Aggregation-Induced Resonance Raman Optical Activity (AIRROA

Article pubs.acs.org/JPCB

Aggregation-Induced Resonance Raman Optical Activity (AIRROA) and Time-Dependent Helicity Switching of Astaxanthin Supramolecular Assemblies Monika Dudek,† Grzegorz Zajac,† Agnieszka Kaczor,†,‡ and Malgorzata Baranska*,†,‡ †

Faculty of Chemistry, Jagiellonian University, Ingardena 3, Krakow 30-060, Poland Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, Krakow 30-348, Poland

‡

ABSTRACT: New methods for enhancing the Raman optical activity (ROA) signal are desirable due to the low efficiency of ROA, demanding otherwise high sample concentrations, high laser powers, and/or long acquisition times. Previously, we have demonstrated a new phenomenon, aggregation-induced resonance ROA (AIRROA), that produces significant enhancement of the ROA signal provided that the excitation wavelength coincides with the absorption of the measured species and that the electronic circular dichroism (ECD) signal in the range of this absorption is nonzero. In this work, analyzing three very different supramolecular astaxanthin aggregates (H1, H2, and J), we confirm the phenomenon and demonstrate that aggregation itself is not enough to enhance the ROA signal and that the above-mentioned conditions are necessary for induction of the resonance ROA effect. Additionally, by analyzing the changes in the ECD spectra of the H1 assembly, we demonstrate that the supramolecular helicity sign switches with time, which is dependent on the prevalence of kinetic or thermodynamic stabilization of the obtained aggregates.

1. INTRODUCTION Raman optical activity (ROA) spectroscopy based on differences in the Raman scattering intensity between right and left circulated polarized light enables the absolute configuration and structure of many biologically important chiral molecules to be probed.1 There are four circular polarized ROA forms that can be performed: incident circular polarization, scattered circular polarization (SCP), and in-phase and out-phase dual circular polarization (DCPI and DCPII, respectively). Currently, SCPROA in a backscattered geometry is the most accessible and most popular form (the only commercially available ROA spectrometer). SCP-Raman and SCP-ROA intensities are characterized by the following equations: ISCP‑Raman = IRU + ILU and ISCP‑ROA = IRU − ILU (I denotes the intensities of the right and left SCP radiation, and U denotes unpolarized incident light), and dimensionless circular intensity difference (CID) is defined by2 Δ=

However, because of the weakness of the ROA effect, highly concentrated and very pure samples as well as long scanacquisition times are usually required. Alternatively, there is a possibility for enhancing ROA signals by registration Raman scattering under resonance conditions.3−7 Resonance ROA (RROA) theory limited to the single electronic state (SES) was developed by Nafie.8 The theory predicts that RROA spectra are monosignate, opposite in sign to the electronic circular dichroism (ECD) of the resonant electronic transition, and have bands with the same relative intensities as those in the parent resonance Raman spectrum. Moreover CID values for backscattered SCP-ROA equal minus half of the ECD anisotropy ratio (geg) Δ=

IRU + ILU

=−

geg 2 Deg R eg = − c (Deg )2 2

Received: June 2, 2016 Revised: July 20, 2016

ΔISCP ‐ ROA I U − ILU = RU ISCP ‐ Raman IR + ILU © XXXX American Chemical Society

IRU − ILU

A

DOI: 10.1021/acs.jpcb.6b05514 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B whereas the geg ratio is defined by ΔεECD 4 R eg geg = = c Deg εUV − vis

