J . Phys. Chem. 199498, 1111-1181
7777
Simultaneous Electrochemical and Electron Paramagnetic Resonance Studies of Carotenoids: Effect of Electron Donating and Accepting Substituents A. S. Jeevarajan, M. Khaled, and L. D. Kispert’ Department of Chemistry, University of Alabama. Tuscaloosa, Alabama 35487-0336 Received: December 29, 1993; In Final Form: March 25, 1994’
A series of substituted phenyl-7’-apocarotenoidswith varying electron donating and accepting substituents was examined by cyclic voltammogram (CV) and simultaneous electrochemical electron paramagnetic resonance (SEEPR). Carotenoids substituted with electron donating groups are more easily oxidized than those with electron accepting substituents. Comproportionation constants for the dication and the neutral species were measured by SEEPR techniques and by simulation of the CVs. The AHppof the SEEPR spectrum of the cation radicals varies from 13.2 to 15.6 G, and the g factors are 2.0021 f 0.0002. These EPR parameters suggest a polyene ?r-cation radical structure. The CVs are calculated using DigiSim, a C V simulation program, and the proposed mechanism involves three electrode reactions and two homogeneous reactions.
Introduction Carotenoids are found in the reaction centers of photosynthetic bacteria and plants. Carotenoids are necessary for the survival of photosynthetic organisms, serving as photoprotect devices and as light-harvesting pigments.’-‘ Photoprotection is achieved from quenching the chlorophyll and bacteriochlorophyll triplet by the carotenoid pigment. The carotenoid pigment can also quench any singlet oxygen that might be produced from photoexcited compounds. The light-harvesting role emerges from the fact that carotenoids absorb light energy in the region of thevisible spectrum (480-550 nm) where chlorophyll and bacteriochlorophyll are not efficient absorbers and then transfer this energy for use in photochemical electron transfer events. Carotenoids are also found to play an integral part in the electron transfer mechanism of the artificial photosynthetic systemsS (C-P-Q) made from porphyrin (P) covalently linked to carotenoids (C) and quinones (Q).However, numerous properties of carotenoids and the cation radicals formed upon electron loss are not understood. Carotenoid cation radicals that have been observed optically to form at the photosystem-I1 reaction center can function as an effective radical-trapping antioxidant6 and can be formed photolytically in solution7 and on Nafion films,8chemically on silicaalumina solid supports? by X-ray irradiation of powders, by reaction with iodine,1° and, because of its low oxidation potential (0.5 V for @-caroteneagainst SCE), by electrochemical methods in solution.11 Simultaneous electrochemical electron paramagnetic resonance (SEEPR) studies” of the carotenoids, @-carotene (I), 8’-apo-&caroten-8’-al, and canthaxanthin show that cation radicals and dications are formed in solution by the transfer of one and two electrons, respectively, upon electrochemical oxidation. The cyclic voltammogram (CV) consists of two separate peaks, one for each oxidation wave for canthaxanthin; the peaks coincide for @-carotene. It was demonstrated that the dication decays by loss of a protonI2 and that it undergoes a comproportionation reaction with the parent carotenoid (eq 1) in dichloromethane.
+ C a r + 2 Car*+ KGom
Car++
It has been difficult to determine the carotenoid cation radical structure in solution13 by electron nuclear double-resonance (ENDOR) techniques because the radical cation is not stable, exhibits exchange coupling at higher concentrations, possesses
* Author to whom correspondence should be addressed. 0
Abstract published in Advance ACS Absrmcrs, July 1, 1994.
