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Preparation of a Trp-BODIPY fluorogenic amino acid to label peptides for enhanced live-cell fluorescence imaging

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Original languageEnglish
JournalNature Protocols
Early online date13 Jul 2017
DOIs
Publication statusE-pub ahead of print - 13 Jul 2017

Abstract

Fluorescent peptides are valuable tools for live-cell imaging due to the high specificity of peptide sequences for their biomolecular targets. When preparing fluorescent versions of peptides, labels need to be introduced at appropriate positions of the sequences to provide suitable reporters, while avoiding any impairment in the molecular recognition properties of the peptides. This protocol describes the preparation of the Trp-based fluorogenic amino acid Fmoc-Trp(C2-BODIPY)-OH and its incorporation into peptides for live-cell fluorescence imaging. Fmoc-Trp(C2-BODIPY)-OH contains a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorogenic core attached to Trp via a spacer-free C–C linkage; it thus mimics the molecular interactions of Trp but includes an environmentally sensitive label that enables fluorescence detection and wash-free imaging. Fmoc-Trp(C2-BODIPY)-OH can be prepared in 3–4 d before being employed for the solid-phase synthesis of fluorogenic peptides (6–7 d). We have used this technology to prepare the fluorogenic antimicrobial peptide BODIPY-cPAF26 (3–4 d), which shows high chemical stability and minimal disruption of cellular activity when compared to the unlabeled sequence. We used BODIPY-cPAF26 for wash-free imaging of fungal pathogens, including real-time visualization of Aspergillus fumigatus (5 d for culturing, 1-2 d for imaging). One major advantage of this procedure is the applicability to most peptide sequences, which enables the preparation of many peptide-based probes for enhanced fluorescence imaging. The procedure of this protocol covers the chemical synthesis of the fluorogenic amino acid Fmoc-Trp(C2-BODIPY)-OH, the preparation of the labeled antimicrobial peptide BODIPY-cPAF26 and their spectral and biological characterization as live-cell imaging probes for different fungal pathogens.

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