possible. On the other hand, a large distance between the chiral centers and unsaturated chain in carotenoid molecules makes registration of the (R)ROA effect impossible for AXT monomers, which do not exhibit pronounced rotational strengths for the main electronic transition (polyene chain chromophore).9 It is known that carotenoids tend to self-aggregate in hydrated organic solvents.10−12 Because of their lipophilicity, molecules form supramolecular arrangements under the influence of hydrophobic effects and intermolecular forces. Formation of supramolecular assemblies occurs in biologically important systems, such as the photosynthetic apparatus13 and lipids bilayers.14,15 The phenomenon of carotenoid aggregation is widely reported in the literature; however, a lot of data concerning this subject is rather unclear and misleading, especially data referring to the synthesis as well as stability of AXT aggregates. AXT is an essential red food colorant and important antioxidant, synthesized de novo by some species of yeast and algae.16 In bioorganisms, AXT exists in the form of aggregates, bound to lipids or proteins; therefore, its Raman signature differs from the signature of the pure compound.17,18 AXT generally forms two types of supramolecular arrangements, namely, J and H (Figure 1).11,12 The type of aggregation of carotenoids affects their stability, as well as redox and photochemical properties.19−21 J aggregates of AXT, formerly reported by us,9 are characterized by a red-shifted absorption band in comparison to that in the monomer spectrum and a loose “head-to-tail” formation. In turn, the H aggregates are in a tight “card-pack” association and their polyene chains are more or less parallel to each other. A compact formation of AXT H aggregates results in loss of the vibrational structure of the electronic absorption spectrum and manifests itself as a blue shift in the ultraviolet−visible (UV−vis) band.11,19 Additionally, two types of AXT H assemblies, H1 and H2, were reported.12,22,23 According to the absorption spectra, the UV−vis band of the H1 assembly is considerably blue-shifted in reference to that of H2 and appears above 390 nm. In this work, we have synthesized both H1 and H2 AXT aggregates. Using UV−vis and Raman spectroscopy as well as their optically equivalent techniques, that is, ECD and ROA, we explored further the phenomenon of AIRROA of AXT. The aim of our study was to compare the spectroscopic and optical properties of two kinds of “card-packed” AXT assemblies, that is, H1 and H2, in reference to those of the J aggregates studied previously. On the basis of the obtained results,9 we defined the contribution of a single carotenoid molecule to the supramolecular chirality of the whole aggregate. In principle, aggregates of carotenoids are formed from both optically active and inactive compounds. Nevertheless, only chiral agents contribute to the supramolecular chirality of AXT assemblies.9 It is important to stress that knowledge about the structure and optical behavior of carotenoids in supramolecular assemblies is still limited. This study was conducted to gain a deeper understanding of the aggregation process and optical activities of the formed structures and the AIRROA phenomenon.

where Deg and Reg describe dipole oscillator and rotational strengths, respectively, of the resonant electronic transition between the ground (g) and resonant excited (e) states; ε is the molar absorption coefficient; Δε is the molar circular dichroism defined by Δε = εL − εR; and c denotes the velocity of light. Deg depends on the square of the electronic transition dipole moment (proportional to electronic absorption intensity), whereas Reg, an chiroptical equivalent of Deg, depends on both electronic and magnetic transition dipole moments (proportional to ECD intensity), which vanishes for nonchiral samples.2,8 In our recently published work,9 it was shown that aggregation of carotenoids can lead to RROA of these assemblies and hence may play a significant role in chiroptical studies. We reported previously a new mechanism by which chirality enhancement for astaxanthin (AXT, 3,3′-dihydroxyβ,β-carotene-4,4′-dione; Figure 1) is achieved through

Figure 1. Structure of the AXT monomer and two types of supramolecular aggregates. The sizes of the weakly bonded J aggregates are up to 7 μm, whereas the sizes of the H assemblies are only up to 160 nm.19

resonance due to supramolecular aggregation, and the effect was named aggregation-induced resonance Raman optical activity (AIRROA). Upon aggregation, the exciton energy is divided into nearly parallel xanthophyll chromophores. As a consequence of the exciton-coupling process, positive and negative Cotton effects in the ECD spectra are present.10 The RROA spectrum can be recorded when the AXT polyene chains are arranged in supramolecular chiral structures similar to helical aggregates. Moreover, if the ECD Cotton effect range of the aggregates coincides with the ROA excitation wavelength (in our case 532 nm), measurement of the RROA effect is

