Searching for the Optimal Fluorophore to Label Antimicrobial Peptides

Article abstract of DOI:10.1021/acscombsci.6b00081

With the advent of antimicrobial resistance, there is an urgent need for new strategies to treat infectious diseases. Antimicrobial peptides are considered as promising candidates, and therefore there is a need to understand their mechanism of action in order to exploit their therapeutic potential. To this end, fluorescent analogs are powerful tools to analyze their behavior and subcellular localization in cells and in vivo. However, the conjugation of fluorophores to antimicrobial peptides, especially in short sequences, can impair their biological activity, making the selection of the fluorescent label an essential step in these studies. In the present work, we have systematically modified a model antifungal hexapeptide with a collection of fluorophores covering broad physicochemical and spectral properties. The resulting conjugates have been examined in two different fungal species, in terms of their activity and intracellular localization. The biological results confirm the influence of the different fluorescent moieties on the subcellular localization of antimicrobial sequences, and provides an insight on the optimal fluorophores to be used in the preparation of fluorescent peptides for different bioimaging assays.

Full text of DOI:10.1021/acscombsci.6b00081

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Article
Searching for the optimal fluorophore to label antimicrobial peptides
Can Zhao, Antonio Fernandez, Nicolaos Avlonitis, Greetje
Vande Velde, Mark Bradley, Nick D. Read, and Marc Vendrell
ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.6b00081 • Publication Date (Web): 10 Oct 2016
Downloaded from http://pubs.acs.org on October 13, 2016
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Searching for the optimal fluorophore to label
antimicrobial peptides
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Can Zhao,1 Antonio Fernandez,2 Nicolaos Avlonitis,3 Greetje Vande Velde,4 Mark Bradley,3
Nick D. Read,1,* Marc Vendrell2,*
1
Manchester Fungal Infection Group, Institute of Inflammation and Repair, University of
Manchester. CTF Building, Grafton St., M13 9NT Manchester, United Kingdom.
2
MRC Centre for Inflammation Research, The University of Edinburgh. 47 Little France
Crescent, EH16 4TJ Edinburgh, United Kingdom.
3
EaStCHEM School of Chemistry, The University of Edinburgh. West Mains Road, Edinburgh
EH9 3FJ, United Kingdom.
4
Biomedical MRI Unit/MoSAIC, Department of Imaging and Pathology, KU Leuven, Leuven,
Belgium.
KEYWORDS: fluorescence, labeling, imaging, probes, fungi, infection.
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Abstract
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With the advent of antimicrobial resistance, there is an urgent need for new strategies to treat
infectious diseases. Antimicrobial peptides are considered as promising candidates, and therefore
there is a need to understand their mechanism of action in order to exploit their therapeutic
potential. To this end, fluorescent analogs are powerful tools to analyze their behavior and
subcellular localization in cells and in vivo. However, the conjugation of fluorophores to
antimicrobial peptides, especially in short sequences, can impair their biological activity, making
the selection of the fluorescent label an essential step in these studies. In the present work, we
have systematically modified a model antifungal hexapeptide with a collection of fluorophores
covering broad physicochemical and spectral properties. The resulting conjugates have been
examined in two different fungal species, in terms of their activity and intracellular localization.
The biological results confirm the influence of the different fluorescent moieties on the
subcellular localization of antimicrobial sequences, and provides an insight on the optimal
fluorophores to be used in the preparation of fluorescent peptides for different bioimaging
assays.
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Introduction
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In the past decades, fluorescent dyes have been broadly used as tags to visualize and study the
roles of peptides in cells and in vivo.1 For these studies, live cell imaging has become the
technology of choice as it provides a direct readout of the localization of the peptides with high
spatial and temporal resolution. In the context of antimicrobial peptides, several reports have
described fluorescence labeling as an approach to study their mechanism of action2ꢀ4 as well as to
develop imaging probes for the rapid identification of microbes at infection sites.5 In all cases, it
is often assumed that fluorescently labeled peptides faithfully mimic the spatioꢀtemporal
dynamics of the native peptides, and fluorescent labels are typically chosen on the basis of
excitation/emission wavelength, quantum yield, photobleaching, environmental sensitivity or
chemical conjugation. Little attention has been paid to whether the fluorescent label might alter
the physicochemical properties of the conjugate, and it is often unclear whether the fluorescent
tag influences the uptake and localization of antimicrobial peptides inside living cells. In order to
evaluate the impact that different fluorophores have in the biological properties of antimicrobial
peptides and their application as imaging probes, we have synthesized a library of fluorescent
conjugates based on the Peptide Antifungal 26 (PAF26).6 This rationally designed hexapeptide
exhibits high potency against filamentous fungi (including human and crop pathogens), low
toxicity against bacterial cells and negligible toxicity against mammalian cells. PAF26 is
currently being used a model peptide for the characterization of the modeꢀofꢀaction of small,
cationic, cell penetrating antifungal peptides and for the design and development of synthetic
antifungal peptides with improved properties for therapeutic uses.7
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In the present work, we have conjugated 12 different fluorophores (either inꢀhouse or
commercially available) to the peptide sequence of PAF26 and compared their biological
activity, cellular uptake and intracellular localization using confocal live cell imaging. From
these studies, we have observed that the activity of the conjugates is practically unaffected by the
fluorescent labels whereas they accumulate in different intracellular regions depending on the
fluorophore attached to the sequence. This systematic study will aid chemists working on
peptideꢀbased imaging agents in the selection of the fluorophores for different fluorescenceꢀ
based biological applications.
