Red-emission, photobiological properties and antimicrobial photodynamic assays using mono-substituted corroles contain carboxylic acid and ester units

doi:10.21203/rs.3.rs-3797476/v1

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Red-emission, photobiological properties and antimicrobial photodynamic assays using mono-substituted corroles contain carboxylic acid and ester units

https://doi.org/10.21203/rs.3.rs-3797476/v1

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published 31 May, 2024

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The mono-substituted corrole derivatives COOH-Cor and COOMe-Cor were obtained by Gryko methodology and characterized by microanalysis. In addition, we investigated experimental photophysical properties (absorption, emission by steady-state and time-resolved fluorescence) in some solvents and TDDFT calculations. Photobiological parameters such as aggregation, photostability and reactive oxygen species generation (ROS) was conducted. In addition, we also evaluated the preliminary antimicrobial photodynamic assays (aPDT) between studied corroles and bacteria strains, by white-light conditions.

Corroles

Photophysics

TD-DFT

Photobiology

Microbiology

aPDT

Corroles are known as photosensitizers for use in photophysical and/or photobiological processes and have recently drawn attention for their great capacity to generate reactive oxygen species (ROS), such as singlet oxygen and radical species [,]. In general, theses class of compounds presents low cytotoxicity, photostability and advantageous use of light in wavelengths up to near-infrared, which facilitates the application of these derivatives in photodynamic processes [].

Corroles have a tetrapyrrolic skeleton with a direct bond between two pyrrole rings, as well as an 18π-electron porphyrin-like system and has attracted significant attention as a new photosensitizer []. Understanding the general photophysical nature of corrole derivatives is also necessary for their application in photobiological processes such as photodynamic therapy (PDT) and antimicrobial photodynamic therapy (aPDT), as photophysical parameters such as excited state lifetime, intersystem crossing (ISC), radiative (k_r) and non-radiative (k_nr) rates and different quantum yields of the sensitizers have specific effects on the functionality of corroles, including their ability to ROS formation [,,].

In the present manuscript, free-base meso-10-(4-carboxyphenyl)-5,15-pentafluorophenyl COOH-Cor and meso-10-(4-methylbenzoate)-5,15-pentafluorophenyl COOMe-Cor derivatives were evaluated by photophysical and theoretical TD-DFT analysis. Moreover, aggregation, photostability, ROS generation and preliminary microorganism photo-inactivation properties were evaluated. Also, the influence of the unprotected (COOH) and protected (COOMe) group will also be discussed and analyzed (Fig. 1).

General

All reagents used were of analytical grade and purchased from Sigma‐Aldrich^® or national suppliers, with no preliminary purification. Column chromatography was executed using silica‐flash 230–400 mesh from Sigma‐Aldrich^®. Analytical preparative TLC was performed on aluminum sheets (1.0 mm thick) with Merck^® TLC silica gel 60.

Corroles COOH-Cor and COOMe-Cor were prepared according to Gryko’s methodology [⁸,⁹] and used as molecules to evaluate their photophysical/photobiological processes and microorganism photo-inactivation. The CHN% elemental analysis of derivatives are listed in the Supplementary information section (Table S1).

Photophysical

Absorption UV-Vis spectra were obtained using the Shimadzu UV-2600 spectrophotometer (1.0 cm optical path length) and measured in the 250–800 nm region for studied corroles COOH-Cor and COOMe-Cor in several solvents such as acetonitrile (MeCN), dichloromethane (DCM), methanol (MeOH), dimethyl sulfoxide (DMSO) and DMSO(5%)/Tris-HCl pH 7.4 mixture buffered solution, with fixed concentrations of 10 μM. For the determination of the steady-state fluorescence emission spectra, we employed a fluorimeter Horiba Fluoromax Plus, where the corroles were dissolved at a fixed concentration of 5.0 μM, in a 1.0 cm optical path length cuvette, excited at Soret transition band (slit 2.0; Em/Exc).

The fluorescence quantum yields (Φ_f) of the corrole derivatives COOH-Cor and COOMe-Cor were measured according to the literature, using meso-tetra(phenyl)porphyrin (TPP) in DMF solution as the standard molecule [¹⁰]. Fluorescence lifetimes (τ_f) were recorded using the Time Correlated Single Photon Counting (TCSPC) method with DeltaHub controller in conjunction with Horiba fluorimeter. Data were processed with DAS6 and Origin^® 8.5 software (Northampton, MA, USA) using mono-exponential fitting of raw data. NanoLED (Horiba) source (1.0 MHz, 441 nm excitation wavelength) was used as an excitation source. The instrumental response function (IRF) was collected using a Ludox® dispersion (Sigma-Aldrich^®, St. Louis, MO, USA). Radiative (k_r), non-radiative (k_nr), radiative (τ_r) and non-radiative (τ_nr) decay values were calculated by equations according to the current literature [¹¹].

Theoretical calculation by DFT

Geometries optimizations and harmonic frequency calculations of COOH-Cor and COOMe-Cor were carried out using the density functional theory (DFT) under the protocol TPSSh/def2-TZVP; Ahlrich’s triple-zeta basis set were applied to all atoms [¹²,¹³].

