Marilene Silva Oliveira

Singlet molecular oxygen generation by light-activated DHN-melanin of the fungal pathogen Mycosphaerella fijiensis in black Sigatoka disease of bananas.

In pathogenic fungi, melanin contributes to virulence, allowing tissue invasion and inactivation of the plant defence system, but has never been implicated as a factor for host cell death, or as a light-activated phytotoxin. Our research shows that melanin synthesized by the fungal banana pathogen Mycosphaerella fijiensis acts as a virulence factor through the photogeneration of singlet molecular oxygen O2 (1Δg). Using analytical tools, including elemental analysis, ultraviolet/infrared absorption spectrophometry and MALDI-TOF mass spectrometry analysis, we characterized both pigment content in mycelia and secreted to the culture media as 1,8-dihydroxynaphthalene (DHN)-melanin type compound. This is sole melanin-type in M. fijiensis. Isolated melanins irradiated with a Nd:YAG laser at 532 nm produced monomol light emission at 1270 nm, confirming generation of O2 (1Δg), a highly reactive oxygen specie (ROS) that causes cellular death by reacting with all cellular macromolecules. Intermediary polyketides accumulated in culture media by using tricyclazole and pyroquilon (two inhibitors of DHN-melanin synthesis) were identified by ESI-HPLC-MS/MS. Additionally, irradiation at 532 nm of that mixture of compounds and whole melanized mycelium also generated O2 (1Δg). A pigmented-strain generated more O2 (1Δg) than a strain with low melanin content. Banana leaves of cultivar Cavendish, naturally infected with different stages of black Sigatoka disease, were collected from field. Direct staining of the naturally infected leaf tissues showed the presence of melanin that was positively correlated to the disease stage. We also found hydrogen peroxide (H2O2) but we cannot distinguish the source. Our results suggest that O2 (1Δg) photogenerated by DHN-melanin may be involved in the destructive effects of Mycosphaerella fijiensis on banana leaf tissues. Further studies are needed to fully evaluate contributions of melanin-mediated ROS to microbial pathogenesis.

Quenching of Singlet Molecular Oxygen, O2(1Δg), by Dipyridamole and Derivatives

Dipyridamole (DIP) is known for its vasodilating and antiplatelet activity, exhibiting also a potent antioxidant effect, strongly inhibiting lipid peroxidation. This effect has been studied in mitochondria and a correlation between the DIP derivatives’ structure, the ability to bind to micelles and biological activity has been suggested. In the present work, the quenching of singlet molecular oxygen, O2(1Δg), by DIP and RA47 and RA25 derivatives was analyzed in acetonitrile (ACN) and aqueous acid solutions. Laser flash photolysis excitation of methylene blue (MB) was made at 532 nm and monomol light emission of O2(1Δg) was monitored at 1270 nm. Bimolecular quenching constants in ACN are consistent with an efficient physical quenching, presenting values a bit lower than the diffusion limit (kt = 3.4–6.8 × 108 m−1 s−1). The quenching process probably occurs via reversible charge transfer with the formation of an exciplex. Calculation of ΔGet associated with O2(1Δg) quenching corroborates with uncompleted electron transfer. In aqueous acid solutions (pH = 3.0), the kt values for DIP and derivatives are 20‐fold smaller when compared with ACN. The electrochemical properties of DIP in ACN are characterized by two consecutive one‐electron processes with half‐wave oxidation potentials of 0.30 and 0.67 V vs saturated calomel electrode (SCE). However, in an aqueous acid medium, a single oxidation wave is observed involving a two‐electron process (0.80 V vs SCE). Therefore, O2(1Δg) quenching is consistent with electrochemical data.

On the Thermal Decomposition of Dipyridamole: Thermogravimetric, Differential Scanning Calorimetric and Spectroscopic Studies

Abstract Thermal decomposition of dipyridamole was followed by analysis of the residue by spectroscopic methods. The loss of mass observed in thermogravimetric (TG) experiments in N2 atmosphere occurs in essentially three steps. The first step, corresponding to 35% of mass loss, was monitored in an isothermal process, and the solid residue was analyzed by proton and carbon NMR, optical absorption, and fluorescence emission. Heating at 305°C leads to new products with optical absorption bands shifted to lower wavelengths relative to dipyridamole. The broad emission band is also shifted to lower wavelengths. NMR analysis demonstrates that the piperidine groups are probably one of the sites of modification because the corresponding resonance peaks are not present in proton or carbon spectra. Preliminary high‐pressure chromatography shows that two main compounds appear at significantly higher polarity as compared with dipyridamole. Isothermal decomposition leaves the pyrimido‐pyrimidine central ring essentially unchanged, and the products involve changes at the peripheral substituent positions. Our results further contribute to elucidate the chemistry of this class of compounds.

Experimental and DFT Computational Insight into Nitrosamine Photochemistry—Oxygen Matters

A nitrosamine photooxidation reaction is shown to generate a peroxy intermediate by experimental physical-organic methods. The irradiation of phenyl and methyl-substituted nitrosamines in the presence of isotopically labeled 18-oxygen revealed that an O atom was trapped from a peroxy intermediate to trimethylphosphite or triphenylphosphine, or by nitrosamine itself, forming two moles of nitramine. The unstable peroxy intermediate can be trapped at low temperature in postphotolyzed solution in the dark. Chemiluminescence was also observed upon thermal decomposition of the peroxy intermediate, that is, when a postphotolysis low-temperature solution is brought up to room temperature. A DFT study provides tentative information for cyclic nitrogen peroxide species on the reaction surface.