Rationales Design von Phe‐BODIPY‐Aminosäuren als fluorogene Bausteine für den peptidbasierten Nachweis von Candida‐Infektionen im Harntrakt


 Pilzinfektionen, die durch Candida‐Arten verursacht werden, gehören zu den häufigsten Infektionen bei Krankenhauspatienten. Die derzeitigen Methoden zum Nachweis von Candida‐Pilzzellen in klinischen Proben beruhen jedoch auf zeitaufwändigen Analysen, die eine schnelle und zuverlässige Diagnose erschweren. In diesem Beitrag beschreiben wir die rationale Entwicklung neuer Phe‐BODIPY‐Aminosäuren als kleine fluorogene Bausteine und ihre Anwendung zur Erzeugung fluoreszierender antimikrobieller Peptide für die schnelle Markierung von Candida‐Zellen im Urin. Mit Hilfe von computergestützten Berechnungen haben wir das fluorogene Verhalten von BODIPY‐substituierten aromatischen Aminosäuren analysiert und Bioaktivitäts‐ und konfokale Mikroskopieexperimente bei verschiedenen Stämmen durchgeführt, um den Nutzen und die Vielseitigkeit von Peptiden mit Phe‐BODIPYs zu bestätigen. Schließlich haben wir einen einfachen und sensitiven fluoreszensbasierten Test zum Nachweis von Candida albicans in menschlichen Urinproben entwickelt.



