期刊
NATURE PROTOCOLS
卷 9, 期 7, 页码 1592-1606出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2014.106
关键词
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资金
- National Health and Medical Research Council (Australia)
- Australia Research Council
- Australian Cancer Research Foundation
- Ramaciotti Foundation
- Children's Medical Research Institute
- Newcastle Innovation Ltd.
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-naphthoic 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-bromobenzadehyde 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.
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