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

Journal name:
Nature Protocols
Volume:
12,
Pages:
1588–1619
Year published:
DOI:
doi:10.1038/nprot.2017.048
Published online

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).

At a glance

Figures

  1. Flowchart outlining all the experimental procedures described in this protocol, which includes chemical synthesis (yellow), in vitro characterization (blue) and fluorescence imaging assays (pink).
    Figure 1: Flowchart outlining all the experimental procedures described in this protocol, which includes chemical synthesis (yellow), in vitro characterization (blue) and fluorescence imaging assays (pink).
  2. Retrosynthetic analysis of the amino acid Fmoc-Trp(C2-BODIPY)-OH (1).
    Figure 2: Retrosynthetic analysis of the amino acid Fmoc-Trp(C2-BODIPY)-OH (1).
  3. Synthesis and chemical characterization of amino acid Fmoc-Trp(C2-BODIPY)-OH (1).
    Figure 3: Synthesis and chemical characterization of amino acid Fmoc-Trp(C2-BODIPY)-OH (1).

    (a) Preparation of amino acid 1 from Fmoc-Trp-OH (2) and m-iodophenyl-BODIPY (3). (b) Spectral properties of amino acid 1. (c) Long-term stability (as determined by HPLC analysis) of amino acid 1 as a solid and in solution (0.1 M) at different temperatures. *Determined after re-dissolution due to solvent evaporation. Mw, microwave.

  4. Synthetic scheme for the preparation of BODIPY-cPAF26 on solid phase.
    Figure 4: Synthetic scheme for the preparation of BODIPY-cPAF26 on solid phase.
  5. BODIPY-cPAF26, a fluorogenic peptide with high affinity for fungal cells.
    Figure 5: BODIPY-cPAF26, a fluorogenic peptide with high affinity for fungal cells.

    (a) Chemical structure of BODIPY-cPAF26. (b) Cell viability plots and nonlinear regressions for BODIPY-cPAF26 and unlabeled PAF26 in N. crassa. The two peptides were incubated at the same concentrations with N. crassa conidia, and after 24 h at 25 °C, fungal growth was determined by measuring the OD at 610 nm. Data are represented as means ± s.d. (n = 3). (c) Fluorescence spectra of BODIPY-cPAF26 after incubation with liposome suspensions of PC/cholesterol (7:1) in PBS ranging from 3.75 to 0.007 mg ml−1 of PC in two-fold serial dilutions, λexc.: 450 nm. (d) Viability of human lung A549 epithelial cells after incubation with different concentrations of BODIPY-cPAF26. Individual data points represented together with means ± s.d. (n = 4). Nonsignificant (NS) differences (P > 0.05) were found between untreated cells and any of the treatments.

  6. Live-cell fluorescence imaging of fungal pathogens.
    Figure 6: Live-cell fluorescence imaging of fungal pathogens.

    (ae) Confocal live-cell images (top: fluorescence, bottom: bright-field) of different fungal species after incubation with BODIPY-cPAF26 (2 μM for ad, 10 μM for e) for 10 min, without any washing steps. (a) N. crassa, (b) A. fumigatus, (c) C. neoformans, (d) F. oxysporum and (e) C. albicans. λexc.: 496 nm, λem.: 505–550 nm. Scale bars, 10 μm.

  7. Time-course fluorescence imaging of A. fumigatus with BODIPY-cPAF26.
    Figure 7: Time-course fluorescence imaging of A. fumigatus with BODIPY-cPAF26.

    High-resolution fluorescence confocal images of the fungal pathogen A. fumigatus after incubation with a cell membrane counterstain (red) and following direct addition of BODIPY-cPAF26 (2 μM, green) without any washing steps. BODIPY-cPAF26 initially interacts rapidly with the apical plasma membrane of A. fumigatus, and its staining then moves toward the base of the germling cell within a few minutes (Supplementary Video 1). λexc: 496 nm, λem: 505–550 nm (green); λexc: 570 nm, λem: 585–650 nm (red). Scale bar, 5 μm.

  8. Time-course illustration of the reaction setup for the synthesis of compound 3 under a N2 atmosphere.
    Figure 8: Time-course illustration of the reaction setup for the synthesis of compound 3 under a N2 atmosphere.

