌的方法, 监测肽合成的进展情况。36这些测试提供了对游离初级和次级胺的存在或缺失的定性评估。或者, 大约 2-3 毫克的肽可以从树脂中, 并通过 LC-MS 分析。</li>
</ol>5. 硫醇烯 Hydrothiolation 和硫醇-yne 环合
<ol><li>通过树脂环合来构造硫醚类链接器 (图 3)。<ol>
<li>准备大约50毫升的 Trt 脱解决方案 (TFA/TIS/DCM 0.03/0.06/1.0)。将胱氨酸-和 mS5轴承树脂 [NH2-r-ms5a a a a 胱氨酸 (Trt) r ' 树脂, 50 毫克] 与 1-2 毫升的 Trt 脱溶液在10毫升的专栏。用 N2轻轻搅动溶液10分钟。最后, 用 DCM (1-2 毫升) 清洗, 共3x。 注: MS5代表亚烷基构造块 (参见图 6中描述的结构), 用于肽偶联和硫原子环合38。</li>
		<li>重复上述过程总共约 6x, 以删除半胱氨酸的三苯甲胺保护组, 直到解决方案颜色不再是黄色。</li>
		<li>冒泡与 N2, 洗涤的 r-mS5a a-胱氨酸 (sh) r ' 树脂 [半胱氨酸与游离硫 (SH)] 与 DMF (1-2 毫升) 和 DCM (1-2 毫升) 按此顺序。用甲醇 (1-2 毫升) 收缩树脂2分钟, 然后用过滤法去除溶剂。接下来, 在2气体的蒸汽下依次干燥树脂, 在柱中约5分钟。</li>
		<li>在嘴瓶内预先准备脱气 DMF, 通过将氮气泡在一个长针上, 并将其拉伸到溶剂中, 使其在大约1小时内起泡。</li>
		<li>通过称重纸将树脂转化为10毫升的圆底烧瓶。将树脂悬浮在脱气 DMF 的2毫升内, 然后加入光 2-羟基 1-[4-(2-hydroxyethoxy)-苯基]-2-甲基 1-丙酮 (基质金属蛋白酶; 1 equiv, 5.6 毫克), 4-methoxyacetophenone (地图; 1 equiv, 3.8 毫克)。</li>
		<li>在烧瓶中加入搅拌杆 (0.3 厘米), 用合适的橡皮塞将烧瓶盖上, 然后用油泵将瓶中的空气置换成氮气。 注: 在固相中, 有效的硫-烯 photoreaction 不需要严格的惰性气氛。然而, 在图 5中, 在溶液阶段的硫 yne photoreaction 是非常必要的。否则, 在紫外线照射时硫磺会氧化。</li>
		<li>将反应瓶设置成 photoreactor, 在室温下紫外线照射下将树脂搅拌1小时 (图 2)。 注意: 在切换 photoreactor 紫外线灯之前, 确保 photoreactor 门关闭, 以防紫外线照射有害辐射。 注: 在 photoreactions 中经常取样反应混合物, 建议新的序列, 因为线性肽前驱体具有相同的分子量的产品。一般情况下, 线性和循环多肽的亲水性明显不同。这可以很容易地区分使用 HPLC。另外, 55 '-dithiobis (2 硝基苯甲酸) (DTNB) 试剂也可以用来研究游离硫醇37的存在。</li>
		<li>将树脂从烧瓶中转移到柱上, 用真空过滤去除溶剂。按照步骤5.1.3 中所述, 清洗和烘干树脂。</li>
		<li>准备大约 10 毫升的鸡尾酒 ( TFA / 小贴士 / EDT / H2O 94 / 1 / 2 . 5 / 2 . 5 ) 在通风罩。</li>
		<li>将树脂转移到2毫升的聚丙烯容器中, 加入1毫升的裂解鸡尾酒 (TFA/小贴士/EDT/H2O 94/1/2.5/2.5) 到容器中, 并用螺钉盖紧密封容器。然后, 用 60 rpm 的速度在一个轨道振动筛上轻轻搅动 1.5-2 h 的容器。 注意: TFA 具有很强的腐蚀性。穿着防护服, 在有效的油烟机中工作。EDT 是一种高度恶臭的物质, 必须处理在一个有效的油烟机。</li>
		<li>在通风罩中的 N2气体流下蒸发, 除去 TFA 鸡尾酒。接下来, 用冷二乙基醚 (1 毫升) 在三十年代沉淀残余物, 并通过离心 1.2万 x g 将其分离2分钟。离心后, 轻轻地将乙醚组件倒出来。重复2x 的沉淀和离心步骤。把残渣蒸发乾燥。</li>
		<li>最后, 用 C18 分析柱 (4.6 x 250 毫米, 流速1.0 毫升/分) 溶解1毫升 H2/乙腈 (2:1) 中的残留物, 并用 HPLC 纯化。使用 H2O (含 0.1% TFA) 和纯乙腈作为溶剂在 2%/分钟线性梯度从20% 到70% 乙腈超过25分钟. 用紫外 280 nm 和 220 nm 波长 (表 2) 监测 HPLC 谱。</li>