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Results and Discussion
Design and synthesis of a library of fluorescent antifungal peptides
For many years, the synthesis of antimicrobial peptides has been adapted to combinatorial
chemistry. However, libraries of antimicrobial peptides have been mainly focused on the
optimization of the peptide sequences, either to enhance their antimicrobial activity, improve
their selectivity against specific strains or pathogens, or to reduce their cytotoxicity in
mammalian cells.8ꢀ10 Some antimicrobial peptides have been labeled with fluorophores in order
to visualize their localization in cells by confocal microscopy.4,11 More recently, the high
selectivity of antimicrobial sequences has been exploited to develop imaging contrast agents for
the rapid identification of infection sites.12,13 In most reports, only one fluorophore is employed
on the basis of its compatibility with the experimental procedures. However, conventional
fluorophores differ largely in their physicochemical features,14,15 and therefore it is reasonable to
expect that different fluorophores could induce variability in the activity and cellular localization
properties of peptide sequences. For instance, Baker et al. reported the influence of different
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fluorescent labels in a combinatorial library of A1R adenosine inhibitors, where significant
differences were observed in the binding properties of several fluorescentlyꢀlabeled inhibitors.16
In the context of short antimicrobial peptides, there are no reports of any systematic evaluation of
their biological properties after conjugation to various fluorescent labels. Herein, we have
designed a library based on the antimicrobial peptide sequence PAF26, which was modified with
a total of 12 fluorophores covering the whole spectral range (from blue to nearꢀinfrared
emission) and diverse physicochemical properties (Chart 1). In addition to conventional
fluorophores (e.g. carboxyfluorescein, carboxyrhodamine, BODIPY, cyanine), we also included
small fluorophores (e.g. NBD, dansyl), compounds with environmentallyꢀsensitive properties
(e.g. Nile Blue, malachite green, styryl), pHꢀsensitivity (e.g. naphthalimide) and esteraseꢀ
activatable fluorophores (e.g. fluorescein diacetate). Overall, these fluorophores constitute a
representative collection of the fluorescent labels that most chemists would employ for labeling
any defined peptide sequence.
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Chart 1. Fluorescent labels for the derivatization of the antimicrobial peptide PAF26. We
employed 12 different fluorophores with suitable reactive groups (in red) for peptide labeling
(e.g. carboxylic acids, sulfonyl chlorides, anhydrides, alkynes). Fluorophores 1, 2, 3 and 7 are
commercially available, whereas the synthesis and characterization of all other fluorophores is
described in the Experimental Procedures and the Electronic Supplementary Information (ESI).
PAF26 (HꢀRKKWFWꢀNH2) is a short antimicrobial peptide which, like many other
antimicrobial peptides, contains both cationic and hydrophobic residues,6,7 and therefore it is an
excellent model for examining the influence of fluorophores in the properties of antimicrobial
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peptides. We designed the synthesis of the hexapeptide using conventional solidꢀphase protocols
in Rink amide polystyrene resin (Scheme 1). In order to incorporate suitable conjugation groups
within the sequence of PAF26, we first synthesized two derivatives containing aminohexanoic or
azidohexanoic spacers directly attached to the Nꢀterminal group of PAF26. Through these
spacers, fluorophores could be readily conjugated using their reactive groups (i.e., carboxylic
acids, chlorides, anhydrides, sulfonyl chlorides or alkynes) without affecting the main
recognition features of the antimicrobial peptide. Most library members were fully synthesized
on solid support, including the coupling of the fluorophore, to render the final fluorescent PAF26
analogues upon acidic cleavage from the resin using reported procedures.17 Due to the lability of
the fluorophores 11 and 12 (i.e., BODIPY and fluorescein diacetate, respectively) to acidic
media, the corresponding derivatives PAF26ꢀ11 and PAF26ꢀ12 were prepared by 1,3ꢀHuisgen
dipolar cycloaddition using the azidoꢀmodified PAF26 and the fluorescent alkynes in solution
phase (Scheme 1). All 12 fluorescent derivatives of PAF26 were purified by semiꢀpreparative
HPLC to isolate the final conjugates (PAF26ꢀ1 to PAF26ꢀ12) in high purities (for
characterization data, see ESI).
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Scheme 1. Solidꢀphase synthesis of a library of fluorescent antimicrobial peptides based on
the hexapeptide PAF26. The peptide sequence of PAF26 was modified with aminoꢀ or
azidohexanoic spacers at the Nꢀterminus to enable the conjugation of different fluorophores,
either by formation of amide bonds in solidꢀphase or by ‘click’ chemistry in solution. All 12
conjugates PAF26ꢀ1 to PAF26ꢀ12 were isolated in good yields and high purities. For the
chemical structures of the fluorophores 1ꢀ12, see Chart 1.
In vitro biological activity of fluorescent antifungal peptides
After completing the synthesis of the library of fluorescent antimicrobial conjugates (PAF26ꢀ1 to
PAF26ꢀ12), we measured their spectral properties and determined their IC50 values in two
different fungal species (i.e., Neurospora crassa and Aspergillus fumigatus) as a measure of their
cellular activity (Table 1). N. crassa has been widely used as an experimental model organism
for filamentous fungi.18 A. fumigatus is a very important opportunistic pathogen responsible for
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multiple human diseases, the most lethal of which, invasive pulmonary aspergillosis, causes
around 200,000 deaths per year.19
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Conjugates incorporating small fluorophores (e.g., NBD (PAF26ꢀ3), naphthalimide (PAF26ꢀ4),
dansyl (PAF26ꢀ7)) showed very similar antifungal activity when compared to the unlabeled
sequence PAF26 in both fungal species. This observation is in line with previous reports where
NBD or dansyl have been described as highly tolerated fluorescence labels for the derivatization
of biomolecules.20 Larger fluorophores, including positivelyꢀcharged fluorophores [i.e.
rhodamine (PAF26ꢀ1), Nile Blue (PAF26ꢀ6), styryl dyes (PAF26ꢀ8 and PAF26ꢀ9)] and
fluoresceinꢀbased structures (PAF26ꢀ2 and PAF26ꢀ12) induced only minor differences in the
antifungal activity of PAF26, with all IC50 values in the low micromolar range. Two conjugates
(PAF26ꢀ10 and PAF26ꢀ11) showed slightly higher activity, being the two corresponding
fluorophores [i.e. cyanine (10) and BODIPY (11)] permeable structures that have been reported
for the preparation of a range of fluorescent probes.21ꢀ25 Altogether, these results corroborate the
minor influence of the fluorescence labels on the antifungal activity of fluorescent PAF26
conjugates, with the smallest impairment being observed for conjugates including small
fluorophores.
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Table 1. Spectral properties and antifungal activities of the fluorescent antimicrobial sequences.
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IC50 (N. crassaǂ) IC50 (A. fumigatusǂ)
MW
Compound
PAF26
λ
exc. (nm)
λem. (nm)
n.d.
