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acceptor substituents
Journalof ElectroanalyticalChemisQ 41I (I 996) 57-66
ELSEVIER
Electrochemical oxidation of carotenoids containing donor/acceptor substituents J.A. Jeevarajan, L.D. Kispert * Dqxumwnr
of Ctwmi.wy.
Uruversi~
of’Alohomu.
Tusroloosu.
AL 35487-0336.
USA
Received20 September1995;in revised form 17 January1996
Abstract
Cyclic voltammetry and squarewave voltammetry of the carotenoidsS-carotene(I), 7’,7’-dimethyl-7’-apo-p-carotene (II), 7’,7’hexadeutercdimethyl-7’-apo-P-carotene (III), 7’-cyano-7’-ethoxycarbonyl-7’-apo-S-carotene (IV), 7’,7’-dicyano-7’-apo-B-carotene (V) and canthaxanthin(VI) showthat the electrochemical propertiesof the syntheticdimethyl andhexadeuterodimethyl compounds aresimilarto thoseof the naturally occurringB-carotene,while the propertiesof the syntheticdicyanocarotenoidare similarto thoseof the naturally occurringcanthaxanthin,andthoseof the cyanoestercompoundareof an intermediatenature.The differencein oxidationpotentialsfor the formationof the cationradicaland the dication is smallfor the first set of compounds(lessthan 36 mV) and large(up to about200 mV>for the second.Simulationof the CVs with the DigiSim@programled to evaluationof the half-wave potentials,heterogeneous rate constantsandhomogeneous equilibriumconstantswhich describethe oxidation propertiesof the carotenoids. Keyword.s: Carotenoids; Cyclic voltammetry; Osteryouna squarewavevoltammetry;DigiSim *’, Electrondonor/acceptor property;Oxidation;Cation radial;Dication;Deprotonation; AMl; Excess chargedensity 1. Introduction
Carotenoids are widely present in photosynthetic membranes. These hydrophobic molecules (a) protect the photosynthetic organismsagainst the damaging effects of singlet oxygen and (b) absorb light and transmit excitation energy to a discrete site in the membrane where charge separation takes place. Owing to their extended conjugation and easeof oxidation, carotenoids may be expected to readily undergo electron-transfer reactions[l-4]. Carotenes lie close to the chlorophyll stacks in photosynthetic membranes and could thus be involved in electron transfer as well as in excited-state reactions. Electrochemical studies show that in the process of oxidation of the carotenoids several speciesare formed 15-81. From electrochemical studiesof B-carotene (I) in aprotic solvent mixtures [6] and of numerouscarotenoids containing various terminal substituents in dichloromethane [5-S], it is known that the oxidation of carotenoids in solution involves the transfer of two electrons. The further conclusion, that the oxidation product Car2+ deprotonates to give a carotenoid cation ‘Car+ (where * denotes a * Corresponding author. 0022.0728/96/$15.00
PII SOO22-0728(96)04572-X
0
1996
ElsevierScience S.A. All
rights
reserved
carotenoid with one less proton) which then undergoes reduction to form a carotenoid radical ( *Car ‘1, was confirmed by later studiesinvolving perdeutero-B-carotene[8]. The carotenoid radical *Car has been shown by EPR measurements[9] to be formed by lossof a proton from an sp3 carbon, most likely the 9, 9’, 13 or 13’ methyl groups. The electrospray massspectrum of B-carotene showed the presenceof peaks for Car’+ and *Car+ when p-carotene was converted into ions in dichloromethane solution using trifluoroacetic acid [ 101.Under appropriate conditions, the species *Car+ is formed by deprotonation of Car*+ (not by loss of H ’ from the molecular ion Car”). However, studies by van Breeman [l 1] have shown that, if the solvent usedwas changedto methanol and rert-butyl ether, only Car+. was observed. Cation radicals have been observed by pulse radiolysis [12], laser ionization [ 131and spectroelectrochemical [ 141 studies. ENDOR measurementsof the cation radicals generatedby different methods [15-171 in solution and in several solid matrices established that the radical electron density is distributed throughout the backbone of the carotenoids. During electrochemical oxidation, dications also undergo a chemical (solution) reaction with the neutral carotenoid to give cation radicals in a compropottionation equilibrium, as
/.A. JCWIWUJLUL
58 Table I Electrochemical
parameters
from simulation
L.D.
Kisprrt/Journnl
of CVs of carotenoids
o~Eleciroancrlyticcll
&carotene
4
411
(19%)
57-66
using DigiSim’ E,/mV ( !k 21
Compound
Chrndry
a
E,/mV
a
A E/mV
K coin
E,/mV f&2)
C&2)
540
545
5
1.2
35
568
602
34
3.8
160 b 90
5.52
588
36
4.1
156 b 68
159
487
172
a
(I)
7’,7’-Dicyano-7’*po-Btarotene
739
916
177
980
238
689
894
205
2915
264
(V)
0
0
Canthaxanthin a Reference b Reduction
(VI)
electrode SCE, relative errors reported. potentials reported for two types of ‘Car’.
demonstrated by simultaneous electrochemistry and EPR (SEEPR) measurements [9,18-211. The present study focuses on the kinetics involved in the electrochemical oxidative process and its dependence on the structure and type of carotenoids. The carotenoids studied were p-carotene (I), 7’,7’-dimethyl-7’-apo+carotene (II), 7’,7’-hexadeuterodimethyl-7’-ape+-carotene (III), 7’-cyano-7’-ethoxycarbonyl-7’-apo+carotene (IV), 7’,7’-dicyano-7’-apo+carotene (V) and canthaxanthin (VI). The two naturally occurring (I and VI) and four synthetic (II to V) carotenoids (see Table 1 for structures) were studied by means of cyclic voltammetry (CV> and Osteryoung square wave voltammetry (OSWV) [22] techniques. Kinetic parameters at the electrode were deduced
from the simulated cyclic voltammograms the DigiSim@ program [23].
with the aid of
2. Experimental CVs and OSWVs were obtained with an EG&G PARC Model 273 potentiostat. The working electrode was a 1.6 mm diameter platinum disk electrode (area 0.02 cm*). The auxiliary electrode was a platinum wire and an SCE was used as reference electrode. In some cases, a silver wire was used as a pseudo-reference electrode. Anhydrous (99 + %1 dichloromethane was obtained from Aldrich Chemical Co. in Sure/SealTM bottles which
J.A. Jreuurujun.
L.D.
Ki.~pert/Journal
oJElrctrounulytrcn1
were opened inside a drybox under a nitrogen atmosphere. The supporting electrolyte, tetra-n-butylammonium hexafluorophosphate (TBAHFP) of polarographic grade, was used as supplied by Fluka and also opened and kept in the dry box under a nitrogen atmosphere. Canthaxanthin (VI) and p-carotene (I) were purchased from Fluka and Sigma respectively. Compounds II to IV [24] and V [25] were synthesized in our laboratory. The purity of all carotenoids was verified by thin-layer chromatography and ‘H NMR spectroscopy. The carotenoids were stored at - 16°C in ampules sealed under vacuum or in vials kept over Drierite, and warmed to room temperature just before use. Care was taken to minimize exposure of the carotenoids to air, moisture or light. Preparation of samplesand the experiments were carried out as rapidly as possible to avoid complications due to decomposition. Sample solutions were 0.1 M in TBAHPP and 0.1 to 3.0 mM in carotenoid. For each concentration of carotenoid. experiments were run at different scan rates, ranging from 50 to 10000 mV s-‘. Simulations using the DigiSim’ program were performed on a Gateway 2000 Pentium P5-60 personal computer. The approach delineated in Ref. [23] was used to achieve the simulations. The DigiSim@ program does not addressadsorption and non-equilibrium processes. The mechanism for the simulation was drafted from previous as well as present work, as given in the Introduction asbackground material. Values for the various parameters were chosen in different ways. For example, the starting values for oxidation potentials were chosen from a rough estimate of the half-wave potential for each oxidation cycle of the CVs. The value of the tranfer coefficient (Y is well known to be 0.5 for most quasi-reversible systemsand this seemedto work very well for our system also. The values for the heterogeneousrate constant were first taken to be an unusually large number (1 X lo4 cm’ s- ’ ). If the simulation is insensitive to changesin values for a particular parameter, it is understood that the values used are wrong. Gradual changes in the values are made until the values start to affect the simulation, in which case we are now getting into the right range of values. For thermodynamically superfluous reactions (TSR& the parametersare derived by the program itself from the values of other parametersentered. For example, the comproportionation equilibrium constant K,,, is derived from the values of the first and second oxidation potentials. Other examples for TSRs are given in the appropriate sections. Chronocoulometric studiesestablisheda rough estimate for the values of the diffusion coefficients for the different carotenoids. The limits given in the tables for the values obtained from the simulations are deduced from a fit of calculated to experimental values. Values outside these limits visually produced changes in the simulated CVs. Only relative errors have been estimated for these values. Once the optimized set of values for all parameters required for the reaction scheme under study had been
Chemistry
411
(1996)
57-66
59
establishedfor a fit to a variety of scan rates and concentrations, then a particular parameter was set to values different from its optimized value while the other parameters were allowed to optimize. The variations in simulated CVs with variations in parametersare dealt with in detail individually under the appropriate sections.