2. EXPERIMENTAL SECTION 2.1. Aggregate Synthesis. The synthesis of AXT supramolecular structures is very sensitive and requires specific conditions, such as stable environmental temperature, pure solvents, low temperature of solutions, and a constant concentration of AXT. Both H1 and H2 AXT aggregates were prepared by mixing AXT/organic solution and water at a B


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The Journal of Physical Chemistry B

Figure 2. UV−vis and ECD spectra of the AXT monomer (black line) and three kinds of (3R,3′R)-AXT aggregates: H1 (green line), H2 (blue line), and J (red line). J and H2 aggregates were measured 2 h after preparation, whereas H1, after 24 h.

temperature of 5 °C in a ratio of 1:9. The AXT H1 aggregate was formed upon adding 2.7 mL of water to 0.3 mL of 10−4 M AXT solution in dimethyl sulfoxide (DMSO). Accordingly, the AXT H2 aggregate was obtained in the same manner by adding 2.7 mL of water to 0.3 mL of 10−4 M AXT solution in acetone. For this purpose, (3S,3′S)-AXT (BASF), (3R,3′R)-AXT (CaroteNature GmbH), spectroscopic grade acetone and DMSO (POCH), as well as 4× distilled water were used. After preparation of AXT aggregates, they were stored in the dark at a constant temperature (20 °C). Acetone and DMSO were chosen because of the good solubility of AXT in those solvents. Furthermore, because of its high viscosity, DMSO stabilizes more coupled H1 aggregates.12 DMSO also enables the observation of higher energy electronic transitions of AXT due to the lower wavelength of the solvent UV cutoff in comparison to that of acetone. Different time periods were needed to stabilize each of the H types of AXT self-assemblies. For this purpose, the samples were stored in a controlled environment, at 20 °C and under dark conditions. Thus, in our experiment, the H2 aggregate was stabilized for 2 h. In turn, because of the chirality-reversal kinetics, the stabilization time for the H1 aggregate was 24 h. In addition, if synthesis is carried out under stable environmental conditions and with the use a very pure chemicals, the aggregates are stable for more than 24 h.

2.2. Measurements. ROA and Raman spectra were obtained using the ChiralRAMAN-2X spectrometer from BioTools Inc. The sample solutions were flowed in ROA optical cells with an antireflective coating and measured using 532 nm excitation wavelength. The ROA/Raman spectra of the aggregates were collected with 7 cm−1 spectral resolution, 10 mW laser power, 2.0580 s integration time, and 24 h acquisition time. In the case of the (3R,3′R)-AXT monomeric solution, an integration time of 4.0424 s was used. Results with artifacts and measurements with incorrect ROA compensation were rejected, and the final ROA/Raman spectra were averaged. UV−vis/ECD spectra were registered using a Jasco J-815 spectrometer, equipped with quartz cells with a path length of 1 cm and the following settings: 330−900 nm spectral range, 100 nm min−1 scanning speed, 1 nm bandwidth, 0.2 nm step size, 0.5 s response time, and an accumulation of one scan. The obtained spectra were baseline and solvent corrected using JASCO software.

3. RESULTS AND DISCUSSION 3.1. Comparison of Aggregates. On the basis of the protocols adopted previously,9,11,12 we have obtained both types of strongly coupled AXT assemblies, that is, H1 and H2. First, AXT aggregates were identified by UV−vis and ECD measurements (Figure 2). Our results are consistent with those reported previously by Olsina, Köpsel, and Fuciman.11,12,22 It is C


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Figure 3. Comparison of UV−vis, ECD, Raman, and ROA spectra of the AXT monomer (black line), (3R,3′R)-AXT H1 aggregate (green line), and (3S,3′S)-AXT H1 aggregate (green dotted line).

