Wash‐free, peptide‐based fluorogenic probes for microbial imaging

Peptides with pan‐antimicrobial affinity were synthesized and decorated with environmentally sensitive fluorophores nitrobenzoxadiazole (green) and merocyanine (red). The labeling efficacies against a range of clinically relevant fungal and Gram‐negative and positive bacterial species (Candida albicans, Escherichia coli, and Staphylococcus aureus) were explored. The fluorogenic probes containing exclusively l or d‐amino acids showed rapid and efficient labeling of all microbial species, whereas probes with a mixture of l or d‐amino acids failed to label fungi or bacteria, highlighting the importance of the α‐helical peptide structure for interaction with the microbial cell membrane. Importantly, the nature of the dye allowed fluorescence detection/labeling without the need for a wash step, paving the way for direct application of the probes.

highlighting the importance of the α-helical peptide structure for interaction with the microbial cell membrane. Importantly, the nature of the dye allowed fluorescence detection/labeling without the need for a wash step, paving the way for direct application of the probes. and misuse of antibiotics that drive antimicrobial resistance. [1] Current diagnostic approaches typically require sampling and culturing, which impose delays in the identification of the specific antimicrobial infection. [2] The polymerase chain reaction is a powerful diagnostic tool, due to its high sensitivity and selectivity, but requires predictive knowledge of the pathogen it will detect and identify, while the sophisticated instrumentation and requirements for coldchains means it will be inaccessible in developing countries where the bulk of microorganism-based infections reside. [3,4] Thus, new, simple, and affordable diagnostic methods are needed to allow rapid and specific diagnosis of bacterial and fungal infections.
Optical molecular imaging has attracted much attention as a method for early stage diagnosis due to its versatility and power for detecting disease-related biomarkers and pathogens both in vitro and in real-time in vivo. Importantly, optical molecular imaging can provide information at a molecular level at low cost and simplicity. [5][6][7] In targeted optical molecular imaging, rationally designed probes, comprised of a fluorescent reporter conjugated to target-specific moiety (e.g., an antibiotic), provide high sensitivity and selectivity. The applied fluorophores typically fall into two categories: so-called "always-on" or those that are "turned-on" upon probe-target interaction. The "turn-on" probes often utilize fluorogenic dyes that are weakly fluorescent until there is a small change in their local environment (eg, polarity, hydrophobicity, or viscosity). [8] Examples of these dyes include nitrobenzoxadiazole (NBD) and merocyanine (MeroCy), which show weak fluorescence in an aqueous environment and produce a much stronger signal, for example, upon binding to a hydrophobic target. This reduces background fluorescence and eliminates the need for wash steps, which is particularly important in diagnostic applications. This environment-based activation strategy makes them outstanding candidates for probes for optical imaging of microorganisms as cell membranes are hydrophobic.
The interest in antimicrobial peptides (AMPs) has increased significantly [9][10][11] with the current AMP database containing over 3150 entries from prokaryotic and eukaryotic organisms, including 1135 naturally isolated antifungal peptides. [12] Most AMPs act by disrupting the cell membrane by various mechanisms, with important structural features including the length of the peptide, overall charge, hydrophobic/hydrophilic properties, and even the length of the side chain. [13] For example, Ng et al. screened 61 short (3-11 residues) AMPs for antifungal activity against fluconazole-resistant strains of Candida albicans and established structure-activity relationships, with the amphipathic peptide H-KKLIKKILKIL-NH 2 (P11-6) [14] showing better antifungal activity than the antifungal drug miconazole. [15] Peptides, including AMPs, have also gained interest as targeting moieties for imaging applications. [16][17][18] Here, we report a series of fluorescent probes based on the AMP P11-6, with the probes carrying an environmentally sensitive fluorophore NBD (green, λ ex/em 485/545 nm) or MeroCy (red, λ ex/em 595/630 nm) that only "turn-on" on/in hydrophobic microbial membranes ( Figure 1). These probes were applied in the imaging of clinically relevant microorganisms including C. albicans (fungus), Escherichia coli (Gram-negative bacteria), and Staphylococcus aureus (Gram-positive bacteria), and their mechanism of action explored by investigating the effect of L and D amino acid residues on selectivity, with the synthesis of six analogues of P11-6.