3. Results and discussion The studies summarized above [5-91 have established the occurrence of reactions (1) to (5) shown in the following scheme. Electrode reactions CAR E, -
CAR+‘+ eE2 CAR+’ -CAR’++eE, *Car+ + e- + *car.
(‘1 (2) (3)
Homogeneousreactions K
CAR* + + CAR a CAR*+ K,, CAR+’ -
K&
2CAR +
(4)
- Car++H+
(5)
*Car’+ H+
(6)
Formation of other minor products CAR+‘+
Products
(7)
CAR*+ + Products
(8)
*;CXf+= Products
(9)
*Car + Products
( 10) Chronocoulometric studies on the newly synthesized compounds II to V show that their oxidation also involves the transfer of two electrons. Other recent studies[26] have indicated that reactions (6) to (10) are also necessaryfor successfulsimulation of the experimental CVS. Figs. 1 to 5 give the experimental and simulated CVs for compoundsI to VI and highlight someinteresting aspectsin the mechanism and parametersused for the fitting of experimental cvs. In a typical CV (Fig. l(a)) the first anodic wave (peak 1) correspondsto the oxidation of neutral carotenoid (VI) to form the cation radical [reaction (111.The secondanodic wave (peak 2) is due to the formation of the dication [reaction (211 by oxidation of the cation radical. In the reverse scan, the dication and the cation radical are reduced (peaks 3 and 4 respectively). The dication by a side reaction undergoesdeprotonation [reaction (511to form a deprotonatedcation *Car+, which in turn undergoesreduction (peak 5) to form the carotenoid radical *Car ’ [reaction (311. As mentioned in the Introduction, it had been shown previously [8] that peak 5 does not appear unless
J.A. Jeeuarujm.
L.D. Kispert/Journul
--------
Experiment Simulation
0.4
0.2
c~~Ei~crrocmcrlyticrr1
Chmzistry
41 I (1996)
57-66
_-------
Experiment Simulation
i.!...!,,,!,..!.,,!,“!.l
1.2
1.0
0.8
0.6
Potential
0.8
0.6
-0.2
-0.4
0.8
0.2
0.4 Potential/
0.4
0.2
0.0
-0.2
-0.4
t6.0
CVs of canthaxanthin
(VI)
at (a) 100
1 I.
1.0
L
0.8
I ”
I.
0.6
0.0
-0.2
V
~ --------
Experiment Simulation
Potential/V Fig. 1. Experimental and simulated and (b) 500 mV se ’ vs. SCE.
0.6
/V
--------
t15.0~,“!“‘!.‘.!“‘!“‘!“‘!“‘!“,~ 1.2 1.0
0.0
‘.
Experiment Simulation
I ”
0.4
0.2
Potential
I ”
0.0
:.
-0.2
IV
Fig. 3. Experimental and simulated CVs of (a) 7’,7’-dimethyl-7’-ape+ carotene (II) and (b) 7’,7’-hexadeuterodimethyl-7’.ape-p-carotene 100 mV s- ’ vs. SCE.
the secondstageof oxidation is reached and that peak 5 is due to a radical and not to the reduction of *Car ‘_ For compoundsI, II and III, the oxidation potentials of reactions (1) and (2) are similar so that the waves overlap completely (Figs. 2 and 3). For the cyanoester compound (Fig. 41, the two oxidation waves overlap partially, and in the case of V and VI (Figs. 1 and 5) the two oxidation waves (peaks 1 and 2) are well separatedand clearly seen. The carotenoids display two general types of redox behavior, which are determined primarily by two factors. The first important factor is their structural symmetry. Thus, reversible CVs were obtained only for the symmetrical compoundsVI and I (Figs. 1 and 2). The unsymmetrical compoundsIV and V show almost irreversible oxida-
L ~.!...!...!...!...!...!.“!.~ 1.o 0.8 0.6
0.4
0.2
0.0
(III) at
-0.2
Potential/V -2o.or -lO.Oi0.0-y 5 2 t10.0~~ ~ -------
Experiment Simulation
5 5 t20.0& 0 t30.0
--------
Experiment Simulation
+40.0 t
t10.0
t 1 .o
.
: ’ .
0.8
: ’
0.6
I
0.4 Potential
Fig. 2. Experimental and simulated of 100 mV s-’ vs. SCE.
:.
0.2
. . : . ’
0.0
I
-0.2
’ !
-0.4
+50.0t.“!““““‘!““““‘!“’ 1.0
0
CVs of p-carotene
0.5
0.0
.i
Potential/V (I) at a scan rate
Fig. 4. Experimental 7’-apo-P-carotene
and simulated
(IV)
at (a)
CVs of 7’-cyano-7’-ethoxycabonyl10000 mV s-’ vs. SCE.
50 and (b)
J.A. Jeeuurujcm.
__ --------
+2.0~,“!“‘!“‘!.“!‘..!“‘!“‘!.‘.1 1.2 1.0 0.6
0.6
! ” 1 .o
”
”
”
! ”
0.5
Potentlal
/ Journd
o~‘&ic)c.~roci,wlltic
0.2
0.0
-0.2
-0.4
Experiment Simulation
“”
.”
! .1
0.0
”
-
/V
Fig. 5. Experimental and simulated CVs of 7’,7’-dicyano-7’.apo-p-carotene (V) (a) with and (b) without deprotonation of the radical canon at 100 mV s-’ vs. SCE.
tion waves, as evidenced by the very low amplitude of peaks 3 and 4 (Figs. 4 and 5). The unsymmetrical hydrocarbons II and III (Figs. 3(a) and 3(b)) exhibit more nearly reversible behavior than compounds IV and V, but less than that of I and VI. This feature will be addressed further in a later section. The secondfeature is the electron donor/acceptor capability of the substituents. Table 1 gives the oxidation potentials for the six carotenoids, the h E values between the first (E,) and second(E,) oxidation steps,the compro-
Table 2 Deprotonation equilibrium constant for the dictation coefticients D for carotenoids I to VI Compound
Kd,
K,,
k,/s ’ a ( zk 0.002)
0.1
II
0.1
0.065 0. I22 b
0.1
(0.122) 0. I22
b
0. I 0. I 0. I
(0.117) 1.65 2.05 0.065
IV V VI
41 I (1996
J 57-66
61
3. I. Deprotonation of the dication Table 2 gives the equilibrium (K,,) and the forward rate constants (k,) for the deprotonation of the dication along with the equilibrium (K&,1 and forward rate constants (k;) for the deprotonation of the radical cation and the diffusion coefficient D for compounds I to VI at a scan rate of 100 mV sP‘. The starting values for these parameters were chosen arbitrarily as described in the Experimental section, and then decreasedin large incre-
K&
and their forward
rate constants
k;/s-
Gp
I
III
and the radical cation
01 Clremtstry
portionation equilibrium constant (K,,,) and the E, [reaction (3)] values. Studies on the oxidation of substituted ethylenes [27] and some aromatic hydrocarbons [28] show that with increasing electron-accepting nature of the substituents. the ease of oxidation decreasesand the compounds exhibit higher oxidation potentials. Similarly, oxidation of the carotenoids containing terminal electronacceptor groups (compounds V and VI) is difficult and furthermore the second oxidation step occurs at quite a different potential to the first oxidation step (Table 1). As the electron-accepting strength of the end groups decreases, the separation h E between the two oxidation waves decreases(compound IV>, and for compounds II and III (which contain electron-donating groups) the two waves merge as for compound I. The formation of a positive charge would not be favorable in the compounds containing electron-withdrawing groups, and hence a higher potential is necessaryfor their oxidation. Subsequentoxidation of the cation radical to the dication would obviously be even more difficult than the first oxidation. This is manifested by the much higher potentials of the second oxidation for V and VI than those observed for the other compounds. The difference in potentials between the first and second oxidations is 205 mV for VI and 177 mV for V, compared with 5 mV for I (Table 1). Conversely, as the strength of the electron-withdrawing groups decreases,the easeof oxidation increasesand the difference in oxidation potentials decreases.
/V
__ --------
t2.0””
Kisprrt
Experiment Simulation
0.4
Potential
L.D.
’ a
X-r and k; respectively IO6 D/cm’
(+7-l
( * 0.2)
2XlO.‘O
5 x 10-6
3.8
2xlo~‘0 (3x10
5 x 10-7
5
Cd) 4x
4
9jc
1.6 x IO- I0
(5x10 9)c 2.7 x lo-” 3.5 x lo-‘” 2XlO~‘?