Time-dependent DFT (TD-DFT) calculations were also carried out, but we have changed the DFT functional to mPWPW, based on the work of benchmark [¹⁴,¹⁵]. These calculations were conducted in dichloromethane (DCM), dimethyl sulfoxide (DMSO), acetonitrile (MeCN) and methanol (MeOH) using the solvation model density (SMD). It uses the conductor-like polarizable continuum model (CPCM) as implemented on ORCA version 5.0.2 to calculate the electrostatic solvation energy [¹⁶,¹⁷,¹⁸].

Spin-orbit coupling effect (SOC) were considered in these calculations to calculate mixed singlets and triplets states after including the coupling effect. Chemcraft program has been used as standard visualization software to help to generate the figures related to this part [¹⁹]. All calculations have been performed at Dell servers on Núcleo de Estudos em Química Computacional (NEQC – UFJF).

Photobiological assays

Aggregation study by UV-Vis absorption

Aggregation by UV-Vis absorption analysis was conducted as a function of successive increase in corrole concentration (0.5 to 60 μM) in DMSO(5%)/Tris-HCl pH 7.4 mixture buffered solution and changes in the Soret band in the 250–800 nm range were monitored. The stability experiments in DMSO and in DMSO(5%)/Tris-HCl pH 7.4 mixture buffered solution of studied corrole was also monitored by absorption UV–Vis measurements at five days. All experiments were performed in duplicate and independently.

Photostability assays

The photostability experiments of corroles COOH-Cor and COOMe-Cor was conducted in DMSO(5%)/Tris-HCl pH 7.4 buffer mixture solution and were also monitored by absorption electronic UV–Vis measurements at different exposure times (0 to 60 min) under white-light LED array system (400 to 800 nm) at irradiance of 50 mW cm⁻² and a total light dosage of 180 J cm⁻². All experiments were performed in duplicate and independently.

Singlet oxygen generation

Singlet oxygen (¹O₂) photo-degradation experiments were performed using the quencher 1,3-diphenylisobenzofuran (DPBF) [²⁰], with a maximum volume of 2.0 mL containing 50 μM DPBF in DMSO, DMF or MeCN, mixed with each corrole as 0.5 mL (50 μM) in the same solvent. To measure ¹O₂ formation, UV-Vis absorption spectra were recorded for each solution (sample and standard) at different exposure times using a green diode laser (TheraLase DMC, 532 nm, São Carlos, Brazil) at a distance of 1.0 cm from the sample, with average power at 100 mW. The ¹O₂ quantum yield (Φ_Δ) and photo-oxidation (k_po) constants generated was calculated according to literature, using meso-tri(phenyl)corrole (TPhCor) dissolved in DMSO (Φ_Δ = 0.67; k_po = 0.654 M^‒1 s^‒1) as a standard molecule [7].

Superoxide generation assays

The superoxide radical (O₂^•‒) species by Nitro Bule Tetrazolium (NBT) reduction, with NADH (reduction agent) assays in DMF solution and this approach was carried out at the same conditions stated in the literature [²¹]. Control experiments were performed in the absence of corroles and all derivatives were irradiated under aerobic conditions with a white-light LED source (irradiance of 50 mW cm⁻² and a total light dosage of 60 J cm⁻²) at a period of 20 min. The progress of the reaction was monitored by following the increase in the absorbance close to 560 nm. The superoxide generation constant (k_SO) values can be obtained according to the literature cited above.

Water/n-octanol partition coefficients (log P_OW)

The log P_OW value was calculated and recorded according to the literature [²²], using the maximum absorbances of the Soret band and volumes in the organic and aqueous phases, respectively.

Photo-inactivation assays in microbiology

Microorganisms

American Type Culture Collection (ATCC) strains were used: Gram negative Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 700603), Pseudomonas aeruginosa (PAO1), Salmonella enteretidis (ATCC 13076) and Gram-positive Staphylococcus aureus (ATCC 29213) and Methicillin-resistant Staphylococcus aureus (MRSA a clinical isolate), respectively. The microorganisms were kept in glycerol culture medium and held to −80°C. The sample was defrosted, inoculated into Brain Heart Infusion (BHI) broth and incubated for 24 h. After, the samples were seeded on Blood Agar Base and incubated for more 24 h at 37°C.

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determination

The MIC determination test was performed by the 96-well microplate broth microdilution method according to CLSI M100-S23 [²³]. The determination of the minimum inhibitory concentration (MIC) was carried out using the microdilution technique in broth with different concentrations of corroles COOH-Cor and COOMe-Cor (220 µM – 25 µM), using to a stock concentration of 440 µM. Different concentrations were used in the assay under dark and white-light LED irradiation conditions at 60 minutes (irradiance of 50 mW cm⁻² and a total light dosage of 180 J cm⁻²) and incubated for 24h at 37ºC. The lowest concentration that did not show visible bacterial growth was considered to MIC value. To determine the minimum bactericidal concentration (MBC), 1.0 μL was removed from each well and plated on a nutrient agar plate, incubated for 24 hours at 37°C. CBM values was considered the lowest concentration that did not show bacterial growth on the plate. All assays were performed in duplicate.

Association with potassium iodide (KI) assays

Experiments for corroles COOH-Cor and COOMe-Cor with KI associations were carried out the same way as described above, although a fixed KI concentration (100 mM) was used. Two plates were separated for each association; the dark/white-light irradiation conditions, incubation, and reading conditions were performed.