0022-3654/94/2098-1111$04.50/0
numerous small couplings, and can only be formed in solution electrochemically for systems where Kwm> 1. These difficulties have been overcome, and the structure of the canthaxanthin cation radical has been determinedl3 by ENDOR. The signal to noise ratio is too small to examine the ENDOR of those radicals with Kwm I 1. Nevertheless, the comproportionation constant and the electrochemical data are useful for studying the stability of the cation radical in dichloromethane solution as a function of substituent. In the present study, a number of synthetic carotenoids bearing substituents of varying electron donating and accepting properties are examined. Values of K L , deduced by SEEPR measurements are used to determine evidence for the cation radical decomposition as a function of donor or acceptor substituents. K,, values deduced from simulation of the CVs are independent of the cation radical decomposition and are measures of the elementary processes indicated by eq 1.
Experimental Section SEEPR experiments were carried out with an IBM Enhanced Electrolytic Cell ER/ 164ECAusinga V-4533 rotating cylindrical cavity on a Varian E-12 EPR spectrometer. A hollow Teflon cylinder was placed along the cavity axis to lower the coaxial resonator frequency to 9.5 GHz. The working electrode was a 6 cm long, 6 mm diameter helical coil of 0.051 cm diameter platinum wire with a surface area of 10 cm2. The auxiliary electrode was also a platinum wire (1 mm thickness), and a silver wire (5 mm thickness) was used as a pseudoreference electrode. The silver wire was left for several hours in the solution of the carotenoid to be measured to ensure more consistent readings. Cyclic voltammograms were obtained by using a BAS-100A electrochemical analyzer and simultaneously recording the EPR spectra during the CV scan. The magnetic field was measured with a Bruker EPR 035M gaussmeter, and the microwave frequency was measured with a Model HP 5245L frequency counter. Thegfactor was measured with reference14to the sulfite anion radical (g = 2.003 16). Cbemicals HPLC grade dichloromethane from unopened bottles (Aldrich) was transferred under nitrogen in a drybox to a septum-stoppered flask. The solvent was transferred by syringe from this flask to a volumetric flask containing a measured amount of carotenoid. The solution was syringed into the IBM electrolytic cell and degassed with dry nitrogen gas. The supporting electrolyte tetrabutylammonium hexafluorophosphate (polarographicgrade) was used as supplied from Fluka. 0 1994 American Chemical Society
Jeevarajan et al.
7778 The Journal of Physical Chemistry, Vol. 98, No. 32, I994
TABLE 1: EPR Parameters and Estimated Electrochemical Values for Carotenoid Cation Radicals in Dichloromethane m 1 / 2
(f10mV) Kjma
compound
.-- 2.4 x 10'
[R'+l'
[Rim gfactor (*0.005) (+ O f G ) (t0.0002)
0.072
13.2
2.0028
0.032
13.7
2.0026
2.5 x 1 0 0.073
13.4
2.0026
0.044
15.2
2.0027
7'-(2,4,6-Trlmethylphenyi)7'-app-carotene @)
7'-(4-&thoxyphenyl)7'-apo-p-carotene
(In)
7'-Phenyl-7'-a~~carotenr (IV)
+co2H 41
4.95
0.040
14.2
2.0028
50
7.04
0.024
14.2
2.0027
0.072
14.3
2.0027
0.114
15.6
2.0027
7'-(eCarboxyphenyl)7'-apo-mro~ne (V)
x4
c
0
2
c
H
3
80
22.7
7'-(Pentafluorophenyi)7'-apo-~~tene (VIII)
a The value of K'EO, depends on the stability of the carotenoid cation radical and is thus not the comproportionationconstant Kmm for the elementary process given in eq 1.
8-Carotene was obtained from the Sigma Chemical Co. The other carotenoids were synthesized as previously described.lS-l7 All carotenoids were purified by column chromatography on silica gel. The 7'-trans/cis isomeric ratio of each was determined by IH-NMR spectroscopy, and the purity was verified by thin-layer chromatography. All carotenoids were stored in the dark at 4 O C in screw-cap vials, sealed with parafilm, and placed in a desiccator containing Drierite. The storage temperature combined with a dry atmosphere is a critical factor in maintaining chemical purity. AM1 (Austin Model 1) calculations18 were done using HyperChem software with a Gateway 2000 486DX2/50 personal computer.
