CHIRALITY 13:739–744 (2001)
Supramolecular Assemblies of Carotenoids 1
´ ZSEF DELI,2 ZSOLT BIKA´DI,1 AND MIKLO ´ S SIMONYI1* FERENC ZSILA,1 JO Department of Molecular Pharmacology, Institute of Chemistry, CRC, Budapest, Hungary 2 Department of Medical Chemistry, University of Pe´cs Medical School, Pe´cs, Hungary
ABSTRACT Carotenoid self-assemblies were formed by aqueous dilution of ethanolic solutions. The four 38,68-epimers of capsanthol ((all-E,3R,58R)-3,38,68-trihydroxy-b,kcarotene) give rise to right- and left-handed card-pack and head-to-tail types of selfassemblies detected by exciton couplets appearing in the CD spectra. Slow kinetics of formation followed for some of the aggregates indicate the complexity of the process. The exciton signals do not appear from equimolar mixtures of related compounds that produce identical type of aggregates of opposite sense on their own. Transformation of self-assembly may reflect the population of k-ring rotamers. Chirality 13:739–744, 2001. © 2001 Wiley-Liss, Inc. KEY WORDS: circular dichroism; exciton signals; kinetics of aggregate formation; compensation of supramolecular chirality The exciton chirality method was not applicable to absolute configurational assignment of 3-hydroxycarotenoids4 because chiral perturbation of the polyene chain by the 3-hydroxyl group is very weak.5 Aggregation of carotenoids, however, gives rise to exciton coupling, as demonstrated for lutein6 and other xanthophylls.7 These studies demonstrated the formation of both card-pack and headto-tail types of aggregates, the first being associated with a large blue shift in the UV absorption spectra while the latter indicated by a red shift to a smaller extent. Structural factors that influence the type and handedness of aggregation of carotenoids remain essentially unknown. In order to get an insight into the relation of molecular structure to supramolecular chirality, four epimer capsanthols ((allE,3R,58R)-3,38,68-trihydroxy-b,k-carotenes) have been selected; they only differ in the configuration of the 38 and 68 positions (Scheme 1). Note that 68 is neighboring the polyene chain. EXPERIMENTAL
Scheme 1. Synthetic route to capsanthol-38,68-epimers.
Water induces the association of natural dyes from a state of molecular distribution, as indicated by spectral changes. The structure of the aggregate is stable even in highly dilute solution owing to hydrophobic association.1 The supramolecular organization is affected by the chirality of monomer molecules resulting in highly ordered assemblies, in which the type and chiral direction of stacking may be influenced by minor structural changes.2 The formation of such aggregates can be detected sensitively by circular dichroism spectrometry1 due to intense exciton coupling.3 © 2001 Wiley-Liss, Inc.
The preparation of capsanthol epimers will be published elsewhere. In short, capsanthin ((all-E,3R,38S,58R)-3,38dihydroxy-b,k-carotene-68-one), the major carotenoid component of ripe Capsicum annuum (red paprika),8 was subjected to Oppenauer oxidation (Al[OPri]3 in acetone) yielding the diketone capsanthone,9 which was subsequently reduced (Scheme 1); the resulting stereoisomers of capsanthol were separated by chromatography (Deli et al., unpublished). 38S,68S-capsanthol-3-38-diacetate was prepared
Contract grant sponsor: Hungarian National Scientific Fund; Contract grant number: OTKA T 030271, OTKA T 033109. *Correspondence to: Miklo´s Simonyi, Department of Molecular Pharmacology, Institute of Chemistry, CRC, Budapest, POB 17, H-1525 Hungary. E-mail:
[email protected] Received for publication 22 November 2000; Accepted 3 May 2001
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Fig. 1. UV/VIS and CD spectra of 68R-capsanthol-38-epimers in ethanolic solution (dotted lines) and upon aqueous dilution (heavy lines). 38R,68R-capsanthol: a and b; 38S,68Rcapsanthol: c and d.
from capsanthin by acetyl chloride followed by NaBH4 reductions.10 Samples for spectrometric analyses were prepared from ethanolic stock solutions containing the given carotenoid
of 1.0 × 10−4 M concentration. One volume of stock solution was diluted by 3 volumes of either ethanol or water, resulting in the final concentration of 2.5 × 10−5 M for the spectroscopic samples. After aqueous dilution, all samples re-
Fig. 2. Spontaneous inversion of 3’S,6’R-capsanthol self-assembly. CD curves were recorded at 5-min intervals for the first hour and at 10-min intervals for the second hour.
Fig. 3. Kinetics of reorganization of 38S,68R-capsanthol self-assembly. Data are intensities read at 401 nm (cf. Fig. 2). Insert: simple scheme of transformation for chiral supramolecules of opposite handedness and rate constants for a single exponential fit (heavy line) and for two different exponentials (dotted lines).