563
498
488
450
635
635
300
465
487
633
505
n.d.
593
529
552
532
n.d.
679
350
706
621
660
540
530
949.6
1530.8
1419.7
1225.6
1285.7
1416.8
1476.8
1295.7
1408.8
1466.9
1547.9
1494.8
1585.7
4.0 ± 0.7
3.8 ± 0.1
5.3 ± 0.2
4.4 ± 0.3
4.2 ± 0.2
4.1 ± 0.4
3.2 ± 0.2
4.0 ± 0.1
3.0 ± 0.1
2.6 ± 0.2
2.0 ± 0.1
2.2 ± 0.2
5.3 ± 0.2
8.0 ± 0.3
6.9 ± 0.2
9.1 ± 0.3
7.9 ± 0.1
8.0 ± 0.2
9.7 ± 0.5
5.8 ± 0.2
7.9 ± 0.2
7.7 ± 0.1
7.2 ± 0.4
4.2 ± 0.4
3.7 ± 0.2
9.9 ± 0.4
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PAF26ꢀ1¥
PAF26ꢀ2¥
PAF26ꢀ3
PAF26ꢀ4
PAF26ꢀ5
PAF26ꢀ6
PAF26ꢀ7
PAF26ꢀ8
PAF26ꢀ9
PAF26ꢀ10
PAF26ꢀ11
PAF26ꢀ12¥ 503
ǂ IC50 values (µM) represented as means ± standard deviations (n=3).
¥ Values obtained for the mixture of 5 and 6ꢀcarboxyisomers.
Fluorescence confocal live cell imaging studies to analyze the localization of PAF26
conjugates in fungal cells
Next we performed live cell imaging experiments to examine the cellular uptake and intracellular
localization of the different conjugates in N. crassa and A. fumigatus by confocal laser scanning
microscopy. Concentrations in the low micromolar range were used, as these have been
previously optimized for the nonꢀpassive uptake of PAF26.16 As shown in Fig 1, the rhodamine
PAF26ꢀ1 derivative localized to the cell envelope (comprising the plasma membrane and the cell
wall) and the intracellular vacuolar/vesicular system of N. crassa conidial germlings, The
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positive charge of the rhodamine fluorophore might favour the interaction with the negatively
charged phospholipids in the plasma membrane. In contrast, the carboxyfluorescein derivative
PAF26ꢀ2 showed a similar pattern of intracellular vacuolar distribution but reduced staining on
the cell envelope (Fig. 1). The negative charge of carboxyfluorescein reduced the affinity of the
conjugate for the cell envelope, with slower internalization (Table 1). Similarly, the diacetylated
derivative (PAF26ꢀ12) showed preferential staining of the vacuolar lumens with no fluorescence
detected in the plasma or intracellular membranes, because its fluorescence emission is triggered
upon reaction with intracellular esterases (Fig. S1 in ESI). We performed coꢀlocalization
experiments between PAF26ꢀ12 and PAF26ꢀ1 and observed that both conjugates displayed a
similar pattern of cellular uptake, first appearing in small vesicles and then accumulating in
larger cell vacuoles (Fig. S2 in ESI).
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Figure 1. Confocal fluorescence microscope images of conidial germlings of N. crassa after
incubation with PAF26ꢀ1, PAF26ꢀ2 or PAF26ꢀ8. A) Fluorescence images of N. crassa after
coꢀstaining with the rhodamine PAF26ꢀ1 derivative (red, 0.5 ꢁM) and the carboxyfluorescein
o
derivative PAF26ꢀ2 (green, 0.5 ꢁM) at 25 C. Scale bar: 2 ꢁm; v: vacuole. B) Imaging of N.
crassa after coꢀstaining with the styryl PAF26ꢀ8 derivative (red, 1 ꢁM) and the rhodamine
PAF26ꢀ1 derivative (yellow, 1 ꢁM) at 25 o C. Scale bar: 5 ꢁm; v: vacuole.
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Conjugates including small fluorophores, such as NBD (PAF26ꢀ3) or naphthalimide (PAF26ꢀ4),
displayed similar intracellular vacuolar/vesicular localization, both in conidial germlings of N.
crassa and A. fumigatus (Fig. S3 in ESI). Notably, the pHꢀsensitive properties of PAF26ꢀ4
enabled preferential imaging of acidified vacuoles as opposed to the general vacuolar and
vesicular structures staining observed with PAF26ꢀ1 (Fig. S4 in ESI), demonstrating the
possibility of monitoring intracellular pH changes in these subcellular structures. Conjugates
including environmentallyꢀsensitive fluorophores such as malachite green, Nile Blue or dansyl
(PAF26ꢀ5, PAF26ꢀ6 and PAF26ꢀ7) showed marginal staining in fungal cells, suggesting the
potential accumulation of these peptide conjugates in neutral hydrophilic microenvironments
(e.g. vacuoles), in which these fluorophores exhibit very low quantum yields.26,27
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On the other hand, cationic styrylꢀbased fluorophores (PAF26ꢀ8 and PAF26ꢀ9) showed
remarkably enhanced staining of the envelope of fungal cells. PAF26ꢀ8 brightly stained the
plasma and intracellular vacuolar/vesicular membranes of N. crassa to a similar extent than
PAF26ꢀ1 (Fig. 1), but without marked accumulation in the vacuole lumens. Imaging experiments
using the PAF26ꢀ9 derivative also showed a similar behavior in both N. crassa and A. fumigatus
(Fig. S5 in ESI). As observed for PAF26ꢀ1, the cationic character of the styryl fluorophores
favoured the rapid interaction with anionic components of the cellular membranes and enhanced
their internalization (Table 1). Interestingly, and in contrast to the results obtained with N. crassa
(Fig. 1), when we incubated PAF26ꢀ8 with A. fumigatus, we did not observe the uptake of the
fluorescent conjugate into healthy cells and hyphae were only stained once the cell membrane
structure was compromised (Fig. S6 and Movie S1 in ESI). These results highlight differences in
the internalization of PAF26ꢀ8 by cells of different fungal species, suggesting the possibility of
using fluorescent labels as means to improve the selectivity of antimicrobial sequences.