times more intense compared to those of individual molecules.25,26 The AXT monomer gives no ECD signal in the range of the main UV−vis band; however, we can observe its low optical activity at 376 nm, derived from the chiral centers of AXT rings. Unlike the monomer, AXT aggregates reveal bisignate couplets in the ECD spectra, according to the positive and negative Cotton effects. The sharp and strongly blue-shifted Cotton couplets at 376 and 396 nm, respectively, are evidence for the formation of the tight H1 aggregate. On the other hand, the broad signal associated with a slightly blueshifted absorption indicates the presence of the H2 assembly. The sequence of Cotton effects results from the exciton chirality of self-assembled chromophores and suggests the direction of packing arrangements.10,25,26 Both J and H2 aggregates of (3R,3′R)-AXT show a negative chirality and most probably display a left-handed helical structure, whereas the stable form of the H1 aggregate exhibits the opposite, that is, a right-handed helical structure. Consequently, aggregates of the (3S,3′S)-AXT enantiomer should exhibit an opposite chirality, that is, a right-handed formation for J and H2 and a left-handed formation for H1. 3.2. H1 Aggregates. H1 aggregates can be characterized by a narrow, nearly 100 nm blue-shifted UV−vis band in reference to the one observed for the monomer. The comparison of UV− vis, ECD, Raman, and ROA spectra of the AXT monomer and H1 aggregates is shown in Figure 3. Cotton effects observed in the ECD spectra of H1 aggregates of AXT enantiomers are mirror images in the region of the main chromophore absorption band (∼385 nm) as well as in the region of chromophore absorption located in the ionone rings of the monomeric subunit of the aggregate (below 340 nm). There are slight differences (shifts in the maxima) in the ECD spectral profiles of the pure monomer solution and the H1 aggregate in the region of chromophore absorption located in the ionone rings (below 340 nm). Those changes are related to differences in the conformations of the monomeric subunits in the aggregate with respect to those of free AXT monomers in

known that the electronic absorption spectra of the aggregates enable us to distinguish J, H1, and H2 AXT self-assemblies. The maximum at 478 nm in the UV−vis spectrum of AXT in acetone is assigned to the S0(1Ag) → S2(1Bu) electronic transition.10 The S0−S2 excitation does not exhibit any vibrational structure, which is specific to carotenoids with conjugated carbonyl groups.24 In reference to the monomer, the J arrangement is characterized by a red-shifted absorption band, whereas H2 and H1 are characterized by blue-shifted bands, with the UV−vis spectrum of H1 exhibiting a stronger hypsochromic effect. Moreover, the absorption profile of J assemblies reveals two maxima located at 553 and 517 nm. The occurrence and overlap of these bands may indicate a mixture of two different J assemblies, which was confirmed by fluorescence studies.19 Self-arrangement of H2 is shaped by an extensive band, with a maximum at 460 nm. Occurrence of a low-intensity shoulder above 550 nm points to some participation of J aggregates. The formation of H1 is defined by a typical narrow absorption band at 385 nm. These apparent dissimilarities between H1, H2, and J arise from the different arrangements in the structure of the aggregates. H aggregates are smaller and more coupled than J aggregates. The size of one H-type supramolecular particle is above 160 nm, whereas the dimension of J increases even up to 7 μm.19 The formation of supramolecular particles depends mainly on intermolecular forces. The “H-bond card-stack” structure of H-type aggregates is determined primarily by hydrogen bonds. In turn, in the “head-to-tail” or “herring-bone” arrangement of J aggregates the weaker van der Waals interactions prevail.10,20 It is evident that the helical aggregates of AXT exhibit strong optical activity, as shown by the chiroptical spectra in Figure 2. In the absence of a chromophore or if the association of the carotenoids is not chiral, no signal will be observed on ECD spectroscopy. Supramolecular arrangements cause a degenerate electronic transition to appear in the exciton coupling. Delocalization of exciton energy over the neighboring chromophores is a source of intensive Cotton effects, several D


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Figure 4. Comparison of the UV−vis and ECD spectra of the AXT H1 aggregate, recorded shortly after sample preparation and after a stabilization time of 24 h. Measurements were made for both AXT enantiomers.

