Investigating Alkylated Prodigiosenes and Their Cu(II)‐Dependent Biological Activity: Interactions with DNA, Antimicrobial and Photoinduced Anticancer Activity

Abstract Prodigiosenes are a family of red pigments with versatile biological activity. Their tripyrrolic core structure has been modified many times in order to manipulate the spectrum of activity. We have been looking systematically at prodigiosenes substituted at the C ring with alkyl chains of different lengths, in order to assess the relevance of this substituent in a context that has not been investigated before for these derivatives: Cu(II) complexation, DNA binding, self‐activated DNA cleavage, photoinduced cytotoxicity and antimicrobial activity. Our results indicate that the hydrophobic substituent has a clear influence on the different aspects of their biological activity. The cytotoxicity study of the Cu(II) complexes of these prodigiosenes shows that they exhibit a strong cytotoxic effect towards the tested tumor cell lines. The Cu(II) complex of a prodigiosene lacking any alkyl chain excelled in its photoinduced anticancer activity, thus demonstrating the potential of prodigiosenes and their metal complexes for an application in photodynamic therapy (PDT). Two derivatives along with their Cu(II) complexes showed also antimicrobial activity against Staphylococcus aureus strains.


S-1 Material and general methods
All reagents were purchased from commercial sources and were used without further purification unless otherwise stated. When noted so, the used solvents were degassed by the "freeze-pump-thaw" technique. 1 H and 13 C NMR spectra were recorded at room temperature on a JeolECX 400 (400 MHz), a JeolECZ 400 (400 MHz) or a Bruker AVANCE III 700 (700 MHz) spectrometer. The chemical shifts (δ) are given in parts Hellma cuvettes with 0.5 or 1 mL sample volume on a Cary 100 Bio UV-VIS spectrophotometer (Agilent).
Fluorescence emission spectra were collected using 1 cm path length Hellma fluorescence cuvettes with 1 mL sample volume on a Cary Eclipse fluorescence spectrophotometer (Varian). pH during titration experiments was adjusted using 10 mM MOPS buffer (with 100 mM NaCl, pH 7.4) or 10 mM potassium tert-butoxide/acetic acid (pH 10) [1] , as noted on the respective measurement. Flash chromatography was performed on silica gel (40-63 μm) or basic aluminum oxide (activity grade I, 50-200 μm) as specified in the respective procedure. RP-HPLC (Chromaster 5000, VWR, Hitatchi) was carried out over a C18 column

UV/VIS melting curves:
CT-DNA (50 µM) melting curves were measured in buffered solution (10 mM MOPS, 0.05% DMSO, pH 7.4) in presence of the ligands or in situ formed Cu(II) complexes (2.5 µM) using a Cary 100 Bio UV/Vis spectrophotometer at 260 nm (heating rate 0.5 °C/min). Melting curves were normalized and used for the calculation of the melting temperatures.

Ethidium bromide (EB) displacement studies:
The fluorescence emission spectra of intercalated EB were recorded using a Cary Eclipse fluorescence spectrophotometer (Varian). A solution of CT-DNA (20 µM) and EB (1.3 µM) in buffered aqueous solution (10 mM MOPS, 100 mM NaCl, pH 7.4) was treated with increasing concentrations of the ligands in presence or absence of equimolar concentration of copper acetate. The fluorescence spectra were collected after each addition between 540 and 730 nm using an excitation wavelength of 518 nm (photomultiplier voltage:

Circular dichroism (CD) spectroscopy:
CD spectra of CT-DNA (100 µM) in buffered aqueous solution (10 mM MOPS, 100 mM NaCl, pH 7.4) were collected on a Jasco J-810 spectrometer in the range of 220 to 320 nm with a measuring velocity of 100 nm/min, and a data point interval of 0.1 nm. Increasing concentrations of the ligands or the in situ formed Cu(II) complexes were used. Final DMSO concentrations remained below 0.4%.

DNA cleavage studies:
Plasmid DNA pBR322 (0.025 µg µL -1 ) was incubated with the prodigiosenes in presence and absence of equimolar concentrations of copper acetate (5-150 µM) for 1 h at 37 °C in MOPS buffer (10 mM, 100 mM NaCl, pH 7.4). The final reaction volume was adjusted to 8 µL by adding deionized water with a final DMSO concentration below 0.8%. After incubation 1.5 μL of loading buffer containing 3.7 mM bromophenol blue and 1.2 M saccharose was added to the incubation solution and loaded onto an agarose gel (1% in 0.5X Tris-borate-EDTA (TBE) buffer) containing EB (0.2 μg mL -1 ). Electrophoresis was carried out with an electrophoresis unit (Carl Roth; power supply: consort EV243) for 2 h at 40 V. DNA bands were visualized by fluorescence imaging of intercalated EB using a Bio-Rad GelDoc EZ Imager. Data analysis was performed with Bio-Rad's Image Lab Software (Version 3.0). Due to the decreased affinity of ethidium bromide to supercoiled DNA a correction factor of 1.22 was used. [2] In order to ensure reproducibility, all DNA cleavage experiments were performed in triplicate, and the standard deviation was used to calculate the error bars.