General experimental information.
Compounds 1b-d, 3, 8 and 9 were synthesized according to modified literature procedures. [1]  Other chemicals were obtained from commercial sources and were used without further purification.Thin-layer chromatography was conducted on Merck silica gel 60 F254 sheets and visualized by UV (254 nm and 365 nm).Reactions were monitored by HPLC-MS analysis using a HPLC Waters Alliance HT comprising a Kinetex C18 column (5 µm, 100 Å, 150 x 4.6 mm), a diode array and a MS detector configured with an electrospray ionization source (micromass ZQ4000).A: H 2O (0.1% HCOOH) and B: CH3CN (0.1% HCOOH) were used as eluents in a gradient from 0-100% B over 8 minutes.Data acquisition was performed with MassLynx software.NMR spectra were recorded on a 500 MHz spectrometer.Chemical shifts (δ) are reported in ppm.Multiplicities are referred by the following abbreviations: s = singlet, d = doublet, t = triplet, dd = double doublet, dt = double triplet and m = multiplet.HRMS (ESI positive) were obtained with a Bruker ESI Micro-TOF mass spectrometer.MALDI analysis was performed on a Bruker UltrafleXtreme MALDI TOF-TOF mass spectrometer.All microwave reactions were carried out in 10 mL sealed glass tubes in a focused mono-mode microwave reactor (Biotage) featured with a surface sensor for internal temperature determination.Cooling was provided by compressed air ventilating the microwave chamber during the reaction.When stated, the final crude was purified via flash column chromatography CombiFlash ISCO RF provided with dual UV detection.
Then, 2-methyl-1H-pyrrole (416 μL, 4.7 mmol) and three drops of TFA were added and the reaction was stirred overnight at r.t in N 2 atmosphere.The complete consumption of the aldehyde was checked by TLC.DDQ (489 mg, 2.2 mmol) dissolved in DCM (20 mL) was added dropwise (10-15 min) to the reaction mixture and the reaction was stirred for 15 min at r.t.Finally, TEA (6.3 mL, 45 mmol) and BF 3•OEt2 (3.7 mL, 30 mmol) were added, and the mixture stirred overnight at r.t.Workup was done by diluting with DCM (20 mL) and washing with H 2O (2 x 50 mL).The organic layers were combined, dried over sodium sulphate, filtered, and concentrated under vacuum.The crude was purified via flash column chromatography S3 using a EtOAc/hexane gradient on silica gel.The desired compound 1a was isolated as a red solid (118 mg, 13%).
The expected adduct was isolated as a dark red solid (44 mg, 65%).
After cleavage as described above, the crude peptide was precipitated by adding cold Et 2O (dropwise) and the resulting precipitate was decanted and dried (x2), obtaining 29 mg of benzyloxycarbonyl (Z) lysine-protected peptide.The crude peptide (24 mg, 0.013 mmol) was deprotected by means of hydrogenation.Peptide was dissolved in HCOOH/DMF/MeOH (0.05:3.3:1) (1.9 mL), followed by addition of 20% Pd(OH)2/C (6.1 mg, ca.quarter of peptide mass).Then, the reaction flask was flushed with N2/vacuum cycles (×3) and filled with H2.The reaction mixture was stirred under H 2 at r.t. for 1 h (monitored by HPLC-MS).The catalyst was removed by filtration under Celite and washed with MeOH.Filtrate was collected in a round bottom flask and solvent was removed under reduced pressure.The residue was dissolved in CH 3CN:H2O and lyophilised.Purification was conducted by semi-Preparative HPLC using a 0-50% gradient over 25 min, with detection at 220 and 280 nm.Pure fractions were collected and lyophilised to afford pure peptide 12 as a white solid (2.3 mg, 10% yield).The synthesis was performed on 52 mg of Sieber Amide resin (0.57 mmol/g).Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Ser(Trt)-OH, Fmoc-Lys(Mmt)-OH, Fmoc-Phe-OH and Fmoc-Pro-OH were used as building blocks, After cleavage as described above, the fully deprotected peptide was precipitated by adding cold Et 2O (dropwise) and the resulting precipitate was decanted and dried (×2).Purification was conducted by semi-Preparative HPLC using a 0-50% gradient over 25 min, with detection at 220 and 260 nm.Pure fractions were collected and lyophilised to afford pure peptide 13 as a white solid (14 mg, 50% yield).
Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Ser(Trt)-OH, Fmoc-Lys(Boc)-OH and Fmoc-Phe-OH were used as building blocks.After cleavage as described above, the peptide crude was precipitated by adding cold Et 2O (dropwise) and the resulting precipitate was decanted and dried to afford 36 mg of a white solid corresponding to the protected peptide.Then, 16 mg of cleaved peptide (1.0 eq.), PyOxim (1.5 eq.) and OxymaPure (1.5 eq.) were dissolved in DMF:ACN (1:1, 0.001 M).After setting the cocktail at -10 ˚C using a salted ice bath, DIPEA (3.0 eq.) was added, and the mixture stirred overnight at r.t.After solvent removal under reduced pressure, the crude peptide was dissolved in TFA:TIS:H2O (95:2.5:2.5) for 1h to remove the side-chain protecting groups.Then, the crude was concentrated under reduced pressure followed by precipitation in cold Et 2O (dropwise).
Purification was conducted by semi-Preparative HPLC using a 0-60% gradient over 25 min, with detection at 220 and 260 nm.Pure fractions were collected and lyophilised to afford pure peptide 14 as a white solid (3.0 mg, 26% yield from cyclization step).
Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH and Fmoc-Trp-OH were used as building blocks.After cleavage as described above, the peptide crude was precipitated by adding cold Et 2O (dropwise) and the resulting precipitate was decanted and dried to afford 36 mg of a white solid corresponding to the protected peptide.Then, 16 mg of cleaved peptide (1.0 eq.), PyOxim (1.5 eq.) and OxymaPure (1.5 eq.) were dissolved in DMF:ACN (1:1, 0.001 M).After setting the cocktail at -10˚C using a salted ice bath, DIPEA (3.0 eq.) was added and the mixture stirred for 2 days at r.t.After solvent removal under reduced pressure, the crude peptide was dissolved in TFA:H 2O:DCM (30:2.5:67.5)for 40 min to remove the side-chain protecting groups.Then, the crude was concentrated under reduced pressure followed by precipitation in cold Et2O (dropwise).Purification was conducted by semi-Preparative HPLC using a 0-50% gradient over 25 min, with detection at 220 and 280 nm.Pure fractions were collected and lyophilised to afford pure peptide 16 as a white solid (1.9 mg, 16% yield from cyclization step).The synthesis was performed on 8.6 mg of Sieber Amide resin (0.57 mmol/g).Fmoc-Leu-OH, Fmoc-His(Mmt)-OH, Fmoc-Ile-OH, Fmoc-Ser(Trt)-OH, Fmoc-Ile-OH, Fmoc-Lys(Mmt)-OH, Fmoc-Phe(pMP-BODIPY)-OH ( 11) and Fmoc-Pro-OH were used as building blocks.After cleavage as described above, the fully deprotected peptide was precipitated by adding cold Et 2O (dropwise) and the resulting precipitate was decanted and dried (x2).Purification was conducted by semi-Preparative HPLC using a 20-60% gradient over 25 min, with detection at 220 and 572 nm.Pure fractions were collected and lyophilised to afford pure peptide 17 as a purple solid (1.7 mg, 26% yield).
Pure fractions were collected and lyophilised to afford pure peptide 18 as an orange solid (3.0 mg, 27% yield).

