Influence of external bacterial structures on the efficiency of photodynamic inactivation by a cationic porphyrin.

The main targets of photodynamic inactivation (PDI) are the external bacterial structures, cytoplasmic membrane and cell wall. In this work it was evaluated how the external bacterial structures influence the PDI efficiency. To reach this objective 8 bacteria with distinct external structures were selected; 4 Gram-negative bacteria (Escherichia coli, with typical Gram-negative external structures; Aeromonas salmonicida, Aeromonas hydrophila both with an S-layer and Rhodopirellula sp., with a peptidoglycan-less proteinaceous cell wall and with cytoplasm compartmentalization) and 4 Gram-positive bacteria (Staphylococcus aureus, with typical Gram-positive external structures; Truepera radiovictrix, Deinococcus geothermalis and Deinococcus radiodurans, all with thick cell walls that give them Gram-positive stains, but including a second complex multi-layered membrane and structurally analogous to that of Gram-negative bacteria). The studies were performed in the presence of 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin tetraiodide (Tetra-Py(+)-Me) at 5.0 µM with white light (40 W m(-2)). The susceptibility of each bacteria to PDI by Tetra-Py(+)-Me was dependent on bacteria external structures. Although all Gram-positive bacteria were inactivated to the detection limit (reduction of ∼8 log) after 60-180 min of irradiation, the inactivation followed distinct patterns. Among the Gram-negative bacteria, E. coli was the only species to be inactivated to the detection limit (∼8 log after 180 min). The efficiency of inactivation of the two species of Aeromonas was similar (reduction of ∼5-6 log after 270 min). Rhodopirellula was less susceptible (reduction of ∼4 log after 270 min). As previously observed, the Gram-positive bacteria are more easily inactivated than Gram-negative strains, and this is even true for T. radiovictrix, D. geothermalis and D. radiodurans, which have a complex multi-layered cell wall. The results support the theory that the outer cell structures are major bacterial targets for PDI. Moreover, the chemical composition of the external structures has a stronger effect on PDI efficiency than complexity and the number of layers of the external coating, and lipids seem to be an important target of PDI.

Porphyrin derivatives as photosensitizers for the inactivation of Bacillus cereus endospores.

AIMS: In this study, we propose (i) to study the photodynamic inactivation (PDI) efficiency of neutral and cationic porphyrin derivatives, (ii) to characterize the kinetics of the inactivation process using Bacillus cereus as a model endospore-producing bacterium and (iii) to conclude on the applicability of porphyrin derivatives in the inactivation of bacterial endospores. METHODS AND RESULTS: The study of PDI of Bacillus cereus endospores, taken as model-endospores, using porphyrin derivatives differing in the number of positive charges and in the meso-substituent groups, showed that neutral, monocationic and dicationic porphyrins are quite ineffective, in contrast with the tri- and tetra-cationic molecules. The most effective porphyrin is a tricationic porphyrin with a meso-pentafluorophenyl group. With this photosensitizer (PS), at 0.5 micromol l(-1), a reduction of 3.5 log units occurs after only 4 min of irradiation. None of the porphyrin derivatives showed toxicity in the absence of light. CONCLUSIONS: Some porphyrin derivatives are efficient PSs for the inactivation of bacterial endospores and should be considered in further studies. Small modifications in the substituent groups, in addition to charge, significantly improve the effectiveness of the molecule as a PS for endospore inactivation. SIGNIFICANCE AND IMPACT OF THE STUDY: Tetrapyrrolic macrocycles should be regarded as worthy to explore for the PDI of spore-producing gram-positive bacteria. The development of molecules, more selective and effective, emerges as a new objective.

Characterization of dinitroporphyrin zinc complexes by electrospray ionization tandem mass spectrometry. Unusual fragmentations of beta-(1,3-dinitroalkyl) porphyrins.

The zinc complexes of diaryl bis(p-nitrophenyl)porphyrins and beta-(1,3-dinitroalkyl)tetraphenylporphyrins were studied by electrospray ionization (ESI) tandem mass spectrometry (MS/MS). All porphyrins showed the protonated molecule under ESI conditions. The protonated molecules were induced to fragment and the corresponding ESI tandem mass spectra were analysed. Porphyrins with two p-nitrophenyl groups showed, as expected, characteristic fragmentations including either loss of one nitro group, as the major fragment of the tandem mass spectra, and loss of both nitro groups. In contrast, MS/MS of the beta-(1,3-dinitroalkyl)porphyrins provided interesting and unexpected results such as the absence (or in insignificant abundance) of the ions formed by loss of one nitro group. However, these porphyrins show an abundant fragment due to combined loss of the two nitro groups. Also, the typical beta-cleavage of the alkyl chain is not observed per se, only when combined with loss of HNO2 or *NO2. Instead, alpha-cleavage, with loss of the beta-pyrrolic substituent, is the most favourable process.