	</ol></li>
	<li>通过分子间硫-烯反应建立硫醚类链接器, 然后通过 macrolactamization cyclize 肽 (图 4)。<ol>
<li>采用标准的 n-芴甲氧羰基基固相肽合成 (许可证), 在步骤 2-4 中所述, 合成了含线性肽 H2n-a a a-a-a-5(2 r ") R ' 树脂 (50 毫克) 的亚烷基残留物。接下来, 洗涤和干燥的树脂, 如步骤5.1.3 所述。</li>
		<li>将树脂悬浮在含有2毫升脱气 DMF 的10毫升圆底烧瓶中, 如步骤5.1.4 所述。</li>
		<li>添加光基质金属蛋白酶和地图 (基质金属蛋白酶: 1 equiv, 5.6 毫克;图: 1 equiv, 3.8 毫克), n-芴甲氧羰基-胱氨酸-OH (3 equiv, 25.7 毫克), 和搅拌棒 (0.3 厘米) 进入烧瓶。用合适的橡皮塞盖住烧瓶, 然后用油泵用氮气代替瓶子里的空气。</li>
		<li>把反应瓶放进 photoreactor。在室温下, 在紫外线照射下搅拌 1-2 小时 (图 2)。</li>
		<li>在 LC-MS 分析下监测反应: 使用裂解鸡尾酒对树脂进行 2-3 毫克的劈裂。然后用冷二乙基醚 (300 µL) 沉淀渣, 通过离心分离残渣, 并将残留物蒸发至干燥, 如步骤5.1.11 所述。然后, 在100µL2/乙腈 (2:1) 中溶解残渣。用 0.22-µm 微孔膜滤过多肽溶液, 并用 LC-MS 与电喷雾电离 (ESI) 中的复合电离进行分析, 并以正向模式进行操作。</li>
		<li>如有必要, 重复步骤 5.2.2 5.2.4 以确保反应完成。</li>
		<li>光反应完成后, 将树脂从烧瓶中转移到柱体中, 用真空过滤去除溶剂。按照步骤5.1.3 中所述, 清洗和烘干树脂。</li>
		<li>添加 benzotriazole-1-yl-oxytripyrrolidinophosphonium 六氟磷酸盐的 DMF 溶液 (PyBob; 2.4 equiv, 31.2 毫克), 1-hydroxybenzotriazole (对 hobt; 2.4 equiv, 8.1 毫克), NMM (4 equiv, 11 µL) 的树脂在列为 macrolactamization。用 N2来泡这个溶液2小时。<ol>
<li>此外, 按照步骤5.2.3 中的描述, 使用 LC MS 来监测这种耦合反应。如有必要, 重复此步骤以确保反应完成。</li>
		</ol>
</li>
		<li>用标准的 n-芴甲氧羰基基许可证延长肽, 如步骤3和4所述。</li>
		<li>在组装所有氨基酸残留物时, 将肽从树脂中分离出来, 如步骤5.1.10 和5.1.11 中所述, 并在步骤5.1.12 中加以纯化。</li>
	</ol></li>
	<li>在溶液阶段构造乙烯基硫化物链接器 (图 5)。<ol>
<li>用标准的 n-芴甲氧羰基基许可证合成炔烃残留量的线性肽, 如步骤 2-4 所述。根据前一项研究20中所述的一项完善的协议, 合成炔烃轴承氨基酸。</li>
		<li>将肽从树脂中劈开, 用冷二乙基醚沉淀, 如步骤 5.1.9-5.1.11 中所述。在树脂的裂解和沉淀之后, 用离心法收集 1.2万 x g 的肽以2分钟。</li>
		<li>在真空中干燥所产生的残余物。将脱气 DMF (50 毫升) 中的残渣溶解在100毫升圆底烧瓶中, 以达到最终浓度0.5 毫米 (根据树脂的负载, 0.025 毫摩尔 (1000 毫升/升/0.5 毫摩尔/升) = 50 毫升)。<ol>
<li>添加光 DMPA (0.5 equiv, 3.2 毫克), 然后德加的反应解决方案10分钟使用 N2通过长针延伸到溶液中。接下来, 在室温下将样品照射到紫外线下 0.5-1 小时, 无需搅拌。</li>
		</ol>
</li>
		<li>在高真空下去除 DMF, 加入二乙醚沉淀粗渣, 以溶解其有机副产品。然后, 用离心法将残渣分离 1.2万 x 克, 2 分钟。离心后, 轻轻地将乙醚组件倒出来。把残渣蒸发乾燥。最后, 在1毫升的 H2/乙腈 (2:1) 中溶解残留物, 并使用液相色谱法对其进行纯化, 如步骤5.1.7 所述。</li>
	</ol></li>
</ol>