formation of carotenoid aggregates, and its value depends on the type of aggregate. For J aggregates, a 5 cm−1 red shift was observed,9 whereas a 3 cm−1 shift was exhibited for H1 aggregates. Generally, in the case of carotenoid aggregates, H aggregates show a less-pronounced red shift of this mode.19 Unfortunately, there are no significant changes in the ROA spectra of H1 aggregates with respect to those of the monomer. As a matter of fact, there are no ROA spectra for both of these forms at all. First, as has been described before, there is no possibility of obtaining an RROA spectrum for the AXT monomer because of the lack of rotational strength of the main electronic absorption band located near the ROA spectrometer excitation line (532 nm).27 Second, nonresonant ROA of the AXT monomer is also impossible to register because of the small number of chiral centers in such a relatively big molecule (almost 100 atoms). Additionally, such a strong Raman intensity enhancement of the modes related to the main chromophore (i.e., a nonchiral polyene chain) influences the ROA spectra, causing the appearance of artifacts related to erroneous compensation of right versus left circular polarization spectra.27,28 Unexpectedly, the helical structure of H1 aggregates of AXT reveals a complicated kinetics, manifested in switching between their supramolecular helicity sign with time. The UV−vis and ECD spectral profiles of H1 aggregates of (3S,3′S)-AXT and (3R,3′R)-AXT optical isomers, recorded 30 min and 24 h after preparation, and their comparison with the spectra for the

DMSO solution. It is possible that the monomer subunits in the aggregate occur in one well-defined conformation of ionone rings with respect to the chain, that is, forced by strong H-bond interactions with other closely packed subunits and surrounding water and DMSO molecules, unlike the monomers in solution, for which a set of conformations is possible, with gauche being the most stable one.17,27,28 The rigidity of the conformation of rings with respect to the chain in the H1 aggregate is supported by the small half-width of the absorption band in the UV−vis spectrum. As the aggregates are a in state in-between solution and solid, they could behave similar to both forms, with a decreased flexibility compared to that of the monomer structures. The Raman spectrum of the AXT monomer in DMSO is strongly enhanced by the resonance effect, related to the main electronic absorption band, located near the excitation line of the spectrometer (532 nm). In the case of H1 aggregates, enhancement of Raman scattering is considerably weaker compared to that for the monomer and it is caused by a blue shift of the aggregate electronic absorption band (492 → 386 nm). Raman spectra of H1 aggregates of AXT revealed some changes in the fluorescence background and position of the CC stretching vibration mode. Similar to that for J aggregates, the fluorescence background is reduced and the band due to CC stretching vibrations is down-shifted (from 1520 cm−1 for the monomer to 1517 cm−1 for the H1 aggregate). The red shift of this mode is characteristic of the E


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Figure 5. Time-dependent UV−vis and ECD spectra of the (3S,3′S)-AXT H1 aggregate. Measurements were recorded shortly after sample preparation (orange line) and then every 30 min (light green lines) until 4 h (dark green line) after the synthesis.