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Finally, both cyanine and BODIPYꢀbased conjugates (PAF26ꢀ10 and PAF26ꢀ11, respectively)
displayed a markedly different staining pattern when compared to all other members of the
PAF26 library. The cyanine derivative (PAF26ꢀ10) brightly stained fungal cells and, besides
localizing within vacuoles and vesicle lumens, it also stained other cytoplasmic organelles. Dual
labeling experiments with the carboxyfluorescein derivative PAF26ꢀ2 confirmed the presence of
PAF26ꢀ10 in the same vacuolar and vesicular organelles but also indicated its localization in
different cytoplasmic organelles (Fig. 2). The BODIPY derivative PAF26ꢀ11 exhibited general
staining of the cytoplasm with no specific accumulation in subcellular organelles, as shown by
coꢀlocalization experiments with PAF26ꢀ1 (Fig. 2).
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Furthermore, we compared the staining of N. crassa cells using the fluorescent peptides PAF26ꢀ
1, PAF26ꢀ2, PAF26ꢀ10 and PAF26ꢀ11 and their corresponding fluorophores alone (Fig S7 in
ESI). In these experiments, we observed that the PAF26 sequence is essential for the interaction,
internalization and accumulation of the fluorescent conjugates in the vacuoles of fungal cells, as
the fluorophores alone were not able to enter the cells or stained nonꢀspecifically intracellular
environments (e.g. the BODIPY fluorophore showed bright, general staining of lipidꢀrich
subcellular structures).
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Figure 2. Fluorescence microscope images of conidial germlings of A. fumigatus after
incubation with PAF26ꢀ10 or PAF26ꢀ11. A) Fluorescence images of A. fumigatus after coꢀ
staining with the cyanine PAF26ꢀ10 (red, 1 ꢁM) and the carboxyfluorescein PAF26ꢀ2 (green, 1
o
ꢁM) derivatives at 37 C. Scale bar: 1 ꢁm. B) Fluorescence images of germinated A. fumigatus
after coꢀstaining with the BODIPY PAF26ꢀ11 (green, 2 ꢁM) and the rhodamine PAF26ꢀ1 (red, 2
ꢁM) derivatives at 37 o C. Scale bar: 1 ꢁm.
Finally, in view of the bright fluorescence signals in fungal cells upon incubation with PAF26ꢀ
10 (Figure 2A) and its emission in the nearꢀinfrared region (λem.: 660 nm, Table 1), we assessed
the suitability of PAF26ꢀ10 for wholeꢀbody in vivo fluorescence imaging experiments in mice
with fungal lung infection. Nearꢀinfrared fluorophores are advantageous for in vivo imaging as
the low autofluorescence background in such region of the spectra enhances the signalꢀtoꢀnoise
ratios.28ꢀ30 We determined the background and fluorescence signals in mice that had been treated
with PAF26ꢀ10, and observed that the cyanine fluorophore was bright enough to provide a
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detectable signal from live animals using wholeꢀbody fluorescence imaging (Fig. S8 in ESI).
These results are in good agreement with previous reports of cyanineꢀlabeled peptides for in vivo
imaging of cancer cells in xenograft models.31,32
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Altogether, the systematic evaluation of a library of fluorescent PAF26ꢀbased antimicrobial
peptides provided important clues of the optimal fluorophores for the preparation of fluorescent
peptides and their application in bioimaging experiments. Whereas positivelyꢀcharged structures
(e.g. rhodamine, styryl) favor the interaction with the anionic membrane components, neutral
(e.g. NBD) and negativelyꢀcharged fluorophores (e.g. fluorescein) preferentially accumulate in
vacuoles, and pHꢀsensitive fluorophores (e.g. naphthalimide) offer the possibility of visualizing
specific intracellular processes in real time, such as vacuolar acidification. Cell permeable
fluorophores (e.g. cyanine) provide bright cytosolic staining and show good discrimination from
background signals in wholeꢀbody in vivo fluorescence imaging. These results suggest that,
whereas the PAF26 sequence is the main component responsible for the antifungal activity of the
fluorescent peptides, the incorporation of different fluorescent moieties into antimicrobial
sequences can lead to major redistributions of the peptide sequences to different subcellular
environments.
Conclusions
Fluorescently labeled peptides are powerful tools for imageꢀbased mechanistic studies in cells.
However, little is known about the influence of fluorescent tags in the uptake and localization of
peptides inside the cells, and fluorophores are primarily selected on the basis of their spectral
properties. We have evaluated the impact of different fluorophores (either commercially
available or synthetically accessible) in the biological properties of a small model antimicrobial
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peptide by synthesizing a library of fluorescent derivatives of the antifungal hexapeptide PAF26.
Solidꢀphase peptide synthesis followed by conventional amide formation or solutionꢀphase
‘click’ conjugation for acidꢀsensitive fluorophores rendered a collection of 12 fluorescent
conjugates with broad spectral and physicochemical properties. The assessment of the
fluorescent peptides in two fungal species (N. crassa and A. fumigatus) indicated the minimal
impact of the fluorescent labels on the antimicrobial activity of the PAF26 sequence (with all
conjugates displaying IC50 values in the low micromolar range) whereas major changes in the
localization pattern were observed upon conjugation to different fluorophores. This systematic
study will aid chemists working on peptideꢀbased imaging agents in the selection of optimal
fluorophores for various fluorescenceꢀbased biological applications.
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Experimental Procedures
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8ꢀ(Dimethylamino)ꢀ3ꢀoxatricyclo[tridecaꢀ1,5,7,9,11ꢀpentaene]ꢀ2,4ꢀdione (4). 2.0 mL of
dimethylamine aqueous solution (19.9 mmol) and a catalytic amount of CuSO4 were added to a
suspension of 4ꢀbromoꢀ1,8ꢀnaphthalenedicarboxylic anhydride (1.9 mmol) in DMF (50 mL). The
mixture was then refluxed for 1.5 h, after which the solvent was evaporated under vacuum.