| General information
Chemicals were purchased from Sigma Aldrich, Merck, Acros, VWR, GL Biochem, and Fisher Scientific and used without further purification. NMR spectra were recorded at 298 K in deuterated solvents using a Bruker AVA500 spectrometer operating at 500 MHz for 1 H and 126 MHz for 13 C, with chemical shifts reported in ppm (referenced to residual non-deuterated solvent for 1 H and deuterated solvent for 13 C). Fluorescence spectra were measured on a Shimadzu RF-6000 spectrofluorophotometer using a quartz cuvette (10 mm).
High-resolution mass spectra (HRMS) were recorded on a Bruker 3.0 T Apex II spectrometer 1.

| Solid-phase synthesis
All the peptides were synthesized on an aminomethyl polystyrene resin (0.745 mmol/g, 1% DVB, 100-200 mesh) functionalized with an Fmoc-Rink linker. The peptides were synthesized using 500 mg of resin, and for each reaction, the resin was preswollen in DCM. The coupling reactions were monitored by the Kaiser test.
The solution was drained and the resin washed thrice with DMF, DCM, and MeOH.   Imaging was performed on a Leica TCS SP8 laser scanning confocal microscope (λ ex = 488 nm for NBD-based probes, λ ex = 561 nm for merocyanine-based probes). Images were analyzed using ImageJ2 (National Institutes of Health).

| Circular dichroism spectroscopy
The circular dichroism (CD) spectra of the peptides (dissolved in 50%

| Design and synthesis of AMP-based fluorescent probes
The AMP H-KKLIKKILKIL-NH 2 (P11-6) [14] and, to improve proteolytic stability, its corresponding enantiomer (D-amino acid sequence) were synthesized on a Rink-amide functionalized polystyrene resin using an Fmoc/ t Bu-based solid-phase peptide synthesis, with DIC/Oxyma as the coupling combination ( Figure 1B)  MeroCy-D) were deprotected and cleaved off the linker (TFA/TIS/H 2 O), purified by semipreparative HPLC, and analyzed by HPLC and HRMS (Table 1).
The environmentally sensitive nature of these probes was demonstrated by fluorescence analysis in PBS and in 99% DMSO, with a 9.2 (λ em = 545 nm) and 8.5-fold (λ em = 630 nm) increase in fluorescence intensities observed for NBD-L and MeroCy-L, respectively, in DMSO compared to PBS (Figure 2A).
3.2 | D and L-analogues of the probes label fungi, Gram-negative, and Gram-positive bacteria The AMP P11-6 has been shown to have an MIC of 3.1 μM against a fluconazole-resistant strain of C. albicans. [15] Thus, the ability of NBD-L and NBD-D (probes with the fluorophore NBD attached to D and L variants of P11-6) to label C. albicans was first investigated with optimization of the probe concentration ( Figure S1). Both the D and L-peptides rapidly labeled the membrane of the fungi, without a need for washing steps, in a concentration-dependent manner with fluorescence saturation observed at <10 μM. Although the NBD fluorophore (λ ex = 485 nm) offers advantages (e.g., environmental sensitivity with a high signal to background ratio) over other always-on reporters (e. g., fluorescein), its emission window is in the green autofluorescence window of tissue. To overcome this, the red emissive (λ ex = 595 nm) environmentally sensitive merocyanine dye [19,20,22] (MeroCy-CO 2 H that bears acceptor and donor moieties connected to each other by double bonds) was also conjugated onto P11-6. Similar labeling efficacies were observed for the merocyanine-conjugated probes with no differences observed between the D and L peptides ( Figure 2B). Next, bacterial labeling was investigated using Gram-positive Methicillin sensitive S. aureus and Gram-negative E. coli. The probes NBD-L, NBD-D, MeroCy-L, and MeroCy-D all efficiently labeled bacteria proving that these probes can be utilized for the broad-spectrum labeling of different microbial species (Figure 3). At 10 μM, all the species were efficiently labeled, whereas at lower concentrations Gram-positive S. aureus showed better binding of the probes ( Figure S4). The probes did not bind to mammalian epithelial cells ( Figure S5).

| α-Helical conformation is required for the microbial labeling
The suggested mechanism of action of P11-6 is based on membrane disruption, explained by the Shai-Matsuzaki-Huang model, [23,24] with initial binding explained by the electrostatic interactions between the positively charged peptide and the negatively charged cell membrane phospholipids. Peptide P11-6 has been shown to exhibit amphipathic behavior and adopt an α-helical conformation, [14] with the α-helical or detection of microbes in other samples, with high sensitivity (compared, e.g., to bright-field imaging).