10-6
Cd) 3 x lo-’ 1.5 X IO.’ I x lomq
4.5 8 7
a All values given are for a scan rate of 100 mV s- ‘, relative errors reported. b Forward rate constant for the deprotonation from a second site in the dication. ’ Equilibrium constant for the deprotonation from a second site in the radical cation. d Values for the forward rate constant for the deprotonation from the first and the second sites in the radical cation are the same.
s- ’
and diffusion
62
J.A. Jeruurujun. a.067
4.q56
L.D. Kisprrt
/ Journal
c~f‘Ele~trocmrrlyriccrl
-0.p54
0.231
Fig. 6. (a) Difference between AMI calculated excess charges of the dication and the neutral species of carotenoids I to VI (III not included). ( ), 7 position; [ 1, 7’ position; ovals, 9 position; rounded rectangles, 13 position; rectangles, 13’ position; circles, 9’ position.
ments until the sensitive range was reached. Once the range is established, minute changes in values were made to obtain the right numbers. Also, increases in values of k, from the values reported in Table 2 increases the height of peak 5 and vice versa. This occurs because, as the extent of deprotonation increases, there is a higher concentration of ‘car+ species formed that get reduced at peak 5. Although the equilibrium constants for the deprotonation of the dication are the same (K,, = 0.11, the value of k, (K,, = kf/kb) increaseswhen the symmetry of the compound is broken. These changes are related to the excess charge densities(Fig. 6) presentat various carbon atoms in the neutral, cation radical and dication species.To obtain the excess charge densities, geometry optimization of the neutral carotenoid. its radical cation and dication was first performed. A single-point AM1 calculation on the geometry optimized structures was then performed to obtain the excess charges on all three species.Values of the difference of the charge densitiesof the dication and the neutral species(Fig. 6) show that for the symmetrical carotenoids I and VI the excesspositive charge is distributed evenly in the two halves of the chain and located primarily at atoms 7, 7’, 9, 9’, 11, 11’ and 13, 13’ (the Fig. 6 caption indicates individual positions). The numbering system for the
Chemistry
41 I f 1996157-66
carotenoids is given in Table 1 for p-carotene. In the unsymmetrical carotenoids IV and V carbon atoms 15, 13’ and 11’ bear a much greater positive charge than in the dications of I and VI, and carbon atoms 11 and 13 are less positive. An asymmetric concentration of the positive charge leads to decreasedstability as reflected by the 20to 30-fold increase in the deprotonation rate constant. It should be noted that the magnitude of the oxidation potential does not correlate with the stability of the dication. Thus, even though the dication of canthaxanthin is formed at very high positive potentials, it is stabilized by equal distribution of the charges over the two halves of the chain. For compounds IV and V, which require similar high positive potentials for oxidation, the dication is highly unstable, and its facile deprotonation leads to the nearly irreversible redox behavior. Since increaseddeprotonation of the dication results in a higher concentration of the *Car’ species,the amplitude of the reduction wave (peak 5) is larger. The redox behavior of the unsymmetrical compoundsII and III is intermediate between that of the symmetrical compoundsand the unsymmetrical compoundscontaining strong electron-withdrawing substituents. For compounds II and III, although the unsymmetrical structure leads to an uneven distribution of the positive charge of the dication on the two halves of the chain, the end methyl groups, which are electron-releasingin nature, stabilize the charge, making its dications more stable than the dications of IV and V. The difference in the excesschargesbetween the 9, 13, 9’ and 13’ positions for II and III is not as great as for IV and V. Consequently, the CVs of II and III show a more reversible wave (peaks 3 and 4) than IV and V, but lessreversible than I and VI. Hence, its k, value is larger than for I and VI, but less than that for IV and V. Earlier studieshave shown that the chain methyl groups are involved in the deprotonation of the dication [9]. Although the symmetrical compounds I and VI have two possible sites of deprotonation (chain 9 and 13 methyls), the unsymmetrical compoundsIV and V have four (chain 9, 13, 9’ and 13’ methyls). In these compoundsonly one proton is lost, their CVs and OSWVs show only one fifth peak, and their CVs can be successfully simulated if only one site of deprotonation is invoked. In addition to the above sitesof deprotonation, compoundsII and III contain two additional terminal methyl groups. The CVs of compoundsII and III show a very broad fifth peak and OSWV clearly shows two fifth peaks at + 0.07 and - 0.17 V (Fig. 7). Evidently, deprotonation of the dications of compounds II and III occurs from two different sites, presumably any one of the methyl groups on the chain and one of the terminal methyl groups forming two types of *Car+ species,the reduction potentials of which are not the same. Irrespective of the particular sites of deprotonation, successful CV simulation (Figs. 3(a) and 3(b)) was achieved by invoking the formation of two different types of *Car+ species.
JA.
Jrtwurctjorr.
LB.
Kqwrr/Jourrwl
c~f’E/r~trourrul~trcul
Chmimy
(3.0 x 10m7 F) had to be included reproduce the experimental CV. 3.3. Deprotonation
0.5: o.o-
! 0.6
”
Fig. 7. OSWV
! ‘. 0.6
. ! “. ! “‘I”’ 0.4 0.2 Potential /V
of II from 0.8 to -0.3
0
! “‘F -0.2
-0.4
V vs. Ag wire.
3.2. CV simulation at higher scan rates One of the criteria that can establish compatibility of a mechanism with the experimental data is successful simulation, in which all calculated parameters have the same magnitude when different experimental CV scan rates are applied. In our work, we noted that the value of k, for the deprotonation of the dication had to be changed by approximately an order of magnitude in order to fit the CVs at higher scan rates. With an increase in scan rate, the extent of deprotonation should decrease because there is not enough time for deprotonation to occur. Figs. 4(a) and 4(b) show the CVs of the cyanoester compound at two different scan rates. The amplitude of peak 5 is less than that of peak 4 at higher scan rates, but the experimental CVs exhibited more deprotonation at higher scan rates than is predicted by the parameters used in the simulation at lower scan rates. All parameters were constant for scan rates of 100 and 10000 mV s- ’ except the forward rate constant for the deprotonation of the dication. The forward rate constant varied from 1.65 SK’ for 100 mV s-’ to 22 s- ’ for 10000 mV s- ’ . This is attributed to adsorption or, in general terms, non-equilibrium conditions. The DigiSim” program does not have provisions for adsorption and it assumes equilibrium conditions for all the reactions used in the mechanism for simulation. Also, at very high scan rates of about IO 000 mV s- ’ the double-layer capacitance Table 3 Decay equilibrium Chemical Car + ‘* Car”
reactlons Products
* Products
‘CX-+
Products
*Car‘*
Products
constants
and their forward
rate constants
63
41 I (1996157-66
in the scheme to
of the radical cation
Although it was possible to simulate the shape of peaks 1 to 4 with reactions (1) to (51, the fifth peak was not satisfactorily reproduced. Table 2 gives the equilibrium constant K&, for the deprotonation of the radical cation and the forward rate constant k; for this equilibrium reaction. Inclusion of the deprotonation of the radical cation [reaction (6)] in the reaction mechanism satisfactorily reproduced experimental behavior (Figs. 5(a) versus 5(b)). Although K& is many orders of magnitude smaller than K,,. the deprotonation of the radical cation was found to be a very important step in the mechanism used for the simulation of the experimental CVs. For compound V. a 44% reduction in the amplitude of the current for the fifth peak was observed when this step was not included in the mechanism used for the simulation. This deprotonation step is a thermodynamically superfluous reaction (TSR), and hence its equilibrium constant K& is calculated from the values given for E, and E, and also the value of K,,. 3.4. Comproportionation
reaction
The comproportionation equilibrium reaction [reaction (4)] is also a TSR and K,,,, is calculated by the program from the difference in E, and E,. With increasing separation in oxidation waves, that is larger AE, the value of K corn increases (Table 1). Canthaxanthin has the largest K corn value, p-carotene has the smallest, and values for the other carotenoids are intermediate. Although K,,, is derived by the program as mentioned above, the values of k, can be adjusted to give the best possible simulations. The values for k, are estimated in a manner similar to that given for the other parameters. Increases or decreases in the value of k, are reflected in the height of peak 4, which reflects the concentration of cation radicals present. A 0.1% increase in k, will result in a 20% increase in the height of peak 4 as more cation radicals are formed by the comproportionation reaction.
(s ’ ) (m parenthesis)
for the carotenoids
I
II
IV
V
VI
0.10 ( I .O) 0.60 (1.4) 0. IO (25) 0.10 (0.1)
0.10 (0.17) 0.60 (0.65) 0.10 a (IO) 0.05 b (0.1)
0.83 (0.65) 0.20 (0.75) 0.75 (7) 0.0 I (0.0001)
0.58 (0.5) 0.20 (0.9) 1.20 (5) 0.0 I (O.oilOl)
0.30 (0.1) 0.75 (0.21) 0.10 (10) 0.10 (0.1)
‘Car represents the carotenoid with one less proton a Value the same for the other -Car+ species. b Value the same for the other ‘Car. species.
64 Table 4 Heterogeneous Electrode
rate constants
(k/cm
sag’ ) for the carotenoids
reactions
El Car+
Car+‘+
6 Car+‘,-Car’++e‘Car++
em I
em
E, ‘Car.