Compound 4 was crystallized from ethanol as a yellow solid (275 mg, 57% yield). HPLC: tR: =
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1
4.74 min; MS (m/z): [M+H]+ calcd for C14H11NO3: 242.1; found: 242.2; H NMR (500 MHz,
DMSO-d6): δ 8.59 (d, J = 8.4 Hz, 1H), 8.46 (d, J = 7.2 Hz, 1H), 8.32 (d, J = 8.4 Hz, 1H), 7.76 (t,
J = 7.9 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 2.51 (s, 6H); 13C NMR (126 MHz, DMSOꢀd6): δ 162.0,
160.8, 157.8, 134.8, 133.6, 133.0, 125.5, 124.1, 119.3, 113.2, 108.4, 44.7.
Nꢀ(4ꢀ((4ꢀcarboxyphenyl)(4ꢀ(dimethylamino)phenyl)methylene)cyclohexaꢀ2,5ꢀdienꢀ1ꢀ
ylidene)ꢀNꢀmethylmethanaminium (5). 4ꢀ(Bis(4ꢀ(dimethylamino)phenyl)methyl)benzoic acid
(0.70 mmol) and pꢀchloranil (1.05 mmol) were dissolved in 30 mL CHCl3, and 0.5 mL AcOH
was added to the solution. The solution was stirred for 1.5 h at 35oC. Compound 5 was
crystallized in water to yield a green solid (176 mg, 70% yield). HPLC: tR: = 4.28 min; MS
+
(m/z): [M+] calcd for C24H25N2O2 : 373.2; found: 373.2; 1H NMR (500 MHz, DMSOꢀd6) δ 10.21
(s, 1H), 8.14 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 9.4 Hz, 2H), 7.11 (d, J =
13
9.5 Hz, 2H), 6.98 (d, J = 9.0 Hz, 2H), 6.64 (d, J = 9.0 Hz, 2H), 3.31 (s, 6H), 3.06 (s, 6H); C
NMR (126 MHz, DMSOꢀd6) δ 166.7, 157.0, 152.9, 151.6, 145.5, 144.6 (x 2), 140.5 (x 2), 132.7,
129.7, 128.9, 126.9 (x 2), 121.2 (x 2), 114.7 (x 2), 111.9 (x 2), 41.1 (x 2), 40.6 (x 2).
Nꢀ(5ꢀ(5ꢀcarboxybutanamido)ꢀ9Hꢀbenzo[a]phenoxazinꢀ9ꢀylidene)ꢀNꢀethylethanaminium (6).
Nile Blue chloride (0.33 mmol) was dissolved in DCM together with glutaric anhydride (1
mmol) and DIPEA (1.14 mmol). The mixture was stirred at r.t. overnight. The crude mixture was
then diluted with DCM and washed twice with water and then evaporated under vacuum. The
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crude was then purified by column chromatography (DCM: MeOH, 9:1) to yield compound 6 as
a dark blue solid (142 mg, 81% yield). HPLC: tR: = 4.05 min; MS (m/z): [M+] calculated for
7
8
+
1
C25H26N3O4 : 432.2; found: 432.2; H NMR (500 MHz, DMSOꢀd6) δ 8.75 (d, J = 8.0 Hz), 8.23
(s), 2.86 (q, J = 7.2 Hz), 2.35 (dt, J = 14.8 Hz, 4.6 Hz), 2.24 (t, J = 7.4 Hz), 1.93 – 1.77 (m), 1.71
(dd, J = 14.8 Hz, 7.4 Hz), 1.15 (d, J = 6.6 Hz); 13C NMR (126 MHz, DMSOꢀd6) δ 178.4, 174.6,
174.6, 164.2, 51.5, 33.3, 21.6, 21.3, 21.3, 20.5, 20.5, 19.1, 19.1, 18.9, 14.5, 14.5, 14.5, 12.3, 12.0.
1ꢀ(5ꢀcarboxypentyl)ꢀ4ꢀ((1E,3E)ꢀ4ꢀ(4ꢀ(Dimethylamino)phenyl)butaꢀ1,3ꢀdienyl)pyridinꢀ1ꢀium
bromide (8). 4ꢀMethylpyridine (5.4 mmol) and ethyl 6ꢀbromohexanoate (6.5 mmol) were
dissolved in 20 mL of ACN, the mixture was refluxed at 90oC overnight. 1ꢀ(6ꢀethoxyꢀ6ꢀ
oxohexyl)ꢀ4ꢀmethylpyridinium was precipitated in cold Et2O:hexane (1:1) to yield a light brown
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+
solid (1.9 g, quantitative yield). HPLC: tR: = 3.58 min; MS (m/z): [M+] calcd for C14H22NO2 :
236.2; found: 236.2. 1ꢀ(6ꢀethoxyꢀ6ꢀoxohexyl)ꢀ4ꢀmethylpyridinium (1.6 mmol) and 3ꢀ(4ꢀ
(dimethylamino)phenyl)acrylaldehyde (2 mmol) were dissolved in 10 mL of EtOH in a
microwave reaction vessel, then pyrrolidine (1.6 mmol) was added to the solution. The mixture
o
was irradiated under microwave for 10 min at 120 C, and the crude was purified by column
chromatography (DCM: MeOH, 9:1) to yield 4ꢀ((1E,3E)ꢀ4ꢀ(4ꢀ(dimethylamino)phenyl)butaꢀ1,3ꢀ
dienꢀ1ꢀyl)ꢀ1ꢀ(6ꢀethoxyꢀ 6ꢀoxohexyl)pyridinꢀ1ꢀium bromide as a red solid (853 mg, quantitative
yield). The ethyl ester (0.25 mmol) was dissolved in 2 mL THF and 1 mL MeOH, mixed with
LiOH (1.25 mmol) in water (1 mL) and stirred overnight at r.t. to yield the carboxylic acid which
was used in situ without any further purification. Characterization data for the ester: HPLC: tR: =
+
1
4.73 min; MS (m/z): [M+] calcd for C25H33N2O2 : 393.3; found: 393.4; H NMR (500 MHz,
DMSOꢀd6) δ 8.80 (d, J = 6.9 Hz, 2H), 8.05 (d, J = 7.0 Hz, 2H), 7.87 – 7.77 (m, 2H), 7.48 (d, J =
8.9 Hz, 2H), 7.02 (d, J = 6.6 Hz, 2H), 6.78 – 6.71 (m, 2H), 4.05 (q, J = 7.1 Hz, 2H), 2.99 (s, 6H),
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2.31 (td, J = 7.3, 2.1 Hz, 2H), 1.95 – 1.83 (m, 2H), 1.61 – 1.52 (m, 2H), 1.38 – 1.25 (m, 2H),
5
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13
1.19 (t, J = 7.1 Hz, 2H), 1.17 (t, J = 7.1 Hz, 3H). C NMR (126 MHz, DMSO) δ 173.2, 153.6,
7
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151.6, 144.1, 142.7, 129.6, 124.0, 123.6, 123.2, 112.5, 60.2, 59.5, 53.6, 33.6, 30.6, 25.3, 24.2,
23.0, 14.6.