Structural characterization of glycoporphyrins by electrospray tandem mass spectrometry.

Porphyrin derivatives having a galactose or a bis(isopropylidene)galactose structural unit, linked by ester or ether bonds, were characterized by electrospray tandem mass spectrometry (ES-MS/MS). The electrospray mass spectra of these glycoporphyrins show the corresponding [M + H](+) ions. For the glycoporphyrins with pyridyl substituents and those having a tetrafluorophenyl spacer, the doubly charged ions [M + 2H](2+) were also observed in ES-MS with high relative abundance. The fragmentation of both [M + H](+) and [M + 2H](2+) ions exhibited common fragmentation pathways for porphyrins with the same sugar residue, independently of the porphyrin structural unit and type of linkage. ES-MS/MS of the [M + H](+) ions of the galactose-substituted porphyrins gave the fragment ions [M + H - C(2)H(4)O(2)](+), [M + H - C(3)H(6)O(3)](+), [M + H - C(4)H(8)O(4)](+) and [M + H - galactose residue](+). The fragmentation of the [M + 2H](2+) ions of the porphyrins with galactose shows the common doubly charged fragment ions [porphyrin + H](2+), [M + 2H - C(2)H(4)O(2)](2+), [M + 2H - C(4)H(8)O(4)](2+), [M + 2H - galactose residue](2+) and the singly charged fragment ions [M + H - C(3)H(6)O(3)](+) and [M + H - galactose residue](+). The fragmentation of the [M + H](+) ions of glycoporphyrins with a protected galactosyl residue leads mainly to the ions [M + H - CO(CH(3))(2)](+), [M + H - 2CO(CH(3))(2)](+), [M + H - 2CO(CH(3))(2) - CO](+), [M + H - C(10)H(16)O(4)](+) and [M + H - protected galactose](+). The doubly charged ions [M + 2H](2+) fragment to give the doubly charged ions [porphyrin + H](2+) and the singly charged ions [M + H - protected galactose residue](+) and [M + H - CO(CH(3))(2)](+). For the porphyrins where the sugar structural unit is linked by an ester bond, [M + 2H](2+), ES-MS/MS showed a major and typical fragmentation corresponding to combined loss of a sugar structural unit and further loss of water, leading to the ion [M + 2H - sugar residue - H(2)O](2+), independently of the structure of the sugar structural unit. These results show that ES-MS/MS can be a powerful tool for the characterization of the sugar structural unit of glycoporphyrins, without the need for chemical hydrolysis.

Electrospray tandem mass spectrometry of new porphyrin amino acid conjugates.

Porphyrin amino acid conjugates with one or two porphyrin units were analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The ESI-MS spectra of all the porphyrins studied, obtained in positive ion mode, show the presence of the corresponding protonated molecule [M+H]+; ESI-MS spectra of diporphyrinyl compounds also show the doubly charged ions [M+2H]2+. The fragmentations of these ions induced by collision with argon were studied (ESI-MS/MS). ESI-MS/MS gives detailed structural information about the amino acids associated with the porphyrin. Cleavage of the bonds in the vicinity of the porphyrin moiety and those involving the side chain of amino acid residues gives structural information about this type of association. A fragmentation common to all derivatives corresponds to the cleavage of the phenyl-CO bond. The expected cleavage of the amide bond, that links the porphyrin to the amino acid moiety, is a minor fragmentation, which in some cases is even absent. The MS/MS spectra of the monoporphyrinyl derivatives show product ions characteristic of the amino acid linked to the porphyrin; the fragmentation also indicates when the amino acids has a terminal carboxylic group or a terminal ester group. The fragmentations of the diporphyrinyl compounds occur mainly by the cleavage of the spacer, leading, in the case of the doubly charged ions, to predominantly mono-charged ions, indicating a preferential location of the two protons in separated porphyrinic units.