meso-Tetraphenylbenzoporphyrin-2(2),2(3)-dicarboxylic anhydride: a platform to benzoporphyrin derivatives.

A method to synthesize meso-tetraphenylbenzoporphyrin-2(2),2(3)-dicarboxylic anhydride is reported. This compound reacts with alkylamines and arylamines to afford the corresponding "phthalimides" in moderate to excellent yields. The reaction of the title compound with benzene-1,4-diamine or with benzene-1,3-diamine yields the corresponding N,N'-(phenylene)bisphthalimides, whereas with benzene-1,2-diamine or naphthalene-1,8-diamine it affords heterocyclic-fused porphyrins. Molecular mechanics simulations elucidates the multiplicity of signals observed in the NMR spectra of the N,N'-(1,4-phenylene)bisphthalimide 11. This molecule exhibits two preferential conformations corresponding to a coplanar and an almost perpendicular arrangement of the benzoporphyrin units relative to the central benzenic ring.

Mechanisms of photodynamic inactivation of a gram-negative recombinant bioluminescent bacterium by cationic porphyrins.

Photodynamic therapy is a very promising approach to inactivate pathogenic microorganisms. The photodamage of cells involves reactive oxygen species (ROS) which are generated in situ by two main mechanisms (type I and/or type II). The mechanism responsible for the photoinactivation (PI) of a bioluminescent recombinant Escherichia coli, induced by three different cationic porphyrins, was identified in this work using a rapid method based on the monitoring of the metabolic activity of this bacterium. The inhibitory effect of the photodynamic process in the presence of a singlet oxygen quencher (sodium azide) or free radical scavengers (d-mannitol and l-cysteine) was evaluated by exposing bacterial suspensions with 0.5 µM Tri-Py(+)-Me-PF, 5.0 µM Tetra-Py(+)-Me or 5.0 µM Tri-SPy(+)-Me-PF to white light. Strong bacterial protection was observed with sodium azide (100 mM) for the three cationic porphyrins. However, in the presence of Tri-Py(+)-Me-PF and Tetra-Py(+)-Me and the free radical scavengers (l-cysteine and d-mannitol) the reduction on the bacterial bioluminescence was significantly higher and similar to that obtained in their absence (5.4-6.0 log reduction). In the case of Tri-SPy(+)-Me-PF two distinct behaviours were observed when l-cysteine and d-mannitol were used as free radical scavengers: while the presence of l-cysteine (100 mM) lead to a bacterial protection similar to the one observed with sodium azide, in the presence of d-mannitol only a small protection was detected. The high inhibition of the PS activity by l-cysteine is not due to its radical scavenger ability but due to the singlet oxygen quenching by the sulfanyl group (-SH). In fact, the photodecomposition of 1,3-diphenylisobenzofuran in the presence of Tri-SPy(+)-Me-PF is completely suppressed when l-cysteine is present. The results obtained in this study suggest that singlet oxygen (type II mechanism) plays a very important role over free radicals (type I mechanism) on the PI process of the bioluminescent E. coli by Tri-Py(+)-Me-PF, Tetra-Py(+)-Me and Tri-SPy(+)-Me-PF. Although the use of scavengers is an adequate and simple approach to evaluate the relative importance of the two pathways, it is important to choose scavengers which do not interfere in both PI mechanisms. Sodium azide and d-mannitol seem to be good oxygen and free radical quenchers, respectively, to study the PI mechanisms by porphyrinic photosensitizers.

Sewage bacteriophage inactivation by cationic porphyrins: influence of light parameters.

Photodynamic therapy has been used to inactivate microorganisms through the use of targeted photosensitizers. Although the photoinactivation of microorganisms has already been studied under different conditions, a systematic evaluation of irradiation characteristics is still limited. The goal of this study was to test how the light dose, fluence rate and irradiation source affect the viral photoinactivation of a T4-like sewage bacteriophage. The experiments were carried out using white PAR light delivered by fluorescent PAR lamps (40 W m(-2)), sun light (600 W m(-2)) and an halogen lamp (40-1690 W m(-2)). Phage suspensions and two cationic photosensitizers (Tetra-Py(+)-Me, Tri-Py(+)-Me-PF) at concentrations of 0.5, 1.0 and 5.0 microM were used. The results showed that the efficacy of the bacteriophage photoinactivation is correlated not only with the sensitizer and its concentration but also with the light source, energy dose and fluence rate applied. Both photosensitizers at 5.0 microM were able to inactivate the T4-like phage to the limit of detection for each light source and fluence rate. However, depending of the light parameters, different irradiation times are required. The efficiency of photoinactivation is dependent on the spectral emission distribution of the light sources used. Considering the same light source and a fixed light dose applied at different fluence rates, phage inactivation was significantly higher when low fluence rates were used. In this way, the light source, fluence rate and total light dose play an important role in the effectiveness of the antimicrobial photodynamic therapy and should always be considered when establishing an optimal antimicrobial protocol.

