Name: constructing thioether/vinyl sulfide-tethered helical peptides via photo-induced thiol-ene/yne hydrothiolation
Uploaded: 2018-08-01T13:30:00.000+00:00
Duration: 11 min 9 s
Description: Watch this Scientific Journal Video about Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation at JoVE.com

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><tbody><tr><td>Rink Amide MBHA resin(0.53 mmol/g)</td><td>HECHENG</td><td>GRM50407</td><td></td></tr><tr><td>Standard Fmoc-protected amino acids</td><td>GL Biochem (Shanghai) Ltd.</td><td></td><td></td></tr><tr><td>N-Methyl-2-pyrrolidinone</td><td>Shenzhen endi Biotechnology Co.Ltd.</td><td>3230</td><td>skin harmful</td></tr><tr><td>N,N-Dimethyl formamide</td><td>Energy</td><td>B020051</td><td>skin harmful</td></tr><tr><td>Dichloromethane</td><td>Energy</td><td>W330229</td><td>skin harmful</td></tr><tr><td>N,N-Diisoproylethylamine</td><td>Aldrich</td><td>9578</td><td>irritant</td></tr><tr><td>Trifluoroacetic acid</td><td>J&amp;amp;K</td><td>101398</td><td>corrosive</td></tr><tr><td>Triisopropylsilane</td><td>J&amp;amp;K</td><td>973821</td><td></td></tr><tr><td>1,2-Ethanedithiol</td><td>J&amp;amp;K</td><td>248897</td><td>Stench</td></tr><tr><td>2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate&amp;nbsp;</td><td>GL Biochem (Shanghai) Ltd.</td><td>851012</td><td></td></tr><tr><td>Morpholine</td><td>Aldrich</td><td>M109062</td><td>irritant</td></tr><tr><td>Diethyl ether</td><td>Aldrich</td><td>673811</td><td>flammable</td></tr><tr><td>Acetonitrile</td><td>Aldrich</td><td>9758</td><td>toxicity</td></tr><tr><td>Methanol</td><td>Aldrich</td><td>9758</td><td>toxicity</td></tr><tr><td>2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone</td><td>Energy</td><td>A050035</td><td></td></tr><tr><td>4-methoxyacetophenone</td><td>Energy</td><td>A050098</td><td></td></tr><tr><td>2,2-dimethoxy-2-phenylacetophenone</td><td>Energy</td><td>D070132</td><td></td></tr><tr><td>5,5&#039;-Dithiobis-(2-nitrobenzoic acid)</td><td>J&amp;amp;K</td><td>281281</td><td></td></tr><tr><td>Benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate</td><td>Energy</td><td>E020172</td><td></td></tr><tr><td>1-Hydroxybenzotriazole</td><td>Energy</td><td>D050256</td><td></td></tr><tr><td>4-Methylmorpholine</td><td>Energy</td><td>W320038</td><td></td></tr><tr><td>High Performance Liquid Chromatography</td><td>SHIMADZU</td><td>LC-30AD</td><td></td></tr><tr><td>Electrospray Ionization Mass</td><td>SHIMADZU</td><td>LCMS-8030</td><td></td></tr><tr><td>Lyophilizer</td><td>Labconco</td><td>FreeZone</td><td></td></tr><tr><td>SpeedVac concentration system</td><td>Thermo</td><td>Savant</td><td></td></tr><tr><td>vacuum manifold</td><td>promega</td><td>A7231</td><td></td></tr><tr><td>three-way stopcocks</td><td>Bio-Rad</td><td>7328107</td><td></td></tr><tr><td>poly-prep chromatography columns&amp;nbsp;</td><td>Bio-Rad</td><td>7311550</td><td></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

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

可逆二硫键交联胶束生产的简便和有效途径

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

科研

生物化学

Synthesis of Programmable Main-chain Liquid-crystalline Elastomers Using a Two-stage Thiol-acrylate Reaction