Relative errors reported. ‘Car represents the carotenoid a Value the same for electron
with one less proton. transfer from the other ‘Car’
species,
3.5. Formation of other minor products Inclusion of reactions (7) to (10) in the mechanistic scheme for the CV simulations was found in previous studies [26] to have a remarkably positive effect in the fitting of the simulated CVs to the experimental ones. The equilibrium constants for these reactions are given in Table 3. It is well known that the intermediate species formed in the oxidation process undergo other chemical reactions such as dimerization, bond breaking followed by cyclization, etc. [29]. Previous SEEPR studies [18] have shown, from the decay of the EPR signal for the cation radicals of carotenoids, that the cation radicals can decay in other ways apart from their oxidation to form the dications. For IV and V. the equilibrium constants for the conversion of the dication into other products are much smaller than those for the other compounds; this means that the forward reaction is very much slower compared with the reverse step, indicating that the loss of dication by this decay step is not as significant for IV and V compared with the other compounds. An increase in the equilibrium constant value from 0.2 to 0.8 for the conversion of dication into other products for compounds IV and V drastically changes the simulated CV. Also, the equilibrium constants for the decay of the other intermediates, namely Car+’ and *Car+, are much larger than that for the dication. The variation of equilibrium constants for the decay of =Car’ species does not significantly affect the simulation. 3.6. Other electrochemical parameters Transfer coefficient (Y values between 0.5 and 0.6, typical for quasi-reversible systems, were used for the simulations. The heterogeneous electron transfer rate constants for reactions (1) to (3) are given in Table 4. These values are very similar to those obtained from previous SEEPR studies performed in our group [ 181. One observes that the rate of electron transfer for the conversion of the cation radical to the dication is in general higher than that for the conversion of the neutral to the cation radical. In general, the rates of electron transfer for the carotenoids reported are relatively high considering the highly resistant solvent (dichloromethane) used. The rate of electron transfer for reaction (3) seems to be much higher than for
reactions (1) and (2) for all the carotenoids. The rate of electron transfer will also depend on the relative diffusion constants of the different species. Increases or decreases in the values of k, affect the height of the peaks. For example, if the value of k, for the cyanoester IV is reduced from 0.0065 (best value) to 0.002 cm s- ‘, a 50% decrease in current for the first electron transfer step given by peak 1 is observed. This reduction is observed because a decrease in the rate of electron transfer would reduce the concentration of the electroactive species reaching the electrode and hence decrease the current observed. The opposite effect on increasing the value of k, also occurs. The diffusion coefficients for carotenoids I to VI are given in Table 2. It is well known that the diffusion of the dication will be much slower than that of the cation radical or the neutral species in aqueous or highly polar solutions. These observations may not be the same in the non-polar medium used in our study; hence the diffusion coefficients of all the species involved in the oxidation process were taken to be the same. The simulated CV was the same regardless of whether the diffusion coefficient of the proton was 9 X 10m5 cm* s- ’ (H+ diffusion coefficient) or the same as that for the neutral carotenoids. Values for diffusion coefficients obtained from chronocoulometric studies [gl were used as starting values, and minor changes were made to fit the exact magnitude of the current. The simulation is very sensitive to changes in values of diffusion coefficients. An 8% increase in the value of the diffusion coefficient for I increased the current at peaks 1 and 2 by 12%. 3.7. Significance of results obtained to photosynthesis The oxidation potentials for the carotenoid and the cation radical and the reduction potential (Table 1) for *Car+ along with the deprotonation equilibrium constants K,, and K& for the dication and cation radical respectively (Table 2), the nature of the substituents and symmetry of the carotenoids are important variables in the electron transfer properties of the carotenoids in the photoprotection of photosynthetic organisms. The fact that most carotenoids get oxidized within 1 V establishes their importance in photosynthesis. For instance, recent studies of
isolated photosystem II reaction centers have shown [3] that when the photosystem II reaction center is photoactivated under conditions where P680’ can photoaccumulate, there is a secondary oxidation of B-carotene by P680f’ to form the carotenoid cation radical. Rapid oxidation and bleaching of one of a pair of excitonically coupled B-carotene molecules occurs, followed by a slower oxidation and bleaching of the second. The different oxidation rates are believed to be due to binding of one carotenoid molecule to the Dl protein and the other to the D2 protein. Photoprotection for P680 is not only provided by B-carotene but also by a cycling of electrons around PSI1 via cytochrome b-559 and a monomeric chlorophyll. The fact that two electrons rather than one are transferred per carotenoid demonstrates its efficiency. Also, the deprotonation of the dication leads to the formation of Ht in the protein matrix and this leads to cis/trans isomerizations [30]. Because of the increased awareness of the involvement of cis/trans isomerization of carotenoids in many biochemical and biological processes [3 1,321, the processes that lead to the formation of these isomers are of interest.
4. Conclusions The experiments performed and the CV simulations support the proposed mechanism. The values of the oxidation potentials suggest that ease of oxidation of the carotenoids depends on the nature of substituents on the carotenoid. Carotenoids with strong electron-withdrawing substituents are very difficult to oxidize, while those with weak electron-withdrawing substituents are easily oxidizable. At faster scan rates the concentration of deprotonated cation formed due to the side reaction decreases significantly, indicating that other chemical and electrochemical reactions compete more effectively with the deprotonation. The fact that near-reversibility of the CVs of IV and V is attained only at high scan rates indicates that the electrochemically active species, namely the cation radical and dication, are highly unstable in solution. From the K,, values we learn that the carotenoids (II to V> which have one of their cyclohexene rings substitutedform the carotene radical more readily than the other two carotenoids (I and VI). The dimethyl compound has two deprotonation sites. indicating that a proton is lost from the chain methyl groups as well as from the end methyl groups. With an increasein electron-withdrawing nature of the substituents, the separationbetween the two oxidation waves increases. thus increasing the comproportionation equilibrium constant K,,,. AM1 calculations of the excess charge distribution in the dications show that in the more stable dications the charge is more evenly distributed in the two halves of the backbone chain, whereas in the highly reactive dications of unsymmetrical carotenoids it is not evenly distributed, thus making them highly unstable. From the data obtained regarding the formation of other minor prod-
ucts, it can be concluded that the dications of IV and V undergo more deprotonation than any other side reaction.
Acknowledgements We would like to thank Dr. S. Feldberg for helpful discussions regarding the simulation program, Mrs. W. Hung for synthesizing carotenoids IV and V, Dr. E.S. Hand for the synthesisof the dimethyl compounds,Drs. A. Jeevarajan and E.S. Hand for their suggestionsand for critically reading the manuscript and Roche Vitamins and Fine Chemicalsfor a gift of the oil suspensionof 8’-apoB-caroten-X’-al, a precursor of compoundsII-V. This work was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences. Department of Energy, under Grant DE-FG05-86ER13465.
References [I] D. Gust. T.A. Moore and A.L. Moore, Act. Chem. Res., 26 (1993) 198. [2] M.P. O’Neil, M.R. Wasielewski, M. Khaled and L.D. Kispert, J. Chem. Phys.. 95 (1991) 7212. [3] J.O.L. Rivas, A. Telfer and J. Barber, B&him. Biophys. Acta, I142 (I 993) 155 and references cited therein. [4] A.L. Moore, A. Joy, R. Tom, D. Gust, T.A. Moore, R.V. Bensasson and E.J. Land. Science, 216 (1982) 982. [S] J.L. Grant, V.J. Kramer, R. Ding and L.D. Kispert, J. Am. Chem. kc.. I IO (1988) 2151. [6] V.G. MairanovsLy, A.A. Engovatov, N.T. Ioffe and (3.1. SamoJ.hvalov. J. Electroanal. Chem., 66 (1975) 123. [7] S.M. Park, J. Electrochem. kc., 125 (1978) 216. [8] M. Khaled, A. Hadjipetrou and L. Kispert, J. Phys. Chem., 94 (1990) 5164. [9] A.S. Jeevarajan, M. Khaled and L.D. Kispert, Chem. Phys. Lett., 22s ( 1994) 340. [IO] G.J. Van Berhel and F. Zhou, Anal. Chem., 66 (1994) 3408. [I I] R.B. van Breemen, Anal. Chem.. 67 (I 995) 2004. [12] J. Lafferty, A.C. Roach, R.S. Sinclair, T.G. Truscott and E.J. Land, J. Chem. Sot. Faraday Trans. I, 71 (I 977) 416. 131 P. Mathis and A. Vermeglto, Photochem. Photobiol., I5 (1972) 157. 141 AS. Jeevarajan, L.D. Kispert and X. Wu. Chem. Phys. Lett., 219 (l9Y.t) 427. 151 Y. Wu, L. Piekara-Sady and L.D. Ktspert, Chem. Phys. Lett., I80 (1991) 573. 161 .4.S. JeevaraJan, L.D. Kispert and L. Piehara-Sady, Chem. Phys. Lett., 209 (I 993) 269. 171 L. Pielara-Sady, A.S. Jeevarajan and L.D. Kispert, Chem. Phys. Lett.. 207 (I 993) 173. [I81 M. Khaled, A. Hadjipetrou, L.D. Kispert and R.D. Allendoerfer, J. Phys. Chem., 95 (1991) 2438. [IO] AS. Jeevarajan, M. Khaled and L.D. Ktspert, J. Phys. Chem., 98 (1994) 7777. [20] M.M. Khaled, Ph.D. Dtssertatton, The University of Alabama, Tuscaloosa, .4L, 1992. [2l] X. Chen. Masters Thesis, The University of Alabama, Tuscaloosa, AL, 1991. [22] J.G. Osteryoung and R.A. Osteryoung, Anal. Chem., 57 (1985) IOIA. [23] M. Rudolph, D.P. Reddy and S.W. Feldberg, Anal. Chem., 66 ts available from Bioanalytical Systems, (1994) 589A (Dtgisim’ Inc., 2701 Kent Ave., West Lafayette. IN 47906).