to differences in their stabilities. The initial form is kinetically favored, whereas the final one is favored thermodynamically. When the concentrations of both forms are the same, the ECD signal for such mixture is close to zero. In our studies, the righthanded aggregate of (3S,3′S)-AXT is stabilized shortly after sample preparation. Approximately 2 h after the synthesis of H1 aggregates, the concentrations of the right- and left-handed structures are similar, and as expected, there is no ECD signal registered. Thereafter, the left-handed form starts to dominate, and consequently, the intensity of the ECD signal increases, but the helicity is reversed in comparison to that of the initial form. For AXT, in contrast to the previously reported work on capsanthol, two forms of H1 aggregates show identical, mirrorimage ECD spectral profiles. Furthermore, it is worth mentioning that the ECD signal assigned to the monomer unit is unchanged with time (see the Cotton effects below 340 nm). 3.3. H2 Aggregates. H2 aggregates of AXT are the second type of supramolecular structure studied in this work. H2 aggregates are characterized by a less-pronounced blue shift of the main absorption band. Comparison of the UV−vis, ECD, Raman, and ROA spectra of the AXT H2 aggregates is shown in Figure 6. The maxima of the obtained electronic absorption spectra of all AXT H2 aggregate forms (both enantiomers and the racemic mixture) exhibit a hypshochromic shift, showing that the obtained supramolecular structures are in definition the H aggregates. The electronic absorption of the obtained aggregates is slightly down-shifted, but the observed shifts are different for assemblies of all forms: 18, 9, and 16 nm for (3R,3′R)-AXT, (3S,3′S)-AXT, and the racemic mixture, respectively. These differences in hypsochromic shift are related to the difficulties in maintaining the same conditions during the synthesis and measurement of all H2 aggregates. It turns out that even minor variations in temperature, concentration, and manner of mixing during the experiments can considerably influence the structure of the final product. It is important to

(3R,3′R)-AXT monomer form obtained under identical conditions are shown in Figure 4. At the beginning of the first ECD experiment, 30 min after preparation, (3S,3′S)-AXT and (3R,3′R)-AXT H1 aggregates possess right- and lefthanded helicities, respectively, whereas after 24 h of stabilization, the handednesses of the studied supramolecular structures are completely opposite. Unlike the ECD bands of the aggregates (∼396, ∼376 nm), the ECD spectral profiles (below 340 nm) of the monomer unit remain stable with time, as during helical rearrangement of the aggregate (built by a given AXT enantiomer) the chirality of the monomer unit does not change. To elucidate more details of this “helicity switch”, time-dependent ECD and UV−vis measurements of H1 aggregates of (3S,3′S)-AXT were performed (Figure 5). On the basis of the UV−vis spectra, it can be observed that just after preparation of the H1 aggregate (time 0 h), the band intensity due to the monomer (∼460 nm) starts to decrease, and at the same time, the band assigned to the aggregate (∼385 nm) appears and shows an increase in its intensity with time (Figure 5). The intensity of the UV−vis band of the aggregate increases in the first hour after sample preparation, and it is stable for several hours. Contrarily, the band at 460 nm, assigned to the monomeric form, decreases in intensity within the first 4 h after sample preparation. Nevertheless, UV−vis spectroscopy is less informative with respect to the structure of the H1 aggregates compared to ECD spectroscopy, as the latter provides information about helicity sign. It reveals that just after the preparation of H1 aggregates the assembly of the (3S,3′S)-AXT enantiomer exhibits righthanded helicity, which is reversed after a few hours but without significant changes in the intensity of the electronic absorption. Such a process was previously described in the case of (3′S,6′R)-capsanthol self-assembly.26,29 Zsila et al. proposed a mechanism for this phenomenon on the basis of the assumption that xanthophylls could simultaneously form two types of H1 aggregates demonstrating mirror-image helicities. The concentrations of these two forms could be different due F


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cm−1 shift in the CC stretching band, which is one of the Raman markers of aggregation. The obtained RROA spectra of AXT enantiomers are mirror images of each other. Furthermore, the RROA spectra are monosignate and opposite in sign to the ECD spectra of the resonant electronic transition, which agrees well with Nafie’s SES theory.8 The CID ratios (ROA/Raman) and g-factors (ECD/UV−vis) are similar in magnitude and are approximately 1.0 × 10−3, which is comparable to the values obtained previously for J aggregates (∼1.5 × 10−3).9 Moreover, only bands related to the chiral structure of the aggregate appeared in the obtained ROA spectrum, and no signal is detected for a racemic mixture, which proves that the registered spectra are not the artifacts. We also observe a mysterious band around 280 cm−1 and an intense, nonfiltered Rayleigh scattering band, with an opposite sign to that of the bands of the aggregates, although they constitute mirror images for enantiomers, which was also revealed previously for the J aggregates.9 We suggest that these bands are possibly related to Rayleigh optical activity. Furthermore, the obtained RROA spectra are so intense that they enable observation of overtones and combinatorial bands, at 2319 cm−1 (2 × the ν(C−C) band at 1161 cm−1) and 2165 cm−1 (combination of the ν(C−C) band at 1161 cm−1 and δ(C−C) band at 1010 cm−1). Figure 6. Comparison of the UV−vis, ECD, Raman, and ROA spectra of the racemic AXT H2 aggregate (black line), (3R,3′R)-AXT H2 aggregate (blue line), and (3S,3′S)-AXT H2 aggregate (blue dotted line).

