Molecular Structure of the Photo-Oxidation Product of Ellagic Acid in Solution.

The photoreaction of the antioxidant ellagic acid (EA) elicits a drastic coloration in solution from colorless to yellow in aerated tetrahydrofuran, which appears as a new absorption band at 405 nm. Analysis of the X-ray crystal structure suggests that the photo-oxidation product of EA is a multiple cleavage π-structure (Ox-EA) that results from the interaction of EA with singlet oxygen followed by sequential cleavage and rearrangement steps.

Introduction

Among a variety of π-conjugated hydrogen-bonded molecules, ellagic acid (EA) has an interesting molecular structure comprising a planar biphenyl moiety bridged by two lactone rings and four hydrogen-bonding −OH groups.[1]EA is mainly found in raspberries, blackberries, strawberries,[2,3] nuts, seeds, and fruit liquors[4,5] as a polyphenol derivative. Its planar π-structure also has both hydrogen-bonding acceptor (lactone) and donor (−OH) sites, and its antioxidant and anticancer effects have attracted much attention. For instance, the high reactivity of EA with the strong carcinogen benzo[a]pyrene-diol epoxide, a metabolic product from benzo[a]pyrene, inhibits DNA mutation through its binding at guanine.[6,7] Although several reports about the antioxidant effects of EA have emerged, a molecular-level understanding of the reaction mechanism is absent.[8]

The antioxidant effects of EA have been reported in terms of active radical oxygen species such as O2• and OH• as scavengers.[9] However, relatively little has been reported on its antioxidant effects against nonfree radical oxygen species such as singlet oxygen (1O2), triplet oxygen (3O2), superoxide (O2–), and peroxide (H2O2). Clearly, understanding the reaction mechanisms of EA with various O2 species is valuable to more fully understand the antioxidant effect of π-molecules. To this end, photocoloration phenomena of EA in aerated tetrahydrofuran (THF) from colorless to yellow following photo-irradiation were observed.

Result and Discussion

The π-planar structure of EA was drastically changed to a twisted Ox-EA structure (Scheme ), as confirmed by single-crystal X-ray diffraction analysis. Broadly, evaluating the photoreaction of EA has the potential to provide new insight into the antioxidant effects of EA.

Scheme 1

Molecular Structures of EA and Its Photoreaction Product (Ox-EA) in THF

The absorption maximum of EA in THF was observed at 367 nm, which could be assigned to a highest occupied molecular orbital (HOMO)–lowest unoccupied molecular orbital (LUMO) transition, whereas the fluorescence spectra also showed an emission band at 376 nm, with a small Stokes shift of 10 nm (Figure S1). Interestingly, photoirradiation of EA at 280–400 nm in aerated THF drastically changed its absorption spectrum (Figure a). The absorption intensity at 367 nm gradually decreased following photoirradiation and a new absorption band around 400 nm appeared, which also changed the solution color from clear to yellow (Figure b). An isosbestic point was observed at 373 nm during the spectral change of EA in THF, suggesting the interconversion of EA and a photoreaction product with a negligible steady-state concentration of reaction intermediates. The completion of the photoreaction was confirmed by time-dependent 1H NMR spectra (Figure S2). The absorption band of the biphenyl π-core at 253 nm was drastically suppressed by the photoreaction; it is expected that the π-conjugated structure of EA would be significantly modulated by photoirradiation with concomitant photo coloration behavior. Notably, such photocoloration behavior of EA was not observed in highly polar solvents like dimethylformamide (DMF) and CH3OH (Figure S3).

Figure 1

Photocoloration behavior of EA in THF. (a) Time-dependent absorption spectra of EA in THF following photoirradiation. (b) Colorless to yellow color change following photoirradiation.

Two types of optical filters—I (λex = 280–400 nm) and II (λex > 400 nm)—were used for the photoreaction in order to evaluate the wavelength dependence of the irradiation light (Figure S4). Utilization of filter I induced a complete conversion of EA to Ox-EA, with saturation of the absorption intensity at 403 nm within 300 min. By comparison, the photoreaction progressed much more slowly with filter II, at approximately 10% conversion under the same condition. Therefore, the optical absorption of EA can effectively indicate the extent of photocoloration and the consequent photoreaction.