Detection of reactive oxygen species (ROS) by quenching of DNA cleavage:
The general DNA cleaving procedure as described before was applied, using a fixed concentration of the in situ formed Cu(II) complexes of 70 μM. The DMSO concentration before further addition of DMSO as scavenger was 0.4% (56 mM). Subsequently one of the following ROS scavengers was added to the incubation solution: DMSO (additional 400 mM), NaN3 (10 mM), pyruvate (2.5 mM) or superoxide dismutase (625 U/mL). One lane containing both, pyruvate and SOD, was also included.

In situ formation of the copper(II) complexes for the cell culture experiments:
For the in situ synthesis of the copper complexes a stock solution (2 mM) of the respective prodigiosene in DMSO was mixed with an equimolar amount of the copper(II) acetate solution in deionized water and diluted with the medium used in the cell culture experiments to the desired final concentrations (2 μM or 10 μM).

Cell viability assays:
Human epidermoid carcinoma A253 cells, human oral adenosquamous carcinoma CAL27 cells, colorectal adenocarcinoma HT29 cells, and mouse fibroblast L929 cells were grown in Dulbecco's modified eagle medium (DMEM) with 10% heat inactivated FCS, 1% penicillin (10,000 IU) and streptomycin (10,000 μg mL −1 ). DMEM (without phenol red) with 10% FCS was used for dilution to reach 2 or 10 μM concentration of the respective compound to be tested. In microplates 2 × 10 4 cells per well were seeded in fresh medium (DMEM without phenol red) containing 10% FCS with 2 μM or 10 μM of the compound and incubated for 24 h. After exchange of medium (to remove any compound not taken up by the cells), the photosensitization was performed at RT with a white light source (Zeiss) for 40 s at a dose rate of approx. 50 J cm −2 . After 24 h recovery, the cell viability of the samples was assessed using the XTT assay [3] and the absorbance was measured with a Tecan Infinite 200 microplate reader, at a wavelength of 490 nm. A wavelength of 690 nm was used to measure the reference absorbance.
For a comparison between cancer cells and healthy cells, both of human epithelial origin in the colon, HT29 cells were grown in DMEM with 10 % FCS, 1% penicillin (10,000 IU) and streptomycin (10,000 μg mL −1 ), and CCD 841 CoN cells (ATCC CRL-1790) were grown in MEM with 10 % FCS and 1% penicillin (10,000 IU) and streptomycin (10,000 μg mL −1 ). 2 × 10 4 HT29 cells or 8.5 × 10 3 CCD 841 CoN cells were seeded in black microwell plates (greiner bio-one). Compounds to be tested were diluted to 2 µM or 10 µM in the respective culture media, added to the plates 24 h after seeding and incubated for 24 h. Medium was exchanged at the end of the incubation time to remove any compound not taken up by the cells and photosensitization was performed at RT with a white light LED source (19 mW) for 43 min at a dose rate of approx. 50 J cm −2 . After 24 h recovery the cell viability was assessed using the XTT assay.

ROS assay in cells:
1.1875 × 10 5 HT29 cells per well or 5.05 × 10 4 CCD 841 CoN cells per well were seeded in 24 well plates.
Compounds to be tested were diluted to 10 µM in the respective culture media, added to the plates 24 h after seeding and incubated for 1 h. Medium was exchanged at the end of the incubation time to remove any compound not taken up by the cells and photosensitization was performed immediately at RT with a white light LED source (19 mW) at a dose rate of approx. 50 J cm −2 . Directly after the photosensitization, 5 µM CellROX ® Orange was added to the cells and incubated for 30 min. Cells were washed twice with PBS, removed from the plates, collected in round bottom tubes, resuspended in 0.5 mL BD FACSFlow™ and fluorescence measured with BD FACSCalibur™. Results were analyzed using the FlowJo software.