Antimicrobial photodynamic therapy: study of bacterial recovery viability and potential development of resistance after treatment.

Antimicrobial photodynamic therapy (aPDT) has emerged in the clinical field as a potential alternative to antibiotics to treat microbial infections. No cases of microbial viability recovery or any resistance mechanisms against it are yet known. 5,10,15-tris(1-Methylpyridinium-4-yl)-20-(pentafluorophenyl)-porphyrin triiodide (Tri-Py(+)-Me-PF) was used as photosensitizer. Vibrio fischeri and recombinant Escherichia coli were the studied bacteria. To determine the bacterial recovery after treatment, Tri-Py(+)-Me-PF (5.0 microM) was added to bacterial suspensions and the samples were irradiated with white light (40 W m(-2)) for 270 minutes. Then, the samples were protected from light, aliquots collected at different intervals and the bioluminescence measured. To assess the development of resistance after treatment, bacterial suspensions were exposed to white light (25 minutes), in presence of 5.0 microM of Tri-Py(+)-Me-PF (99.99% of inactivation) and plated. After the first irradiation period, surviving colonies were collected from the plate and resuspended in PBS. Then, an identical protocol was used and repeated ten times for each bacterium. The results suggest that aPDT using Tri-Py(+)-Me-PF represents a promising approach to efficiently destroy bacteria since after a single treatment these microorganisms do not recover their viability and after ten generations of partially photosensitized cells neither of the bacteria develop resistance to the photodynamic process.

Charge effect on the photoinactivation of Gram-negative and Gram-positive bacteria by cationic meso-substituted porphyrins.

BACKGROUND: In recent times photodynamic antimicrobial therapy has been used to efficiently destroy Gram (+) and Gram (-) bacteria using cationic porphyrins as photosensitizers. There is an increasing interest in this approach, namely in the search of photosensitizers with adequate structural features for an efficient photoinactivation process. In this study we propose to compare the efficiency of seven cationic porphyrins differing in meso-substituent groups, charge number and charge distribution, on the photodynamic inactivation of a Gram (+) bacterium (Enterococcus faecalis) and of a Gram (-) bacterium (Escherichia coli). The present study complements our previous work on the search for photosensitizers that might be considered good candidates for the photoinactivation of a large spectrum of environmental microorganisms. RESULTS: Bacterial suspension (10(7) CFU mL(-1)) treated with different photosensitizers concentrations (0.5, 1.0 and 5.0 microM) were exposed to white light (40 W m(-2)) for a total light dose of 64.8 J cm(-2). The most effective photosensitizers against both bacterial strains were the Tri-Py+-Me-PF and Tri-Py+-Me-CO2Me at 5.0 microM with a light fluence of 64.8 J cm(-2), leading to > 7.0 log (> 99,999%) of photoinactivation. The tetracationic porphyrin also proved to be a good photosensitizer against both bacterial strains. Both di-cationic and the monocationic porphyrins were the least effective ones. CONCLUSION: The number of positive charges, the charge distribution in the porphyrins' structure and the meso-substituent groups seem to have different effects on the photoinactivation of both bacteria. As the Tri-Py+-Me-PF porphyrin provides the highest log reduction using lower light doses, this photosensitizer can efficiently photoinactivate a large spectrum of environmental bacteria. The complete inactivation of both bacterial strains with low light fluence (40 W m(-2)) means that the photodynamic approach can be applied to wastewater treatment under natural light conditions which makes this technology cheap and feasible in terms of the light source.

Photoinactivation of bacteria in wastewater by porphyrins: bacterial beta-galactosidase activity and leucine-uptake as methods to monitor the process.

The increasing interest in photodynamic antimicrobial chemotherapy as a promising technology for wastewater treatment demands more efficient photosensitizers and faster methods for the monitorization of the photoinactivation process. Two sets of neutral and cationic porphyrins were used as photosensitizers for the photoinactivation of sewage bacteria. Two of the cationic porphyrins revealed to be highly active, even against Gram-negative bacteria, inactivating ca. 94-99.8% of these at 5 microM and after 270 min of irradiation with white light (9 mW cm(-2)). In addition, two expeditious methods for the evaluation of the bacteria photoinactivation were developed. These methods are based on beta-galactosidase activity and on leucine incorporation and give an excellent relation with faecal coliform counts. The evaluation of these two descriptors of bacterial activity, directly related with bacterial abundance, is an easy and fast way to monitor the micro-organisms during the photoinactivation process.