    (a) Addition of anhydrous DCM. (b) Addition of 2,4-dimethylpyrrole. (c) Solution resulting after addition of TFA. (d) Slow addition of DDQ. (e) Addition of BF3·OEt2 to the TEA-containing mixture. (f) Isolated pure compound 3.

  9. Time-course illustration of the reaction setup for the synthesis of amino acid 1.
    Figure 9: Time-course illustration of the reaction setup for the synthesis of amino acid 1.

    (a) Reaction vessel after microwave irradiation. (b) Filtration of the crude reaction through Celite under vacuum. (c) Crude mixture before purification. (d) Isolated pure amino acid 1.

  10. Setup for the solid-phase synthesis of BODIPY-cPAF26.
    Figure 10: Setup for the solid-phase synthesis of BODIPY-cPAF26.

    (a) Manifold for manual SPPS. (b) Experimental setup for resin cleavage. (c) Crude protected peptide after lyophilization.

  11. Experimental setup for the hydrogenation of the cyclic protected peptide.
    Figure 11: Experimental setup for the hydrogenation of the cyclic protected peptide.

    (a) Purge under a N2 stream. (b) Hydrogenation reaction under H2 at atmospheric pressure. (c) Filtration of crude solution of the reaction mixture through a glass Pasteur pipette. (d) Pure BODIPY-cPAF26 after semipreparative HPLC purification.

  12. Image showing BODIPY-cPAF26 and fluorescein under excitation with 365-nm light using a hand-held UV lamp.
    Figure 12: Image showing BODIPY-cPAF26 and fluorescein under excitation with 365-nm light using a hand-held UV lamp.

    (i) BODIPY-cPAF26 (10 μM) in a high concentration of liposomes (1.8 mg ml−1). (ii) BODIPY-cPAF26 (10 μM) in low concentration of liposomes (0.03 mg ml−1). (iii) BODIPY-cPAF26 (10 μM) in DPBS. (iv) Fluorescein (4 μM) in basic EtOH.

  13. Image of A549 cells in a 96-well plate after detergent incubation.
    Figure 13: Image of A549 cells in a 96-well plate after detergent incubation.

    Cells were incubated in four replicates with different concentrations of BODIPY-cPAF26 (rows B–E), as well as full DMSO (row F) and DPBS (row G) for positive and negative controls, respectively.

  14. Plate layout for the antifungal activity assays.
    Figure 14: Plate layout for the antifungal activity assays.

    (a) Steps 154–156. (b) Step 157. (c) Step 158. (d) Step 160. Conidia are incubated in six replicates with different concentrations of peptide (rows A–C and E–G) and controls are incubated without peptide (column 12). In addition, 'blank' wells contain Vogel's sucrose minimal medium only (rows D and H).

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

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

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

  18. Electrospray analysis of BODIPY-cPAF26 and unlabeled PAF26 after 24 h incubation in a protease cocktail.
    Supplementary Fig. 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).

Videos

  1. Supplementary Video 1. Time-course high-resolution imaging of A. fumigatus upon treatment with BODIPY-cPAF26.
    Video 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 (2μM, 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, 5μm.

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Author information

  1. These authors contributed equally to this work.

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

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

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 financial interests

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

Corresponding authors

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Analysis of the long-term stability of the amino acid 1 when stored as a solid at different temperatures. (33 KB)

    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.

  2. Supplementary Figure 2: Analysis of the long-term stability of the amino acid 1 when dissolved in organic solvents at different temperatures. (34 KB)

    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.

  3. Supplementary Figure 3: Time-course analysis of the chemical integrity of BODIPY-cPAF26 and unlabeled linear PAF26 in proteolytic environments. (106 KB)

    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.

  4. Supplementary Figure 4: Electrospray analysis of BODIPY-cPAF26 and unlabeled PAF26 after 24 h incubation in a protease cocktail. (113 KB)

    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).

Video

  1. Video 1: Supplementary Video 1. Time-course high-resolution imaging of A. fumigatus upon treatment with BODIPY-cPAF26. (411 KB, Download)
    A. fumigatus cells were pretreated with a cell membrane counterstain (red) and imaged under a confocal microscope. Cells were then treated with BODIPY-cPAF26 (2μM, 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, 5μm.

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  1. Supplementary Text and Figures (796 KB)

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

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