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(E)ꢀ1ꢀ(5ꢀcarboxypentyl)ꢀ4ꢀ(4ꢀ(Dibutylamino)styryl)pyridinꢀ1ꢀium bromide (9). (3E,5E)ꢀ 1ꢀ
(6ꢀethoxyꢀ6ꢀoxohexyl)ꢀ4ꢀmethylpyridinium (1.6 mmol) and 4ꢀ(dibutylamino)benzaldehyde (2
mmol) were dissolved in 10 mL of EtOH in a microwave reaction vessel, then pyrrolidine (1.6
mmol) was added to the solution. The mixture was left to react under microwave irradiation for
10 min at 120oC. The crude was purified by column chromatography (DCM: MeOH, 9:1) to
yield (E)ꢀ4ꢀ(4ꢀ(dibutylamino)styryl)ꢀ1ꢀ(6ꢀethoxyꢀ6ꢀoxohexyl)pyridinꢀ1ꢀium bromide as a purple
solid (703 mg, quantitative yield). The ethyl ester (0.25 mmol) was dissolved in 2 mL THF and 1
mL MeOH, mixed with LiOH (1.25 mmol) in water (1 mL) and stirred overnight at r.t. to yield
the carboxylic acid which was used in situ without any further purification. Characterization data
for the ester: HPLC: tR: = 5.76 min; MS (m/z): [M+] calcd for C29H43N2O2 : 451.3; found: 451.4.
+
1H NMR (500 MHz, DMSOꢀd6) δ 8.76 (d, J = 7.0 Hz, 2H), 8.05 (d, J = 7.1 Hz, 2H), 7.91 (d, J =
16.0 Hz, 1H), 7.57 (d, J = 8.9 Hz, 2H), 7.12 (d, J = 16.0 Hz, 1H), 6.74 (d, J = 9.1 Hz, 2H), 4.41
(t, J = 7.4 Hz, 2H), 4.05 (q, J = 7.1 Hz, 4H), 3.41 – 3.34 (m, 3H), 2.31 (td, J = 7.3, 1.6 Hz, 2H),
1.98 – 1.84 (m, 2H), 1.63 – 1.47 (m, 4H), 1.41 – 1.25 (m, 4H), 1.20 (t, J = 7.1 Hz, 3H), 1.17 (t, J
= 7.1 Hz, 3H), 0.93 (t, J = 7.4 Hz, 6H).13C NMR (126 MHz, DMSO) δ 173.2, 154.3, 150.4,
143.9, 142.8, 139.1, 131.0, 122.7, 122.2, 117.0, 112.0, 60.2, 59.3, 53.6, 50.3, 33.6, 30.6, 29.5,
25.3, 23.1, 20.1, 14.6.
2ꢀ((1E,3Z)ꢀ3ꢀ(5ꢀcarboxypyridinꢀ2ꢀyl)ꢀ5ꢀ((E)ꢀ1,3,3ꢀtrimethylindolinꢀ2ꢀylidene)pentaꢀ1,3ꢀ
dienꢀ1ꢀyl)ꢀ1,3,3ꢀtrimethylꢀ3Hꢀindolꢀ1ꢀium iodide (10). 3HꢀIndoliumꢀ2,3,3ꢀtetramethyl iodide
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(0.72 mmol), NaOAc (0.72 mmol) and 6ꢀ(1,3ꢀdioxopropanꢀ2ꢀyl) nicotinic acid (0.36 mmol) were
added to a microwave reaction vessel with 10 mL Ac2O:AcOH (1:1). The mixture was then
heated up to 160oC under microwave irradiation for 0.5 h, after which the solvent was
evaporated under vacuum. The crude was then purified using column chromatography
(DCM:MeOH, 98:2 to 9:1) to yield compound 10 as a blue solid (144 mg, 80% yield). HPLC: tR:
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= 4.72 min; MS (m/z): [M+] calcd for C33H34N3O2 : 504.3; found: 504.4; H NMR (500 MHz,
DMSOꢀd6) δ 9.26 (d, J = 1.3 Hz, 1H), 8.44 (s, 1H), 8.41 (s, 1H), 8.40 (d, J = 2.2 Hz, 1H), 8.39
(d, J = 2.2 Hz, 1H), 7.65 (d, J = 7.4 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.42 (dd, J = 8.0, 1.2 Hz,
2H), 7.40 (d, J = 1.1 Hz, 3H), 7.28 (ddd, J = 7.4, 6.5, 1.9 Hz, 2H), 5.85 (d, J = 14.3 Hz, 1H), 3.39
(s, 6H), 1.76 (s, 12H). 13C NMR (126 MHz, DMSO) δ 177.5, 152.8, 152.4, 141.4, 139.9, 135.3,
133.7, 131.7, 131.1, 129.3, 128.8, 126.9, 126.3, 123.4, 11.9, 101.4, 52.6, 32.1, 27.5.