Scavengers of reactive oxygen species assays

In an attempt to determine possible ROS produced by the studied corroles, ascorbic acid (AA; Sigma-Aldrich^®), dimethyl sulfoxide (DMSO; Sigma-Aldrich^®), tert-butanol (t-BuOH; Merck^®), ethylenediamine tetra-acetic acid (EDTA; Sigma-Aldrich^®) and N-acetylcysteine (NAC; Sigma-Aldrich^®) ROS scavengers were used to detect oxygen reactive species in the following sequence: singlet oxygen (¹O₂), superoxide radical (O₂^•−), hydroxyl radical (•OH), ion chelators (membrane destabilization) and hydroperoxyl radical (•OOH), respectively.

The dilution of the corrole solutions was maintained under the same conditions as the MIC determination. Sub-inhibitory concentrations of each scavenger (AA = 31.0 mM; DMSO = 3.0%; t-BuOH = 3.0%; EDTA = 31.0 mM and NAC = 31.0 mM) and the standardized inoculum were added to the 96-well microplates. The plates were kept at 37ºC for 24 h with white-light LED irradiation periods of 60 min (irradiance of 50 mW cm⁻² and a total light dosage of 180 J cm⁻²), respectively. The MIC variation values were used to determine the type of ROS produced by the corroles [²⁴].

Checkerboard assay

The interaction of studied corroles with the commercial antimicrobial Imipenen (IMP; Sigma-Aldrich^®) against Pseudomonas aeruginosa PA01 was performed by the checkerboard assay [²⁵]. For this assay, 50 μL of MHB was added to a 96-well plate. Then, an IMP solution four times more concentrated was prepared, and 50 μL was placed in all row 2 performing a serial dilution up to well 10. Separately, twice as concentrated concentrations of corroles COOH-Cor and COOMe-Cor were prepared, and 50 μL of each concentration was arranged horizontally in rows B, C, D, E, F, G, and H. Finally, 10 μL of the bacterial inoculum was added to all wells except for column 11, which received only MHB as a negative control. Column 12 only received MHB and inoculum as a positive control. The plate was irradiated with white-light LED irradiation conditions for 60 min (irradiance of 50 mW cm⁻² and a total light dosage of 180 J cm⁻²) and incubated for 24 h at 37ºC.

The assay was performed in triplicate on different days. The possible interaction of the corroles with the antimicrobial was analyzed using the fractional inhibitory concentration index (FICI), which was calculated using the following formula: FICI_A+ FICI_B= (MIC_A in association/MIC_A alone) + (MIC_B in association/MIC_B alone). Next, the possible interaction was classified as synergistic (FICI ≤ 0.5), indifferent (0.5 < FICI < 4.0), or antagonistic (FICI ≥ 4.0).

Statistical analysis

GraphPad Prism version 5.0 (GraphPad Software, Inc., California, USA) was used to perform the statistical analysis. Statistical analysis and data were expressed as mean ± standard deviation. Experiments were performed in duplicate.

General aspects of corroles

Mono-substituted corroles containing carboxylic acid (COOH) or ester (COOMe) peripheral groups are compounds of photochemical and photobiological interest. These derivatives were obtained using a methodology already described by Gryko and co-workers [8,9], by the reaction between a selected dipyrromethane and an aldehyde, under acidic conditions, followed by oxidation by a quinone (Scheme 1). This interest is because these compounds can be better ROS generators when activated with adequate light irradiation. According to the literature, these derivatives can generate several species, such as ROS by energy transfer processes (Type II) and/or ROS by electron transfer processes (Type I), having photophysical properties suitable for application in photodynamic processes. In addition, these corrole derivatives can be considered stable in solution, do not present aggregation phenomena, and are photo-stable in the presence of white-light irradiation.

Scheme 1. Synthesis of corroles COOH-Cor and COOMe-Cor used in this study. Steps: (i) TFA, Ar atm, 5 min.; (ii) MeOH/H₂O, HCl 37%, r.t., 2 h; (iii) DDQ, DCM, r.t., 15 min.

Photophysical properties of corroles COOH-Cor and COOMe-Cor

The UV-Vis absorption spectra in the visible region of the studied corroles COOH-Cor and COOMe-Cor in some solvents (DCM, MeCN, MeOH, DMSO and DMSO(5%)/Tris-HCl pH 7.4 buffer mixture) are recorded and, as example, absorption spectra of corrole COOH-Cor shown in Figure 2a. The data referring to molar absorptivities (ε) and wavelengths of the main transitions (λ_abs) are listed in Table 1. The absorption spectra of the corrole COOMe-Cor are shown in the Supplementary information section (Figure S1).

In general, all corroles in all solvents used showed typical electronic transitions already predicted for this type of macrocycle, in this case the Soret band and two Q bands (Figure 2a). For both corroles in DMSO solutions, the Soret band splitting and a difference in intensities of Q-bands were noted when compared to the others solution. This fact has been reported in the literature by several authors and can be attributed to the presence of possible tautomeric species in solution, occurring by the possibility of pyrrole nitrogen deprotonation or hydrogen bond interactions [7,²⁶]. In this way, different tautomeric types of the studied corroles are predicted in polar solvents such as DMSO, and thus, can interact with the molecules by solvation and stabilize them, mainly through possible intermolecular interactions such as hydrogen bonds or dipole–dipole forces.