66
J.A. Jeeuurujm.
L.D.
Kispert/Journol
[241 E.S. Hand and L.D. Kispert, unpublished results. i2.51 E.S. Hand, K.A. Belmore and L.D. Kispert, J. Chem. Sot. Perkin Trans. 2, ( 1993) 659. [26] J.A. Jeevarajan, A.S. Jeevarajan and L.D. Kispert, J. Chem. Sot. Faraday Trans., in press. [27] J. Phelps and A.J. Bard, J. Elecuoanal. Chem., 68 (1976) 3 13. [28] H. Volz and W. Lotsch, Tetrahedron Lett., 27 (1969) 2275.
o~~Ele~~trounulyti~~1
[29]
Chemistry
411
(1996)
57-66
W. Vetter, G. Englert, N. Rigassi and U. Schwieter, in 0. Isler (Ed.), Carotenoids, Birkhluser, Base], 1971, p. 249. [30] G. Gao. CC. Wei, AS. Jeevarajan and L.D. Kispert, J. Phys. Chem., in press. (311 I. Ashikawa, A. Miyata, H. Koike, Y. lnoue and Y. Koyama, B&hem., 25 (1986) 6154. [32] Y. Koyama and Y. Mukai, Adv. Spectrosc., 21 (1993) 49.
ELSEVIER
Electrochemical oxidation of carotenoids containing donor/acceptor substituents J.A. Jeevarajan, L.D. Kispert * Dqxumwnr
of Ctwmi.wy.
Uruversi~
of’Alohomu.
Tusroloosu.
AL 35487-0336.
USA
Received20 September1995;in revised form 17 January1996
Abstract
Cyclic voltammetry and squarewave voltammetry of the carotenoidsS-carotene(I), 7’,7’-dimethyl-7’-apo-p-carotene (II), 7’,7’hexadeutercdimethyl-7’-apo-P-carotene (III), 7’-cyano-7’-ethoxycarbonyl-7’-apo-S-carotene (IV), 7’,7’-dicyano-7’-apo-B-carotene (V) and canthaxanthin(VI) showthat the electrochemical propertiesof the syntheticdimethyl andhexadeuterodimethyl compounds aresimilarto thoseof the naturally occurringB-carotene,while the propertiesof the syntheticdicyanocarotenoidare similarto thoseof the naturally occurringcanthaxanthin,andthoseof the cyanoestercompoundareof an intermediatenature.The differencein oxidationpotentialsfor the formationof the cationradicaland the dication is smallfor the first set of compounds(lessthan 36 mV) and large(up to about200 mV>for the second.Simulationof the CVs with the DigiSim@programled to evaluationof the half-wave potentials,heterogeneous rate constantsandhomogeneous equilibriumconstantswhich describethe oxidation propertiesof the carotenoids. Keyword.s: Carotenoids; Cyclic voltammetry; Osteryouna squarewavevoltammetry;DigiSim *’, Electrondonor/acceptor property;Oxidation;Cation radial;Dication;Deprotonation; AMl; Excess chargedensity 1. Introduction
Carotenoids are widely present in photosynthetic membranes. These hydrophobic molecules (a) protect the photosynthetic organismsagainst the damaging effects of singlet oxygen and (b) absorb light and transmit excitation energy to a discrete site in the membrane where charge separation takes place. Owing to their extended conjugation and easeof oxidation, carotenoids may be expected to readily undergo electron-transfer reactions[l-4]. Carotenes lie close to the chlorophyll stacks in photosynthetic membranes and could thus be involved in electron transfer as well as in excited-state reactions. Electrochemical studies show that in the process of oxidation of the carotenoids several speciesare formed 15-81. From electrochemical studiesof B-carotene (I) in aprotic solvent mixtures [6] and of numerouscarotenoids containing various terminal substituents in dichloromethane [5-S], it is known that the oxidation of carotenoids in solution involves the transfer of two electrons. The further conclusion, that the oxidation product Car2+ deprotonates to give a carotenoid cation ‘Car+ (where * denotes a * Corresponding author. 0022.0728/96/$15.00
PII SOO22-0728(96)04572-X
0
1996
ElsevierScience S.A. All
rights
reserved
carotenoid with one less proton) which then undergoes reduction to form a carotenoid radical ( *Car ‘1, was confirmed by later studiesinvolving perdeutero-B-carotene[8]. The carotenoid radical *Car has been shown by EPR measurements[9] to be formed by lossof a proton from an sp3 carbon, most likely the 9, 9’, 13 or 13’ methyl groups. The electrospray massspectrum of B-carotene showed the presenceof peaks for Car’+ and *Car+ when p-carotene was converted into ions in dichloromethane solution using trifluoroacetic acid [ 101.Under appropriate conditions, the species *Car+ is formed by deprotonation of Car*+ (not by loss of H ’ from the molecular ion Car”). However, studies by van Breeman [l 1] have shown that, if the solvent usedwas changedto methanol and rert-butyl ether, only Car+. was observed. Cation radicals have been observed by pulse radiolysis [12], laser ionization [ 131and spectroelectrochemical [ 141 studies. ENDOR measurementsof the cation radicals generatedby different methods [15-171 in solution and in several solid matrices established that the radical electron density is distributed throughout the backbone of the carotenoids. During electrochemical oxidation, dications also undergo a chemical (solution) reaction with the neutral carotenoid to give cation radicals in a compropottionation equilibrium, as
/.A. JCWIWUJLUL
58 Table I Electrochemical
parameters
from simulation
L.D.
Kisprrt/Journnl
of CVs of carotenoids
o~Eleciroancrlyticcll
&carotene
4
411
(19%)
57-66
using DigiSim’ E,/mV ( !k 21
Compound
Chrndry
a
E,/mV
a
A E/mV
K coin
E,/mV f&2)
C&2)
540
545
5
1.2
35
568
602
34
3.8
160 b 90
5.52
588
36
4.1
156 b 68
159
487
172
a
(I)
7’,7’-Dicyano-7’*po-Btarotene
739
916
177
980
238
689
894
205
2915
264
(V)
0
0
Canthaxanthin a Reference b Reduction
(VI)
electrode SCE, relative errors reported. potentials reported for two types of ‘Car’.
demonstrated by simultaneous electrochemistry and EPR (SEEPR) measurements [9,18-211. The present study focuses on the kinetics involved in the electrochemical oxidative process and its dependence on the structure and type of carotenoids. The carotenoids studied were p-carotene (I), 7’,7’-dimethyl-7’-apo+carotene (II), 7’,7’-hexadeuterodimethyl-7’-ape+-carotene (III), 7’-cyano-7’-ethoxycarbonyl-7’-apo+carotene (IV), 7’,7’-dicyano-7’-apo+carotene (V) and canthaxanthin (VI). The two naturally occurring (I and VI) and four synthetic (II to V) carotenoids (see Table 1 for structures) were studied by means of cyclic voltammetry (CV> and Osteryoung square wave voltammetry (OSWV) [22] techniques. Kinetic parameters at the electrode were deduced
from the simulated cyclic voltammograms the DigiSim@ program [23].
with the aid of
2. Experimental CVs and OSWVs were obtained with an EG&G PARC Model 273 potentiostat. The working electrode was a 1.6 mm diameter platinum disk electrode (area 0.02 cm*). The auxiliary electrode was a platinum wire and an SCE was used as reference electrode. In some cases, a silver wire was used as a pseudo-reference electrode. Anhydrous (99 + %1 dichloromethane was obtained from Aldrich Chemical Co. in Sure/SealTM bottles which
J.A. Jreuurujun.
L.D.
