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Background: Photodynamic inactivation (PDI) is an attractive alternative to treat Candida albicans infections, especially considering the spread of resistant strains. The combination of the photophysical advantages of Zn(II) porphyrins (ZnPs) and the plasmonic effect of silver nanoparticles (AgNPs) has the potential to further improve PDI. Here, we propose the novel association of polyvinylpyrrolidone (PVP) coated AgNPs with the cationic ZnPs Zn(II) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin or Zn(II) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin to photoinactivate C. albicans. Methods: AgNPs stabilized with PVP were chosen to allow for (i) overlap between the NP extinction and absorption spectra of ZnPs and (ii) favor AgNPs-ZnPs interaction; prerequisites for exploring the plasmonic effect. Optical and zeta potential (ζ) characterizations were performed, and reactive oxygen species (ROS) generation was also evaluated. Yeasts were incubated with individual ZnPs or their respective AgNPs-ZnPs systems, at various ZnP concentrations and two proportions of AgNPs, then irradiated with a blue LED. Interactions between yeasts and the systems (ZnP alone or AgNPs-ZnPs) were evaluated by fluorescence microscopy. Results: Subtle spectroscopic changes were observed for ZnPs after association with AgNPs, and the ζ analyses confirmed AgNPs-ZnPs interaction. PDI using ZnP-hexyl (0.8 µM) and ZnP-ethyl (5.0 µM) promoted a 3 and 2 log10 reduction of yeasts, respectively. On the other hand, AgNPs-ZnP-hexyl (0.2 µM) and AgNPs-ZnP-ethyl (0.6 µM) systems led to complete fungal eradication under the same PDI parameters and lower porphyrin concentrations. Increased ROS levels and enhanced interaction of yeasts with AgNPs-ZnPs were observed, when compared with ZnPs alone. Conclusion: We applied a facile synthesis of AgNPs which boosted ZnP efficiency. We hypothesize that the plasmonic effect combined with the greater interaction between cells and AgNPs-ZnPs systems resulted in an efficient and improved fungal inactivation. This study provides insight into the application of AgNPs in PDI and helps diversify our antifungal arsenal, encouraging further developments toward inactivation of resistant Candida spp.

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Candida albicans is the main cause of superficial candidiasis. While the antifungals available are defied by biofilm formation and resistance emergence, antimicrobial photodynamic inactivation (aPDI) arises as an alternative antifungal therapy. The tetracationic metalloporphyrin Zn(II) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (ZnTnHex-2-PyP4+) has high photoefficiency and improved cellular interactions. We investigated the ZnTnHex-2-PyP4+ as a photosensitizer (PS) to photoinactivate yeasts and biofilms of C. albicans strains (ATCC 10231 and ATCC 90028) using a blue light-emitting diode. The photoinactivation of yeasts was evaluated by quantifying the colony forming units. The aPDI of ATCC 90028 biofilms was assessed by the MTT assay, propidium iodide (PI) labeling, and scanning electron microscopy. Mammalian cytotoxicity was investigated in Vero cells using MTT assay. The aPDI (4.3 J/cm2) promoted eradication of yeasts at 0.8 and 1.5 µM of PS for ATCC 10231 and ATCC 90028, respectively. At 0.8 µM and same light dose, aPDI-treated biofilms showed intense PI labeling, about 89% decrease in the cell viability, and structural alterations with reduced hyphae. No considerable toxicity was observed in mammalian cells. Our results introduce the ZnTnHex-2-PyP4+ as a promising PS to photoinactivate both yeasts and biofilms of C. albicans, stimulating studies with other Candida species and resistant isolates.

