5362
J. Phys. Chem. 1996, 100, 5362-5366
Geometrical Isomerization of Carotenoids Mediated by Cation Radical/Dication Formation G. Gao, C. C. Wei, A. S. Jeevarajan, and L. D. Kispert* Department of Chemistry, The UniVersity of Alabama, Tuscaloosa, Alabama 35487-0336 ReceiVed: September 29, 1995; In Final Form: NoVember 28, 1995X
Electrochemical oxidation of all-trans-canthaxanthin and β-carotene in dichloromethane leads to significant trans-to-cis isomerization, with cis isomers accounting for about 40% of the products formed. The electrochemically generated isomers were separated by reverse-phase high-performance liquid chromatography and identified as 9-cis, 13-cis, 15-cis, and 9,13-di-cis isomers of the carotenoids by 1H-NMR spectroscopy and optical spectroscopy (Q ratio). The results of simultaneous bulk electrolysis and optical absorption spectroscopy indicate the following isomerization mechanism: the all-trans cation radicals and/or dications formed by electrochemical oxidation of all-trans-carotenoids can easily undergo geometrical isomerization to form cis cation radicals and/or dications. The latter are converted by the comproportionation equilibrium to cation radicals which are then transformed to neutral cis-carotenoids by exchanging one electron with neutral carotenoids. AM1 molecular orbital calculations, which show that the energy barriers of configurational transformation from trans to cis are much lower in the cation radical and dication species than in the neutral molecule, strongly support the first step of this mechanism.
Introduction Carotenoids are widely distributed among living organisms of plants, animals, and certain bacteria. Around 600 naturally occurring carotenoids have been isolated and identified.1 It is well-known that certain carotenoids have important biochemical and biological functions as light harvesting and photoprotection in photosynthesis in plants2,3 and have nutritional importance of provitamin A activity in man.4 Some additional functions which have been suggested include cancer prevention and treatment,5 which are perhaps due to their antioxidant and free radical quenching activity,6 immune enhancement effect,7 and in vivo antioxidants.8 Although carotenoids are generally present in their most stable form, where all double bonds in the backbone have the trans configuration, individual carotenoids are capable of forming
different mono- or di-cis geometrical isomers which may involve one or two double bonds at positions 7, 9, 13, 15, 13′, and 9′. Together with trans isomers, these cis isomers are also present in human tissue and pigment-protein complexes of photosynthesizing plants and bacteria and are known to affect the biochemistry of carotenoids in certain situations. For example, (a) the β-carotene in thylakoid membranes is about 80% alltrans and about 20% of various cis isomers, and most of the * To whom correspondence should be addressed. X Abstract published in AdVance ACS Abstracts, February 15, 1996.
0022-3654/96/20100-5362$12.00/0
cis isomers originate from photosystem I.9 (b) Natural selection of certain types of cis/trans-carotenoid configurations has been found in purple photosynthetic bacteria: the all-trans configuration is selected by the light-harvesting complex for the lightharvesting function, while the 15-cis configuration is selected by the reaction center (RC) in the photosynthetic organisms for the photoprotective function.2,10 The photoprotective function is related to the transformation between the 15-cis and the alltrans configurations of the RC-bound carotenoid Via the formation of the triplet state.11 (c) Animal studies indicate differences in provitamin activity among the cis and trans isomers of provitamin A carotenoids.12 (d) The configuration change is also found in spinach leaves and the thermophilic cyano bacterium, Synechococcus Valcunus copeland.10,13 (e) Mono-cis-canthaxanthin isomers localized in the ovaries, eggs, and haemolymph of the female brine shrimp Artemia are observed for the first time in animals. The presence of these cis-canthaxanthins at specific sites of only reproductively active females suggests that they serve an important function in reproduction and/or embryonic development.14 Because of the increased awareness of the involvement of trans/cis isomerization of carotenoids in many biochemical and biological processes, numerous methods of isolation and detection of trans/cis isomers,15,16 as well as their preparation, have been developed. The methods leading to formation of mixtures of cis and trans isomers include refluxing in organic solvents, melting of crystals, contact for prolonged periods with certain active surfaces, treatment with acids, and irradiation of solutions (catalyzed by iodine).17,18 Here we report for the first time that electrochemical oxidation of all-trans-canthaxanthin and β-carotene in dichloromethane also leads to significant trans/cis isomerization. The results of optical absorption spectroscopy and HPLC suggest that trans/cis isomerization takes place Via the cation radical and dication. The significance of this work is as follows: (a) Any method that could form cation radicals and/or dications of carotenoids would lead to geometrical isomerization as well; e.g., experiments have proven that treatment of all-trans-carotenoids with FeCl3 generates cation radicals and dications and can result in efficient formation of cis isomers. (b) Since carotenoid cation radicals have been © 1996 American Chemical Society
Geometrical Isomerization of Carotenoids
Figure 1. Cyclic voltamograms of (a) all-trans-canthaxanthin and (b) all-trans-β-carotene versus Ag/AgCl. The scan rate is 0.1 V s-1.