作者没有什么可透露的。

在图 3所描述的树脂内分子内硫-烯环化中, 发现半胱氨酸残留物的三苯甲酯的去除是随后 photoreaction 的关键步骤。此外, 在反应之前和之后的肽分子量被发现是相同的, 如图 6B所示。因此, 需要使用 HPLC 鉴定或 DTNB 化验, 以监测反应。如图 4所述的分子间硫-烯反应, MS 监测是必要的。虽然发现内酰胺耦合的进一步步骤是需要建造一个?...

配体对蛋白质-蛋白质相互作用的调节 (PPIs) 的发展为现代药物发现提供了一种有吸引力的方法。因此, 我们投入了大量的精力研究新的化学模式, 可以有效地调节 PPIs1,2,3。PPIs 一般由浅, 大, 和/或停止相互作用的表面组成, 小分子通常被认为是不合适的配体为 PPIs4,5的调制。利用合适的暴露交互表面积, 模拟蛋白质界面结构特征的短肽代表了解决这个问题6,7的理想候选者。然而, 短肽通常是非结构化的水溶液。这是由于水分子与肽骨干和定义构象的分子内氢键网络竞争, 在水8中由熵不利。此外, 肽的固有低稳定性和细胞渗透性的特性在很大程度上限制了它们在生物应用中的应用9,10。根据蛋白质数据库 (PDB) 分析, > 50% 的 PPIs 涉及短α螺旋相互作用11。因此, 在螺旋稳定方面发展了不同的化学方法。这些包括二硫化物或硫醚类债券形成12,13,14, 圆环闭合置换15, 内酰胺圆环形成16, "点击" 化学17, 加法perfluoroarenes18,19, 和乙烯基硫化物形成20。
稳定的螺旋肽广泛用于各种细胞内靶, 包括 p53, 雌激素受体, Ras, BCL-2 家族蛋白, 其他21,22,23,24。ALRN-6924 是 MDM2 和 MDMX 的全烃类钉式双肽抑制剂, 目前正被用于临床研究25。在过去的几年中, 我们的小组重点研究了使用硫醇烯和硫醇-yne 反应26,27,28的新的肽稳定方法的发展。总的来说, 我们已经证明, 这些照片引发的反应是有效的温和条件下, 自然丰富的半胱氨酸使用。此外, 我们已经表明, 这些反应具有良好的功能组耐受性, 是生物正交, 并已被证明适用于肽和蛋白质的修改29。由此产生的硫醚类/乙烯基硫化物栓肽在很大程度上改善了约束肽的化学空间, 提供了一种不稳定的系绳修饰中心, 并被证明适用于许多生物应用30 ,31,32。迄今为止, 只有有限的报告被描述的硫醇烯/硫醇-yne 肽环合。在2009年发表的一项研究中, Anseth 了活性烯烃与半胱氨酸之间的肽环合的树脂内分子硫醇烯反应.在 2015年, 秋等。描述了一个双组分基启动的硫醇烯反应的肽吻合34和随后的, 顺序硫醇-yne/烯偶联反应35。最近, 我们描述了一系列的工作基于硫醚类/乙烯基硫化物栓肽20,26,27。本议定书描述了上述硫醚类/乙烯基硫化物栓肽的详细合成, 希望这将有助于更广泛的研究社区。