The steady-state fluorescence emission analysis of corroles COOH-Cor and COOMe-Cor in the selected solvents (Soret band excitation; emission at 600 to 800 nm range), using COOH-Cor derivative as an example, all spectra are shown in Figure 2b and the photophysical data are reported in Table 1. The steady-state fluorescence emission spectra of the corrole COOMe-Cor are shown in the Supplementary information section (Figure S2).

The emission quantum yield values (Φ_f) were calculated according to the reference tetra(phenyl)porphyrin (TPP) standard in DMF solution (Φ_f = 11.0%). In general, the values of the emission quantum yield of the studied corrole derivatives are low to moderate, with shorter Stokes Shifts (SS) values and agree with the predicted structure (Table 1). More differences that are notable in the corrole COOMe-Cor in both solutions, where the compound showed the lowest Φ_f values, probably due to a poor stabilization of the excited states by the ester unit. In the less polar solvents (such as DCM and MeCN), both corroles presents the highest quantum fluorescence yield values, a fact that can be attributed to more effective interactions and stabilization in the singlet excited state (Table 1). Data referring to the frontier molecular orbitals from theoretical calculations by TD-DFT analysis of these corroles will help a better interpretation of the presented results.

Finally, with regard to the lifetime decay (τ_f) values of the corroles COOH-Cor (Figure 2c) and COOMe-Cor, small differences are observed according to the polarity of the solvent, with the derivative COOH-Cor, the lifetime values being longer (between 4.0 to 6.0 ns) than the COOMe-Cor ester compound (between 3.0 to 4.50 ns) (Table 1). The non-radiative (k_nr) rates higher than the radiative (k_r) values and this fact may be attributed to the low fluorescence values of the derivatives, concomitantly with which the compounds have high values of radiative lifetimes (τ_r). The fluorescence decay plots of the corrole COOMe-Cor are shown in the Supplementary information section (Figure S3).

Figure 2. (a) Normalized absorption spectra of corrole COOH-Cor, (b) steady-state fluorescence emission spectra of corrole COOH-Cor, (c) lifetime decay plots of corrole COOH-Cor and (d) photography of solution of corroles COOH-Cor and COOMe-Cor under natural light and UV lamp (365 nm) conditions.

Table 1. Photophysical parameters of porphyrins COOH-Cor and COOMe-Cor in some solvents.

	COOH-Cor
Solvent	λ_Abs, nm (log ε)^a	λ_Em, nm (QY; %)^b	SS (nm/cm⁻¹)^c	τ_f (ns)^d; χ²	k_r (x 10⁸ s⁻¹)^e	k_nr (x 10⁸ s⁻¹)^e	τ_r (ns)^f	τ_nr (ns)^f
DCM	423 (70220), 559 (6760), 613 (6620)	656 (21.0)	233/840	4.70 ± 0.004; 0.97769	0.45	1.70	22.5	5.95
MeCN	423 (77095), 583 (7760), 622 (20415)	635 (25.0)	212/790	4.30 ± 0.007; 0.96234	0.60	1.75	17.0	5.75
MeOH	409 (65200), 564 (10520), 613 (7370)	661 (18.0)	252/930	4.18 ± 0.006; 0.94069	0.45	1.95	23.0	5.10
DMSO	429 (66075), 583 (8385), 626 (20400)	637 (6.00)	208/760	5.52 ± 0.006; 0.97460	0.10	1.70	92.0	5.85
DMSO(5%)/Tris-HCl	409 (65710), 567 (11390), 615 (7125)	-----	-----	-----	-----	-----	-----	-----
	COOMe-Cor
Solvent	λ_Abs, nm (log ε)^a	λ_Em, nm (QY; %)^b	SS (nm/cm⁻¹)^c	τ_f (ns)^d; χ²	k_r (x 10⁸ s⁻¹)^e	k_nr (x 10⁸ s⁻¹)^f	τ_r (ns)^f	τ_nr (ns)^f
DCM	425 (56290), 575 (11820), 630 (12710)	668 (22.0)	243/855	3.06 ± 0.003; 0.94072	0.70	2.55	14.0	3.90
MeCN	432 (70830), 586 (20820), 633 (25895)	631 (3.0)	199/730	-----	-----	-----	-----	-----
MeOH	414 (51780), 576 (12035), 622 (112505)	653 (16.0)	239/885	3.82 ± 0.003; 0.97548	0.40	2.20	24.0	4.55
DMSO	433 (57080), 589 (10000), 630 (16655)	637 (7.0)	204/740	4.48 ± 0.004; 0.97191	0.15	2.05	64.0	4.80
DMSO(5%)/Tris-HCl	418 (51110), 583 (13600), 622 (11360)	-----	-----	-----	-----	-----	-----	-----

^aConcentration at 10 µM; ^bConcentration at 5.0 µM and quantum yields (QY) are measure using TPP as standard reference (11% in DMF), with an error close to 5.0%; ^cStokes Shifts (SS) = (1/λ_abs ×10⁻⁷) – (1/λ_em ×10⁻⁷), using Soret absorption and first emission bands; ^dConcentration at 5.0 µM, using excitation at 441 nm by NanoLED source, with an error close to 5.0%; ^eUsing the same equations by reference [11]; ^fUsing the same equations by reference [11];

Theoretical calculations

Looking for structural comparisons between COOH-Cor and COOMe-Cor derivatives we can find many similarities since the only difference lies on the exchange of hydrogen atom by a methyl group. Table S2 provides all essential data for such comparison based on the label numbers of each atom on each corrole given in the Supplementary information section (Figures S4-S5). The bond lengths match perfectly until bond distance given by R61-R66. From that to the end of the table one can be seen some expected differences mainly caused by the bulky of methyl group attached to the carboxylate. Figure 3 shows the optimized geometries of each corrole.

