1] Department of Chemistry, Centre for Chemical Biology, School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales, Australia. [2].
Leibniz Institut für Molekulare Pharmakologie & Freie Universität Berlin, Berlin, Germany.
Nat Protoc. 2014 Jul;9(7):1592-606. doi: 10.1038/nprot.2014.106. Epub 2014 Jun 12.
This protocol describes the synthesis of two classes of clathrin inhibitors, Pitstop 1 and Pitstop 2, along with two inactive analogs that can be used as negative controls (Pitstop inactive controls, Pitnot-2 and Pitnot-2-100). Pitstop-induced inhibition of clathrin TD function acutely interferes with clathrin-mediated endocytosis (CME), synaptic vesicle recycling and cellular entry of HIV, whereas clathrin-independent internalization pathways and secretory traffic proceed unperturbed; these reagents can, therefore, be used to investigate clathrin function, and they have potential pharmacological applications. Pitstop 1 is synthesized in two steps: sulfonation of 1,8-naphthalic anhydride and subsequent reaction with 4-amino(methyl)aniline. Pitnot-1 results from the reaction of 4-amino(methyl)aniline with commercially available 4-sulfo-1,8-naphthalic anhydride potassium salt. Reaction of 1-naphthalene sulfonyl chloride with pseudothiohydantoin followed by condensation with 4-bromobenzaldehyde yields Pitstop 2. The synthesis of the inactive control commences with the condensation of 4-bromobenzaldehyde with the rhodanine core. Thioketone methylation and displacement with 1-napthylamine affords the target compound. Although Pitstop 1-series compounds are not cell permeable, they can be used in biochemical assays or be introduced into cells via microinjection. The Pitstop 2-series compounds are cell permeable. The synthesis of these compounds does not require specialist equipment and can be completed in 3-4 d. Microwave irradiation can be used to reduce the synthesis time. The synthesis of the Pitstop 2 family is easily adaptable to enable the synthesis of related compounds such as Pitstop 2-100 and Pitnot-2-100. The procedures are also simple, efficient and amenable to scale-up, enabling cost-effective in-house synthesis for users of these inhibitor classes.
本方案描述了两类笼形蛋白抑制剂（Pitstop1 和 Pitstop2）以及两种无活性类似物（Pitstop 无活性对照物 Pitnot-2 和 Pitnot-2-100）的合成。Pitstop 诱导的笼形蛋白 TD 功能抑制会急性干扰笼形蛋白介导的胞吞作用（CME）、突触小泡再循环和 HIV 的细胞进入，而无网格蛋白的内吞作用途径和分泌运输则不受干扰；因此，这些试剂可用于研究网格蛋白的功能，并且具有潜在的药理学应用。Pitstop1 通过两步合成：1,8-萘二甲酸酐的磺化和随后与 4-氨基（甲基）苯胺的反应。Pitnot-1 是由 4-氨基（甲基）苯胺与市售的 4-磺基-1,8-萘二甲酸酐钾盐反应得到的。1-萘磺酰氯与假硫代海因反应，然后与 4-溴苯甲醛缩合得到 Pitstop2。无活性对照物的合成始于 4-溴苯甲醛与缩硫酮核心的缩合。硫酮甲基化并用 1-萘胺取代得到目标化合物。虽然 Pitstop1 系列化合物不能穿透细胞，但它们可以用于生化测定或通过显微注射引入细胞。Pitstop2 系列化合物可穿透细胞。这些化合物的合成不需要特殊设备，可在 3-4 天内完成。微波辐射可用于缩短合成时间。Pitstop2 家族的合成很容易适应，可用于合成相关化合物，如 Pitstop2-100 和 Pitnot-2-100。该方法简单、高效且易于放大，可实现这些抑制剂类别的用户的成本效益内部合成。
