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.

Photoinactivation of Escherichia coli (SURE2) without intracellular uptake of the photosensitizer.

AIM: This study was performed to investigate the possibility to photodynamically inactivate Gram-negative bacteria without intracellular uptake of the photosensitizer. The efficiency of the photodynamic growth inhibition of Escherichia coli (SURE2) was proved in a comparative study of a neutral and a cationic photosensitizer. METHODS AND RESULTS: We used confocal laser scanning microscopy (CLSM) to investigate the uptake of the photosensitizer by the bacteria to show that both chlorin e(6) and TMPyP are not accumulated in the cells. Fluorescence lifetime imaging (FLIM) and phototoxicity experiments were used to investigate the photodynamic inactivation of the Gram-negative bacteria. The phototoxicity experiments were carried out using a white light LED-setup to irradiate the bacterial suspensions. The viability of the bacteria was obtained by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-assay. For the cationic TMPyP, photodynamic inactivation without intracellular uptake was observed, whereas for chlorin e(6) such behaviour was not found. CONCLUSIONS: It was proven that in general, it is possible to photodynamically inactivate Gram-negative bacteria without photosensitizer accumulation in the bacterial cells. This fact is especially interesting, considering that the development of resistances may be prevented, leaving the active components outside the bacterium. SIGNIFICANCE AND IMPACT OF THE STUDY: In a world with bacteria that gain the ability to withstand the effects of antibiotics and are able to transmit on these resistances to the next generation, it becomes necessary to develop new approaches to inhibit the growth of multi-resistant bacteria. The photodynamic inactivation of bacteria is based on a three-component system by which the growth of the bacterial cells is inhibited. The well-directed oxidative damage is initiated by visible light of a certain wavelength, which excites a nontoxic photoactive molecule, called photosensitizer. Its reaction with oxygen causes the generation of cytotoxic species like singlet oxygen, which is highly reactive and causes the inactivation of the growth of bacteria.

Differentiation of aminomethyl corrole isomers by mass spectrometry.

Two new isomeric aminomethyl corrole derivatives of [5,10,15-tris(pentafluorophenyl)corrolato]gallium(III) were synthesized with pyridine (py) molecules as axial ligands. When investigated by electrospray ionization mass spectrometry, in the positive and the negative ion modes, these compounds showed an unusual gas-phase behavior that could be used for their differentiation. In the positive ion mode, the differentiation was achieved through the formation of diagnostic fragment ions formed from [M-py + H](+) precursors, by (CH(3) )(2) NH and HF losses. An unusual addition of water to the main fragment ions provides an alternative route for isomer identification. Semi-empirical calculations were performed to elucidate the structures and stabilities of the main ionic species formed in the positive ion mode. In the negative ion mode isomer discrimination is accomplished via the fragmentation of the methoxide adduct ions [M-py + CH(3) O](-) through (CH(3) )(2) N(.) and HF losses.

New porphyrin amino acid conjugates: synthesis and photodynamic effect in human epithelial cells.

The efficacy of new porphyrin amino acid conjugates as photosensitizers for photodynamic therapy (PDT) were assayed in vitro on tumoral (HeLa) and on non tumoral (HaCaT) human cell lines. The conjugates stable in liposomes are able to penetrate efficiently in the cytoplasm of cultured cancer and normal cells. No dark cytotoxicity is observed at the same concentration used for PDT cell treatment and during long incubation time (24h). The cell survival after the PDT treatment with visible light is dependent upon light exposure level and compound concentration. The tested compounds show higher photocytotoxicity in tumoral HeLa cells than in no tumoral HaCaT cells. The results suggest that these amino acid porphyrin conjugates are potential photosensitizers for PDT.

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.

Reduction and adduct formation from electrosprayed solutions of porphyrin salts.

The solutions of four meso-tetrakis(N-alkylpyridinium-4-yl)porphyrin salts and of the p-toluenesulfonate salt of meso-tetrakis(4-trimethylammoniumphenyl)porphyrin, in methanol, were studied by electrospray mass spectrometry, in order to investigate the influence of the type of counter ion, the length of the substituent N-alkyl groups of the four (N-alkylpyridinium-4-yl)porphyrins and the presence of an aromatic (alkylpyridinium) or aliphatic (trimethylammonium) nitrogen, in ion formation. In our experimental conditions, adducts with the counter ions were formed only for the meso-tetrakis(4-trimethylammoniumphenyl)porphyrin and were not observed for the other porphyrins, even when the counter ion was the same. In contrast, formation of reduced species, such as the [M(4+) + e(-)]3+, [M(4+) + 2e(-)]2+, [M(4+) + 4e(-) + 2H(+)]2+, and [M(4+) + 5e(-) + 2H(+)]+ ions was observed only for the (N-alkylpyridinium-4-yl)porphyrins and the appearance of these species is apparently solvent related and may occur via counter ion/solvent adducts.