Antimicrobial Susceptibility testing (AST):
The antimicrobial susceptibility of C0, C6 and C16 as well as their copper complexes CuC0, CuC6 and CuC16 was investigated according to the standards of the Clinical and Laboratory Standards Institute. [4,5] For the antimicrobial susceptibility testing two-fold dilution series of the prodigiosenes were prepared in 50 µL cation-adjusted Mueller-Hinton broth (CAMHB) in the double concentration of the test concentration required in the rows 1 to 10 of the microtiter plates. Row 11 was used as growth control and was filled with 50 µL of CAMHB and row 12 was used as sterility control and filled with 100 µL of CAMHB. For the first investigation of the antimicrobial activity six reference strains commonly used for AST and biocide efficacy

S-2 Synthesis of ligands
Ligands were synthesized based on procedures adapted from the literature [6] (Scheme S-1). Modifications and peculiarities of the syntheses are described in the following. The mono-alkylated pyrroles 3-5 were prepared from 1H-pyrrole 2 and the corresponding alkyl bromide, as it is described in the literature procedure. [7] The carboxaldehydes 6-9 were thereupon easily accessible via a Vilsmeier-Haackformylation. It is to be noted that, especially in the case of the hexadecyl derivative 9, the treatment with saturated Na2CO3 solution during workup was insufficient to completely hydrolyze the intermediately generated dimethyl iminium salt 9b. The air stable iminium salt could, however, be further hydrolyzed to Our attempts to synthesize and isolate the boronic acid 18 following the procedure established in the literature, [8] and subsequently using it in the cross-coupling reaction only afforded the desired product in low yields (29%). We attribute the low yield in part to the instability of the pyrrole boronic acid 18, which is known to be prone to protodeboronation, [9][10][11][12] but also to the low thermal stability of the triflates 14-17 under the reaction conditions (90 °C). The thermal decomposition of 14-17 into a complex product mixture can be observed when exposing the triflates to the coupling conditions in absence of the boronic acid. The use of the MIDA-boronate, an air-stable precursor of the boronic acid as described by Burke et al., [8] did not improve the overall yield of the coupling step (14-24%). In our experience, an improvement of the yield for the cross-coupling reaction was only achieved by using a larger excess of the boronic acid (8-10 eq.) and when performing the synthesis of the boronate and the ensuing cross-coupling in a one-pot fashion at room temperature, without isolation of the sensitive boronic acid. Final Boc-deprotection occurs simultaneously during purification of the desired prodigiosene due to basic conditions. The different reaction conditions used for the cross-coupling step are described in the following section. This section also includes the NMR spectroscopic characterization of precursors 9b, 10, 11, 13, 14, 15, and 17, which as such are not described in the literature. Scheme S-1. Synthesis pathway used for the preparation of prodigiosenes C0, C6, C11, and C16.
The crude product was purified by column chromatography over aluminum oxide eluting with a hexane:ethyl acetate 4:1 mixture. The fraction containing the title compound was dissolved in 1-2 mL of ethanol and treated with three drops of HCl (25% in water), to yield the hydrochloric salt of C11 as a red solid (90 mg, 14%).
Following the same procedure as for C11, although using 15 as a precursor and tetrakis(triphenylphosphine)palladium(0) (5 mol%) as catalyst, C6 was obtained as a red solid (24%).

Boc-pyrrole-2-boronic acid
A solution of the triflate 16 (418 mg, 0.877 mmol) in abs. degassed THF (30 mL) under argon atmosphere was treated in sequence with N-Boc-pyrrole-2-boronic acid (740 mg, 3.51 mmol), tetrakis(triphenylphosphine)palladium(0) (50 mg, 0.044 mmol), and potassium carbonate (969 mg, 7.02 mmol) and stirred for 6 h at 90 °C. After extraction with ethyl acetate (3 x 50 mL), the combined organic layers were washed with water (30 mL) and brine (50 mL), dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified by column chromatography on basic aluminum oxide, eluting with a hexane:ethyl acetate 4:1 mixture to yield C11 as a red solid (100 mg, 29%). Further purification of the title compound before the biological studies was achieved through precipitation of the hydrochloric salt from a solution in ethanol after the addition of three drops of HCl (25% in water), followed by purification through HPLC over a C18 column (mobile phase: ethanol:water 8:2).

Synthesis of prodigiosenes C0, C6, and C16 through a one-pot reaction with pyrrole boronate
A solution of 2,2,6,6-tetramethylpiperidine (0.34 mL g, 2.00 mmol) in abs. degassed THF (10 mL) was Following the one-pot procedure as for C6, although using triflate 17, C16 was obtained and purified by column chromatography on basic aluminum oxide eluting with a hexane:ethyl acetate 4:1 mixture. The fraction containing the title compound was dissolved in 1 mL of ethanol and treated with three drops of HCl (25% in water) to yield the hydrochloric salt of C16 as a deep red-violet solid (73%). Further purification of the title compound before the biological studies was achieved through HPLC over a C18-column (mobile phase: ethanol:water 95:5).
Following the one-pot procedure as for C6, although using triflate 14, C0 was obtained and purified by column chromatography on basic aluminum oxide eluting with a hexane:ethyl acetate 4:1 mixture to yield The 1 H NMR data for C6 are reported in the literature, [14] but deviate significantly from our results.

S-6 Ethidium bromide displacement studies
The ethidium bromide (EB) displacement data were evaluated by using the Stern-Volmer equation (1) where I0