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Química

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

Published: August 01, 2018
doi:
10.3791/57356
, , ,
1Key Laboratory of Chemical Genomics, School of Chemical Biology and Biotechnology,Peking University Shenzhen Graduate School

Summary

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。

Abstract

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。

Introduction

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。

Protocol

1. 设备准备 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。 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…

Representative Results

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。 <p…

Discussion

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?…

Declarações

The authors have nothing to disclose.

Acknowledgements

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)。

Materials

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

ThioetherVinyl SulfideHelical PeptidesPhoto-induced Thiol-ene/yne Hydro-thiolationRink Amide MBHA ResinFmoc DeprotectionHCTUDIPEATrityl DeprotectionSolid-phase Peptide Synthesis

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Shi, X., Liu, Y., Zhao, R., Li, Z. Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation. J. Vis. Exp. (138), e57356, doi:10.3791/57356 (2018).
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