Computational details
DFT and TD-DFT calculations were performed with the M06-2X hybrid exchange-correlation functional [3] and the 6-311+G(2d,p) Pople basis set as implemented in the Gaussian 09 [4]   package.This choice is supported by previous benchmarks performed on aza-BODIPY and BODIPY dyes, [5] which demonstrate that this level of theory provides good consistency with experimental trends for optical spectra, yet a systematic overshooting of the transition energies (by c.a. 0.4 eV).However, this systematic error is not a concern for the present study as we are not interested in theoretically reproducing the experimental spectra, rather than comparing the different molecules studied (e.g., transition state barriers) on the same footing.Numerical frequency calculations were used to ascertain the nature of the stationary points and the same increased integration grid (i.e., ultrafine (99,590)) respect to the default setting was used in all computations as this is recommended for describing correctly very low frequency modes.

Culture of fungal strains.
All strains used in this experiment were grown on SAB agar at 37 °C for 3 days.Cells were harvest using sterile inoculation loop by taking a single colony and resuspending in PBS supplemented with 0.1% tween-20 (PBST).The concentration of cells was then quantified with a haemocytometer.For the determination of the minimum inhibitory concentration, cell density was adjusted to 10 6 cells mL -1 with 20% liquid Vogel's medium.

Culture of E. coli.
E. coli was grown on Lysogeny Broth (LB) agar at 37 °C for 1 day and harvested using sterile inoculation loop by taking a single colony and resuspending in PBST.The number of cells was quantified using a haemocytometer.For the determination of the minimum inhibitory concentration, cell density was adjusted to 10 6 cells mL -1 with 20% liquid LB medium.

In vitro measurements of minimum inhibitory concentrations.
Minimum inhibition concentration (MIC) measurements were performed as described previously with minor changes. [7]Each compound was dissolved in DMSO at concentration of 100 mM, this was used as the stock solution.For testing the MIC, the stock solution was further diluted in water to reach a concentration of 1 mM and was added in to a 96-well plate cell culture plate.A serial dilution was then performed within the 96-well plate, and the compound solutions at different concentrations were then mixed with conidia suspended in 20% Vogel's medium to reach a final volume of 100 µL per well.The final conidia concentration was 5 × 10 5 cells mL -1 in 20% Vogel's medium, the highest tested concentration of each compound was at 50 mg mL -1 .After 48 h incubation at 37°C, MIC was determined by brightfield microscopy from three independent experiments (n = 3).For testing the MIC against E. coli, the same protocol was followed apart from using liquid LB as the medium at a final concentration of 10%.

Confocal live-cell microscopy.
Probe 17 was mixed with Candida spp. to reach a final concentration of 10 µM and a final cell concentration of 5 × 10 5 cells/mL in PBS.The cells combined with the peptide were dispensed into the wells of Ibidi μ-slide 8 well (Ibidi GmbH, Germany) and incubated for 10 min at r.t.
Live cell imaging of the germinated spores was performed using a Leica TCS SP8 confocal laser scanning microscope equipped with photomultiplier tubes, hybrid GaAsP detectors and a 63× water immersion objective and white light laser (575 nm was used for excitation S13 wavelength and 600-650 nm was used for emission).Images were taken at 15, 30 and 60 min timepoints.Images were processed using Imaris software 8.0 developed by Bitplane (Zurich, Switzerland).

Measurements of the limit of detection.
The limit of detection (LoD) was determined by fluorescence titration of serial dilutions of C. albicans cells spiked into human urine samples from healthy donors.For probe 17, samples were previously incubated with probe 17 for 1 h at 37 °C.The colony forming units (CFU) were determined from the interpolation of a standard curve from the plotting of the CFU counting of a series of C. albicans cultures vs. their associated OD 600 measurements.The LOD was calculated using the equation LoD = (3 x σ)/k, were σ is the standard deviation of blank solutions and k is the slope of the linear regression fit from the fluorescence emission vs.