Results and Discussion The CVs of the carotenoids I-VI11 (Table 1) were measured using the BAS lOOA with a silver wire as a pseudoreference electrode. The results are arranged in Table 1 according to the increase in the separation of the two oxidation waves ( A E l / z ) that form the carotenoid cation radical (Car*+)and the dication-
(Car++). The separation of the two oxidation waves for Car*+ and Car++can be seen in Figure la. As the difference in the two oxidation waves decreases (IV, Figure 1b), the separation appears as a broadening of the CV oxidation wave, and ultimately the two waves completely merge for I1 (Figure IC). This difference is attributed to the presence of the electron donating trimethyl substituent in 11 (although NMR measurements indicate the trimethyl-substituted phenyl group is not coplanar with the chain) and the electron withdrawing substituent, perfluorophenyl, in VIII. A CV similar to that of I1 consisting of two overlapping one-electron oxidation waves has been reported for 8-carotene.11 For 1-111, the CV waves were too overlapped to measure A E l p directly and so are not given in Table 1. It is evident that the absolute value of the first oxidation wave and the separation of the two oxidation waves (IV-VU) decrease when electron donating terminal substituents are present. The comproportionation constants (KL,)given in Table 1 for carotenoids IEVIII were calculated using the previously described
7780
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The Journal of Physical Chemistry, Vol. 98, No. 32, 1994
TABLE 2
SCHEME 1 Electrode Reactions Car
->
Car.+
->
*Car+ + e- ->
I
+ eCar++ + eCar.+
II III IV
V VI
'Car.
w
Homogeneous Reactions Car++
+
Car
Kcom
2
car.+
the bottom of Figure 3, indicate that the positive charges are delocalized in the two halves of the molecule. This suggests that the intrinsic polyene structure of the carotenoids can favor the formation of a dication. If an electron withdrawing group is present a t one end of the carotenoid molecule, as in compound VIII, formation of the dication is more difficult. As a result, in the cyclic voltammogram, two separate peaks are observed for such carotenoids. This is even more pronounced when both ends of the carotenoid contain an electron withdrawing group, such as for canthaxanthinll and rhodoxanthin.22 One of the consequences of this behavior is that the equilibrium concentration of the cation radical of VI11 is much higher than that for I or IV (Figure 2).
Simulation of Cyclic Voltammograms The cyclic voltammograms of the carotenoids I-VI11 were simulated using the DigiSim software.23 This was necessary if fundamental values of Eo and K,, are to be obtained. The experimental (solid line) and simulated (dashed line) CVs are given in Figure 1 for representative carotenoids: an electron withdrawing substituted phenyl carotenoid (VIII, Figure la), a phenyl-substituted carotenoid (IV,Figure 1b), and an electron donating, but nonplanar, substituted phenyl carotenoid (11,Figure IC). The three electrode reactions with their formal potentials (Eol, E02, E03) and the homogeneous reactions with their comproportionation constant (K,,) and deprotonation equilibrium constant (Kdp)used for the simulation are given in Scheme 1, where *Car represents the carotenoid with one less proton. The formal potentials (EO1, E O Z , Eo3), referenced to a silver wire pseudo electrode and obtained from the simulated CVs, are given in Table 2. The Kwm values were deduced from the difference between E O 1 and E02, and &p equals 0.02 for 11-VIII. The transfer coefficient is 0.5, and the heterogeneous electron transfer rate constant is 0.02 cm/s, which suggests a favorable electron transfer. The scan rate is 50 mV/s. The surface area of the electrode is 0.02 cm2. The concentration of the carotenoid varies from 0.5 to 1 mM. The diffusion coefficient ( D ) of the carotenoid is 1.O X 10-5 cm2/s. The hydrogen ion concentration in dichloromethane is 0 . 1 4 2 mM.24 The Koomvalues listed in Table 2 are much larger than the K L m values listed in Table 1. The Kcomvalues in Table 2 are deduced from the E O 1 and E O 2 values of the simulated CVs. The K',, values reported in Table 1 for compounds I-IV are derived from the steady state concentration of carotenoid cation radicals measured by SEEPR spectroscopy. For compounds IV-VI11 in Table 1, the K L , values are estimated from the difference in peak potentials of the cation radical and the dication obtained by experiment and not from thesimulation. Thus, the K',,values in Table 1 reflect the stability of the cation radical during electrolysis and the inability to estimate the difference in peak potential and do not reflect the value for the elementary process given in eq 1. If a carotenoid is difficult to oxidize (higher oxidation potentials for dication formation), it is easier to reduce the deprotonated
Simulated CV Parameters for Carotenoids I-VI11 5 30 560 550 550 550 575 605 635
560 640 630 680 685 720 755 795
3.5 22.5 22.5 157 191 283 343 507
35 60 90 90 90 120 150 160
0.05 0.02 0.02 0.02 0.02 0.02 0.02 0.02
VIn Silver wire reference electrode in dichloromethane. Experiments done with a bridged SCE reference electrode gave Eo values within h10 mV. Variation of D and Kdp gives a better cv fit.