Yellow-colored single crystals isolated from the photoreaction products were utilized for the X-ray single-crystal structural analysis (Figures S5 and S6). The relatively low yield of 26% was due to a loss of the reaction product during the purification. Both the time-dependent UV–vis and 1H NMR spectra supported the existence of clear and complete photoreaction (Figures a, S2, and S7). The planar π-conjugated structure of EA was transformed to an asymmetrical π-structure (Ox-EA) by multiple bond cleavages (Figure a). The absorption spectrum of the yellow crystals in THF was completely consistent with that of the photoreaction products indicated by the absorption band at 403 nm (Figure b). As seen in Scheme , one O2 molecule was formally added to EA to generate the Ox-EA photoreaction product, which drastically changed the electronic structure of EA. The C–O bond lengths in Ox-EA were consistent with the formations of ether (C–O–C), carbonyl (C=O), and carboxylic acid (−COOH) groups (Figure S6). During the photoreaction, one lactone ring was cleaved and twisted to form Ox-EA, and the biphenyl π-core within EA was broken to form a new lactone ring. In addition, the four terminal −OH groups of EA were partially transformed to −COOH. Both the 1H and 13C NMR spectra of the photoreaction product also support the formation of Ox-EA (Figures S8 and S9). Only one aromatic signal, with a chemical shift of δ = 7.35 ppm, was observed in the 1H NMR spectrum of EA, whereas two types of aromatic signals were confirmed at δ = 7.21 and 7.54 ppm in the Ox-EA spectrum. By comparison, 14 distinct independent signals caused by the low molecular symmetry of Ox-EA were apparent in its 13C NMR spectrum. These signals were consistent with simulated chemical shifts based on density functional theory (DFT) calculations at the B3LYP/6-311+G(2d,p) level of theory.[10]

Figure 2

Molecular structure of the photoreaction products of EA. (a) Molecular structure of Ox-EA based on single-crystal X-ray structural analysis at 100 K. (b) Absorption spectra of EA, EA after the photoreaction (P.R. of EA), and Ox-EA in THF.

The decrease of the absorption intensity at 254 nm was consistent with the formation of Ox-EA in the absence of a biphenyl π-core. DFT calculations of EA and Ox-EA at the B3LYP/6-31G(d,p) level of theory also supported their different HOMO–LUMO gaps of 4.21 eV (EA) and 3.33 eV (Ox-EA), where the LUMO of EA was approximately 1.2 eV higher in energy than that of Ox-EA (Figure S10).

The molecular formula of Ox-EA could be formally determined by the addition of one oxygen molecule to the EA, suggesting that molecular oxygen in THF served as a necessary input for this photoreaction. To this end, the photoreaction of EA was examined in both aerated and nitrogen-purged THF (Figure a).[11] Notably, no photoreaction of EA occurred in the absence of O2 in THF. Therefore, dissolved O2 is needed for the photo-oxidation reaction of EA in THF. Because EA itself is stable under ambient conditions (Figure a), its reaction with O2 should initiate this photoreaction.

Figure 3

Absorption spectral changes of EA following photoirradiation. (a) Spectral changes with and without dissolved O2 in THF. (b) Linear relation between the reaction product concentration and reaction time.

Two types molecular oxygen, either singlet 1O2 or triplet 3O2, possibly contributed to the photoreaction of EA in THF. Di(tert-butyl)hydroxytoluene (BHT) has previously been used as a reaction inhibitor of 3O3 in THF.[12] On this basis, the photoreaction efficiency of EA was examined in photoreaction solvents with and without BHT in THF. A slight broadening of the absorption band at 403 nm of Ox-EA was confirmed in the THF solution without BHT during a 24 h photoirradiation period (Figure S12), suggesting decomposition of the reaction products caused by the presence of 3O2. Therefore, the photoreaction proceeds in the presence of 1O2 as EA + 1O2 = Ox-EA. Photoreaction of EA was not observed in either DMF or CH3OH. A similar solvent-dependent inactivation of the photoreaction of the bacteriorhodopsin dimer (BC1) has been reported in 1O2-containing DMF and CH3OH.[13] The formation of 1O2 species by the excited-state BC1* was suppressed in highly polar solvents. These results also support our observations that singlet oxygen generated by the photosensitizing reaction of EA promoted this photoreaction. The reaction rate of EA + 1O2 = Ox-EA in THF was determined by a spectroscopic method with an initial [EA0] concentration of 500 μM, where the photoreaction, which occurs under ambient conditions of the instrument, is supplied with a sufficient amount of O2. Time-dependent spectral changes of the absorption band at 403 nm by the photoirradiation decrease the concentration of the photoreaction product [Ox-EA], which gives a reaction rate of k = 7.8 × 10–5 s–1 assuming a first-order reaction.

The photoreaction of π-molecules with 1O2 has been identified as [4 + 2] or [2 + 2] cyclization reactions (Scheme ).[14−17] The stabilities of possible [4 + 2] and [2 + 2] cyclization products of EA with oxygen were evaluated using DFT calculations based on the B3LYP/6-31G(d,p) level of theory (Scheme and Figure S13). The most stable 1O2 adduct for EA was the reaction intermediate of the [2 + 2]-1 structure, whose energy was 61.0 and 73.6 kJ mol–1 lower than those of the [2 + 2]-2 and [4 + 2] cyclization products, respectively (Scheme ). Effective conjugation in [2 + 2]-1 imparts it with relatively high stability, whereas destruction of this π-conjugation in the [4 + 2] cyclization process destabilizes the intermediate structure. Although no reaction intermediates were detected, a plausible mechanism for this photoreaction is proposed in Scheme b. Specifically, the photogenerated dioxetane intermediate [2 + 2]-1, which is unstable, is easily cleaved to form a tricycle intermediate A with a terminal conjugated enol carboxylic acid group. Sequential ring-rearrangement reactions between the terminal enol and the reactive acid anhydride then produce the stable Ox-EA product. Overall, then, selective formation of dioxetane followed by structural rearrangements affords Ox-EA.

