학술논문
Synthesis of the Pitstop family of clathrin inhibitors
Document Type
Report
Author
Source
Nature Protocols. July 1, 2014, Vol. 9 Issue 7, p1592, 15 p.
Subject
Language
English
ISSN
1754-2189
Abstract
INTRODUCTION The importance of clathrin as a target Clathrin is a protein complex of three identical 190-kDa clathrin heavy chains (CHCs) arranged in a trimer (called a triskelion) of three [...]
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.
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.