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Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin chloride (MnTE-2-PyPCl5, BMX-010, and AEOL10113) is among the most studied superoxide dismutase (SOD) mimics and redox-active therapeutics, being currently tested as a drug candidate in a phase II clinical trial on atopic dermatitis and itch. The thermal stability of active pharmaceutical ingredients (API) is useful for estimating the expiration date and shelf life of pharmaceutical products under various storage and handling conditions. The thermal decomposition and kinetic parameters of MnTE-2-PyPCl5 were determined by thermogravimetry (TG) under nonisothermal and isothermal conditions. The first thermal degradation pathway affecting Mn-porphyrin structural integrity and, thus, activity and bioavailability was associated with loss of ethyl chloride via N-dealkylation reaction. The thermal stability kinetics of the N-dealkylation process leading to MnTE-2-PyPCl5 decomposition was investigated by using isoconversional models and artificial neural network. The new multilayer perceptron (MLP) artificial neural network approach allowed the simultaneous study of ten solid-state kinetic models and showed that MnTE-2-PyPCl5 degradation is better explained by a combination of various mechanisms, with major contributions from the contraction models R1 and R2. The calculated activation energy values from isothermal and nonisothermal data were about 90 kJ mol-1 on average and agreed with one another. According to the R1 modelling of the isothermal decomposition data, the estimated shelf life value for 10% decomposition (t 90%) of MnTE-2-PyPCl5 at 25°C was approximately 17 years, which is consistent with the high solid-state stability of the compound. These results represent the first study on the solid-state decomposition kinetics of Mn(III) 2-N-alkylpyridylporphyrins, contributing to the development of this class of redox-active therapeutics and SOD mimics and providing supporting data to protocols on purification, handling, storage, formulation, expiration date, and general use of these compounds.

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BACKGROUND: Photodynamic inactivation (PDI) is emerging as a promising alternative for cutaneous leishmaniasis (CL). The chemotherapy currently used presents adverse effects and cases of drug resistance have been reported. ZnTnHex-2-PyP4+ is a porphyrin with a high potential as a photosensitizer (PS) for PDI, due to its photophysical properties, structural stability, and cationic/amphiphilic character that can enhance interaction with cells. This study aimed to investigate the photodynamic effects mediated by ZnTnHex-2-PyP4+ on Leishmania parasites. METHODS: ZnTnHex-2-PyP4+ stability was evaluated using accelerated solvolysis conditions. The photodynamic action on promastigotes was assessed by (i) viability assays, (ii) mitochondrial membrane potential evaluation, and (iii) morphological analysis. The PS-promastigote interaction was studied. PDI on amastigotes and the cytotoxicity on macrophages were also analyzed. RESULTS: ZnTnHex-2-PyP4+, under submicromolar concentration, led to immediate inactivation of more than 95% of promastigotes. PDI promoted intense mitochondrial depolarization, loss of the fusiform shape, and plasma membrane wrinkling in promastigotes. Fluorescence microscopy revealed a punctate PS labeling in the parasite cytoplasm. PDI also led to reductions of ca. 64% in the number of amastigotes/macrophage and 70% in the infection index after a single treatment session. No noteworthy toxicity was observed on mammalian cells. CONCLUSIONS: ZnTnHex-2-PyP4+ is stable against demetallation and more efficient as PS than the ethyl analogue ZnTE-2-PyP4+, indicating readiness for evaluation in in vivo studies as an alternative approach to CL. GENERAL SIGNIFICANCE: This report highlighted promising photodynamic effects mediated by ZnTnHex-2-PyP4+ on Leishmania parasites, opening up perspectives for applications in CL pre-clinical assays and PDI of other microorganisms.

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Two classes of heterogenized biomimetic catalysts were prepared and characterized for hydrocarbon oxidations: (1) by covalent anchorage of the three Mn(iii) meso-tetrakis(2-, 3-, or 4-pyridyl)porphyrin isomers by in situ alkylation with chloropropyl-functionalized silica gel (Sil-Cl) to yield Sil-Cl/MnPY (Y = 1, 2, 3) materials, and (2) by electrostatic immobilization of the three Mn(iii) meso-tetrakis(N-methylpyridinium-2, 3, or 4-yl)porphyrin isomers (MnPY, Y = 4, 5, 6) on non-modified silica gel (SiO2) to yield SiO2/MnPY (Y = 4, 5, 6) materials. Silica gel used was of column chromatography grade and Mn porphyrin loadings were deliberately kept at a low level (0.3% w/w). These resulting materials were explored as catalysts for iodosylbenzene (PhIO) oxidation of cyclohexane, n-heptane, and adamantane to yield the corresponding alcohols and ketones; the oxidation of cyclohexanol to cyclohexanone was also investigated. The heterogenized catalysts exhibited higher efficiency and selectivity than the corresponding Mn porphyrins under homogeneous conditions. Recycling studies were consistent with low leaching/destruction of the supported Mn porphyrins. The Sil-Cl/MnPY catalysts were more efficient and more selective than SiO2/MnPY ones; alcohol selectivity may be associated with hydrophobic silica surface modification reminiscent of biological cytochrome P450 oxidations. The use of widespread, column chromatography, amorphous silica yielded Sil-Cl/MnPY or SiO2/MnPY catalysts considerably more efficient than the corresponding, previously reported materials with mesoporous Santa Barbara Amorphous No 15 (SBA-15) silica. Among the materials studied, in situ derivatization of Mn(iii) 2-N-pyridylporphyrin by covalent immobilization on Sil-Cl to yield Sil-Cl/MnP1 showed the best catalytic performance with high stability against oxidative destruction and reusability/recyclability.