Ki.~pert/Journal
oJElrctrounulytrcn1
were opened inside a drybox under a nitrogen atmosphere. The supporting electrolyte, tetra-n-butylammonium hexafluorophosphate (TBAHFP) of polarographic grade, was used as supplied by Fluka and also opened and kept in the dry box under a nitrogen atmosphere. Canthaxanthin (VI) and p-carotene (I) were purchased from Fluka and Sigma respectively. Compounds II to IV [24] and V [25] were synthesized in our laboratory. The purity of all carotenoids was verified by thin-layer chromatography and ‘H NMR spectroscopy. The carotenoids were stored at - 16°C in ampules sealed under vacuum or in vials kept over Drierite, and warmed to room temperature just before use. Care was taken to minimize exposure of the carotenoids to air, moisture or light. Preparation of samplesand the experiments were carried out as rapidly as possible to avoid complications due to decomposition. Sample solutions were 0.1 M in TBAHPP and 0.1 to 3.0 mM in carotenoid. For each concentration of carotenoid. experiments were run at different scan rates, ranging from 50 to 10000 mV s-‘. Simulations using the DigiSim’ program were performed on a Gateway 2000 Pentium P5-60 personal computer. The approach delineated in Ref. [23] was used to achieve the simulations. The DigiSim@ program does not addressadsorption and non-equilibrium processes. The mechanism for the simulation was drafted from previous as well as present work, as given in the Introduction asbackground material. Values for the various parameters were chosen in different ways. For example, the starting values for oxidation potentials were chosen from a rough estimate of the half-wave potential for each oxidation cycle of the CVs. The value of the tranfer coefficient (Y is well known to be 0.5 for most quasi-reversible systemsand this seemedto work very well for our system also. The values for the heterogeneousrate constant were first taken to be an unusually large number (1 X lo4 cm’ s- ’ ). If the simulation is insensitive to changesin values for a particular parameter, it is understood that the values used are wrong. Gradual changes in the values are made until the values start to affect the simulation, in which case we are now getting into the right range of values. For thermodynamically superfluous reactions (TSR& the parametersare derived by the program itself from the values of other parametersentered. For example, the comproportionation equilibrium constant K,,, is derived from the values of the first and second oxidation potentials. Other examples for TSRs are given in the appropriate sections. Chronocoulometric studiesestablisheda rough estimate for the values of the diffusion coefficients for the different carotenoids. The limits given in the tables for the values obtained from the simulations are deduced from a fit of calculated to experimental values. Values outside these limits visually produced changes in the simulated CVs. Only relative errors have been estimated for these values. Once the optimized set of values for all parameters required for the reaction scheme under study had been
Chemistry
411
(1996)
57-66
59
establishedfor a fit to a variety of scan rates and concentrations, then a particular parameter was set to values different from its optimized value while the other parameters were allowed to optimize. The variations in simulated CVs with variations in parametersare dealt with in detail individually under the appropriate sections.
3. Results and discussion The studies summarized above [5-91 have established the occurrence of reactions (1) to (5) shown in the following scheme. Electrode reactions CAR E, -
CAR+‘+ eE2 CAR+’ -CAR’++eE, *Car+ + e- + *car.
(‘1 (2) (3)
Homogeneousreactions K
CAR* + + CAR a CAR*+ K,, CAR+’ -
K&
2CAR +
(4)
- Car++H+
(5)
*Car’+ H+
(6)
Formation of other minor products CAR+‘+
Products
(7)
CAR*+ + Products
(8)
*;CXf+= Products
(9)
*Car + Products
( 10) Chronocoulometric studies on the newly synthesized compounds II to V show that their oxidation also involves the transfer of two electrons. Other recent studies[26] have indicated that reactions (6) to (10) are also necessaryfor successfulsimulation of the experimental CVS. Figs. 1 to 5 give the experimental and simulated CVs for compoundsI to VI and highlight someinteresting aspectsin the mechanism and parametersused for the fitting of experimental cvs. In a typical CV (Fig. l(a)) the first anodic wave (peak 1) correspondsto the oxidation of neutral carotenoid (VI) to form the cation radical [reaction (111.The secondanodic wave (peak 2) is due to the formation of the dication [reaction (211 by oxidation of the cation radical. In the reverse scan, the dication and the cation radical are reduced (peaks 3 and 4 respectively). The dication by a side reaction undergoesdeprotonation [reaction (511to form a deprotonatedcation *Car+, which in turn undergoesreduction (peak 5) to form the carotenoid radical *Car ’ [reaction (311. As mentioned in the Introduction, it had been shown previously [8] that peak 5 does not appear unless
J.A. Jeeuarujm.
L.D. Kispert/Journul
--------
Experiment Simulation
0.4
0.2
c~~Ei~crrocmcrlyticrr1
Chmzistry
41 I (1996)
57-66
_-------
Experiment Simulation
i.!...!,,,!,..!.,,!,“!.l
1.2
1.0
0.8
0.6
Potential
0.8
0.6
-0.2
-0.4
0.8
0.2
0.4 Potential/
0.4
0.2
0.0
-0.2
-0.4
t6.0
CVs of canthaxanthin
(VI)
at (a) 100
1 I.
1.0
L
0.8
I ”
I.
0.6
0.0
-0.2
V
~ --------
Experiment Simulation
Potential/V Fig. 1. Experimental and simulated and (b) 500 mV se ’ vs. SCE.
0.6
/V
--------
t15.0~,“!“‘!.‘.!“‘!“‘!“‘!“‘!“,~ 1.2 1.0
0.0
‘.
Experiment Simulation
I ”
0.4
0.2
Potential
I ”
0.0
:.
-0.2
IV
Fig. 3. Experimental and simulated CVs of (a) 7’,7’-dimethyl-7’-ape+ carotene (II) and (b) 7’,7’-hexadeuterodimethyl-7’.ape-p-carotene 100 mV s- ’ vs. SCE.
the secondstageof oxidation is reached and that peak 5 is due to a radical and not to the reduction of *Car ‘_ For compoundsI, II and III, the oxidation potentials of reactions (1) and (2) are similar so that the waves overlap completely (Figs. 2 and 3). For the cyanoester compound (Fig. 41, the two oxidation waves overlap partially, and in the case of V and VI (Figs. 1 and 5) the two oxidation waves (peaks 1 and 2) are well separatedand clearly seen. The carotenoids display two general types of redox behavior, which are determined primarily by two factors. The first important factor is their structural symmetry. Thus, reversible CVs were obtained only for the symmetrical compoundsVI and I (Figs. 1 and 2). The unsymmetrical compoundsIV and V show almost irreversible oxida-
L ~.!...!...!...!...!...!.“!.~ 1.o 0.8 0.6
0.4
0.2
0.0
(III) at
-0.2
Potential/V -2o.or -lO.Oi0.0-y 5 2 t10.0~~ ~ -------
Experiment Simulation
5 5 t20.0& 0 t30.0
--------
Experiment Simulation
+40.0 t
t10.0
t 1 .o
.
: ’ .
0.8
: ’
0.6
I
0.4 Potential
Fig. 2. Experimental and simulated of 100 mV s-’ vs. SCE.
:.
0.2
. . : . ’
0.0
I
-0.2
’ !
-0.4
+50.0t.“!““““‘!““““‘!“’ 1.0
0
CVs of p-carotene
0.5
0.0
.i
Potential/V (I) at a scan rate
Fig. 4. Experimental 7’-apo-P-carotene
and simulated
(IV)
at (a)
CVs of 7’-cyano-7’-ethoxycabonyl10000 mV s-’ vs. SCE.
50 and (b)
J.A. Jeeuurujcm.
__ --------
+2.0~,“!“‘!“‘!.“!‘..!“‘!“‘!.‘.1 1.2 1.0 0.6
0.6
! ” 1 .o
”
”
”
! ”
0.5
Potentlal
/ Journd
o~‘&ic)c.~roci,wlltic
0.2
0.0
-0.2
-0.4
Experiment Simulation
“”
.”
! .1
0.0
”
-
/V
Fig. 5. Experimental and simulated CVs of 7’,7’-dicyano-7’.apo-p-carotene (V) (a) with and (b) without deprotonation of the radical canon at 100 mV s-’ vs. SCE.