<table border="0" cellpadding="0" cellspacing="0" width="644">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rink Amide MBHA resin(0.53 mmol/g)</td>
			<td style="width:143px;">HECHENG</td>
			<td style="width:119px;">GRM50407</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Standard Fmoc-protected amino acids</td>
			<td style="width:143px;">GL Biochem (Shanghai) Ltd.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">N-Methyl-2-pyrrolidinone</td>
			<td style="width:143px;">Shenzhen endi Biotechnology Co.Ltd.</td>
			<td style="width:119px;">3230</td>
			<td style="width:167px;">skin harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N,N-Dimethyl formamide</td>
			<td style="width:143px;">Energy</td>
			<td style="width:119px;">B020051</td>
			<td style="width:167px;">skin harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dichloromethane</td>
			<td style="width:143px;">Energy</td>
			<td style="width:119px;">W330229</td>
			<td style="width:167px;">skin harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N,N-Diisoproylethylamine</td>
			<td style="width:143px;">Aldrich</td>
			<td style="width:119px;">9578</td>
			<td style="width:167px;">irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trifluoroacetic acid</td>
			<td style="width:143px;">J&#38;K</td>
			<td style="width:119px;">101398</td>
			<td style="width:167px;">corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triisopropylsilane</td>
			<td style="width:143px;">J&#38;K</td>
			<td style="width:119px;">973821</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1,2-Ethanedithiol</td>
			<td style="width:143px;">J&#38;K</td>
			<td style="width:119px;">248897</td>
			<td style="width:167px;">Stench</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate&#160;</td>
			<td style="width:143px;">GL Biochem (Shanghai) Ltd.</td>
			<td style="width:119px;">851012</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Morpholine</td>
			<td style="width:143px;">Aldrich</td>
			<td>M109062</td>
			<td style="width:167px;">irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Diethyl ether</td>
			<td style="width:143px;">Aldrich</td>
			<td style="width:119px;">673811</td>
			<td style="width:167px;">flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acetonitrile</td>
			<td style="width:143px;">Aldrich</td>
			<td style="width:119px;">9758</td>
			<td style="width:167px;">toxicity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:143px;">Aldrich</td>
			<td style="width:119px;">9758</td>
			<td style="width:167px;">toxicity</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone</td>
			<td style="width:143px;">Energy</td>
			<td style="width:119px;">A050035</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4-methoxyacetophenone</td>
			<td style="width:143px;">Energy</td>
			<td style="width:119px;">A050098</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2,2-dimethoxy-2-phenylacetophenone</td>
			<td style="width:143px;">Energy</td>
			<td style="width:119px;">D070132</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">5,5&#39;-Dithiobis-(2-nitrobenzoic acid)</td>
			<td style="width:143px;">J&#38;K</td>
			<td style="width:119px;">281281</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate</td>
			<td style="width:143px;">Energy</td>
			<td style="width:119px;">E020172</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1-Hydroxybenzotriazole</td>
			<td style="width:143px;">Energy</td>
			<td style="width:119px;">D050256</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4-Methylmorpholine</td>
			<td style="width:143px;">Energy</td>
			<td style="width:119px;">W320038</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">High Performance Liquid Chromatography</td>
			<td style="width:143px;">SHIMADZU</td>
			<td style="width:119px;">LC-30AD</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electrospray Ionization Mass</td>
			<td style="width:143px;">SHIMADZU</td>
			<td style="width:119px;">LCMS-8030</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lyophilizer</td>
			<td style="width:143px;">Labconco</td>
			<td style="width:119px;">FreeZone</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SpeedVac concentration system</td>
			<td style="width:143px;">Thermo</td>
			<td style="width:119px;">Savant</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">vacuum manifold</td>
			<td style="width:143px;">promega</td>
			<td style="width:119px;">A7231</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">three-way stopcocks</td>
			<td style="width:143px;">Bio-Rad</td>
			<td style="width:119px;">7328107</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">poly-prep chromatography columns&#160;</td>
			<td style="width:143px;">Bio-Rad</td>
			<td style="width:119px;">7311550</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

constructing thioether/vinyl sulfide-tethered helical peptides via photo-induced thiol-ene/yne hydrothiolation