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BACKGROUND: Cardiomyopathies remain among the leading causes of death worldwide, despite all efforts and important advances in the development of cardiovascular therapeutics, demonstrating the need for new solutions. Herein, we describe the effects of the redox-active therapeutic Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin, AEOL10113, BMX-010 (MnTE-2-PyP5+), on rat heart as an entry to new strategies to circumvent cardiomyopathies. METHODS: Wistar rats weighing 250-300 g were used in both in vitro and in vivo experiments, to analyze intracellular Ca2+ dynamics, L-type Ca2+ currents, Ca2+ spark frequency, intracellular reactive oxygen species (ROS) levels, and cardiomyocyte and cardiac contractility, in control and MnTE-2-PyP5+-treated cells, hearts, or animals. Cells and hearts were treated with 20 µM MnTE-2-PyP5+ and animals with 1 mg/kg, i.p. daily. Additionally, we performed electrocardiographic and echocardiographic analysis. RESULTS: Using isolated rat cardiomyocytes, we observed that MnTE-2-PyP5+ reduced intracellular Ca2+ transient amplitude, without altering cell contractility. Whereas MnTE-2-PyP5+ did not alter basal ROS levels, it was efficient in modulating cardiomyocyte redox state under stress conditions; MnTE-2-PyP5+ reduced Ca2+ spark frequency and increased sarcoplasmic reticulum (SR) Ca2+ load. Accordingly, analysis of isolated perfused rat hearts showed that MnTE-2-PyP5+ preserves cardiac function, increases SR Ca2+ load, and reduces arrhythmia index, indicating an antiarrhythmic effect. In vivo experiments showed that MnTE-2-PyP5+ treatment increased Ca2+ transient, preserved cardiac ejection fraction, and reduced arrhythmia index and duration. MnTE-2-PyP5+ was effective both to prevent and to treat cardiac arrhythmias. CONCLUSION: MnTE-2-PyP5+ prevents and treats cardiac arrhythmias in rats. In contrast to most antiarrhythmic drugs, MnTE-2-PyP5+ preserves cardiac contractile function, arising, thus, as a prospective therapeutic for improvement of cardiac arrhythmia treatment.

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The quantification of zinc in over-the-counter drugs as commercial propolis extracts by molecular fluorescence technique using meso-tetrakis(4-carboxyphenyl)porphyrin (H2 TCPP4 ) was developed for the first time. The calibration curve is linear from 6.60 to 100 nmol L-1 of Zn2+ . The detection and quantification limits were 6.22 nmol L-1 and 19.0 nmol L-1 , respectively. The reproducibility and repeatability calculated as the percentage variation of slopes of seven calibration curves were 6.75% and 4.61%, respectively. Commercial propolis extract samples from four Brazilian states were analyzed and the results (0.329-0.797 mg/100 mL) obtained with this method are in good agreement with that obtained with the Atomic Absorption Spectroscopy (AAS) technique. The method is simple, fast, of low cost and allows the analysis of the samples without pretreatment. Moreover the major advantage is that Zn-porphyrin complex presents fluorescent characteristic promoting the selectivity and sensitivity of the method.

