Synthesis of the Pitstop family of clathrin inhibitors
| Autor: | Volker Haucke, Lisa von Kleist, Mark J. Robertson, Adam McCluskey, Wiebke Stahlschmidt, Phillip J. Robinson, Fiona M. Deane |
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| Rok vydání: | 2014 |
| Předmět: |
Sulfonamides
biology media_common.quotation_subject Chemistry Techniques Synthetic Naphthalenes Bioinformatics Endocytosis Clathrin Combinatorial chemistry General Biochemistry Genetics and Molecular Biology chemistry.chemical_compound Aniline Rhodanine chemistry Reagent biology.protein Thiazolidines Synaptic vesicle recycling Thioketone Internalization media_common |
| Zdroj: | Nature Protocols. 9:1592-1606 |
| ISSN: | 1750-2799 1754-2189 |
| DOI: | 10.1038/nprot.2014.106 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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