4ꢀ(1,3,5,5,7,9ꢀHexamethylꢀ5Hꢀ4λ4,5λ4ꢀdipyrrolo[1,2ꢀc:2',1'ꢀf][1,3,2]diazaborininꢀ10ꢀyl)ꢀNꢀ
(propꢀ2ꢀynꢀ1ꢀyl)benzamide (11). 2,4ꢀDimethylpyrrole (4.5 mmol) and 4ꢀcarboxybenzaldehyde
(2 mmol) were dissolved in DCM under Ar. One drop of TFA was added and the solution was
stirred at r.t. until complete consumption of the aldehyde. Then, a solution of 2,3ꢀdichloroꢀ5,6ꢀ
dicyanoꢀ1,4ꢀbenzoquinone (2 mmol) in DCM was added, and the stirring continued for 10 min
followed by addition of triethylamine (4 mL) and BF3Et2O (4 mL). After stirring for another 2 h
the reaction mixture was washed with water and dried, then solvent was evaporated under
vacuum. The carboxylic acid was then purified by column chromatography (DCM: MeOH,
95:5). The crude product was then concentrated and precipitated (AcOEt: hexane, 1:1) to yield a
dark red oil (160 mg, 20 % yield). HPLC: tR: = 5.81 min; MS (m/z): [M+] calcd for
C20H19BF2N2O2: 368.2; found: 368.2. Then, the carboxylic acid (0.19 mmol) was dissolved with
oxyma (0.57 mmol) in DCM and stirred for 5 min. DIC (1.14 mmol) was added and stirred for
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another 2 min. Propargylamine (0.21 mmol) was then added and whole mixture was stirred at r.t.
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for 3 h. Compound 11 was isolated as a bright orange solid (17 mg, 22% yield over all steps).
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HPLC: tR: = 5.80 min; MS (m/z): [M+H]+ calcd for C23H22BF2N3O: 406.2; found: 406.2; H
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NMR (500 MHz, DMSOꢀd6) δ 8.05 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 6.20 (s, 2H),
5.76 (s, 1H), 4.09 (dd, J = 5.6, 2.5 Hz, 2H), 2.47 (s, 6H), 1.35 (s, 6H); 13C NMR (126 MHz,
DMSO) δ 165.7, 143.2, 135.8, 134.8, 130.9, 128.7, 122.0, 112.1, 111.6, 98.8, 73.4, 29.1, 14.7,
14.6.
3ꢀOxoꢀ6ꢀ(propꢀ2ꢀynꢀ1ꢀylcarbamoyl)ꢀ3Hꢀspiro[isobenzofuranꢀ1,9'ꢀxanthene]ꢀ3',6'ꢀdiyl
diacetate (12). 5,6ꢀcarboxyfluorescein diacetate (0.43 mmol) and oxyma (1.29 mmol) were
dissolved in DCM and stirred for 5 min. Then DIC (2.58 mmol) was added to the mixture, stirred
for 2 min and propargylamine (1.29 mmol) was added. The whole mixture was stirred at r.t. for 3
h then evaporated under vacuum. Compound 12 was purified by column chromatography (DCM:
MeOH: AcOH, 98:1:1) to yield a yellow oil (175 mg, 82% yield); HPLC: tR: = 5.36 min; MS
(m/z): [M+H]+ calcd for C28H19NO8: 497.1; found: 497.8; 1H NMR (500 MHz, DMSOꢀd6) δ 9.31
(t, J = 5.5 Hz, 1H), 9.13 (t, J = 5.5 Hz, 1H), 8.54 (dd, J = 1.6, 0.7 Hz, 1H), 8.29 (dd, J = 8.1, 1.6
Hz, 1H), 8.22 (d, J = 1.4 Hz, 1H), 8.16 (dd, J = 8.1, 0.7 Hz, 1H), 7.83 (t, J = 1.0 Hz, 1H), 7.55
(dd, J = 8.0, 0.7 Hz, 1H), 7.37 – 7.28 (m, 2H), 7.02 – 6.90 (m, 6H), 4.12 (dd, J = 5.5, 2.5 Hz,
2H), 4.00 (dd, J = 5.5, 2.5 Hz, 2H), 3.16 (t, J = 2.5 Hz, 1H), 3.08 (t, J = 2.5 Hz, 1H), 2.29 (s,
3H), 2.29 (s, 3H).13C NMR (126 MHz, DMSO) δ 170.3, 169.0, 167.4, 155.5, 152.4, 150.8,
135.5, 134.9, 130.0, 129.4, 119.1, 115.9, 110.6, 82.4, 81.4, 73.5, 29.6, 20.9.
Solidꢀphase peptide synthesis. All peptides were manually synthesized in polystyrene syringes
fitted with a polyethylene porous disc using Fmocꢀbased SPPS. Solvents and soluble reagents
were removed by suction. The Fmoc group was removed with piperidine: DMF (1:4) (1 × 1 min,
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2 × 10 min). Peptide synthesis transformations and washings were performed at r.t. For peptide
elongation, after the Fmoc group was removed, resins were washed with DMF (4 × 1 min), DCM
(3 × 1 min) and DMF (4 x 1 min). Unless otherwise noted, standard coupling procedures were
used with DIC (3 eq.) and OxymaPure (3 eq.) in DMF for 1ꢀ2 h and 5ꢀmin of preꢀactivation. The
completion of the coupling was monitored by the Kaiser test.33 Then, resins were filtered and
washed with DCM (4 x 1 min) and DMF (4 x 1 min).
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Synthesis of the peptides PAF26ꢀ1/10. For conjugates PAF26ꢀ1, PAF26ꢀ2, PAF26ꢀ5, PAF26ꢀ
6, PAF26ꢀ8, PAF26ꢀ9, PAF26ꢀ10, standard amide bond formation coupling conditions with
fluorophoreꢀCOOH (3 eq.), DIC (3 eq.) and OxymaPure (3 eq.) in DMF were used. For
compounds PAF26ꢀ3, PAF26ꢀ4 and PAF26ꢀ7, coupling of the dyes was accomplished by
incubation of the peptide with the fluorophore (3 eq.) and DIPEA (3 eq.) in DMF. All resins
were then washed with DMF (4 × 1 min) and DCM (3 × 1 min). For the cleavages, resins were
stirred in 5 mL of TFA: H2O (95:5) for 2 h at r.t. as previously reported.34 The beads were
washed with TFA thoroughly and the peptides precipitated with iceꢀcold Et2O after rotary
evaporation before final purification by semiꢀpreparative HPLC. Characterization details can be
found in the ESI.