Figure 3. DFT optimized geometries for (a) COOH-Cor and (b) COOMe-Cor derivatives.

As previously studied the Soret and Q bands are quite remarkable to characterize absorption spectra of corroles. TD-DFT calculations can access excitations from ground state and make the assignments of molecular orbitals possible. TD-DFT for compound COOH-Cor in DCM shows differences of 11 and 19 nm when comparing the calculated wavelength with the experimental ones, which are quite reasonable values. Soret bands were encountered in 411.7, 463 and 492.6 nm, while the Q band should be the ones in 577.9 and 591.7 nm (please refer to Figure S6 in theSupplementary information section). This band are attributed to the very first excited state (S₁), the one at 2.095 eV above the ground state (S₀) with main transitions assigned to the key frontier molecular orbitals 188 ® 191, among others. One can be mentioned that OM189 is HOMO and OM190 is LUMO. The second excited state S₂ is 2.145 eV above S₀ and once again such transitions, among others are quite expressive, involving once again OM191 (HOMO+1) as well as OM190 (HOMO). The other Soret band is suggested to be the one at 412 nm which may correspond to the sixth excitation from S₀. The four main molecular orbitals concerning such transitions are displayed in Figure 4 with their energy differences. It is important to notice that such energies are not the same discussed above. This happens due to the composition of each electronic state, not only constituted by the molecular orbitals showed. One can be seen that HOMO are mainly delocalize through the corrole ring with some contribution from its meso substituents. The HOMO-1 on the other hand has no contribution from such ligands. If we look at LUMO and the one above, LUMO+1, there are also an interesting difference: the 4-carboxyphenyl group does not contributes to LUMO, while LUMO+1 does have a large involvement of this part of the molecule. Finally, it is trustable to say that the nature of such transitions is mainly p ® p* as expected due to the corrole ring.

Figure 4. Frontier molecular orbitals for corrole COOH-Cor. HOMO-1, HOMO, LUMO and LUMO+1 are displayed along the energy differences between these molecular orbitals.

A corresponding analysis also in DCM was made for COOMe-Cor; its molecular orbitals are presented in the Supplementary information section (Figure S7). These orbitals are, as expected, quite similar to those of COOH-Cor and the energy gap between them are also comparable. Its calculated UV-Vis spectrum is shown in Figure S8 in the Supplementary information section. For this corrole the Soret bands lie in 411.5, 451.1 and 574.3 nm, while the Q bands are probably the ones in 574.3 and 590.6 nm. The same analysis made for COOH-Cor can be established here since the orbitals involved in each transition seems to have quite the same contribution and energies are comparable. Table 2 shows a comparison between the main transitions and electronic assignments of both corroles simulated at different dielectric constants.

Table 2. Main UV–vis absorptions (Soret and Q bands) and their respective transition energies for corroles COOH-Cor and COOMe-Cor in different solvents using CPCM, SMD. Dielectric constant are also shown in parenthesis. l is the wavelength in nm and f stands for the oscillator strength. E_gap is given in eV.

TD-DFT data	COOH-Cor			COOMe-Cor
Solvent (CPCM, SMD)	l / nm	f	E_gap / eV	l / nm	f	E_gap / eV
DCM (9.08)	591.7	0.108	2.095	590.6	0.146	2.099
	577.9	0.229	2.145	574.3	0.196	2.159
	492.6	0.442	2.517	481.4	0.517	2.579
	463.0	0.420	2.678	451.1	0.508	2.748
	411.7	0.602	3.012	411.5	0.563	3.013
MeCN (36.6)	591.1	0.087	2.097	589.7	0.122	2.102
	577.0	0.224	2.149	573.4	0.192	2.162
	493.5	0.389	2.512	482.0	0.455	2.572
	464.4	0.377	2.670	451.8	0.452	2.744
	409.6	0.559	3.027	409.6	0.527	3.027
MeOH (32.63)	592.8	0.060	2.092	591.0	0.086	2.098
	579.9	0.250	2.149	576.9	0.225	2.149
	501.1	0.350	2.474	493.7	0.391	2.511
	472.8	0.336	2.622	463.5	0.367	2.675
	409.5	0.570	3.027	409.6	0.559	3.027
DMSO (47.2)	592.0	0.104	2.094	590.7	0.147	2.099
	578.7	0.228	2.142	574.6	0.189	2.158
	494.7	0.423	2.505	482.4	0.502	2.570
	465.6	0.407	2.663	452.1	0.500	2.742
	411.5	0.614	3.013	411.2	0.573	3.015

In all parameters shown in Table 2, the general behavior of each corrole is very similar. One would expect that DCM could present the most different performance due to its most distant dielectric constant (9.08) comparable to the other solvents. However, this was not observed at least in this level of theory. To check the comparison established between all solvents in each corrole, Figures S9 and S10 in the Supplementary information section shows the behavior of each corrole studied here in a dependence of E_gapversus. the wavelength. One can be seen that there is basically no difference seen for COOH-Cor in all four solvents used. The same trend is observed for COOMe-Cor. Nonetheless, it is important to mention that there is a higher inclination for the graphs of COOMe-Cor when compared to its parent compound. Also, the adjusted R-squared is a bit larger for all solvents of COOMe-Cor. This graphical analysis may suggest that the overall profile seen in COOMe-Cor spectra may have a better description of the energy levels for the main excitations found.

