title: Synthesis of the Pitstop family of clathrin inhibitors.
abstract: 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.
authors.affiliation: 1] Department of Chemistry, Centre for Chemical Biology, School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales, Australia. [2].
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Robertson
authors.affiliation: 1] Department of Chemistry, Centre for Chemical Biology, School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales, Australia. [2].
authors.forename: Fiona M
authors.initials: FM
authors.lastname: Deane
authors.affiliation: Leibniz Institut für Molekulare Pharmakologie & Freie Universität Berlin, Berlin, Germany.
authors.forename: Wiebke
authors.initials: W
authors.lastname: Stahlschmidt
authors.affiliation: Leibniz Institut für Molekulare Pharmakologie & Freie Universität Berlin, Berlin, Germany.
authors.forename: Lisa
authors.initials: L
authors.lastname: von Kleist
authors.affiliation: Leibniz Institut für Molekulare Pharmakologie & Freie Universität Berlin, Berlin, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Haucke
authors.affiliation: Cell Signalling Unit, Children's Medical Research Institute, The University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Phillip J
authors.initials: PJ
authors.lastname: Robinson
authors.affiliation: Department of Chemistry, Centre for Chemical Biology, School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales, Australia.
authors.forename: Adam
authors.initials: A
authors.lastname: McCluskey
mesh_terms: D060326:Chemistry Techniques, Synthetic; D002966:Clathrin / Q000037:antagonists & inhibitors*; D009281:Naphthalenes / Q000737:chemistry; D013449:Sulfonamides / Q000138:chemical synthesis*; D053778:Thiazolidines / Q000138:chemical synthesis*
chemical_list: C542420:4-sulfo-1,8-naphthalic anhydride; D002966:Clathrin; D009281:Naphthalenes; D013449:Sulfonamides; D053778:Thiazolidines; C578512:pitstop 2; C012514:1,8-naphthalenedicarboxylic acid anhydride