Scheme 2

Photoreaction Mechanism of EA in THF; (a) Relative Stability of Three Possible Intermediate EA–Oxygen Adducts; (b) Photoinduced Oxidation Mechanism of EA to Form Ox-EA

In conclusion, the photoabsorption of EA in aerated THF generated excited-state 1EA*, which was transformed from the singlet to the triplet state by intersystem crossing. The reaction of triplet 3EA* with O2 generated dissolved singlet 1O2 in THF, which reacted with the π-planar structure of EA and formed an unstable dioxetane intermediate by a [2 + 2] cyclization reaction. Stepwise cleavages of the biphenyl π-core formed a dioxetane structure with concomitant intramolecular ring rearrangement between the enol oxygen and acid anhydride, which drastically modified the molecular and electronic structure of EA to form the photo-oxidation product Ox-EA. A possible antioxidant reaction mechanism of EA via its photoreaction in THF is proposed.

Experimental Section

General Methods

Commercially available reagents and solvents were used without further purification. Chemical shifts (δ) in ppm were referenced to residual nondeuterated solvent (1H 1.72 ppm and 13C 67.2 ppm for THF-d8). Mass spectra were obtained in the FAB negative mode with a magnetic sector mass spectrometer. Infrared (IR, 400–4000 cm–1) spectra were measured with a resolution of 4 cm–1.

Photocoloration of EA

Photocoloration of EA was monitored with the UV–vis spectrum. UV-light (280–400 nm) or visible light (>400 nm) was irradiated to a solution of EA (3.78 mg, 12.5 μL) in dry THF [50 mL, aerated or degassed (N2 bubbling)] with a 150 W mercury xenon lamp through a filter. The time course of the UV–vis spectrum of this solution was monitored every 30 min after dilution of the solution to 50 μM.

Photoreaction of EA and Isolation of EAO Single Crystal

UV-light (280–400 nm, 150 W mercury xenon lump) was irradiated to a stirred solution of EA (30.2 mg, 100 μmol) in dry THF (100 mL) at room temperature for 24 h. The solvent was evaporated, and the resulting solid was suspended with CH2Cl2 to give an orange solid (28.3 mg), which contains Ox-EA as a major product. A part of the solid (10.5 mg) was suspended in a small amount of acetone, and the insoluble material was removed by filtration. Toluene was added to the filtrate, and slow evaporation of the solvents gave Ox-EA single crystals (2.7 mg, 26%).

mp 275–280 °C (decomposition).

1H NMR (400 MHz, THF-d8): δ 9.52 (s, OH, 1H), 9.31 (s, OH, 1H), 7.54 (s, Ar-H, 1H), 7.21 (s, Ar-H, 1H).

13C NMR (100 MHz, THF-d8): δ 167.7, 160.3, 158.6, 158.2, 149.3, 147.5, 144.0, 135.0, 130.6, 127.3, 124.7, 115.6, 107.2, 107.1.

IR: 3541, 3280–2930 (br), 1902, 1734, 1687, 1591, 1549, 1514, 1462, 1369, 1192, 1117, 1082, 1063, 1005, 966, 906, 847 cm–1.

HRMS (FAB): calcd for C14H6O10, 332.9883 [(M – H)−]; found, 332.9880.

X-ray Structural Analysis

Single crystals of Ox-EA·(acetone)·(H2O)2 were obtained by slow evaporation from acetone–toluene. Temperature-dependent crystallographic data were collected using a diffractometer equipped with a rotating anode fitted with a multilayer confocal optic using Cu Kα (λ = 1.54187 Å) radiation. Structure refinements were carried out using the full-matrix least-squares method on F2. Calculations were performed using the Crystal Structure and SHELX software packages.[18] Parameters were refined using anisotropic temperature factors except for the hydrogen atom. Crystal data for Ox-EA: formula C17H16O13, orthorhombic, space group Pca21 (no. 29), a = 19.3428(5), b = 6.4501(2), c = 28.2467(7) Å, V = 3524.14(17) Å3, Z = 8, Dcalcd = 1.614 g cm–3, Cu Kα radiation (1.54187 Å), μ = 1.49 cm–1, T = 100 K, 36 016 reflections measured, 6440 independent reflection used, Rall = 0.143, wR2 = 0.219, GOF = 1.033.

Computational Methods

DFT calculations were performed with the Gaussian 09 program package.[19] The geometries of the molecules were optimized using the B3LYP/6-31G(d,p) basis set. Stationary points were assessed by a vibration frequency analysis.