found to play an active role in photodriven electron transport processes in synthetic triad and pentad molecules,19,20 cis isomers of carotenoids may also be formed in those systems. Experimental Section all-trans-Canthaxanthin, β-carotene, and tetrabutylammonium hexafluorophosphate (TBAHFP) were purchased from Fluka. Anhydrous dichloromethane, HPLC grade acetonitrile, and ferric chloride were obtained from Aldrich. Cyclic voltammetry and bulk electrolysis were carried out using the Bio Analytical Systems BAS-100W electrochemical analyzer. For CV measurements, a platinum disk electrode (diameter ) 1.6 mm) was used as the working electrode, the auxiliary electrode was a platinum wire, and the reference electrode was a Ag/AgCl electrode. For bulk electrolysis, a platinum coil was used as the working electrode, a silver wire was the pseudo reference electrode, and a platinum wire was the auxiliary electrode. All solutions were prepared in a drybox under a nitrogen atmosphere. Except as specifically noted, 1 mM solutions of canthaxanthin or β-carotene and of TBAHFP (0.1 M) in dichloromethane were used. A Vydac 201TP54 polymeric C18 column (250 × 4.6 mm i.d.) packed with 5 µm particles (Hesperia, CA) and a Shimadzu LC-600 pump with a SPD-6AV UV-vis detector were used for the HPLC separation and detection. Acetonitrile was used as the mobile phase. The detector was set at 465 and 450 nm for canthaxanthin and β-carotene, respectively, near the maximum absorbance of the neutral cis-carotenoids. The optical absorption spectra in the range of 190 to 1100 nm were measured using a Shimadzu UV-1601 UV-vis spectrophotometer. The quartz cell used for simultaneous bulk electrolysis and optical absorption spectroscopy measurements was described in refs 21 and 22. AM1 (Austin Model 1) semiempirical molecular orbital calculations23 were carried out using HyperChem software24 running on a Gateway 2000 P5-60 (Pentium) personal computer. Results and Discussion Cyclic Voltammetry. Previous investigations21, 25-28 have established that the electrode and homogeneous reactions shown in eqs 1-6 take place during the electrochemical oxidation of
J. Phys. Chem., Vol. 100, No. 13, 1996 5363
Figure 2. Simultaneous bulk electrolysis with optical absorption spectra of (a) all-trans-canthaxanthin and (b) all-trans-β-carotene. Bulk electrolysis at 0.9 V for 5 min for I and at 0.6 V for 2 min for II. The measurements were carried out at 23 °C. Spectra were obtained before (dashed line) and after (solid line) electrolysis.
most carotenoids. The redox reactions (eqs 1-3) can be detected by cyclic voltammetry (CV), as is illustrated in Figure 1 for canthaxanthin (I) and β-carotene (II) in dichloromethane. For canthaxanthin, peak 1 corresponds to the one-electron oxidation of the neutral species (eq 1), and peak 2 results from the subsequent one-electron oxidation of the cation radical (eq 2). Peaks 3 and 4 are due to sequential one-electron reductions of the dication and the cation radical, respectively. Peak 5 results from the reduction (reverse of eq 3) of the transient intermediate, *Car+, which is formed by loss of a proton from the dication (eq 5). For β-carotene, E°1 and E°2 are very similar, so peaks 1 and 2 overlap.