tion waves, as evidenced by the very low amplitude of peaks 3 and 4 (Figs. 4 and 5). The unsymmetrical hydrocarbons II and III (Figs. 3(a) and 3(b)) exhibit more nearly reversible behavior than compounds IV and V, but less than that of I and VI. This feature will be addressed further in a later section. The secondfeature is the electron donor/acceptor capability of the substituents. Table 1 gives the oxidation potentials for the six carotenoids, the h E values between the first (E,) and second(E,) oxidation steps,the compro-
Table 2 Deprotonation equilibrium constant for the dictation coefticients D for carotenoids I to VI Compound
Kd,
K,,
k,/s ’ a ( zk 0.002)
0.1
II
0.1
0.065 0. I22 b
0.1
(0.122) 0. I22
b
0. I 0. I 0. I
(0.117) 1.65 2.05 0.065
IV V VI
41 I (1996
J 57-66
61
3. I. Deprotonation of the dication Table 2 gives the equilibrium (K,,) and the forward rate constants (k,) for the deprotonation of the dication along with the equilibrium (K&,1 and forward rate constants (k;) for the deprotonation of the radical cation and the diffusion coefficient D for compounds I to VI at a scan rate of 100 mV sP‘. The starting values for these parameters were chosen arbitrarily as described in the Experimental section, and then decreasedin large incre-
K&
and their forward
rate constants
k;/s-
Gp
I
III
and the radical cation
01 Clremtstry
portionation equilibrium constant (K,,,) and the E, [reaction (3)] values. Studies on the oxidation of substituted ethylenes [27] and some aromatic hydrocarbons [28] show that with increasing electron-accepting nature of the substituents. the ease of oxidation decreasesand the compounds exhibit higher oxidation potentials. Similarly, oxidation of the carotenoids containing terminal electronacceptor groups (compounds V and VI) is difficult and furthermore the second oxidation step occurs at quite a different potential to the first oxidation step (Table 1). As the electron-accepting strength of the end groups decreases, the separation h E between the two oxidation waves decreases(compound IV>, and for compounds II and III (which contain electron-donating groups) the two waves merge as for compound I. The formation of a positive charge would not be favorable in the compounds containing electron-withdrawing groups, and hence a higher potential is necessaryfor their oxidation. Subsequentoxidation of the cation radical to the dication would obviously be even more difficult than the first oxidation. This is manifested by the much higher potentials of the second oxidation for V and VI than those observed for the other compounds. The difference in potentials between the first and second oxidations is 205 mV for VI and 177 mV for V, compared with 5 mV for I (Table 1). Conversely, as the strength of the electron-withdrawing groups decreases,the easeof oxidation increasesand the difference in oxidation potentials decreases.
/V
__ --------
t2.0””
Kisprrt
Experiment Simulation
0.4
Potential
L.D.
’ a
X-r and k; respectively IO6 D/cm’
(+7-l
( * 0.2)
2XlO.‘O
5 x 10-6
3.8
2xlo~‘0 (3x10
5 x 10-7
5
Cd) 4x
4
9jc
1.6 x IO- I0
(5x10 9)c 2.7 x lo-” 3.5 x lo-‘” 2XlO~‘?
10-6
Cd) 3 x lo-’ 1.5 X IO.’ I x lomq
4.5 8 7
a All values given are for a scan rate of 100 mV s- ‘, relative errors reported. b Forward rate constant for the deprotonation from a second site in the dication. ’ Equilibrium constant for the deprotonation from a second site in the radical cation. d Values for the forward rate constant for the deprotonation from the first and the second sites in the radical cation are the same.
s- ’
and diffusion
62
J.A. Jeruurujun. a.067
4.q56
L.D. Kisprrt
/ Journal
c~f‘Ele~trocmrrlyriccrl
-0.p54
0.231
Fig. 6. (a) Difference between AMI calculated excess charges of the dication and the neutral species of carotenoids I to VI (III not included). ( ), 7 position; [ 1, 7’ position; ovals, 9 position; rounded rectangles, 13 position; rectangles, 13’ position; circles, 9’ position.
ments until the sensitive range was reached. Once the range is established, minute changes in values were made to obtain the right numbers. Also, increases in values of k, from the values reported in Table 2 increases the height of peak 5 and vice versa. This occurs because, as the extent of deprotonation increases, there is a higher concentration of ‘car+ species formed that get reduced at peak 5. Although the equilibrium constants for the deprotonation of the dication are the same (K,, = 0.11, the value of k, (K,, = kf/kb) increaseswhen the symmetry of the compound is broken. These changes are related to the excess charge densities(Fig. 6) presentat various carbon atoms in the neutral, cation radical and dication species.To obtain the excess charge densities, geometry optimization of the neutral carotenoid. its radical cation and dication was first performed. A single-point AM1 calculation on the geometry optimized structures was then performed to obtain the excess charges on all three species.Values of the difference of the charge densitiesof the dication and the neutral species(Fig. 6) show that for the symmetrical carotenoids I and VI the excesspositive charge is distributed evenly in the two halves of the chain and located primarily at atoms 7, 7’, 9, 9’, 11, 11’ and 13, 13’ (the Fig. 6 caption indicates individual positions). The numbering system for the
Chemistry
41 I f 1996157-66
carotenoids is given in Table 1 for p-carotene. In the unsymmetrical carotenoids IV and V carbon atoms 15, 13’ and 11’ bear a much greater positive charge than in the dications of I and VI, and carbon atoms 11 and 13 are less positive. An asymmetric concentration of the positive charge leads to decreasedstability as reflected by the 20to 30-fold increase in the deprotonation rate constant. It should be noted that the magnitude of the oxidation potential does not correlate with the stability of the dication. Thus, even though the dication of canthaxanthin is formed at very high positive potentials, it is stabilized by equal distribution of the charges over the two halves of the chain. For compounds IV and V, which require similar high positive potentials for oxidation, the dication is highly unstable, and its facile deprotonation leads to the nearly irreversible redox behavior. Since increaseddeprotonation of the dication results in a higher concentration of the *Car’ species,the amplitude of the reduction wave (peak 5) is larger. The redox behavior of the unsymmetrical compoundsII and III is intermediate between that of the symmetrical compoundsand the unsymmetrical compoundscontaining strong electron-withdrawing substituents. For compounds II and III, although the unsymmetrical structure leads to an uneven distribution of the positive charge of the dication on the two halves of the chain, the end methyl groups, which are electron-releasingin nature, stabilize the charge, making its dications more stable than the dications of IV and V. The difference in the excesschargesbetween the 9, 13, 9’ and 13’ positions for II and III is not as great as for IV and V. Consequently, the CVs of II and III show a more reversible wave (peaks 3 and 4) than IV and V, but lessreversible than I and VI. Hence, its k, value is larger than for I and VI, but less than that for IV and V. Earlier studieshave shown that the chain methyl groups are involved in the deprotonation of the dication [9]. Although the symmetrical compounds I and VI have two possible sites of deprotonation (chain 9 and 13 methyls), the unsymmetrical compoundsIV and V have four (chain 9, 13, 9’ and 13’ methyls). In these compoundsonly one proton is lost, their CVs and OSWVs show only one fifth peak, and their CVs can be successfully simulated if only one site of deprotonation is invoked. In addition to the above sitesof deprotonation, compoundsII and III contain two additional terminal methyl groups. The CVs of compoundsII and III show a very broad fifth peak and OSWV clearly shows two fifth peaks at + 0.07 and - 0.17 V (Fig. 7). Evidently, deprotonation of the dications of compounds II and III occurs from two different sites, presumably any one of the methyl groups on the chain and one of the terminal methyl groups forming two types of *Car+ species,the reduction potentials of which are not the same. Irrespective of the particular sites of deprotonation, successful CV simulation (Figs. 3(a) and 3(b)) was achieved by invoking the formation of two different types of *Car+ species.
JA.
Jrtwurctjorr.
LB.
Kqwrr/Jourrwl
c~f’E/r~trourrul~trcul
Chmimy
(3.0 x 10m7 F) had to be included reproduce the experimental CV. 3.3. Deprotonation
0.5: o.o-
! 0.6
”
Fig. 7. OSWV
! ‘. 0.6
. ! “. ! “‘I”’ 0.4 0.2 Potential /V
of II from 0.8 to -0.3
0
! “‘F -0.2
-0.4
V vs. Ag wire.
3.2. CV simulation at higher scan rates One of the criteria that can establish compatibility of a mechanism with the experimental data is successful simulation, in which all calculated parameters have the same magnitude when different experimental CV scan rates are applied. In our work, we noted that the value of k, for the deprotonation of the dication had to be changed by approximately an order of magnitude in order to fit the CVs at higher scan rates. With an increase in scan rate, the extent of deprotonation should decrease because there is not enough time for deprotonation to occur. Figs. 4(a) and 4(b) show the CVs of the cyanoester compound at two different scan rates. The amplitude of peak 5 is less than that of peak 4 at higher scan rates, but the experimental CVs exhibited more deprotonation at higher scan rates than is predicted by the parameters used in the simulation at lower scan rates. All parameters were constant for scan rates of 100 and 10000 mV s- ’ except the forward rate constant for the deprotonation of the dication. The forward rate constant varied from 1.65 SK’ for 100 mV s-’ to 22 s- ’ for 10000 mV s- ’ . This is attributed to adsorption or, in general terms, non-equilibrium conditions. The DigiSim” program does not have provisions for adsorption and it assumes equilibrium conditions for all the reactions used in the mechanism for simulation. Also, at very high scan rates of about IO 000 mV s- ’ the double-layer capacitance Table 3 Decay equilibrium Chemical Car + ‘* Car”
reactlons Products
* Products
‘CX-+
Products
*Car‘*
Products
constants
and their forward
rate constants
63
41 I (1996157-66
in the scheme to
of the radical cation
Although it was possible to simulate the shape of peaks 1 to 4 with reactions (1) to (51, the fifth peak was not satisfactorily reproduced. Table 2 gives the equilibrium constant K&, for the deprotonation of the radical cation and the forward rate constant k; for this equilibrium reaction. Inclusion of the deprotonation of the radical cation [reaction (6)] in the reaction mechanism satisfactorily reproduced experimental behavior (Figs. 5(a) versus 5(b)). Although K& is many orders of magnitude smaller than K,,. the deprotonation of the radical cation was found to be a very important step in the mechanism used for the simulation of the experimental CVs. For compound V. a 44% reduction in the amplitude of the current for the fifth peak was observed when this step was not included in the mechanism used for the simulation. This deprotonation step is a thermodynamically superfluous reaction (TSR), and hence its equilibrium constant K& is calculated from the values given for E, and E, and also the value of K,,. 3.4. Comproportionation
reaction
The comproportionation equilibrium reaction [reaction (4)] is also a TSR and K,,,, is calculated by the program from the difference in E, and E,. With increasing separation in oxidation waves, that is larger AE, the value of K corn increases (Table 1). Canthaxanthin has the largest K corn value, p-carotene has the smallest, and values for the other carotenoids are intermediate. Although K,,, is derived by the program as mentioned above, the values of k, can be adjusted to give the best possible simulations. The values for k, are estimated in a manner similar to that given for the other parameters. Increases or decreases in the value of k, are reflected in the height of peak 4, which reflects the concentration of cation radicals present. A 0.1% increase in k, will result in a 20% increase in the height of peak 4 as more cation radicals are formed by the comproportionation reaction.