1,3-Dioxopyrrolo[3,4-b]porphyrins: synthesis and chemistry.

A novel 1,3-dioxopyrrolo[3,4-b]porphyrin (2) has been synthesized in 70% yield following a [4 + 2] cycloaddition reaction of pyrrolo[3,4-b]porphyrin 1 with (1)O(2). The new imide was used as a template to other 1,3-dioxopyrrolo[3,4-b]porphyrins and to the corresponding open counterparts. The UV/vis absorption spectra of the new compounds show significant red-shifts when compared with those of the nonsubstituted analogues. The structure of an imide derivative was confirmed by single-crystal X-ray diffraction.

Functional cationic nanomagnet-porphyrin hybrids for the photoinactivation of microorganisms.

Cationic nanomagnet-porphyrin hybrids were synthesized and their photodynamic therapy capabilities were investigated against the Gram (-) Escherichia coli bacteria, the Gram (+) Enterococcus faecalis bacteria and T4-like phage. The synthesis, structural characterization, photophysical properties, and antimicrobial activity of these new materials are discussed. The results show that these new multicharged nanomagnet-porphyrin hybrids are very stable in water and highly effective in the photoinactivation of bacteria and phages. Their remarkable antimicrobial activity, associated with their easy recovery, just by applying a magnetic field, makes these materials novel photosensitizers for water or wastewater disinfection.

Photodynamic inactivation of recombinant bioluminescent Escherichia coli by cationic porphyrins under artificial and solar irradiation.

A faster and simpler method to monitor the photoinactivation process of Escherichia coli involving the use of recombinant bioluminescent bacteria is described here. Escherichia coli cells were transformed with luxCDABE genes from the marine bioluminescent bacterium Vibrio fischeri and the recombinant bioluminescent indicator strain was used to assess, in real time, the effect of three cationic meso-substituted porphyrin derivatives on their metabolic activity, under artificial (40 W m(-2)) and solar irradiation (approximately 620 W m(-2)). The photoinactivation of bioluminescent E. coli is effective (>4 log bioluminescence decrease) with the three porphyrins used, the tricationic porphyrin Tri-Py+-Me-PF being the most efficient compound. The photoinactivation process is efficient both with solar and artificial light, for the three porphyrins tested. The results show that bioluminescence analysis is an efficient and sensitive approach being, in addition, more affordable, faster, cheaper and much less laborious than conventional methods. This approach can be used as a screening method for bacterial photoinactivation studies in vitro and also for the monitoring of the efficiency of novel photosensitizer molecules. As far as we know, this is the first study involving the use of bioluminescent bacteria to monitor the antibacterial activity of porphyrins under environmental conditions.

Sewage bacteriophage photoinactivation by cationic porphyrins: a study of charge effect.

Photodynamic therapy has been used to inactivate microorganisms through the use of targeted photosensitizers. Recently the inactivation of bacteria in residual waters has been reported, but nothing is known about photoinactivation of environmental bacteriophages, which are often used as indicators of human enteric viruses. In this study we tested the effect of six cationic porphyrin derivatives with two to four charges on the photoinactivation of a sewage bacteriophage. A phage suspension of 5 x 10(7) PFU mL(-1) was exposed to white light (40 W m(-2)), during 270 min, at three photosensitizer concentrations (0.5, 1.0 and 5.0 microM). Tetra- and tricationic porphyrins inactivated the T4-like sewage phage to the limits of detection, but dicationic porphyrins did not lead to a significant decrease in phage viability. At the highest photosensitizer concentration (5.0 microM), the phage was completely inactivated (>99.9999% of inactivation, reduction of 7.2 log) after 270 min by the tetracationic porphyrin. Two of the tricationic derivatives also led to phage inactivation to the limit of detection. The rate of bacteriophage photoinactivation and the efficiency of the photosensitizer appeared to vary with the charge and with the substituents in the meso-positions of the porphyrin macrocycle. Tetra- and tricationic porphyrins can, therefore, be used as a new method for inactivating sewage bacteriophages that are frequently used as human enteric virus indicators. The complete inactivation of viruses with low light intensity means that this methodology can be used even on cloudy days and during winter, opening the possibility to develop new technologies for wastewater treatment.