electrode reactions E°1
Car y\z Car•+ + eE°2
Car•+ y\z Car2+ + eE°3
*Car• y\z *Car+ + e-
(1) (2) (3)
homogeneous reactions Kcom
Car2+ + Car y\z 2Car•+ Kdp
Car2+ y\z *Car+ + H+ K′dp
Car•+ y\z *Car• + H+
(4) (5) (6)
Simultaneous Bulk Electrolysis and Optical Absorption Spectroscopy. Carotenoid cation radicals formed by bulk electrolysis in dichloromethane can be observed by simultaneous optical absorption spectroscopy.22 The optical absorption spectra of all-trans-canthaxanthin and β-carotene are shown in Figure 2, a and b, respectively. In both parts a and b, dashed lines are the spectra of all-trans neutral carotenoids (obtained before bulk electrolysis). For all-trans-carotenoid, it is known that its spectrum contains an absorption maximum (λ1) in the visible region, but the λ2 band in the UV region is absent. In
5364 J. Phys. Chem., Vol. 100, No. 13, 1996
Gao et al.
Figure 3. HPLC chromatograms at 23 °C of (a) all-trans-canthaxanthin and (b) all-trans-β-carotene (both 0.3 mM in CH2Cl2 with 0.05 M TBAHFP), measured before (dashed line) and after (solid line) bulk electrolysis at 0.9 V for 5 min for I and 0.6 V for 4 min for II, respectively. Flow rate ) 1 mL/min; sample injected ) 10 µL; detection at 465 and 450 nm for I and II, respectively.
SCHEME 1 trans-Car
–e–
trans-Car•+
–e–
trans-Car2+
cis-Car•+
cis-Car2+ + trans-Car cis-Car•+ + trans-Car
trans-*Car+
isomerization
isomerization
(8)
cis-Car
–H+
Kcom
(7)
cis-Car2+ cis-Car•+ + trans-Car•+
electron exchange
Figure 4. AM1 calculation of backbone bond lengths of all-transcanthaxanthin. Dashed lines are neutral carotenoids in both (a) and (b); solid line is cation radical in (a) and dication in (b).
cis-Car + trans-Car•+
(7) (8)
the case of cis isomers, a strong λ1 band and a weak λ2 band “cis peak” are present. Solid lines in Figure 2 are optical spectra obtained during simultaneous anodic bulk electrolysis. The additional absorption at 875 nm for I and 990 nm for II is attributed to cation radicals that are generated during the electrolysis. As determined from the rate of decay of the long-wavelength maximum absorption, the half-life of the cation radical of I is 570 s and that for II is 2100 s; i.e., these species are quite stable in dichloromethane. High-Performance Liquid Chromatography. Figure 3a shows the chromatograms of all-trans-canthaxanthin before (dashed line) and after (solid line) bulk electrolysis at 0.9 V for 5 min in dichloromethane. After HPLC separation, four new components were identified to be the 15-cis, 13-cis, 9-cis, and 9,13-di-cis isomers of I according to 1H NMR spectroscopy and/ or values of the Q ratio29 (absorbance of the “cis peak”/ absorbance of the most intense maximum), which has been previously established. Approximately 40% of I was converted to cis isomers. The mixture obtained by bulk electrolysis of II (Figure 3b) was considerably more complex, and only three components could be definitively identified. Isomerization Mechanism. The above results are consistent with the reactions shown in Scheme 1. Electrochemical oxidation of the all-trans-carotenoid generates the corresponding cation radical and dication species, which can then isomerize to various cis species. Any cis dications are expected to react
with neutral carotenoid in the comproportionation equilibrium (eq 7) to give cis cation radicals. Subsequent electron exchange between the cis cation radicals and the neutral trans-carotenoid results in the formation of the neutral cis isomers (eq 8). According to Scheme 1, any method in which dications and/ or cation radicals are generated is expected to lead to geometrical isomerization. This hypothesis is confirmed by the fact that treatment of I with ferric chloride results in the formation of the same isomers, in the same relative amounts, as were obtained by electrochemical oxidation,29 and the fact that cation radicals and dications of carotenoids are formed by oxidation with ferric chloride in dichloromethane solution.30 HPLC analysis of mixtures of II and FeCl3 showed that analogous transformations occurred. It should be mentioned that there have been several papers discussing the mechanism of cis/trans isomerization of carotenoids by methods other than electrochemical isomerization. Koyama et al.31-33 reported that trans-to-cis, cis-to-trans, and cis-to-cis geometrical isomerization processes occur in tripletsensitized