(s ’ ) (m parenthesis)
for the carotenoids
I
II
IV
V
VI
0.10 ( I .O) 0.60 (1.4) 0. IO (25) 0.10 (0.1)
0.10 (0.17) 0.60 (0.65) 0.10 a (IO) 0.05 b (0.1)
0.83 (0.65) 0.20 (0.75) 0.75 (7) 0.0 I (0.0001)
0.58 (0.5) 0.20 (0.9) 1.20 (5) 0.0 I (O.oilOl)
0.30 (0.1) 0.75 (0.21) 0.10 (10) 0.10 (0.1)
‘Car represents the carotenoid with one less proton a Value the same for the other -Car+ species. b Value the same for the other ‘Car. species.
64 Table 4 Heterogeneous Electrode
rate constants
(k/cm
sag’ ) for the carotenoids
reactions
El Car+
Car+‘+
6 Car+‘,-Car’++e‘Car++
em I
em
E, ‘Car.
Relative errors reported. ‘Car represents the carotenoid a Value the same for electron
with one less proton. transfer from the other ‘Car’
species,
3.5. Formation of other minor products Inclusion of reactions (7) to (10) in the mechanistic scheme for the CV simulations was found in previous studies [26] to have a remarkably positive effect in the fitting of the simulated CVs to the experimental ones. The equilibrium constants for these reactions are given in Table 3. It is well known that the intermediate species formed in the oxidation process undergo other chemical reactions such as dimerization, bond breaking followed by cyclization, etc. [29]. Previous SEEPR studies [18] have shown, from the decay of the EPR signal for the cation radicals of carotenoids, that the cation radicals can decay in other ways apart from their oxidation to form the dications. For IV and V. the equilibrium constants for the conversion of the dication into other products are much smaller than those for the other compounds; this means that the forward reaction is very much slower compared with the reverse step, indicating that the loss of dication by this decay step is not as significant for IV and V compared with the other compounds. An increase in the equilibrium constant value from 0.2 to 0.8 for the conversion of dication into other products for compounds IV and V drastically changes the simulated CV. Also, the equilibrium constants for the decay of the other intermediates, namely Car+’ and *Car+, are much larger than that for the dication. The variation of equilibrium constants for the decay of =Car’ species does not significantly affect the simulation. 3.6. Other electrochemical parameters Transfer coefficient (Y values between 0.5 and 0.6, typical for quasi-reversible systems, were used for the simulations. The heterogeneous electron transfer rate constants for reactions (1) to (3) are given in Table 4. These values are very similar to those obtained from previous SEEPR studies performed in our group [ 181. One observes that the rate of electron transfer for the conversion of the cation radical to the dication is in general higher than that for the conversion of the neutral to the cation radical. In general, the rates of electron transfer for the carotenoids reported are relatively high considering the highly resistant solvent (dichloromethane) used. The rate of electron transfer for reaction (3) seems to be much higher than for
reactions (1) and (2) for all the carotenoids. The rate of electron transfer will also depend on the relative diffusion constants of the different species. Increases or decreases in the values of k, affect the height of the peaks. For example, if the value of k, for the cyanoester IV is reduced from 0.0065 (best value) to 0.002 cm s- ‘, a 50% decrease in current for the first electron transfer step given by peak 1 is observed. This reduction is observed because a decrease in the rate of electron transfer would reduce the concentration of the electroactive species reaching the electrode and hence decrease the current observed. The opposite effect on increasing the value of k, also occurs. The diffusion coefficients for carotenoids I to VI are given in Table 2. It is well known that the diffusion of the dication will be much slower than that of the cation radical or the neutral species in aqueous or highly polar solutions. These observations may not be the same in the non-polar medium used in our study; hence the diffusion coefficients of all the species involved in the oxidation process were taken to be the same. The simulated CV was the same regardless of whether the diffusion coefficient of the proton was 9 X 10m5 cm* s- ’ (H+ diffusion coefficient) or the same as that for the neutral carotenoids. Values for diffusion coefficients obtained from chronocoulometric studies [gl were used as starting values, and minor changes were made to fit the exact magnitude of the current. The simulation is very sensitive to changes in values of diffusion coefficients. An 8% increase in the value of the diffusion coefficient for I increased the current at peaks 1 and 2 by 12%. 3.7. Significance of results obtained to photosynthesis The oxidation potentials for the carotenoid and the cation radical and the reduction potential (Table 1) for *Car+ along with the deprotonation equilibrium constants K,, and K& for the dication and cation radical respectively (Table 2), the nature of the substituents and symmetry of the carotenoids are important variables in the electron transfer properties of the carotenoids in the photoprotection of photosynthetic organisms. The fact that most carotenoids get oxidized within 1 V establishes their importance in photosynthesis. For instance, recent studies of
isolated photosystem II reaction centers have shown [3] that when the photosystem II reaction center is photoactivated under conditions where P680’ can photoaccumulate, there is a secondary oxidation of B-carotene by P680f’ to form the carotenoid cation radical. Rapid oxidation and bleaching of one of a pair of excitonically coupled B-carotene molecules occurs, followed by a slower oxidation and bleaching of the second. The different oxidation rates are believed to be due to binding of one carotenoid molecule to the Dl protein and the other to the D2 protein. Photoprotection for P680 is not only provided by B-carotene but also by a cycling of electrons around PSI1 via cytochrome b-559 and a monomeric chlorophyll. The fact that two electrons rather than one are transferred per carotenoid demonstrates its efficiency. Also, the deprotonation of the dication leads to the formation of Ht in the protein matrix and this leads to cis/trans isomerizations [30]. Because of the increased awareness of the involvement of cis/trans isomerization of carotenoids in many biochemical and biological processes [3 1,321, the processes that lead to the formation of these isomers are of interest.
4. Conclusions The experiments performed and the CV simulations support the proposed mechanism. The values of the oxidation potentials suggest that ease of oxidation of the carotenoids depends on the nature of substituents on the carotenoid. Carotenoids with strong electron-withdrawing substituents are very difficult to oxidize, while those with weak electron-withdrawing substituents are easily oxidizable. At faster scan rates the concentration of deprotonated cation formed due to the side reaction decreases significantly, indicating that other chemical and electrochemical reactions compete more effectively with the deprotonation. The fact that near-reversibility of the CVs of IV and V is attained only at high scan rates indicates that the electrochemically active species, namely the cation radical and dication, are highly unstable in solution. From the K,, values we learn that the carotenoids (II to V> which have one of their cyclohexene rings substitutedform the carotene radical more readily than the other two carotenoids (I and VI). The dimethyl compound has two deprotonation sites. indicating that a proton is lost from the chain methyl groups as well as from the end methyl groups. With an increasein electron-withdrawing nature of the substituents, the separationbetween the two oxidation waves increases. thus increasing the comproportionation equilibrium constant K,,,. AM1 calculations of the excess charge distribution in the dications show that in the more stable dications the charge is more evenly distributed in the two halves of the backbone chain, whereas in the highly reactive dications of unsymmetrical carotenoids it is not evenly distributed, thus making them highly unstable. From the data obtained regarding the formation of other minor prod-
ucts, it can be concluded that the dications of IV and V undergo more deprotonation than any other side reaction.
Acknowledgements We would like to thank Dr. S. Feldberg for helpful discussions regarding the simulation program, Mrs. W. Hung for synthesizing carotenoids IV and V, Dr. E.S. Hand for the synthesisof the dimethyl compounds,Drs. A. Jeevarajan and E.S. Hand for their suggestionsand for critically reading the manuscript and Roche Vitamins and Fine Chemicalsfor a gift of the oil suspensionof 8’-apoB-caroten-X’-al, a precursor of compoundsII-V. This work was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences. Department of Energy, under Grant DE-FG05-86ER13465.
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