dication (electrode reaction 3) to form the carotenoid neutral radical. This trend is clearly seen in the values of E O 3 listed in Table 2. For the substituted phenyl compounds, it is observed that the intensity of the peak for the third electrode reaction is higher for the electron donating substituents, even though the deprotonation equilibrium value (KdP) is constant for all the carotenoids (II-MII). This observation suggests that the dication stability increases as the electron donating capability of the substituent increases. It is not clear why I1 (Table 2) should be harder to oxidize than IV, because electron donation by the methyl groups should make it easier to oxidize 11. However, N M R measurements indicate that the trimethyl-substituted phenyl group is not coplanar with the chain, which may reduce the electron donation effect of the terminal substituent. It is also interesting to note that the radical cation of VI11 is the most stable of all carotenoids in Table 1, but the highest oxidation potential (Table 2) is required for formation. The electron withdrawing property of the substituted phenyl in VI11 stabilizes the cation radical of MIL However, the presence of the electron withdrawing group requires a higher oxidation potential for radical formation.
Conclusions Electron donating and accepting groups present in a terminal phenyl-substituted carotenoid strongly influence the oxidation potentialofthesecarotenoidsand the stability of thecation radicals and dications in dichloromethane solution. The stronger the electron accepting nature of the substituent, the more stable are the cation radicals and the less stable are the dications in dichloromethane solution. The magnitudes of the EPR parameters of AHppand g factors for the carotenoid cation radicals suggest a polyene a-cation radical structure for the carotenoids with little unpaired electron density present on the donor or acceptor substituent. In the dication, the two positive charges are delocalized in the two halves of the molecule with the stability of the dication increasing with electron donating character of the substituent. The stability of the carotenoid cation radicals varies with substituent; the perfluorophenyl species appears to be the most stable, occurring at the highest oxidation potential. Acknowledgment. We thank Dr. S.Feldberg of Brookhaven National Laboratory and Dr. Adrian Bott of Bioanalytical Systems for helpful discussions regarding the DigiSim software and Dr. Elli Hand for reading the manuscript. This work was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, Office of Energy Research of the US. Department of Energy under Grant No. DE-FG05-86ER13465. References and Notes (1) Koyama, Y. J. Photochem. Photobiol. 1991,B9,265and references therein. (2) Frank, H.A,; Violette, C.A.; Trautman, J. K.;Shreve, A. P.;Owens, T. G.; Albrecht, A. C. Pure Appl. Chem. 1991,63,109. (3) Mathis,P.;Schenck,C. C. InCwotenoidChemistryandBiochemistry; Britton, G., Goodwin, T. W., Eds.; Pergamon Press: New York, 1981; pp 339-351. (4) Mimuro, M.; Katoh, T. Pure Appl. Chem. 1991, 63, 123.
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