Original Contribution EPR SPIN TRAPPING DETECTION OF CARBON-CENTERED CAROTENOID AND b -IONONE RADICALS TATYANA A. KONOVALOVA ,* L OWELLD. KISPERT ,* N IKOLAI E. P OLYAKOV , † andTATYANA V. LESHINA † *Department of Chemistry, University of Alabama, Tuscaloosa, AL, USA and † The Institute of Chemical Kinetics and Combustion SB RAS, Novosibirsk, Russia (Received 22 October 1999; Revised 18 January 2000; Accepted 1 February 2000) Abstract—Free radical intermediates were detected by the electron paramagnetic resonance spin trapping technique upon protonation/deprotonation reactions of carotenoid and b -ionone radical ions. The hyperfine coupling constants of their spin adducts obtained by spectral simulation indicate that carbon-centered radicals were trapped. The format these species was shown to be a result of chemical oxidation of neutral compounds by Fe 31 or I 2 followed by deprotonation of the corresponding radical cations or addition of nucleophilic agents to them. Bulk electrolysis red of b -ionone and carotenoids also leads to the formation of free radicals via protonation of the radical anions. Two differentspin adducts were detected in the reaction of carotenoid polyenes with piperidine in the presence of 2-methyl-2-nitroso-propane (MNP). One is attributable to piperidine radicals (C 5 H 10 N • ) trapped by MNP and the other was identified as trapped neutral carotenoid ( b -ionone) radical produced via protonation of the radical anion. Formation of these radical anions was confirmed by ultraviolet-visible spectroscopy. It was found that the ability of carotenoid radical anions/cations to produce neutral radicals via protonation/deprotonation is more pronounced for unsymmet carotenoids with terminal electron-withdrawing groups. This effect was confirmed by the radical cation deprotonat energy (H D ) estimated by semiempirical calculations. The results indicate that the ability of carotenoid radical cation to deprotonate decreases in the sequence: b -ionone . unsymmetrical carotenoids . symmetrical carotenoids. The minimum H D values were obtained for proton abstraction from the C(4) atom and the C(5)-methyl group ofthe cyclohexene ring. It was assumed that deprotonation reaction occurs preferentially at these positions. © 2000 Elsevier Science Inc. Keywords—Carotenoid, b -ionone, Protonation, Deprotonation, Free radical, Spin trapping INTRODUCTION Carotenoid polyenes are present in photosynthetic mem- branes. They serve as light-harvesting accessory pig- mentsand protectors againstthe damaging effect of singletoxygen [1–3]. Due to their extended p -electron systems, carotenoids are involved in the transport of charge across biological membranes. Transient absorp- tion studies have demonstrated that lightexcitation of chloroplasts leads to the formation of a carotenoid radi- cal cation Car •1 at the PSII reaction centers [4,5]. More- over,carotenoids can function as effective biological antioxidants preventing degenerative diseases such as cardiovascular disease and cancer. High reactivity of caro- tenoids with a variety of free radical species (peroxyl, alkoxyl, phenoxyl, thiyl,and sulfoxyl radicals) has been established [6 – 8]. The antioxidant activity of the carote- noids is determined by their ability to participate in elec- tron-transfer reactions in which the carotenoid can be oxi- dized to its radical cation and the resulting electron can be used to deactivate free radicals: R • 1 e 2 3 R 2 (1) Deactivation of free radicals (for example, lipid peroxyl radicals RO 2 • that arise during the process of lipid per- oxidation) is also affected by radical addition to carot- enoid chains of conjugated double bonds [9]: RO 2 • 1 Car 3 @RO 2 –Car# • (2) Address Correspondence to: Dr.LowellD. Kispert, University of Alabama, Department of Chemistry, Box 870336, Tuscaloosa, AL, USA; 35487;Tel: (205)348-5954; Fax: (205)348-9104; E-Mail: lkispert@bama.ua.edu. Free Radical Biology & Medicine, Vol. 28,No.7, pp.1030 –1038, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/00/$–see front matter PII S0891-5849(00)00192-1 1030