在这里, 我们描述了一个详细的协议, 以制备硫醚类栓肽使用在树脂内分子内/分子间硫醇-烯 hydrothiolation。此外, 本议定书还描述了用溶液中内硫醇-yne hydrothiolation 在 i、i+4 位置上具有烯烃/炔烃侧链和半胱氨酸残留物的乙烯基硫化物栓系肽的制备。采用标准的 n-芴甲氧羰基基固相肽合成 (许可证) 合成了线性多肽。硫醇烯 hydrothiolation 是使用分子内的硫原子-烯反应或分子间硫-烯反应, 取决于肽的长度。在本研究中, 在对短肽的情况下, 用脱的三苯甲酯的树脂在全合成了线性肽后, 对其进行了分子内硫-烯反应。然后将树脂设置为紫外线照射, 使用光 4-methoxyacetophenone (MAP) 和 2-羟基 1-[4-(2-hydroxyethoxy)-苯基]-2-甲基-1-丙酮 (基质)。在n-、n-甲基酰胺 (DMF) 溶剂中溶解 n-芴甲氧羰基胱氨酸-OH, 进行分子间硫醇烯反应。然后用树脂上的烯烃残留物对肽进行反应。之后, macrolactamization 使用 benzotriazole-1-yl-oxytripyrrolidinophosphonium 六氟磷酸盐 (PyBop)、1-hydroxybenzotriazole (对 hobt) 和 4-甲基啉) (NMM) 作为活化试剂在树脂上进行。随着 macrolactamization, 肽的合成继续使用标准许可证。在硫 yne hydrothiolation 的情况下, 线性肽从树脂中分解, 干燥后溶解在脱气 DMF 中。然后使用紫外光与光 22-藜-2-phenylacetophenone (DMPA) 进行辐照。反应后, 采用高效液相色谱法 (HPLC) 对 DMF 进行了蒸发, 并对粗渣进行沉淀纯化。这些方法可用于简化硫醚类栓循环多肽的产生, 因为使用的硫烯/yne 单击化学具有优越的功能群耐受性和良好的产量。将硫醚类键引入多肽, 利用了半胱氨酸残留物的亲核性质, 并与二硫键的氧化还原惰性有关。

用树脂内分子内硫醇-烯 photoreaction YmS5AAAC-nh2及其环化烯产品 ac Y (环-15)-[MS5AAAC]-nh2的 HPLC 和 MS 谱, 在图中描述6B. 循环肽与其线性前体相比具有相同的分子量。然而, 在相同的分离条件下, 其液相色谱保留时间比其前体早约2分钟。有不同序列的短肽都被观察到有一个良好的转换, 如图 6C所示。
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Watch this Scientific Journal Video about Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation at JoVE.com

Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation

利用光致硫醇-烯/yne Hydrothiolation 构造硫醚类/乙烯基硫化物栓系螺旋肽

本文提出了一种利用光致硫醇烯/硫醇-yne hydrothiolation 构建硫醚类/乙烯基硫化物栓系螺旋肽的协议。

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene ...

constructing-thioethervinyl-sulfide-tethered-helical-peptides-via

Research

JoVE Journal

Chemistry

10.6K 次观看.  Peking University Shenzhen Graduate School. 本文提出了一种利用光致硫醇烯/硫醇-yne hydrothiolation 构建硫醚类/乙烯基硫化物栓系螺旋肽的协议。

视频: Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

保利的微波辅助功能化（乙二醇）和On-树脂链聚合反应，并形成水凝胶的肽使用

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

科研

化学

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

生物分子有序结构由短肽的自组装形成

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Preparing Silica Aerogel Monoliths via a Rapid Supercritical Extraction Method