photoisomerization and direct photoexcitation of β-carotene and 8′-apo-β-caroten-8′-al. Their Raman spectra and Pariser-Parr-Pople calculations of the bond order of both S0 and T1 states predicted that the triplet state is responsible for the isomerization mechanism. Sundquist et al.34 reported that incubation of the all-trans isomers of β-carotene, lycopene, and canthaxanthin with 3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxetane (HTMD, a thermodissociable source of electronically triplet excited ketone) led to significant trans-to-cis isomerization also by a triplet-sensitized mechanism. Isomerization35 of all-trans-carotenoids to cis isomers (primarily the 15,15′-cis isomer), which takes place at the earliest stages of oxidation with oxygen in the dark, has been attributed to facile formation of singlet biradical,36 which is thermodynamically stabilized by extensive delocalization of the two unpaired electrons in a perpendicular transition state. AM1 MO Calculations. An attempt to calculate the energetics involved in the transformation of trans to cis isomers
Geometrical Isomerization of Carotenoids
J. Phys. Chem., Vol. 100, No. 13, 1996 5365
TABLE 1: Bond Lengths (Å) of Carotenoids Using AM1 Calculation bond length compd
bond
neutral
cation radical
dication
I
C6sC7 C7dC8 C8sC9 C9dC10 C10sC11 C11dC12 C12sC13 C13dC14 C14sC15 C15dC15′ C15′sC14′ C14′dC13′ C13′sC12′ C12′dC11′ C11′sC10′ C10′dC9′ C9′sC8′ C8′dC7′ C7′sC6′ C6sC7 C7dC8 C8sC9 C9dC10 C10sC11 C11dC12 C12sC13 C13dC14 C14sC15 C15dC15′ C15′sC14′ C14′dC13′ C13′sC12′ C12′dC11′ C11′sC10′ C10′dC9′ C9′sC8′ C8′dC7′ C7′sC6′
1.457 1.342 1.458 1.353 1.445 1.346 1.451 1.358 1.441 1.349 1.442 1.354 1.457 1.347 1.443 1.351 1.459 1.343 1.457 1.458 1.343 1.458 1.352 1.444 1.347 1.451 1.356 1.443 1.350 1.441 1.354 1.456 1.348 1.444 1.352 1.461 1.343 1.458
1.457 1.346 1.452 1.368 1.423 1.372 1.418 1.400 1.396 1.394 1.398 1.394 1.424 1.370 1.424 1.365 1.454 1.345 1.457 1.455 1.348 1.448 1.371 1.420 1.375 1.416 1.399 1.397 1.394 1.398 1.395 1.422 1.373 1.422 1.367 1.453 1.348 1.455
1.446 1.363 1.426 1.405 1.379 1.419 1.374 1.448 1.350 1.447 1.349 1.453 1.369 1.420 1.378 1.409 1.423 1.365 1.445 1.424 1.382 1.402 1.423 1.366 1.427 1.363 1.454 1.347 1.446 1.346 1.451 1.366 1.428 1.366 1.421 1.405 1.383 1.422
II
via neutral, cation radical, and dication of carotenoids was made. AM1 semiempirical molecular orbital calculations of total energies and bond lengths of neutral, cation radical, and dication of all-trans-canthaxanthin and β-carotene were carried out on structures that were optimized by the AM1-RHF method before a single-point calculation. As illustrated in Figure 4 for compound I, in the neutral species the bond lengths between the backbone carbon atoms alternate in a regular manner. In the cation radical (Figure 4a), the five central bonds have the same length, and the change in bond length gradually decreases and is about the same as in the neutral species for the terminal bonds. Even more dramatic changes occur in the dication (Figure 4b) where, compared to the neutral species, the bond orders are inverted for most of the backbone bonds. The same trends were observed for the neutral, cation radical, and dication of β-carotene (Table 1). Previously calculated bond lengths in excited triplet (T1) docosaundecaene and nonadecanonaen-1-al (model compounds for β-carotene and 8′-apo-β-caroten-8′-al, respectively) showed large changes32,34 similar to those of the carotenoid cation radical. Since isomerization requires a 180° twist of a double bond in the neutral species and since the bond orders are different in the charged species, it was expected that the energy barrier to rotation would also be different. To determine this, fixed geometry AM1 calculations of total energy as a function of the dihedral angle of the groups attached to certain adjacent carbon atoms, such as C9dC10, C13dC14, C15dC15′, and C7dC8, were carried out. The results for the neutral, cation radical, and
Figure 5. Fixed-geometry calculations of total energy of all-transcanthaxanthin as a function of the dihedral angle around double bonds: (a) C9dC10, (b) C13dC14, (c) C15dC15′, and (d) C7dC8, using AM1-RHF single-point calculation. The all-trans molecules were optimized by AM1 geometry optimization. (*) Optimized geometrical cis isomer is presumed. (**) Optimized geometry with presumed 127° dihedral angle.
