Cecília P. Sár

Selective Reduction of Nitrones and Nitroxides to Functionalized Secondary Amines

Abstract Nitrones and nitroxides were selectively reduced with Fe/AcOH to secondary amines in the presence of different functional groups (aldehyde, nitrile, carboxylic ester, activated and nonactivated double or triple bonds).

Utilizing new adamantyl spin traps in studying UV-B-induced oxidative damage of photosystem II

Abstract Two recently synthesized adamantyl nitrone spin traps, α-(4-pyridyl-N-oxide)-(1-adamantyl)-N-nitrone (POAN) and α-(4-pyridyl)- (I-adamantyl)-N-nitrone (PyAN), have been found to be useful for detecting reactive oxygen species in thylakoid membranes. Both show lower inhibition of photosystem II (PSII) electron transport and higher stability of their radical adducts than those of the widely used corresponding phenyl nitrones. Both spin traps are suitable for detecting hydroxyl radicals in UV-B-exposed thylakoid membranes when applied at 800 μM. Moreover, at 2 mM, POAN markedly retards UV-B-induced D1 protein degradation. No such effect of PyAN is observed, and neither trap shows marked protection against UV-B-induced electron-transport inactivation. The different chemical structures of the two traps imply different penetration into the thylakoid membrane, suggesting that UV-B-induced D1 protein degradation is brought about by hydroxyl radicals produced at special locations within the thylakoid membrane: these sites are accessible for POAN but inaccessible for the less membrane-penetrating PyAN.

Synthesis of α-aryl N-adamant-1-yl nitrones and using them for spin trapping of hydroxyl radicals

alpha-Aryl N-adamant-1-yl nitrones were synthesized and evaluated with respect to the stability of the hydroxyl radical adduct. The polarity and water solubility of nitrones were altered with changing the alpha-aryl groups. Introduction of adamantane ring instead of tert-butyl group resulted in a reasonable good stability of hydroxyl radical adduct for biological measurements.

Synthesis of new pyrrolidine nitroxide epoxides as versatile paramagnetic building blocks

Abstract Pyrrolidine nitroxide epoxides were synthesized by oxidation of the corresponding alkene, diene and α,β-unsaturated aldehyde with m -CPBA or H 2 O 2 to provide new spin labels and paramagnetic synthons.

Indanedione spin labelling of Na,K-ATPase

The indanedione series of vinyl ketone spin-labelling reagents has been extended in two ways: by increasing the length of the rigid spacer between the reactive centre and the nitroxide ring, or by introducing an electrophilic substituent (that could also hinder its rotation) at the bridge head position of the nitroxide ring. Three reagents of this new series have been used to spin label the Class II thiol groups of membranous Na,K-ATPase from Squalus acanthias. With a conjugated diene spacer, the majority of spin labels are strongly held but a minor population is relatively mobile at 37 degrees C. With a conjugated triene spacer, the nitroxide is still strongly held but a portion of the label is non-covalently bound. The 4-bromo-pyrroline derivative (with short vinyl spacer) is tightly held at the attachment site, and the conventional electron paramagnetic resonance (EPR) spectra distinguish between the two enantiomeric structures which differ in their mobility at 37 degrees C. Saturation transfer EPR (ST-EPR) spectra of this label at 4 degrees C have been used to determine the dependence of the protein rotational mobility on ionic strength. Electrostatic repulsion contributes to the lateral interactions between Na,K-ATPase molecules.

New sensors for monitoring reactive oxygen in plants under light stress

Reactive oxygen species (ROS) play a key role in a variety of biotic and abiotic stress conditions. Double (spin and fluorescent) ROS sensors contain a fluorophore and a spin trap: conversion of the latter to an EPR active nitroxide also causes partial fluorescence quenching. In this way, double sensors can be applied for monitoring ROS in experiments when EPR measurements are hard to carry out [1]. Using a dansyl-based double sensor, DanePy, we detected singlet oxygen in vivo, in leaves exposed to photoinhibition by excess photosynthetically active radiation [2]. Because the stress-inducible blue-green fluorescence may obstruct the detection of DanePy fluorescence quenching at 535-545 nm in leaves, we also investigated the application of phtalimide and rhodamine fluorophores in double sensors. Besides these molecules, we also studied the versatility (specificity, penetration, stability) of double sensors in which the fluorophore was combined with a spin trap instead of the singlet oxygen reactive moiety. These latter enabled us to compare oxygen (superoxide, hydroxyl) radical and singlet oxygen production in leaves exposed a variety of biotic stress conditions, such as light- (UV-B, photoinhibition), water- or temperature-stress. This work was supported by the Hungarian National Research Foundation (OTKA T030362) and the Hungarian Ministry of Education (FKFP 0252/1999). [1] Hideg E, Vass I, Kalai T and Hideg K (2000) Meth. Ezymol., 319, 77-85. [2] Hideg E, Kalai T, Hideg K and Vass I (1998) Biochemistry 37, 11405-11411.