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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- 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.
- 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.
- 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.
- 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: 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 (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.
- Supplementary Text and Figures (796 KB)
Supplementary Figures 1–4 and Supplementary Tables 1 and 2.