Protocol | Published:

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

Nature Protocols volume 12, pages 15881619 (2017) | Download Citation

Abstract

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(C2-BODIPY)-OH and its incorporation into peptides for live-cell fluorescence imaging—an approach that is applicable to most peptide sequences. Fmoc-Trp(C2-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(C2-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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Acknowledgements

L.M.-T. acknowledges the support of MECD-Spain for an FPU Scholarship. R.S.-F. acknowledges the support of an MSCA Individual Fellowship (659046). R.L. acknowledges the support of DGICYT-Spain (CTQ2015-67870-P) and Generalitat de Catalunya (2014 SGR 137). M.V. acknowledges the support of the Medical Research Council, a Marie Curie Integration Grant (333847) and the Biotechnology and Biological Sciences Research Council (BB/M025160/1). The authors thank Luxembourg Bio Technologies (Rehovot) for kindly supplying OxymaPure and derived reagents.

Author information

Author notes

    • Lorena Mendive-Tapia
    • , Ramon Subiros-Funosas
    •  & Can Zhao

    These authors contributed equally to this work.

Affiliations

  1. Department of Inorganic and Organic Chemistry, University of Barcelona, Barcelona, Spain.

    • Lorena Mendive-Tapia
    •  & Fernando Albericio
  2. Medical Research Council/University of Edinburgh Centre for Inflammation Research, The University of Edinburgh, Edinburgh, UK.

    • Ramon Subiros-Funosas
    •  & Marc Vendrell
  3. Manchester Fungal Infection Group, Division of Infection, Immunity and Respiratory Medicine, University of Manchester, Manchester, UK.

    • Can Zhao
    •  & Nick D Read
  4. Networking Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Barcelona, Spain.

    • Fernando Albericio
    •  & Rodolfo Lavilla
  5. Laboratory of Organic Chemistry, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain.

    • Rodolfo Lavilla

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Contributions

L.M.-T. performed all compound syntheses and chemical characterization; R.S.-F. performed in vitro spectral and biological characterization; C.Z. and N.D.R. designed and performed the experiments with fungal cells; F.A., R.L. and M.V. designed the chemical syntheses; R.L. and M.V. supervised the project; M.V. analyzed the data and wrote the paper. All authors discussed the results and commented on the manuscript.

Competing interests

The University of Edinburgh has filed an invention disclosure form to protect part of the technology described in this study.

Corresponding authors

Correspondence to Rodolfo Lavilla or Marc Vendrell.

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    Supplementary Text and Figures

    Supplementary Figures 1–4 and Supplementary Tables 1 and 2.

Videos

  1. 1.

    Supplementary Video 1. Time-course high-resolution imaging of A. fumigatus upon treatment with BODIPY-cPAF26.

    A. fumigatus cells were pretreated with a cell membrane counterstain (red) and imaged under a confocal microscope. Cells were then treated with BODIPY-cPAF26 (2 μM, green) and further imaged without any washing steps. The video shows the rapid fluorogenic response of BODIPY-cPAF26 upon interaction with the cell membrane of A. fumigatus. Scale bar, 5 μm.

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DOI

https://doi.org/10.1038/nprot.2017.048

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