Reduction of cationic free-base meso-tris-N-methylpyridinium-4-yl porphyrins in positive mode electrospray ionization mass spectrometry.

Reductions involving more than one electron with formation of the M+ and [M+2H]+ ions were observed for electrosprayed meso-tris(N-methylpyridinium-4-yl)porphyrin iodides, MI3. These reductions were studied by using different solvents and flow rates. Formation of the [M+2H]+ ions occurred only for protic solvents and to a larger extent at lower flow rates. The type of the fourth substituent does not seem to affect the reduction processes. Formation of the two reduced species, M+ and [M+2H]+ ions, may occur through the participation of counter ion/solvent clusters. Reduction of multiply charged, non-metallated species with formation of [M+nH]+ ions (n > 1) was not observed before in positive mode electrospray mass spectrometry.

Study by liquid secondary ion and electrospray mass spectrometry of synthesized and formed-in-source metallocorroles.

Liquid secondary ion and electrospray mass spectrometry were used to study the complexation in-source of 5,10,15-tris(pentafluorophenyl)corrole with several divalent transition-metal ions. The metallocorrole ions formed in-source were identified by comparing their product ion mass spectra with the spectra of the same ions formed from metallocorroles obtained from classical procedures. Positive metallocorrole ion formation is accompanied by oxidation of the metal centre. Mechanisms were proposed for the oxidation processes, and data from negative-ion spectra reinforced these mechanisms.

Characterization of isomeric cationic porphyrins with beta-pyrrolic substituents by electrospray mass spectrometry: the singular behavior of a potential virus photoinactivator.

Electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS) have been used to differentiate the 2- and 4-methylpyridyl isomers of free-base and metallated cationic beta-vinylpyridylporphyrins. The analysis by ESI-MS/MS of the deuterated analogs and semiempirical calculations of structural and electronic parameters were also undertaken. The two free-base isomers are easily differentiated by ESI-MS/MS but the presence of a metallic center renders differentiation of the metallated isomers less effective. The data acquired show that of all the studied compounds, the free-base 2-methylpyridyl isomer, which was operative in the in vitro photoinactivation of Herpes simples virus, has a different gas-phase behavior. Local distortion of the macrocycle due to the presence of the beta-vinylpyridyl substituent occurs for all the compounds, but a different electron density distribution can account for the observed gas-phase behavior of this potential virus photoinactivator.

Intra- and intermolecular heavy-atom effects on the fluorescence properties of brominated C60 polyads.

A series of brominated mono-methano[60]fullerene malonate derivatives (two diads and one triad) are investigated for intramolecular and external heavy-atom effects on their fluorescence. Significant internal and external heavy-atom effects are observed in the three cases. It is shown that the internal effect doubles when going from the diads to the triad. In bromobenzene and in iodobenzene, the external effect is predominant, and diads and triad behave identically.

Interactions of cationic porphyrins with double-stranded oligodeoxynucleotides: a study by electrospray ionisation mass spectrometry.

Electrospray ionisation mass spectrometry (ESI-MS), electrospray ionisation tandem mass spectrometry (ESI-MS/MS) and Ultraviolet-visible (UV-vis) spectroscopy were used to investigate the non-covalent interactions between small oligonucleotide duplexes with the GC motif and a group of cationic meso(N-methylpyridynium-4-yl)porphyrins (four free bases with one to four positive charges, and the zinc complex of the tetracationic free base). The results obtained point to outside binding of the porphyrins, with the binding strength increasing with the number of positive charges. Fragmentations involving losses from both chains were observed for the porphyrins with N-methylpyridinium-4-yl groups in opposite meso positions.

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.

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.

Cycloreversion and other gas-phase reactions of neutral and cationic pyrrolidine-fused chlorins and isobacteriochlorins under ion bombardment and electrospray.

Neutral and cationic pyrrolidine-fused chlorins and isobacteriochlorins derived from meso-tetrakis(pentafluorophenyl)porphyrin undergo cycloreversion reactions in the gas phase, either when desorbed from a liquid matrix by ion bombardment or when electrosprayed. Cycloreversion occurs through loss of either neutral or charged moieties, with and without hydrogen and methyl radical migration, and both as high- and low-energy collision processes. For the doubly charged isobacteriochlorin, one-electron reduction with methyl loss occurs under ion bombardment and electrospray, through hypervalent pyrrolidinium radical formation.