4. CONCLUSIONS Here, we report the second example of a new mechanism of ROA signal enhancement that is based on observation of the resonance effect induced by aggregation of carotenoids (AIRROA). It has been shown undoubtedly that occurrence of the ECD signal in the region coinciding with the ROA excitation line is crucial for obtaining resonance. Only one of the H aggregates of AXT studied here (H2) exhibits the resonance effect in the ROA spectra. Aggregation of H1 AXT self-assemblies leads to a rise in the strong Cotton effect shifted to the UV region, which is too far away from the 532 nm excitation line of the ROA spectrometer for resonance to take place. For H2 self-assemblies, aggregation leads to a rise in the ECD signal in the main electronic absorption band region in the range, but contrarily to that in H1 aggregates, the observed hypsochromic shift is not so pronounced; consequently, the resonance ROA signal is detectable. Moreover, the handedness of the H1 AXT aggregates changes with time, which is caused by the simultaneous formation of two opposite (left- and right-handed) H1 aggregates (in the case of one AXT enantiomer). The initial form is kinetically favored, whereas the final one is favored thermodynamically; therefore, a switch between the sign of the helices is observed in the spectra with time, manifested as a change in the sign of the Cotton effects. Actually, the AIRROA phenomenon, as well as the supramolecular chirality switch of aggregates, can be explained in a limited way. For that reason, a lot of experimental work has been undertaken. We study various carotenoids and their aggregates to understand better the nature of the AIRROA phenomenon. The results obtained for chiral assemblies of lutein, zeaxanthin, and fucoxanthin are very promising and are prepared for publication. The AIRROA effect has great potential and opens new perspectives for general applicability in supramolecular chemistry. For example, new medical diagnostic factors, biosensors, or even alternatively functionalized materials can be explored in the near future.

stress that the electronic absorption band around 380 nm appears a few seconds after the preparation of H2 aggregates, but it is observed for only a few minutes. Acetone−water mixtures probably possess a reduced ability to stabilize H1 aggregates compared to DMSO, which is related to the better H-bond acceptor and donor properties of the latter. As the assembly built from the racemic mixture of AXT (i.e., the mixture of (3R,3′R)-AXT, (3S,3′S)-AXT, and the meso form (3S,3′R and 3R,3′S) in the ratio 1:1:2) exhibits the same UV− vis spectral profile as that of assemblies of pure enantiomers, it is supposed to form structurally identical aggregates. Moreover, the absorption profile of H2 aggregates reveals a shoulder located at around 540 nm, which is related to minor amounts of other structures (e.g., the monomer and J aggregate) appearing during H2 aggregate preparation. The ECD profiles of (3R,3′R)-AXT and (3S,3′S)-AXT H2 aggregates are mirror images of each other, which means that the structures of the obtained aggregates are also mirror images. The racemic mixture of AXT (3R,3′R/meso/3S,3′S; 1:2:1) does not possess any rotational strength because Cotton effects from enantiomers of opposite sign are canceled out,30 whereas the meso compound is supposed to form nonchiral aggregates. Exciton splitting of the obtained ECD bands suggests that (3R,3′R)-AXT and (3S,3′S)-AXT H2 aggregates are left- and right-handed helices, respectively.11 Here, we report the second example of the AIRROA effect from supramolecular, chiral aggregates of carotenoids. In contrast to H1 aggregates, H2 aggregates possesses a high rotational strength in the region of ROA spectrometer excitation (532 nm), which is critical when RROA is considered. The Raman spectra of (3R,3′R)-AXT, (3S,3′S)AXT, and rac-AXT are identical and exhibit most intense bands at 1515 cm−1 (ν(CC)), 1161 cm−1(ν(C−C)), and 1010 cm−1(δ(C−C)). Similar to that for J aggregates, we observe a 5 G


