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

Fluorescent peptides with excellent target specificity are useful imaging probes. This protocol describes the synthesis of a tryptophan-based fluorogenic amino acid (Fmoc-Trp(C 2 -BODIPY)-OH) and its incorporation into peptides for live-cell imaging. Fluorescent peptides are valuable tools for live-...

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Bibliographic Details
Published in:Nature protocols Vol. 12; no. 8; pp. 1588 - 1619
Main Authors: Mendive-Tapia, Lorena, Subiros-Funosas, Ramon, Zhao, Can, Albericio, Fernando, Read, Nick D, Lavilla, Rodolfo, Vendrell, Marc
Format: Journal Article
Language:English
Published: London Nature Publishing Group UK 01.08.2017
Nature Publishing Group
Subjects:
14/10
14/34
14/63
631/1647/328/1978
631/92/611
639/624/1107/510
639/638/403/933
Amino acids
Analysis
Analytical Chemistry
Antiinfectives and antibacterials
Antimicrobial peptides
Aspergillus fumigatus - cytology
Biological Techniques
Boron Compounds - analysis
Boron Compounds - chemical synthesis
Chemical synthesis
Complications and side effects
Computational Biology/Bioinformatics
Dosage and administration
Fluorescence
Fungi
Health aspects
Imaging
Life Sciences
Lipophilic
Microarrays
Microbiological Techniques - methods
Microscopy
Molecular interactions
Optical Imaging - methods
Organic Chemistry
Pathogenic microorganisms
Pathogens
Peptides
protocol
Solid phase methods
Solid phase synthesis
Spacer
Staining and Labeling - methods
Substrates
Tryptophan
Tryptophan - analysis
Tryptophan - chemical synthesis
ISSN:1754-2189, 1750-2799, 1750-2799
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Summary:Fluorescent peptides with excellent target specificity are useful imaging probes. This protocol describes the synthesis of a tryptophan-based fluorogenic amino acid (Fmoc-Trp(C 2 -BODIPY)-OH) and its incorporation into peptides for live-cell imaging. Fluorescent peptides are valuable tools for live-cell imaging because of the high specificity of peptide sequences for their biomolecular targets. When preparing fluorescent versions of peptides, labels must be introduced at appropriate positions in the sequences to provide suitable reporters while avoiding any impairment of the molecular recognition properties of the peptides. This protocol describes the preparation of the tryptophan (Trp)-based fluorogenic amino acid Fmoc-Trp(C 2 -BODIPY)-OH and its incorporation into peptides for live-cell fluorescence imaging—an approach that is applicable to most peptide sequences. Fmoc-Trp(C 2 -BODIPY)-OH contains a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza- s -indacene) fluorogenic core, which works as an environmentally sensitive fluorophore, showing high fluorescence in lipophilic conditions. It is attached to Trp via a spacer-free C–C linkage, resulting in a labeled amino acid that can mimic the molecular interactions of Trp, enabling wash-free imaging. This protocol covers the chemical synthesis of the fluorogenic amino acid Fmoc-Trp(C 2 -BODIPY)-OH (3–4 d), the preparation of the labeled antimicrobial peptide BODIPY-cPAF26 by solid-phase synthesis (6–7 d) and its spectral and biological characterization as a live-cell imaging probe for different fungal pathogens. As an example, we include a procedure for using 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).
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ISSN:1754-2189
1750-2799
1750-2799
DOI:10.1038/nprot.2017.048