Triplet states were also considered in TD-DFT calculations including spin-orbit coupling effect (SOC). The coupling between the first excited triplet state T₁ and S₀ is quite strong through the y component of the SOC operator and strong with the z component of the operator . Spin substates are considered and show different weights in this mixture. The spectrum generated from SOC inclusion on TD-DFT embraces all singlets and triplets’ states, but still the transition from triplet to the ground state is not strong (oscillator strengths are nearly null) compatible with no phosphorescence, as expected and confirmed by photophysical analysis.

Aggregation and stability assays by UV-Vis analysis

The evaluation of the aggregation behavior of corroles COOH-Cor and COOMe-Cor in buffered solution was studied by UV-Vis analysis in DMSO(5%)/Tris-HCl buffer (pH 7.4) mixture solution and as example, compound COOH-Cor is presented in the Figure 5. In general, no shift at the maximum Soret absorbance wavelength was observed as a function of thevariation of the concentration from 0.5 to 60 μM. Corrole UV-Vis aggregation spectra of compound COOMe-Cor can be found in the Supplementary information material (Figure S11). Regarding stability in solution, the compounds were monitored over a period of 5 days and it was possible to notice greater stability of corrole in buffered solution (see Supplementary information material - Figure S12-S13).

Figure 5. (a) Aggregation study for COOH-Cor, using DMSO(5%)/Tris-HCl buffer (pH 7.4) mixture solution and (b) graph plots show the linear behavior of the Soret band absorbance as a function of the concentration (0.5 to 60 μM).

Photo-stability behavior of corroles

In terms of photo-stability, some dyes need to remain stable when exposed to light doses for a long time. Therefore, photo-bleaching analysis is an important parameter in biologic media (buffered solution) to study the photo-degradation process after light source irradiation. Based on the small changes in the absorption spectra as a function of time, it was confirmed that the studied corroles COOH-Cor and COOMe-Cor exhibit good stability when irradiated with a white-light LED array system for 60 min at irradiance of 50 mW cm⁻² and a total light dosage of 180 J cm⁻² (see Supplementary Information - Figures S14–S15).

Photobiological parameters of corroles

The ability of each corrole derivative to produce ROS was investigated and for singlet oxygen generation species (¹O₂) was using 1,3-diphenylisobenzofuran (DPBF) in DMF, DMSO and MeCN solutions by UV-Vis spectroscopy. Using as example, Figure 6a-b show the singlet oxygen production parameters for corrole COOH-Cor in DMF solution. All DPBF photo-oxidation spectra of the corroles are shown in the Supplementary information section (Figure S16-S20).

The photo-oxidation profile was monitored at DPBF absorbance transition band decrease in all solvents tested during irradiation with a green-diode laser at 532 nm. The singlet oxygen quantum yield (Φ_Δ) and photo-oxidation constant (k_po) values for both corroles in all solvents are listed in Table 3 and compounds are with TPhCor molecule standard(Φ_Δ^std = 67.0% in DMSO). In this way, it is possible to note that the COOH-Cor compound produces more ¹O₂ species in both solutions than the COOMe-Cor derivative, which is a better generator of singlet oxygen in these solutions, favoring the Type I ROS generation mechanism.

The capacity of corroles COOH-Cor and COOMe-Cor to generate superoxide species (O₂^•−) was investigated in DMF solution, using corrole COOMe-Cor as example (Figure 6c-d). For this application, solutions of corroles containing NBT reagent and the reducing agent NADH were irradiated with a white-light LED source (irradiance of 50 mW cm⁻² and a total light dosage of 60 J cm⁻²) in aerobic conditions, at a period of 20 min. The reaction of NBT with superoxide radical species produced diformazan, which can be monitored following the absorption band of this product (see Supplementary information section - Figure S21). The superoxide generation constant (k_SO) by the NBT reduction assays is shown in Table 3. Contrary to what was observed for the generation of singlet oxygen species, after white-light irradiation conditions, corrole COOMe-Cor forming more O₂^•− species (Table 3). The generation of superoxide is also dependent on the substituent inserted in corrole, since ester unit groups favor the formation of these species in a solution.

In addition, the partition coefficients (log P_OW) were measured for each corrole derivative and the values found for the neutral corroles are in accordance with the literature [4], with corrole COOHe-Cor showing a more hydrophobic character when compared to the COOMe-Cor derivative. This fact is assigned to the presence of more polar group (carboxylic acid) in the periphery of corroles (Table 3).