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The application of fluorescent II-VI semiconductor quantum dots (QDs) as active photosensitizers in photodymanic inactivation (PDI) is still being evaluated. In the present study, we prepared 3 nm size CdTe QDs coated with mercaptosuccinic acid and conjugated them electrostatically with Zn(II) meso-tetrakis (N-ethyl-2-pyridinium-2-yl) porphyrin (ZnTE-2-PyP or ZnP), thus producing QDs-ZnP conjugates. We evaluated the capability of the systems, bare QDs and conjugates, to produce reactive oxygen species (ROS) and applied them in photodynamic inactivation in cultures of Candida albicans by irradiating the QDs and testing the hypothesis of a possible combined contribution of the PDI action. Tests of in vitro cytotoxicity and phototoxicity in fibroblasts were also performed in the presence and absence of light irradiation. The overall results showed an efficient ROS production for all tested systems and a low cytotoxicity (cell viability >90%) in the absence of radiation. Fibroblasts incubated with the QDs-ZnP and subjected to irradiation showed a higher cytotoxicity (cell viability <90%) depending on QD concentration compared to the bare groups. The PDI effects of bare CdTe QD on Candida albicans demonstrated a lower reduction of the cell viability (~1 log10) compared to bare ZnP which showed a high microbicidal activity (~3 log10) when photoactivated. The QD-ZnP conjugates also showed reduced photodynamic activity against C. albicans compared to bare ZnP and we suggest that the conjugation with QDs prevents the transmembrane cellular uptake of the ZnP molecules, reducing their photoactivity.

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Azanone ((1)HNO, nitroxyl) is a highly reactive molecule with interesting chemical and biological properties. Like nitric oxide (NO), its main biologically related targets are oxygen, thiols, and metalloproteins, particularly heme proteins. As HNO dimerizes with a rate constant between 10(6) and 10(7) M(-1) s(-1), reactive studies are performed using donors, which are compounds that spontaneously release HNO in solution. In the present work, we studied the reaction mechanism and kinetics of two azanone donors Angelís Salt and toluene sulfohydroxamic acid (TSHA) with eight different Mn porphyrins as trapping agents. These porphyrins differ in their total peripheral charge (positively or negatively charged) and in their Mn(III)/Mn(II) reduction potential, showing for each case positive (oxidizing) and negative (reducing) values. Our results show that the reduction potential determines the azanone donor reaction mechanism. While oxidizing porphyrins accelerate decomposition of the donor, reducing porphyrins react with free HNO. Our results also shed light into the donor decomposition mechanism using ab initio methods and provide a thorough analysis of which MnP are the best candidates for azanone trapping and quantification experiments.

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In situ (1)H NMR data are reported for 106 Ru(porp)(RSH)(2) species, where porp is the dianion of ß-octaethylporphyrin (OEP), meso-tetraphenylporphyrin (TPP), and its para-substituted tetraphenyl analogues (T-p-XPP; X = OMe, Me, F, Cl, CO(2)Me, CF(3)), meso-tetrakis(3,5-dimethylphenyl)porphyrin (T-m,m'-Me(2)PP), and meso-tetramesitylporphyrin (TMP), and R = Me, Et, (n)Pr, (i)Pr, (n)Bu, (t)Bu, (n)Hex, Bn (benzyl), Ph, and p-MeOC(6)H(4). The upfield shifts in the SH resonances upon coordination of the thiol reflect changes in the porphyrin ring current and are analyzed using an empirical model that depicts quantitatively the nonbonding, electronic, and steric interactions between the thiol ligands, where steric factors dominate, and the porphyrin plane, where electronic factors dominate; such interactions are typically involved in small-molecule recognition within metalloporphyrin systems. Implications of the findings to hemethiolate proteins and surface coordination chemistry are also briefly presented.