Synthesis of the peptides PAF26ꢀ11/12. Azidoꢀderivatized peptides were cleaved from the resin
by cleavage with 5 mL of TFA: H2O: TIS (95:2.5:2.5) for 2 h at r.t. The beads were washed with
TFA thoroughly and the peptides precipitated with iceꢀcold Et2O after rotary evaporation. For the
conjugation was performed, the peptide (1 eq.), ascorbic acid (1 eq.), tris[(1ꢀbenzylꢀ1Hꢀ1,2,3ꢀ
triazolꢀ4ꢀyl)methyl] amine (TBTA) (0.3 eq.) and CuSO4 (0.3 eq.) were dissolved in H2O, and the
corresponding alkynes (2 eq.) were added using a minimum amount of MeOH, leaving the
reaction for aprox. 4 h at r.t. The reaction was monitored with HPLC, and upon completion,
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peptides were purified by semiꢀpreparative HPLC. Characterization details can be found in the
ESI.
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Measurement of spectral properties. Spectroscopic data were recorded on a Synergy HT
spectrophotometer (Biotek). Compounds were dissolved at concentrations around 10 ꢀMꢀ50 ꢀM
in phosphate buffer saline (PBS) and spectra were recorded at r.t.
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Culturing of N. crassa and A. fumigatus. Cultures of N. crassa (wild type strain # 2489 from
the Fungal Genetics Stock Center) and A. fumigatus (CEA10 strain from the fungal stock
collection of the Manchester Fungal Infection Group) were grown on solid 100% Vogel’s agar
o
medium35 at 25 °C for 3ꢀ5 days under constant light (N. crassa) or 37 C for 4ꢀ5 days (A.
fumigatus). The spores (conidia) were then harvested from the plates with sterile deionized water
(N. crassa) or 0.05% Tween 80 in sterile deionized water (A. fumigatus), and the spore density
was determined with a haemocytometer.
Antifungal activity measurements. The BacTiterꢀGloTM Microbial Cell Viability Assay kit
(Promega), with CELLSTAR white polystyrene flat bottomed 96ꢀwell plates (Greiner), were
used for these assays. Each antifungal peptide, at an appropriate serial diluted concentrations,
was mixed with conidia of N. crassa or A. fumigatus to reach a final volume of 100 ꢁL per well
and a final conidial concentration of 5 × 105 cells/mL in 10% liquid Vogel’s medium. After 24 h
of incubation (at 25 oC for N. crassa or 37 oC for A. fumigatus), the amount of fungal growth was
determined by measurement of the luminescence in a TriStar LB 941 multimode microplate
reader. IC50 values were determined using four parameter logistic regression. Data analysis was
performed using Sigma Plot 10.0. Values are represented as means ± s.e.m. from two
independent experiments (n = 3).
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Confocal live cell microscopy. Conidia at a concentration of 5 × 105 cells/mL were dispensed
into the wells of an 8ꢀwell slide culture chamber (ibidi USA Inc., Madison, WI) and incubated
for 3 h at 25 oC (N. crassa) or for 16 h at 37 oC (A. fumigatus) in 10% Vogel’s medium in order
to allow germination to occur before the addition of antifungal peptide. The peptide was added to
produce the appropriate final concentration immediately before the liveꢀcell imaging was
initiated. Individual germinated spores of each species were imaged for periods of up to 1 h at
the same temperature as they had previously been incubated at in a temperatureꢀcontrolled
chamber mounted on the microscope stage. The liveꢀcell imaging was performed with a Leica
TCS SP8 confocal laser scanning microscope equipped with photomultiplier tubes, hybrid
GaAsP detectors and a 63× water immersion objective and. White light (450ꢀ750 nm), argon ion
(458 nm, 476 nm, 488 nm and 496 nm) and blue diode lasers (405 nm) were used for excitation.
Images were processed and analyzed using Imaris 3D/4D image processing and analysis
software v 8.0 (Bitplane, Zurich, Switzerland). Details on the excitation and emission
wavelengths used for all of the PAF26ꢀ1/12 conjugates are shown in Table S2 in the ESI.
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In vivo fluorescence imaging. Animal experiments were carried out in compliance with national
and European regulations and were approved by the animal ethics committee of KU Leuven
(p103ꢀ2012).
9ꢀweekꢀold female Balb/C mice of (Balb/cAnNCrl from Charles River
Laboratories, bred at the KU Leuven animal facility, Leuven, Belgium) were kept in individually
ventilated cages with free access to food and water. Immune competent mice (n = 3) were
infected by inhalation of a fungal yeast cell suspension (C. gattii R265, 50,000 CFUs in 20 µL
PBS, 10 µL per nostril) under transient isoflurane gas anesthesia (2 % in pure O2). Three weeks
after infection, wholeꢀbody fluorescence imaging was carried out under isoflurane anesthesia on
an IVIS Spectrum system with Living Image software (version 4.5.2, Perkin Elmer). The
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fluorescence signal intensity was measured using the excitation/emission 640/680 filter pair
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before and 5 min after intratracheal administration of 7.5 nmol of the PAF26ꢀ10 derivative.
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Supporting Information. NMR spectra and additional characterization data for the fluorescent
PAF26 conjugates, additional imaging data. This material is available free of charge via the
Internet at http://pubs.acs.org.
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Author Information
The manuscript was written with contributions of all authors. All authors have given approval to
the final version of the manuscript.
* Corresponding authors: nick.read@manchester.ac.uk; marc.vendrell@ed.ac.uk
Acknowledgements
A.F. acknowledges funding from the Foundation Alfonso Martin Escudero (FAME, Spain).
G.V.V. acknowledges a postdoctoral fellowship of the Flemish Research Foundation (FWO).
N.D.R. acknowledges funding from the Scottish Universities Life Sciences Alliance (SULSA).
M.V. acknowledges funding from the Medical Research Council and the Marie Curie Career
Integration Grant (333487).
Abbreviations
PAF26, Peptide Antifungal 26; NBDꢀCl, 4ꢀchloroꢀ7ꢀnitroꢀ2,1,3ꢀbenzoxadiazole; BODIPY, 4,4ꢀ
difluoroꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀindacene;
TFA,
trifluoroacetic
acid;
DIC,
diisopropylcarbodiimide; DCM, dichloromethane, TBTA, tris(benzyltriazolylmethyl)amine.
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