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

Nature Protocols volume 12, pages 1588–1619 (2017)Cite this article

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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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Figure 1
Figure 2
Figure 3: Synthesis and chemical characterization of amino acid Fmoc-Trp(C2-BODIPY)-OH (1).
Figure 4
Figure 5: BODIPY-cPAF26, a fluorogenic peptide with high affinity for fungal cells.
Figure 6: Live-cell fluorescence imaging of fungal pathogens.
Figure 7: Time-course fluorescence imaging of A. fumigatus with BODIPY-cPAF26.
Figure 8: Time-course illustration of the reaction setup for the synthesis of compound 3 under a N2 atmosphere.
Figure 9: Time-course illustration of the reaction setup for the synthesis of amino acid 1.
Figure 10: Setup for the solid-phase synthesis of BODIPY-cPAF26.
Figure 11: Experimental setup for the hydrogenation of the cyclic protected peptide.
Figure 12: Image showing BODIPY-cPAF26 and fluorescein under excitation with 365-nm light using a hand-held UV lamp.
Figure 13: Image of A549 cells in a 96-well plate after detergent incubation.
Figure 14: Plate layout for the antifungal activity assays.

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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

  1. Lorena Mendive-Tapia, Ramon Subiros-Funosas and Can Zhao: These authors contributed equally to this work.

Authors and 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. Division of Infection, Manchester Fungal Infection Group, Immunity and Respiratory Medicine, University of Manchester, Manchester, UK

    Can Zhao & Nick D Read

  4. Networking Centre on Bioengineering,

    Fernando Albericio & Rodolfo Lavilla

  5. , Biomaterials and Nanomedicine (CIBER-BBN), Barcelona, Spain

    Fernando Albericio & Rodolfo Lavilla

  6. Laboratory of Organic Chemistry, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain

    Rodolfo Lavilla

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  1. Lorena Mendive-Tapia
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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.

Corresponding authors

Correspondence to Rodolfo Lavilla or Marc Vendrell.

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

Integrated supplementary information

Supplementary Figure 1 Analysis of the long-term stability of the amino acid 1 when stored as a solid at different temperatures.

HPLC-MS traces of the amino acid 1 after being stored in the dark for 4 months at r.t., 4 ºC, and -20 ºC. UV detection: 500 nm.

Supplementary Figure 2 Analysis of the long-term stability of the amino acid 1 when dissolved in organic solvents at different temperatures.

HPLC-MS traces of the amino acid 1 after being stored in the dark for 4 months in: a) DCM at -20 ºC, b) MeOH at 4 ºC, c) DMF at r.t. In c), the green arrow points at the remaining amino acid 1 and the main peaks correspond to Fmoc-removed side products. UV detection: 500 nm.

Supplementary Figure 3 Time-course analysis of the chemical integrity of BODIPY-cPAF26 and unlabeled linear PAF26 in proteolytic environments.

HPLC traces of BODIPY-cPAF26 (a) and unlabeled PAF26 (b) before incubation (top) and after incubation (bottom) at a concentration of 200 μM in a protease cocktail (1 mg L-1). Green arrows point at the peaks of intact BODIPY-cPAF26 and red arrows point at intact PAF26. UV detection: 280 nm. Purities were determined by integration of the peak areas in respective HPLC chromatograms at 280 nm.

Supplementary Figure 4 Electrospray analysis of BODIPY-cPAF26 and unlabeled PAF26 after 24 h incubation in a protease cocktail.

Both peptides (200 μM) were incubated in 1 mg L-1 of the protease cocktail, and their respective mass spectra were recorded on a Waters Micromass ZQ mass spectrometer (ESI positive mode). a) MS analysis of BODIPY-cPAF26 (exact mass: 1311 Da). b) MS analysis of unlabeled PAF26 (exact mass: 949 Da).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Tables 1 and 2. (PDF 777 kb)

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. (AVI 410 kb)

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Mendive-Tapia, L., Subiros-Funosas, R., Zhao, C. et al. Preparation of a Trp-BODIPY fluorogenic amino acid to label peptides for enhanced live-cell fluorescence imaging. Nat Protoc 12, 1588–1619 (2017). https://doi.org/10.1038/nprot.2017.048

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