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Cationic Mn porphyrins are among the most potent catalytic antioxidants and/or cellular redox modulators. Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin chloride (MnTE-2-PyPCl(5)) is the Mn porphyrin most studied in vivo and has successfully rescued animal models of a variety of oxidative stress-related diseases. The stability of an authentic MnTE-2-PyPCl(5) sample was investigated hereon by thermogravimetric, derivative thermogravimetric, and differential thermal analyses (TG/DTG/DTA), under dynamic air, followed by studies at selected temperatures to evaluate the decomposition path and appropriate conditions for storage and handling of these materials. All residues were analyzed by thin-layer chromatography (TLC) and UV-vis spectroscopy. Three thermal processes were observed by TG/DTG. The first event (endothermic) corresponded to dehydration, and did not alter the MnTE-2-PyPCl(5) moiety. The second event (endothermic) corresponded to the loss of EtCl (dealkylation), which was characterized by gas chromatography-mass spectrometry. The residue at 279°C had UV-vis and TLC data consistent with those of the authentic, completely dealkylated analog, MnT-2-PyPCl. The final, multi-step event corresponded to the loss of the remaining organic matter to yield Mn(3)O(4) which was characterized by IR spectroscopy. Isothermal treatment at 188°C under static air for 3h yielded a mixture of partially dealkylated MnPs and traces of the free-base, dealkylated ligand, H(2)T-2-PyP, which reveals that dealkylation is accompanied by thermal demetallation under static air conditions. Dealkylation was not observed if the sample was heated as a solid or in aqueous solution up to ∼100°C. Whereas moderate heating changes sample composition by loss of H(2)O, the dehydrated sample is indistinguishable from the original sample upon dissolution in water, which indicates that catalytic activity (on Mn basis) remains unaltered. Evidently, dealkylation at high temperature compromises sample activity.

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Thirty-two Ru(porp)L(2) complexes have been synthesized, where porp = the dianion of meso-tetramesitylporphyrin (TMP) or meso-tetrakis(4-methylphenyl)porphyrin (H(2)T-pMe-PP), and L = a thiol, a sulfide, a disulfide, or a trisulfide. Species studied were with RSH [R = Me, Et, (n)Pr, (i)Pr, (t)Bu, Bn (benzyl), and Ph], RSR (R = Me, Bn), RSSR (R = Me, Et, (n)Pr, Bn) and MeSS(t)Bu, and RSSSR (R = Me, Bn). All the species except two, which were the isolated Ru(T-pMe-PP)((t)BuSH)(2) and Ru(TMP)(MeSSMe)(2), were characterized in situ. The disulfide complex was characterized by X-ray analysis. (1)H NMR data for the coordinated thiols are the first reported within metalloporphyrin systems, and are especially informative because of the upfield shifts of the axial sulfur-containing ligands due to the porphyrin π-ring current effect, which is also present in the di- and trisulfide species. The disulfide in the solid state structure of Ru(TMP)(MeSSMe)(2) is η(1)(end-on) coordinated, the first example of such bonding in a nontethered, acyclic dialkyl disulfide; (1)H-(1)H EXSY NMR data in solution show that the species undergoes 1,2-S-metallotropic shifts. Stepwise formation of the bis(disulfide) complex from Ru(TMP)(MeCN)(2) in solution occurs with a cooperativity effect, resembling behavior of Fe(II)-porphyrin systems where crystal field effects dominate, but ligand trans-effects are more likely in the Ru system. The η(1)(end-on) coordination mode is also favored for the trisulfide ligand. Discussed also are the remarkable linear correlations that exist between the ring-current shielding shifts for the axial ligand C(1) protons of Ru(porp)(RS(x)R)(2) and x (the number of S atoms). The Introduction briefly reviews literature on Ru- and Fe porphyrins (including heme proteins) with sulfur-containing ligands or substrates, and relationships between our findings and this literature are discussed throughout the paper.