This study presents a novel two-stage thiol-acrylate Michael addition-photopolymerization (TAMAP) reaction to prepare main-chain liquid-crystalline elastomers (LCEs) with facile control over network structure and programming of an aligned monodomain. Tailored LCE networks were synthesized using routine mixing of commercially available starting materials and pouring monomer solutions into molds to cure. An initial polydomain LCE network is formed via a self-limiting thiol-acrylate Michael-addition reaction. Strain-to-failure and glass transition behavior were investigated as a function of crosslinking monomer, pentaerythritol tetrakis(3-mercaptopropionate) (PETMP). An example non-stoichiometric system of 15 mol% PETMP thiol groups and an excess of 15 mol% acrylate groups was used to demonstrate the robust nature of the material. The LCE formed an aligned and transparent monodomain when stretched, with a maximum failure strain over 600%. Stretched LCE samples were able to demonstrate both stress-driven thermal actuation when held under a constant bias stress or the shape-memory effect when stretched and unloaded. A permanently programmed monodomain was achieved via a second-stage photopolymerization reaction of the excess acrylate groups when the sample was in the stretched state. LCE samples were photo-cured and programmed at 100%, 200%, 300%, and 400% strain, with all samples demonstrating over 90% shape fixity when unloaded. The magnitude of total stress-free actuation increased from 35% to 115% with increased programming strain. Overall, the two-stage TAMAP methodology is presented as a powerful tool to prepare main-chain LCE systems and explore structure-property-performance relationships in these fascinating stimuli-sensitive materials.

A novel methodology is presented to synthesize and program main-chain liquid-crystalline elastomers using commercially available starting monomers. A wide range of thermomechanical properties was tailored by adjusting the amount of crosslinker, while the actuation performance was dependent on the amount of applied strain during programming.

的可编程主链液晶性弹性体用双级硫醇丙烯酸酯反应合成

This study presents a novel two-stage thiol-acrylate Michael addition-photopolymerization (TAMAP) reaction to prepare main-chain liquid-crystalline ...

化学

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

产生和回收的β-细胞的细胞球体从逐步增长的PEG-肽水凝胶

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

生物工程

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 ...

Synthesis of a Thiol Building Block for the Crystallization of a Semiconducting Gyroidal Metal-sulfur Framework

We present a method for preparing thioester molecules as the masked form of the thiol linkers and their utilization for accessing a semiconducting and porous metal-dithiolene network in the highly ordered single crystalline state. Unlike the highly reactive free-standing thiols, which tend to decompose and complicate the crystallization of metal-thiolate open frameworks, the thioester reacts in situ to provide the thiol species, serving to mitigate the reaction between the mercaptan units and the metal centers, and to improve crystallization consequently. Specifically, the thioester was synthesized in a one-pot procedure: an aromatic bromide (hexabromotriphenylene) reacted with excess sodium thiomethoxide under vigorous conditions to first form the thioether intermediate product. The thioether was then demethylated by the excess thiomethoxide to provide the thiolate anion that was acylated to form the thioester product. The thioester was conveniently purified by standard column chromatography, and then used directly in the framework synthesis, wherein NaOH and ethylenediamine serve to revert in situ the thioester to the thiol linker for assembling the single-crystalline Pb(II)-dithiolene network. Compared with other methods for thiol synthesis (e.g., by cleaving alkyl thioether using sodium metal and liquid ammonia), the thioester synthesis here uses simple conditions and economical reagents. Moreover, the thioester product is stable and can be conveniently handled and stored. More importantly, in contrast to the generic difficulty in accessing crystalline metal-thiolate open frameworks, we demonstrate that using the thioester for in situ formation of the thiol linker greatly improves the crystallinity of the solid-state product. We intend to encourage broader research efforts on the technologically important metal-sulfur frameworks by disclosing the synthetic protocol for the thioester as well as the crystalline framework solid.

Here, we present a one-pot, transition-metal-free synthesis of thiols and thioesters from aromatic halides and sodium thiomethoxide, followed by the preparation of single crystals of a metal-dithiolene network using thiol species generated in situ from the more stable and tractable thioester.

半导体回转金属硫框架结晶的硫醇构建块的合成

We present a method for preparing thioester molecules as the masked form of the thiol linkers and their utilization for accessing a semiconducting and ...