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(19) Köhn, S.; Kolbe, H.; Korger, M.; Köpsel, C.; Mayer, B.; Auweter, H.; Lüddecke, E.; Bettermann, H.; Martin, H.-D. Aggregation and Interface Behaviour of Carotenoids. In Carotenoids; Britton, G., Liaaen-Jensen, S., Pfander, H., Eds.; Birkhäuser Basel, 2008; Vol. 4, pp 53−98. (20) Hempel, J.; Schädle, C. N.; Leptihn, S.; Carle, R.; Schweiggert, R. M. Structure Related Aggregation Behavior of Carotenoids and Carotenoid Esters. J. Photochem. Photobiol., A 2016, 317, 161−174. (21) Polyakov, N. E.; Magyar, A.; Kispert, L. D. Photochemical and Optical Properties of Water-Soluble Xanthophyll Antioxidants: Aggregation vs Complexation. J. Phys. Chem. B 2013, 117, 10173− 10182. (22) Fuciman, M.; Durchan, M.; Šlouf, V.; Keşan, G.; Polívka, T. Excited-State Dynamics of Astaxanthin Aggregates. Chem. Phys. Lett. 2013, 568−569, 21−25. (23) Giovannetti, R.; Alibabaei, L.; Pucciarelli, F. Kinetic Model for Astaxanthin Aggregation in Water−Methanol Mixtures. Spectrochim. Acta, Part A 2009, 73, 157−162. (24) Zigmantas, D.; Hiller, R. G.; Sharples, F. P.; Frank, H. A.; Sundstrom, V.; Polivka, T. Effect of a Conjugated Carbonyl Group on the Photophysical Properties of Carotenoids. Phys. Chem. Chem. Phys. 2004, 6, 3009−3016. (25) Zsila, F.; Bikádi, Z.; Deli, J.; Simonyi, M. Chiral Detection of Carotenoid Assemblies. Chirality 2001, 13, 446−453. (26) Simonyi, M.; Bikádi, Z.; Zsila, F.; Deli, J. Supramolecular Exciton Chirality of Carotenoid Aggregates. Chirality 2003, 15, 680− 698. (27) Zajac, G.; Kaczor, A.; Buda, S.; Młynarski, J.; Frelek, J.; Dobrowolski, J. C.; Baranska, M. Prediction of ROA and ECD Related to Conformational Changes of Astaxanthin Enantiomers. J. Phys. Chem. B 2015, 119, 12193−12201. (28) Zajac, G.; Kaczor, A.; Chruszcz-Lipska, K.; Dobrowolski, J. C.; Baranska, M. Bisignate Resonance Raman Optical Activity: A Pseudo Breakdown of the Single Electronic State Model of RROA? J. Raman Spectrosc. 2014, 45, 859−862. (29) Zsila, F.; Deli, J.; Bikádi, Z.; Simonyi, M. Supramolecular Assemblies of Carotenoids. Chirality 2001, 13, 739−744. (30) Müller, R. K.; Bernhard, K.; Mayer, H.; Rüttimann, A.; Vecchi, M. Beitrag Zur Analytik Und Synthese von 3-Hydroxy-4-Oxocarotinoiden. Helv. Chim. Acta 1980, 63, 1654−1664.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +48 12 663 2253. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Science Centre (grant no. DEC-2012/07/B/ST5/00889). REFERENCES

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Aggregation-Induced Resonance Raman Optical Activity (AIRROA

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