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Mn porphyrins are among the most efficient SOD mimics with potency approaching that of SOD enzymes. The most potent ones, Mn(III) N-alkylpyridylporphyrins bear positive charges in a close proximity to the metal site, affording thermodynamic and kinetic facilitation for the reaction with negatively charged superoxide. The addition of electron-withdrawing bromines onto beta-pyrrolic positions dramatically improves thermodynamic facilitation for the O2*- dismutation. We have previously characterized the para isomer, Mn(II)Br(8)TM-4-PyP(4+) [Mn(II) beta-octabromo-meso-tetrakis(N-methylpyridinium-4-yl)porphyrin]. Herein we fully characterized its meta analogue, Mn(II)Br(8)TM-3-PyP(4+) with respect to UV/vis spectroscopy, electron spray mass spectrometry, electrochemistry, O2*- dismutation, metal-ligand stability, and the ability to protect SOD-deficient Escherichia coli in comparison with its para analogue. The increased electron-deficiency of the metal center stabilizes Mn in its +2 oxidation state. The metal-centered Mn(III)/Mn(II) reduction potential, E((1/2))=+468 mV vs NHE, is increased by 416 mV with respect to non-brominated analogue, Mn(III)TM-3-PyP(5+) and is only 12 mV less positive than for para isomer. Yet, the complex is significantly more stable towards the loss of metal than its para analogue. As expected, based on the structure-activity relationships, an increase in E((1/2)) results in a higher catalytic rate constant for the O2*- dismutation, log k(cat)> or =8.85; 1.5-fold increase with respect to the para isomer. The IC(50) was calculated to be < or =3.7 nM. Manipulation of the electron-deficiency of a cationic porphyrin resulted, therefore, in the highest k(cat) ever reported for a metalloporphyrin, being essentially identical to the k(cat) of superoxide dismutases (log k(cat)=8.84-9.30). The positive kinetic salt effect points to the unexpected, unique and first time recorded behavior of Mn beta-octabrominated porphyrins when compared to other Mn porphyrins studied thus far. When species of opposing charges react, the increase in ionic strength invariably results in the decreased rate constant; with brominated porphyrins the opposite was found to be true. The effect is 3.5-fold greater with meta than with para isomer, which is discussed with respect to the closer proximity of the quaternary nitrogens of the meta isomer to the metal center than that of the para isomer. The potency of Mn(II)Br(8)TM-3-PyP(4+) was corroborated by in vivo studies, where 500 nM allows SOD-deficient E. coli to grow >60% of the growth of wild type; at concentrations > or =5 microM it exhibits toxicity. Our work shows that exceptionally high k(cat) for the O2*- disproportionation can be achieved not only with an N(5)-type coordination motif, as rationalized previously for aza crown ether (cyclic polyamines) complexes, but also with a N(4)-type motif as in the Mn porphyrin case; both motifs sharing "up-down-up-down" steric arrangement.

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Beta-hexabromo-5,10,15,20-tetrakis(4-carbomethoxyphenyl)porphyrinatomanganese(III) chloride (Mn(III)(Br6TCMPP)Cl) was prepared by selective Br2-hexabromation of its parent non-brominated manganese complex (Mn(III)(TCMPP)Cl), whereas the octabrominated analogue beta-octabromo-5,10,15,20-tetrakis(4-carbomethoxyphenyl)porphyrinatomanganese(III) chloride (Mn(III)(Br8TCMPP)Cl) was synthesized via metallation of the corresponding free-base. Beta-octabromo-5,10,15,20-tetrakis(4-carbomethoxyphenyl)porphyrin was obtained by demetallation of its brominated Cu(II) derivative, which, in its turn, was prepared by either a Br2 or an N-bromosuccinimide protocol. Relative to Mn(III)(TCMPP)Cl (E(1/2) = -0.16 V vs. normal hydrogen electrode, CH2Cl2), the Mn(III)/Mn(II) reduction potential of Mn(III)(Br8TCMPP)Cl and Mn(III)(Br6TCMPP)Cl showed anodic shifts of 0.43 and 0.33 V, respectively, which corresponded to a linear shift of 0.05 V per bromine added. These manganese complexes were evaluated as cytochrome P450 mimics in catalytic iodosylbenzene (PhIO)-oxidations of cyclohexane and cyclohexene. In aerobic PhIO-oxidation of cyclohexene, epoxidation and allylic autoxidation reactions were inversely related, competitive processes; the most efficient P450-mimics were the least effective autoxidation catalysts. Mn(III)(Br6TCMPP)Cl was more efficient as epoxidation or hydroxylation catalyst than both its fully and non-beta-brominated counterparts were. There was no linear relationship between the catalytic efficiency and both the number of bromine substituents and the Mn(III)/Mn(II) potential; these observations were compared to Lyons system literature data and discussed. Analogously to enzymatic optimum pH effects, an optimum redox potential effect is suggested as relevant in designing and understanding cytochrome P450 biomimetic catalysts.

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