Figure 6. (a) DPBF photo-oxidation UV-Vis spectra by irradiation with green-diode laser (532 nm; 100 mW) at a period of 20 min. in DMF solution, in the presence of corrole COOH-Cor and (c) NBT reduction assays UV-Vis spectra by irradiation with white-light LED source (irradiance of 50 mW cm⁻² and a total light dosage of 60 J cm⁻²) at a period of 20 min. in DMF solution, in the presence of corrole COOMe-Cor. Graph plots (b) and (d) show the kinetic profile.

Table 3. Photobiological data of corroles COOH-Cor and COOMe-Cor.

	k_po (M^‒1 s^‒1)^a			¹O₂ (Φ_Δ; %)^b			O₂^•‒ (k_SO; min^‒1)	Log P_OW
Corrole	DMF	DMSO	MeCN	DMF	DMSO	MeCN	DMF	Octanol/Water
COOH-Cor	0.798	0.812	0.524	82.0	89.0	48.0	0.0016	+1.29
COOMe-Cor	0.352	0.482	0.371	36.0	53.0	34.0	0.024	+1.75

^aUsing tri(phenyl)corrole TPhCor as standard in DMSO (k_po = 0.654 M^‒1 s^‒1) [4]; ^b Using tri(phenyl)corrole TPhCor as standard in DMSO (Φ_Δ = 67.0%) [4];

Microorganism photoinactivation by corroles

The MIC and MBC values of derivatives COOH-Cor and COOMe-Cor against four Gram-negative and two Gram-positive bacteria are listed in Table 4. Corroles were similar effective against all microorganisms tested and corrole COOMe-Cor showed no difference in activity under dark or white-light conditions.

Corrole COOH-Cor showed good photo-inactivation results under white-light LED irradiation against P. aeruginosa PA01, however it did not show satisfactory photo-inactivation results against other bacteria strains. The structural composition of teichoic and lipoteichoic acids (in Gram positive bacteria) probably provide low porosity to the cell wall of this bacteria, in addition to the outer membrane, difficult the penetration of neutral corroles into the bacterial cell, which explains our findings. The evolution of microbial resistance increased the demand for efficient alternative therapies, such as aPDT, which has shown positive possibilities in antimicrobial treatment. Porphyrin-like photosensitizers such as corroles, especially cationic compounds, present desirable activity by producing ROS and better interacting with the microbial cell membrane [²⁷]. According to the best MIC results, the compound COOH-Cor, the P. aeruginosa PA01 strain were chosen for the subsequent assays.

Table 4. MIC and MBC values (in μM) of corroles COOH-Cor and COOMe-Cor against ATCC bacteria strains when exposed to dark and white-light LED irradiation conditions at irradiance of 50 mW cm⁻² and a total light dosage of 180 J cm⁻² (total time of 60 min).

	MIC COOH-Cor (µM)			MIC COOMe-Cor (µM)
Microorganism	Dark	Light	PI index*	Dark	Light	PI index*
Escherichia coli ATCC 25922	55.5	55.5	1.00	110.8	110.8	1.00
Klebsiella pneumoniae ATCC 700603	55.5	55.5	1.00	110.8	55.5	2.00
Pseudomonas aeruginosa 01	27.7	13.8	2.00	27.7	27.7	1.00
Salmonella enteretidis ATCC 13076	55.5	55.5	1.00	110.8	110.8	1.00
Staphylococcus aureus ATCC 29213	55.5	27.7	2.00	55.5	55.5	1.00
MRSA Clinical isolate	55.5	27.7	2.00	55.5	55.5	1.00
	MBC COOH-Cor (µM)			MBC COOMe-Cor (µM)
Microorganism	Dark	Light	PI index*	Dark	Light	PI index
Escherichia coli ATCC 25922	55.5	55.5	1.00	55.5	55.5	1.00
Klebsiella pneumoniae ATCC 700603	55.5	55.5	1.00	55.5	55.5	1.00
Pseudomonas aeruginosa 01	55.5	55.5	1.00	55.5	55.5	1.00
Salmonella enteretidis ATCC 13076	55.5	55.5	1.00	55.5	55.5	1.00
Staphylococcus aureus ATCC 29213	55.5	55.5	1.00	55.5	55.5	1.00
MRSA Clinical isolate	55.5	55.5	1.00	55.5	55.5	1.00

*PI index: phototoxic index (calculated by μM values) = dark/white-light ratio;

Association with potassium iodide (KI)

In an attempt to improve the photodynamic activity of corroles against the strains used here, association tests with potassium iodide were conducted. A fixed concentration of KI (100 mM) was used in combination with corroles COOH-Cor and COOMe-Cor to assess the MIC variability of the studied strains. Dark/white-light irradiation, incubation and reading conditions were performed as described above, in duplicate. After the incubation period, it was observed that there was no variation in the MIC for any of the bacteria tested, therefore, the KI test did not generate a photo-oxidative response.

ROS scavenger tests

To evaluate the photo-inactivation mechanism of corrole COOH-Cor, the MIC of ROS was performed: Ascorbic acid (AA; singlet oxygen species), dimethyl sulfoxide (DMSO; superoxide radical species), tert-Butanol (t-BuOH; hydroxyl radical species), N-acetylcysteine (NAC; hydroxyperoxyl radical species) and EDTA (Ca²⁺ and Mg²⁺ ion chelators), with the data are described in Table 5.

