title: Novel trans-Stilbene-based Fluorophores as Probes for Spectral Discrimination of Native and Protofibrillar Transthyretin.
abstract: Accumulation of misfolded transthyretin (TTR) as amyloid fibrils causes various human disorders. Native transthyretin is a neurotrophic protein and is a putative extracellular molecular chaperone. Several fluorophores have been shown in vitro to bind selectively to native TTR. Other compounds, such as thioflavin T, bind TTR amyloid fibrils. The probe 1-anilinonaphthalene-8-sulfonate (ANS) binds to both native and fibrillar TTR, becoming highly fluorescent, but with indistinguishable emission spectra for native and fibrillar TTR. Herein we report our efforts to develop a fluorescent small molecule capable of binding both native and misfolded protofibrillar TTR, providing distinguishable emission spectra. We used microwave synthesis for efficient production of a small library of trans-stilbenes and fluorescence spectral screening of their binding properties. We synthesized and tested 22 trans-stilbenes displaying a variety of functional groups. We successfully developed two naphthyl-based trans-stilbenes probes that detect both TTR states at physiological concentrations. The compounds bound with nanomolar to micromolar affinities and displayed distinct emission maxima upon binding native or misfolded protofibrillar TTR (>100 nm difference). The probes were mainly responsive to environment polarity providing evidence for the divergent hydrophobic structure of the binding sites of these protein conformational states. Furthermore, we were able to successfully use one of these probes to quantify the relative amounts of native and protofibrillar TTR in a dynamic equilibrium. In conclusion, we identified two trans-stilbene-based fluorescent probes, (E)-4-(2-(naphthalen-1-yl)vinyl)benzene-1,2-diol (11) and (E)-4-(2-(naphthalen-2-yl)vinyl)benzene-1,2-diol (14), that bind native and protofibrillar TTR, providing a wide difference in emission maxima allowing conformational discrimination by fluorescence spectroscopy. We expect these novel molecules to serve as important chemical biology research tools in studies of TTR folding and misfolding.
authors.affiliation: IFM-Department of Chemistry, Linköping University , Linköping 581 83, Sweden.
authors.forename: Raúl I
authors.initials: RI
authors.lastname: Campos
authors.affiliation: IFM-Department of Chemistry, Linköping University , Linköping 581 83, Sweden.
authors.forename: Xiongyu
authors.initials: X
authors.lastname: Wu
authors.affiliation: IFM-Department of Chemistry, Linköping University , Linköping 581 83, Sweden.
authors.forename: Mathias
authors.initials: M
authors.lastname: Elgland
authors.affiliation: IFM-Department of Chemistry, Linköping University , Linköping 581 83, Sweden.
authors.forename: Peter
authors.initials: P
authors.lastname: Konradsson
authors.affiliation: IFM-Department of Chemistry, Linköping University , Linköping 581 83, Sweden.
authors.forename: Per
authors.initials: P
authors.lastname: Hammarström
mesh_terms: D000682:Amyloid / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D055592:Biophysical Phenomena; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D011228:Prealbumin / Q000737:chemistry* / Q000378:metabolism*; D017510:Protein Folding; D013050:Spectrometry, Fluorescence
chemical_list: D000682:Amyloid; D005456:Fluorescent Dyes; D011228:Prealbumin
keywords: amyloid; fluorescence; probe; spectrum; stilbene; transthyretin