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting self-assembled supramolecular structures. The synthetic route takes advantage of solid-phase peptide synthesis with the on-resin coupling of the dithiolane precursor molecule, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the microwave-assisted thioacetate deprotection of the peptide N-terminus before final cleavage from the resin to yield the 1,2-dithiolane modified peptide. After the high-performance liquid chromatography (HPLC) purification of the 1,2-dithiolane peptide, derived from the nucleating core of the A&#946; peptide associated with Alzheimer&#39;s disease, the peptide is shown to self-assemble into cross-&#946; amyloid fibers. Protocols to characterize the amyloid fibers by Fourier-transform infrared spectroscopy (FT-IR), circular dichroism spectroscopy (CD) and transmission electron microscopy (TEM) are presented. The methods of N-terminal modification with a 1,2-dithiolane moiety to well-characterized self-assembling peptides can now be explored as model systems to develop post-assembly modification strategies and explore dynamic covalent chemistry on supramolecular peptide nanofiber surfaces.

A protocol for the synthesis of a 1,2-dithiolane modified peptide and the characterization of the supramolecular structures resulting from the peptide self-assembly.

12-Dithiolane 改性自组装肽的合成与表征

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

硫醇反应试剂的合成与生物共轭作用--用于原位选择性修饰免疫共轭的生成

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded oligomeric strands mediated by paired dynamic covalent interactions in a manner mimicking the thermal cycling commonly employed for the self-assembly of complementary nucleic acid sequences. Primary amine monomers bearing aldehyde and amine pendant moieties are functionalized with orthogonal protecting groups for use as dynamic covalent reactant pairs. Using a modified automated peptide synthesizer, the primary amine monomers are encoded into oligo(peptoid) strands through solid-phase submonomer synthesis. Upon purification by high-performance liquid chromatography (HPLC) and characterization by electrospray ionization mass spectrometry (ESI-MS), sequence-specific oligomers are subjected to high-loading of a Lewis acidic rare-earth metal triflate which both deprotects the aldehyde moieties and affects the reactant pair equilibrium such that strands completely dissociate. Subsequently, a fraction of the Lewis acid is extracted, enabling annealing of complementary sequence-specific strands to form information-encoded molecular ladders characterized by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The simple procedure outlined in this report circumvents kinetic traps commonly experienced in the field of dynamic covalent assembly and serves as a platform for the future design of robust, complex architectures.

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

信息承载Peptoids及其序列定向动态共价自组装的合成

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded ...

Constructing Cyclic Peptides Using an On-Tether Sulfonium Center

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as a consequence, they are now used clinically. It is, therefore, critical to seek effective strategies for synthesizing cyclic peptides to promote their application in the field of drug discovery. This paper reports a detailed protocol for the efficient synthesis of cyclic peptides using on-resin or intramolecular (intermolecular) bisalkylation. Using this protocol, linear peptides were synthesized by taking advantage of solid-phase peptide synthesis with cysteine (Cys) and methionine (Met) coupled simultaneously on the resin. Further, cyclic peptides were synthesized via bisalkylation between Met and Cys using a tunable tether and an on-tether sulfonium center. The whole synthetic route can be divided into three major processes: the deprotection of Cys on the resin, the coupling of the linker, and the cyclization between Cys and Met in a trifluoroacetic acid (TFA) cleavage solution. Furthermore, inspired by the reactivity of the sulfonium center, a propargyl group was attached to the Met to trigger thiol-yne addition and form a cyclic peptide. After that, the crude peptides were dried and dissolved in acetonitrile, separated, and then purified by high-performance liquid chromatography (HPLC). The molecular weight of the cyclic peptide was confirmed by liquid chromatography-mass spectrometry (LC-MS), and the stability of the cyclic peptide combination with the reductant was further confirmed using HPLC. In addition, the chemical shift in the cyclic peptide was analyzed by 1H nuclear magnetic resonance (1H NMR) spectra. Overall, this protocol aimed to establish an effective strategy for synthesizing cyclic peptides.

This protocol presents the synthesis of cyclic peptides via bisalkylation between cysteine and methionine and the facile thiol-yne reaction triggered by the propargyl sulfonium center.

使用系绳上的锍中心构建环肽

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

在肽膜前体的维西图洛合成中,用于自主囊泡生长

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...