A procedure for the fabrication of monolithic silica aerogels in eight hours or less via a rapid supercritical extraction process is described. The procedure requires 15-20 min of preparation time, during which a liquid precursor mixture is prepared and poured into wells of a metal mold that is placed between the platens of a hydraulic hot press, followed by several hours of processing within the hot press. The precursor solution consists of a 1.0:12.0:3.6:3.5 x 10-3 molar ratio of tetramethylorthosilicate (TMOS):methanol:water:ammonia. In each well of the mold, a porous silica sol-gel matrix forms. As the temperature of the mold and its contents is increased, the pressure within the mold rises. After the temperature/pressure conditions surpass the supercritical point for the solvent within the pores of the matrix (in this case, a methanol/water mixture), the supercritical fluid is released, and monolithic aerogel remains within the wells of the mold. With the mold used in this procedure, cylindrical monoliths of 2.2 cm diameter and 1.9 cm height are produced. Aerogels formed by this rapid method have comparable properties (low bulk and skeletal density, high surface area, mesoporous morphology) to those prepared by other methods that involve either additional reaction steps or solvent extractions (lengthier processes that generate more chemical waste).The rapid supercritical extraction method can also be applied to the fabrication of aerogels based on other precursor recipes.

This article describes a rapid supercritical extraction method for fabricating silica aerogels. By utilizing a confined mold and hydraulic hot press, monolithic aerogels can be made in eight hours or less.

通过快速超临界萃取法制备二氧化硅气凝胶巨石

A procedure for the fabrication of monolithic silica aerogels in eight hours or less via a rapid supercritical extraction process is described. The ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

蛋白质生物缀合物的合成<em&gt;通过</em&gt;半胱氨酸马来酰亚胺化学

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Epitaxial Growth of Perovskite Strontium Titanate on Germanium via Atomic Layer Deposition

Atomic layer deposition (ALD) is a commercially utilized deposition method for electronic materials. ALD growth of thin films offers thickness control and conformality by taking advantage of self-limiting reactions between vapor-phase precursors and the growing film. Perovskite oxides present potential for next-generation electronic materials, but to-date have mostly been deposited by physical methods. This work outlines a method for depositing SrTiO3 (STO) on germanium using ALD. Germanium has higher carrier mobilities than silicon and therefore offers an alternative semiconductor material with faster device operation. This method takes advantage of the instability of germanium's native oxide by using thermal deoxidation to clean and reconstruct the Ge (001) surface to the 2&#215;1 structure. 2-nm thick, amorphous STO is then deposited by ALD. The STO film is annealed under ultra-high vacuum and crystallizes on the reconstructed Ge surface. Reflection high-energy electron diffraction (RHEED) is used during this annealing step to monitor the STO crystallization. The thin, crystalline layer of STO acts as a template for subsequent growth of STO that is crystalline as-grown, as confirmed by RHEED. In situ X-ray photoelectron spectroscopy is used to verify film stoichiometry before and after the annealing step, as well as after subsequent STO growth. This procedure provides framework for additional perovskite oxides to be deposited on semiconductors via chemical methods in addition to the integration of more sophisticated heterostructures already achievable by physical methods.

This work details the procedures for the growth and characterization of crystalline SrTiO3 directly on germanium substrates by atomic layer deposition. The procedure illustrates the ability of an all-chemical growth method to integrate oxides monolithically onto semiconductors for metal-oxide semiconductor devices.

锗通过原子层沉积钙钛矿型钛酸锶的外延生长

Atomic layer deposition (ALD) is a commercially utilized deposition method for electronic materials. ALD growth of thin films offers thickness control and ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

洞察氨基酸和多肽与无机材料的使用相互作用的单分子力谱

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Self-assembling Morphologies Obtained from Helical Polycarbodiimide Copolymers and Their Triazole Derivatives