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3 volumes of water in order to ensure the simultaneous induction of aggregation for both components. UV/VIS and CD spectra were taken on a Jasco J-715/150S spectrometer at 25°C, with 0.5 cm pathlength. Temperature control was provided by a Peltier thermostat. For molecular model calculations, the initial coordinates of capsanthol atoms were modeled using Sybyl 6.6 program (Tripos, St. Louis, MO) on a Silicon Graphics Octane workstation under Irix 6.5 operation system. Structures were minimized using the Powell Conjugate Gradient method until the convergence was less than 0.001 kcal/ (molÅ). Finally, a grid search by MMFF94 force field was performed and fully minimized structures were obtained at each point of a 10° search over the t5-6-7-8 and the t18-58-68-78 torsion angles. RESULTS AND DISCUSSION Fig. 4. Extinction of exciton couplets of self-assemblies in time for an equimolar mixture of 38R,68R-capsanthol and 38S,68R-capsanthol.
mained clear, with no sign of opalescence or precipitation. Samples of two carotenoids in equimolar extent were prepared by mixing their ethanolic stock solutions in equal volumes; one volume of the mixed solution was diluted by
UV/VIS and CD spectra of 68R-capsanthol-38-epimers are shown in Figure 1. Dotted lines represent spectra taken in ethanol, which are very similar for the epimers. Absorption in the visible range is assigned to electronic transition from the 1Ag (ground) to the 1Bu (excited) state of conjugated p-electrons; differences of wavelengths between neighboring extrema/shoulders refer to different vibrational levels that are superimposed on the electronic excitation.7 The
Fig. 5. UV/VIS and CD spectra of 68S-capsanthol-38-epimers in ethanolic solution (dotted lines) and upon aqueous dilution (heavy lines). 38R,68S-capsanthol: a and b; 38S,68Scapsanthol: c and d.
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Fig. 6. CD curves of self-assemblies of 38S,68S-capsanthol-3,38-diacetate (A) and 38S,68S-capsanthol (B). Heavy line is the CD spectrum appearing immediately upon aqueous dilution of an equimolar mixture of ethanolic solutions of the two compounds.
CD intensity of samples in ethanol is so weak that they appear as baseline for both epimers in the given layout. Hence, chiral perturbation even by the 68 position does not provide a strong CD signal. Upon aqueous dilution the absorption suffers a blue shift of ∼100 nm for both 38epimers with the disappearance of vibrational superposition. It indicates tight stacking of molecules to such an extent that individual vibrations of the single molecules could no more influence the electronic excitation; this is indicative of the formation of card-pack aggregates.7 Dramatic changes also occur in the CD spectra: a sharp exciton couplet appears immediately upon aqueous dilution for the 38R-epimer showing positive sign for higher wave-
Fig. 7. Kinetics of time-dependent transformation of 38S,68S-capsanthol3,38-diacetate self-assembly. Data are intensities read at 434 nm (cf. Fig. 6) at 2-min intervals for the first 20 min and at 4-min intervals between 20 and 60 min. Insert: simple scheme of transformation and rate constants for a single exponential fit (heavy line) and for two different exponentials (dotted lines).
Fig. 8. Grid search for energy minima at 10° intervals over b-ring rotation around the C6–C7 bond.
length (Fig. 1b), an indication of right-handed angle between the stacked neighboring chromophores. The CD spectrum of 38R-epimer is invariant over time. This is not the case for 38S,68R-capsanthol: the CD spectra given in Figure 1d was recorded 2 h after aqueous dilution. Another difference between the two 38-epimers is the lefthandedness of the 38S,68R-capsanthol self-assembly, as spectra of b and d in Figure 1 are in mirror image relation. The fate of 38S,68R-capsanthol self-assembly during the first 2 h is demonstrated in Figure 2. Characteristically, the CD spectrum recorded immediately after aqueous dilution is right-handed and it spontaneously changes handedness by transforming into its final, left-handed arrangement. This supramolecular phenomenon is analogous to a unidirectional chiral inversion at the molecular level. A kinetic analysis of the above transformation was attempted by recording intensities of the CD curves at a
Fig. 9. Grid search for energy minima at 10° intervals over k-ring rotation in 38-epimers of 68R-capsanthol around the C58–C68 bond.