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.

Ion-selective electrodes based on metalloporphyrins for gibberellic acid determination in agricultural products.

This work describes the construction, evaluation and analytical application of electrodes selective to the gibberellate anion for the determination of gibberellic acid in agricultural products. Several types of PVC membrane electrodes without internal reference solution were prepared using the manganese(III) complex of meso-tetraphenylporphyrin (TPP) as ionophore and dibutyl phthalate (DBP), as plasticizer. The incorporation of lipophilic chemical species as additives, was also carried out aiming the evaluation of the response characteristics of the electrodes. To accomplish the analysis of commercial agricultural products a selective membrane composed of 28.0% (w/w) of PVC, 66.0% (w/w) of plasticizer and 6% (w/w) of ionophore was used, with no additive. This potentiometric unit presented a linear response between 10(-4) and 10(-1) mol L(-1) in gibberellate, a slope of about -69 mV dec(-1) and a reproducibility of about +/-1 mV day(-1). The potentiometric analysis of gibberellic acid in commercial products was carried out by direct potentiometry and the results obtained were compared with those provided by HPLC.

Unexpected fragmentation of beta-substituted meso-tetraphenylporphyrins induced by high-energy collisional activation.

The protonated molecules and radical cations of meso-tetraphenylporphyrins with beta-pyrrolic substituents, when formed by fast atom bombardment (FAB) and subjected to high-energy collisions, give rise to unexpected fragment ions. The reaction involves hydrogen migration from the ortho position of the phenyl ring to the a atom of the substituent, with formation of an intramolecular, six-membered ring. The process is analogous to condensed-phase cyclizations described for the same type of compounds. The fragmentation requires the presence of a double bond in the substituent group attached to the pyrrolic ring. A rearrangement process involving anchimeric assistance by the phenyl group (analogous to an ortho effect) is proposed for the formation of these ions.

Lignanamides and other phenolic constituents from the bark of kenaf (Hibiscus cannabinus).

Two new acyclic phenylpropane lignanamides, grossamide K and erythro-canabisine H, and the naphthol glucoside 2,5-dimethyl-3-O-beta-D-glucopyranosylnaphthol, along with six known compounds were isolated from the acetone extract of bark of Hibiscus cannabinus. All structures were established by spectroscopic methods including 2D NMR techniques, which allowed the correction of certain previously reported 13C NMR assignments of grossamide.

Differentiation of positional isomers of nitro meso-tetraphenylporphyrins by tandem mass spectrometry.

We studied by tandem mass spectrometry two isomers of nitro meso-tetraphenylporphyrin, one with a nitro group in the para position of a phenyl ring and the other with the same group in a beta-pyrrolic position, and their copper complexes. Collisional activation of the molecular ions of both free-base porphyrins and of their copper complexes produces an array of product ions that permit ready differentiation of the two positional isomers. The diagnostic ions, when the nitro group is in a beta-pyrrolic position, may be produced through intramolecular and double cyclization processes, triggered by the interaction of the nitro substituent with the neighboring meso-phenyl ring. These diagnostic ions do not form when the nitro group is in the para position. The gas-phase processes have precedents in solution chemistry.

Phenolic constituents from the core of kenaf (Hibiscus cannabinus).

Four lignans, boehmenan H [2-(4-hydroxy-3-methoxyphenyl)-5-[3-(4-hydroxy-3-methoxycinnamoyloxy)propyl]-3-hydroxymethyl-7-methoxybenzodihydrofuran], boehmenan K [2-(4-hydroxy-3-methoxyphenyl)-5-[3-(4-hydroxycinnamoyloxy)-1-propenyl]-3-(4-hydroxy-3-methoxycinnamoyloxymethyl)-7-methoxybenzodihydrofuran], threo-carolignan H [threo-1-(4-hydroxy-3-methoxyphenyl)-2-[4-[3-(4-hydroxy-3-methoxycinnamoyloxy)propyl]-2-methoxyphenoxy]-1,3-propanodiol], and threo-carolignan K [threo-1-(4-hydroxy-3-methoxyphenyl)-3-(4-hydroxy-3-methoxycinnamoyloxy)-2-[4-[3-(4-hydroxycinnamoyloxy)-1-propenyl]-2-methoxyphenoxy]-1-propanol] as well as several other lignans, aldehydes and a tyramine derivative were isolated from the acetone extract of core of kenaf (Hibiscus cannabinus). All the structures were established by spectroscopic methods. The hitherto unreported 13C NMR spectra of some compounds are also presented and discussed. 2D NMR techniques have allowed the revision of certain previously reported 13C NMR assignments of some scarce naturally occurring compounds.