A facile method for the preparation of polycarbodiimide-based secondary structures (e.g., nano-rings, &#34;craters,&#34; fibers, looped fibers, fibrous networks, ribbons, worm-like aggregates, toroidal structures, and spherical particles) is described. These aggregates are morphologically influenced by extensive hydrophobic side chain-side chain interactions of the singular polycarbodiimide strands, as inferred by atomic force microscopy (AFM) and scanning electron microscopy (SEM) techniques. Polycarbodiimide-g-polystyrene copolymers (PS-PCDs) were prepared by a combination of synthetic methods, including coordination-insertion polymerization, copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) &#34;click&#34; chemistry, and atom transfer radical polymerization (ATRP). PS-PCDs were found to form specific toroidal architectures at low concentrations in CHCl3. To determine the influence of a more polar solvent medium (i.e., THF and THF/EtOH) on polymer aggregation behavior, a number of representative PS-PCD composites have been tested to show discrete concentration-dependent spherical particles. These fundamental studies are of practical interest to the development of experimental procedures for desirable architectures by directed self-assembly in thin film. These architectures may be exploited as drug carriers, whereas other morphological findings represent certain interest in the area of novel functional materials.

Here, we present a protocol to prepare and visualize secondary structures (e.g., fibers, toroidal architectures, and nano-spheres) derived from helical polycarbodiimides. The morphology characterized by both atomic force microscopy (AFM) and scanning electron microscopy (SEM) was shown to depend on molecular structure, concentration, and the solvent of choice.

从螺旋聚碳化二亚胺共聚物及其三唑衍生物获得自组装形貌

A facile method for the preparation of polycarbodiimide-based secondary structures (e.g., nano-rings, &#34;craters,&#34; fibers, looped fibers, fibrous ...

Synthesizing Sodium Tungstate and Sodium Molybdate Microcapsules via Bacterial Mineral Excretion

We present a method, the bacterial mineral excretion (BME), for synthesizing two kinds of microcapsules, sodium tungstate and sodium molybdate, and the two metal oxides&#39; corresponding nanoparticles&#8212;the former being as small as 22 nm and the latter 15 nm. We fed two strains of bacteria, Shewanella algae and Pandoraea sp., with various concentrations of tungstate or molybdate ions. The concentrations of tungstate and molybdate were adjusted to make microcapsules of different length-to-diameter ratios. We found that the higher the concentration the smaller the nanoparticles were. The nanoparticles came in with three length-to-diameter ratios: 10:1, 3:1 and 1:1, which were achieved by feeding the bacteria respectively with a low concentration, a medium concentration, and a high concentration. The images of the hollow microcapsules were taken via the scanning electron microsphere (SEM). Their crystal structures were verified by X-ray diffraction (XRD)&#8212;the crystal structure of molybdate microcapsules is Na2MoO4 and that of tungstate microcapsules is Na2WO4 with Na2W2O7. These syntheses all were accomplished under a near ambient condition.

This work presents a protocol for manufacture sodium tungstate and sodium molybdate microcapsules via bacteria and their corresponding nanoparticles.

通过细菌矿物排泄合成钨酸钠和钼酸钠微胶囊

We present a method, the bacterial mineral excretion (BME), for synthesizing two kinds of microcapsules, sodium tungstate and sodium molybdate, and the ...

Fabrication and Testing of Catalytic Aerogels Prepared Via Rapid Supercritical Extraction

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three preparation methods are described: (a) the incorporation of metal salts into silica or alumina wet gels using an impregnation method; (b) the incorporation of metal salts into alumina wet gels using a co-precursor method; and (c) the addition of metal nanoparticles directly into a silica aerogel precursor mixture. The methods utilize a hydraulic hot press, which allows for rapid (&#60;6 h) supercritical extraction and results in aerogels of low density (0.10 g/mL) and high surface area (200-800 m2/g). While the work presented here focuses on the use of copper salts and copper nanoparticles, the approach can be implemented using other metal salts and nanoparticles. A protocol for testing the three-way catalytic ability of these aerogels for automotive pollution mitigation is also presented. This technique uses custom-built equipment, the Union Catalytic Testbed (UCAT), in which a simulated exhaust mixture is passed over an aerogel sample at a controlled temperature and flow rate. The system is capable of measuring the ability of the catalytic aerogels, under both oxidizing and reducing conditions, to convert CO, NO and unburned hydrocarbons (HCs) to less harmful species (CO2, H2O and N2). Example catalytic results are presented for the aerogels described.

Here we present protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms. Methods for preparing materials using copper salts and copper-containing nanoparticles are featured. Catalytic testing protocols demonstrate the effectiveness of these aerogels for three-way catalysis applications.

快速超临界萃取制备催化气凝胶的制备与试验研究

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three ...

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.