The method used was broth microdilution already described previously, with a serial dilution of the corroles in the 96-well plate and 10 µL of scavenger substances were added at fixed concentrations of 10×1/4 of the MIC isolated from these substances. Finally, 15 µL of inoculum was added to all wells, except in the negative control that received only culture medium, the plates were irradiated with white-light LED irradiation source for 60 minutes and incubated for 24h at 37ºC. The ROS mechanism was determined when the MIC of corrole with the scavenging substances increased in their presence.

In the presence of AA (¹O₂ scavenger), DMSO (O₂^•− scavenger), and EDTA (ion chelator), the MIC values of corrole COOH-Cor tested against PA01 strain were higher than the value obtained in the MIC of corrole in the absence of ROS scavengers. These results show that singlet oxygen and superoxide radicals might participate in a simultaneous oxidation mechanism (Types I and II). One way to increase the photosensitization of Gram-negative bacteria can be achieved by adding biological or chemical molecules, which modify the native consistency of the cell membrane [25]. In this way, EDTA molecule is considered an ionic chelator of the cell membrane, which allows an increase in the photodynamic activity of derivatives in its presence. This molecule promotes the capture of photosensitizer, since it removes the ions present in the membrane, thus avoiding neutralization of negative charges and producing electrostatic repulsion between the lipopolysaccharides. In addition, molecules can penetrate the inner cytoplasmic membrane more easily. In this study, neutral corroles were used and, due to the results obtained, EDTA can be possible cause a increase in photo-inactivation activity.

Table 5. MIC values (in μM) of corrole COOH-Cor against P. aeruginosa PA01 strain when exposed in the absence and in the presence of ROS scavengers under white-light LED irradiation conditions (irradiance of 50 mW cm⁻² and a total light dosage of 180 J cm⁻², at 60 min).

	MIC COOH-Cor (µM)
Microorganism	Absence	AA	DMSO	t-BuOH	NAC	EDTA
P. aeruginosa PA01	13.8	55.5	55.5	13.8	13.8	27.7

Checkerboard test

The synergistic activity between selected corrole COOH-Cor and Imipenen (IMP) against the P. aeruginosa PA01 strain was performed using the checkerboard assay according the literature [25]. The synergistic potential between the antimicrobial and corrole molecule was evaluated by the value obtained in the FICI values. The synergistic potential was observed when the antimicrobial was associated with the corrole photosensitizer (Table 6). The antimicrobial Imipenem is a carbapenem belonging to the β-lactam class, which has inhibitory action against peptidoglycan crosslinking during cell wall synthesis. Imipenem is the first-choice antimicrobial agent for the treatment of Pseudomonas aeruginosa infections.

Corrole COOH-Cor with white-light irradiation can interfere with β-lactamases enzymes, aminoglycoside modifying enzymes, thus damaging the antimicrobial efflux pump or increase the binding uptake of antimicrobial targets, thus making the bacteria less resistant to the commercial antimicrobials. However, the antimicrobial may also play a role in photo-inactivation, increasing the permeability of the bacterial outer membrane, thereby increasing the affinity of compound for the bacterial cell and consequently a better photodynamic effect of corrole. This is a promising discovery, as it enables one to associate antimicrobials with corrole contain carboxylic acid peripheral units in order to be an alternative for treatment against P. aeruginosa strains.

Table 6. MIC values (in μM) of corrole COOH-Cor against P. aeruginosa PA01 strain in the abscence and in the presence of Imipenen (IMP) under white-light LED irradiation conditions (irradiance of 50 mW cm⁻² and a total light dosage of 180 J cm⁻², at 60 min).

	MIC values (µM)
Microorganism	COOH-Cor	IMP	COOH-Cor + IMP	IMP + COOH-Cor	FICI	Interaction
P. aeruginosa PA01	13.8	12.6	0.23	2.43	0.187	Synergism

Final Remarks

In this work, we investigated and studied the photophysical/photobiological properties of mono-substituted corroles containing carboxylic acid or ester group at meso-10-position in different solvents. Photobiological parameters such as ROS generation and photostability were evaluated and it was found that these compounds are good for use in photoinduced processes, by singlet or superoxide reactive species generation. Furthermore, the antimicrobial properties of corroles against bacteria strains were evaluated, and the corrole derivative COOH-Cor had a preference for photoinactive process, mainly in the presence of imipenem antibiotic.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Acknowledgments

The authors would like to thank the CAPES (Finance code 001), CNPq and FAPERGS agencies. B.A.I. thanks CNPq (409.150/2018-5, 304.711/2018-7 and 305.458/2021-3) and FAPERGS (21/2551-0002114-4). L.A.S.C thanks to CNPq (310365/2021-0) and FAPEMIG (CEX-APQ-02244-18). R.C.V.S. thanks to CNPq (305914/2022-0).

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Scheme 1 is available in the Supplementary Files section

No competing interests reported.

Scheme1.png
Scheme 1. Synthesis of corroles COOH-Cor and COOMe-Cor used in this study. Steps: (i) TFA, Ar atm, 5 min.; (ii) MeOH/H₂O, HCl 37%, r.t., 2 h; (iii) DDQ, DCM, r.t., 15 min.
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Red-emission, photobiological properties and antimicrobial photodynamic assays using mono-substituted corroles contain carboxylic acid and ester units

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