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given wavelength; 401 nm was selected where the largest variation is seen. The simplest scheme of transformation is when A represents the initially formed self-assembly (kinetically favored), B is the final form (thermodynamically favored), and reversibility is assumed between the two (cf. insert Fig. 3). The kinetic solution of this scheme in terms of D« values gives a single exponential dependence with a rate constant that is the sum of single constants referring to the opposite directions (insert Fig. 3, the meaning of subscripts are: t variable, i infinite, 0 initial). As seen, a single exponential curve does not fit well with the experimental values, which can be much better described by two exponentials; the slowest rate refers to a half-life of 55 min. Hence, the reorganization in the cardpack assembly is rather slow and its mechanism is more complex than a single process. The time-dependence of formation for the 38S,68R-capsanthol self-assembly is a sharp contrast between the 38S- and 38R-epimers of 68Rcapsanthol. As the CD curves in Figure 1b,d are mirror images, and the two samples placed in different cuvettes one behind the other would to a large extent cancel each other, it is of interest to see what happens if the two epimers are mixed in ethanol solution (molecular state) and the selfassemblies of the two-handedness are allowed to develop simultaneously in the same phase. The result is shown in Figure 4. It can be seen that the extinction reflects the immediate formation of right-handed 38R,68R- and the time dependent building-up of left-handed 38S,68R-capsanthol selfassembly. It gives an indication of the simultaneous existence of the two right- and left-handed aggregates, the crystallographic analog of which is a conglomerate. Hence, card-pack assemblies of the same handedness are stable enough to coexist in the liquid phase. The 38-epimers of 68S-capsanthol fall into a different class. The ethanolic spectra are similar to those of the 38-epimers of 68R-capsanthol, but the self-assemblies are different (Fig. 5). Upon aqueous dilution a red shift is seen in the visible absorption and the superposition of vibrational levels are retained, indicating the formation of headto-tail assemblies in which the molecules are packed loosely enough to allow vibrational transitions to influence electronic excitation. The CD spectra of the 38-epimers of 68S-capsanthol display almost identical, wide, structured and left-handed exciton couplets, in accordance with the conclusion drawn from the visible spectra on the formation of head-to-tail type of self-assemblies. The CD spectra of both epimers are established immediately and remain constant in time. Hence, the 38-epimers of 68S-capsanthol form aggregates of identical kind, stability, and sign of the chiral angle. Interestingly, the handedness of the head-to-tail assembly is reversed by acetylation at the 3- and 38-positions, as shown in Fig. 6 for 38S,68S-capsanthol-3-38-diacetate (A), recorded 1 h after aqueous dilution. This is another example of mirror image-related spectra, one of which is formed immediately, while the other develops over time. Contrary to the earlier time-dependent extinction of the exciton signals of card-pack aggregates (Fig. 4), the simul-
taneous development of head-to-tall assemblies of 38S,68Scapsanthol and its 3-38-diacetate gives the CD curve, demonstrating immediate compensation of supramolecular chirality (Fig. 6, A+B). It indicates that the weaker interaction operative between neighboring molecules is insufficient for the creation of the characteristic assembly of component molecules. The crystallographic analog of this compensation is a racemic crystal. It is of interest to apply the simple kinetic analysis shown above (Fig. 3) to the time-dependent formation of head-totail assembly. Data read at 434 nm and plotted against time demonstrate the same type of deviation from a single exponential dependence (Fig. 7) as found for the card-pack assembly. Hence, the development of the head-to-tail aggregate does not involve a single process, either. In order to find a structural distinction between the timedependent and immediate formation of aggregates, energy profiles were computed around the torsion angles of bonds linking the cyclic end groups to the polyene chain. The b-ring is connected by a bond between the C6–C7 atoms of sp2 geometry; accordingly, two minima were found around + and −70° (Fig. 8). These minima are of equal energy. In contrast, the k-ring can rotate around the C58–C68 atoms of sp3 geometry. Accordingly, three minima were found roughly 120° apart (Fig. 9). An interesting difference was found between the 38R,68R- and 38S,68R-capsanthol epimers; while the former has a global minimum at 180°, the latter is characterized by two lower minima equally deep. It seems reasonable to assume the immediate formation of card-pack self-assembly from 38R,68R-capsanthol, the molecules of which populate essentially one rotamer, while 38S,68R-capsanthol molecules start to assemble from two different conformations. In conclusion, spectroscopic experiments with 38,68capsanthol-epimers have demonstrated a variety of supramolecular phenomena. Subtle structural changes were found to alter the kind (card-pack or head-to-tail), handedness, and the rate of formation of chiral self-assemblies. The kinetic analysis of aggregate reorganization is incompatible with a simple mechanism. The simultaneous formation of like type assemblies of opposite handedness from stoichiometric mixtures led to extinction of supramolecular chirality in two different ways: either by conglomerate type racemization for card-pack, or by intercalation for head-totail assemblies. The time-dependence of assembly formation may be related to differential population of rotamers in monomer molecules. Finally, the exciton chirality method is very useful for the study of noncovalent chemical interactions in the formation of chiral self-assemblies. ACKNOWLEDGMENTS
The authors thank Koji Nakanishi and Nina Berova (Columbia University), Nobuyuki Harada (Tohoku University), and Laurence Nafie (Syracuse University) for helpful discussions. LITERATURE CITED 1. Hoshino T, Matsumoto U, Goto T. Evidences of the self-association of anthocyanins I. Circular dichroism of cyanin anhydrobase. Tetrahedron Lett 1980;21:1751–1754.
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