dication species of canthaxanthin, shown in Figure 5, can be summarized as follows: (1) in the transition state between trans/ cis isomerization, two planar portions of the chain are oriented perpendicular to one another (dihedral angle 90°); (2) as expected, much lower energy barriers to trans/cis isomerization exist in the cation radical and the dication than in neutral canthaxanthin; (3) the energy barriers for rotation about the C13dC14 and C15dC15′ bonds in the cation radical and the dication are the lowest, followed by the barriers for rotation about the C9dC10 and the C7dC8 bonds. This sequence is consistent with the fact that the largest changes of bond length occur in the central portion of the chain. The AM1 calculation results strongly support the proposal that isomerization occurs Via the cation radical and/or the dication in the electrochemical and chemical oxidation process. The energy/angle relationships as the C7dC8 bond is twisted (Figure 5d) differ considerably from those of rotation about other
5366 J. Phys. Chem., Vol. 100, No. 13, 1996
Gao et al. References and Notes
TABLE 2: Energies of Transition States Using Fixed Geometry AM1 Calculation ∆Eb compd
transition statea
neutral
cation radical
dication
I
C13dC14 C9dC10 C15dC15′ C7dC8 C13dC14 C9dC10 C15dC15′ C7dC8
55.03 57.20 56.26 57.22 54.58 57.26 56.87 64.39
24.67 39.17 24.42 54.84 24.33 37.39 24.65 56.08
2.82 13.53 3.34 34.16 3.27 9.19 3.91 22.55
II
a Perpendicularly twisted structure with 90° dihedral angle. b ∆E ) E90° dihedral angle - Eall-trans, E ) total energy (kcal mol-1).
bonds (Figure 5a-c), which results in planar cis isomers (180° twist). The energy barrier at ca. 90° rotation is the same in the cation radical as in the neutral compound and remains relatively high in the dication. Further, an energy minimum is observed when the dihedral angle of the substitutents at C7 and C8 is ca. 130°. More extensive calculations in which the specified angular change was 1° (rather than the 10° change generally used) showed that the energy minimum occurs at a twist of 127°. As the angle is increased, repulsion between the ring 5-methyl and the chain 9-methyl groups rapidly escalates. If rotation of the C8-C9 bond is allowed in the 7-cis isomer, the steric repulsion may be lowered. Calculations for β-carotene (Table 2) show the same trend of smaller energy barriers to trans/cis isomerization in the cation radical and dication than in the neutral species, as was observed for canthaxanthin. Conclusions Electrochemical oxidation of all-trans-canthaxanthin and β-carotene in dichloromethane leads to significant formation of cis isomers, primarily the 9-cis, 13-cis, 15-cis, and 9,13-dicis isomers. AM1 molecular orbital calculations of total energies and bond lengths show that the energy barriers of configurational transformation are much lower in the cation radical and dication than in the neutral carotenoids in accord with changes in the chain carbon-carbon bond lengths in these species. It is possible that cis isomers of carotenoids could be formed while irradiating the artificial photosynthetic triad and pentad compounds in which cation radicals are known19,20 to be formed. Acknowledgment. Dr. Elli Hand is thanked for useful discussions. This work was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, Office of Energy Research of the U.S. Department of Energy under Grant DE-FG05-86ER13465.
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