From vine to wine: photophysics of a pyranoflavylium analog of red wine pyranoanthocyanins

Introduction

The anthocyanins are the major class of natural plant pigments responsible for most of the red, blue and purple colors of flowers and fruits [1], [2], [3]. In nature, these colors provide contrast with the green-brown background of leaves and branches and thus serve to attract pollinators to flowers and to signal the ripeness of fruit to seed dispersal agents such as herbivores. In plants, anthocyanins are usually localized in vacuoles, where the color can be additionally stabilized and/or modulated by copigmentation, i.e. inter- and intramolecular complex formation between the anthocyanin and colorless electron-rich molecules (copigments) also present in the vacuoles or covalently attached to the sugar residues [4], [5]. Anthocyanins with vicinal OH groups in the B-ring can chelate metal ions like Al(III), resulting in a change in the color from red-purple for the free anthocyanin to blue in the metal complex [6] (the chemical basis of the red/blue color change in Hydrangea flowers [7]). The blue pigments of some flowers are even more complex, the colored species being a self-organizing metal-ion-stabilized supramolecular complex of defined stoichiometry formed between anthocyanins and copigment molecules (e.g. six of each, plus four metal cations) [6].

In the ground electronic state, anthocyanins and 7-hydroxyflavylium cations are weak acids, with pK_a values for the acid (AH⁺)-base (A) equilibrium of the 7-hydroxy group typically in the range of ca. 4–5. Above about pH 3, however, most natural anthocyanins begin to lose their intense visible color due to the attack of water at the 2-position of the cationic form (AH⁺) of the anthocyanin to form the colorless or pale yellow hemiketal B (on the time scale of seconds to minutes). This is then followed by ring-opening tautomerism (on the time scale of minutes) to the (E)-chalcone (EC), which can then slowly (hours to days) equilibrate with the (Z)-chalcone (ZC) [1], [2], [3], as outlined in Scheme 1 for malvidin 3-O-glucoside, the predominant anthocyanin present in purple Vitis vinifera grapes. Given the wide range of colors of the anthocyanins present in nature and the fact that they are known to be safe food additives with good properties as anti-oxidants, they might potentially be excellent candidates for applications as coloring agents in food or other consumer products. Nonetheless, in practice, the color loss due to the pH-dependent multiequilibria inherently limits their utility to relatively acidic media, where the cation AH⁺ is the dominant form present, as well as the absence of nucleophilic agents such as sulfite [8].

Scheme 1:

The pH-dependent anthocyanin multiequilibria [1], [2], [3], illustrated with malvidin 3-O-glucoside.

Work over the last few decades has also provided a rather clear picture of the photophysics of the excited singlet state of anthocyanins. Upon absorption of light in aqueous media, the excited singlet state AH⁺* of anthocyanins undergoes ultrafast adiabatic proton transfer to water (in ca. 5–15 ps) to produce the excited conjugate base A*, which then relaxes back to the ground state in about 200 ps without significant net photochemistry [1], [3], [9]. In anthocyanin-copigment complexes, electronic excitation is followed by even faster charge-transfer-mediated internal conversion back to the ground state in several hundred femtoseconds [10]. The absorption of UV light by the colorless forms of anthocyanins also results in fast photoprocesses [1], [11], specifically photoinduced ring-opening tautomerism in the case of the excited hemiketal B* (reflecting the fact that it is a chromene, a chromophore known to be photochromic) and photoisomerization in the case of the chalcones. All of these photoprocesses are consistent with the proposed role of anthocyanins in red-purple leaves of providing the photosynthetic apparatus with photoprotection against excess solar radiation [1], [3].

In young red wines, the color is due primarily to free anthocyanins and/or anthocyanin complexes. However, during the ageing of red wines, the anthocyanins are slowly degraded or transformed by condensation with yeast metabolites or copigments into a variety of more complex pigment molecules whose colors are much more pH-stable and, in addition, much less susceptible to bleaching by sulfite [8], [12], [13], [14], [15], [16]. Of particular importance for the color of mature wines are the pyranoanthocyanins, which differ from the anthocyanins by the presence of an additional pyran ring bridging between the 4-carbon and the 5-hydroxy group of the anthocyanin precursor [13], [14], [15], as shown in Scheme 2 for the transformation of malvidin 3-O-glucoside into a corresponding pyranoanthocyanin related to Vitisin B (R=H) found in mature red wine.

Scheme 2:

Transformation of malvidin 3-O-glucoside into a Vitisin B type pyranoanthocyanin during the ageing of red wine.

Essentially all of the features of the chemistry and photochemistry of naturally-occurring anthocyanins are mimicked by the synthetically more accessible 7-hydroxyflavylium cations, in which the type and position of additional substituents can be varied in order to modulate the reactivity or selectively block one or more of the pH-dependent multiequilibria [1], [2], [3]. In this context, Chassaing et al. [17] recently reported a convenient transformation of the 5,7-dihydroxy-4-methylflavylium cation (readily obtained from the acid-catalyzed condensation of phloroglucinol with benzoylacetone) into the corresponding pyranoflavylium cations by reaction with benzaldehydes bearing electron-donating substituents, such as the p-methoxyphenyl-substituted pyranoflavylium cation (I). In this work, we present results for the photophysics of I, which, by analogy with flavylium cations, should provide insight into the fundamental aspects of the photochemical behavior to be expected for natural pyranoanthocyanins.

Experimental section

Materials and sample preparation

The p-methoxyphenyl-substituted pyranoflavylium chloride (I) was prepared (Scheme 3) by the condensation of 5,7-dihydroxy-4-methylflavylium chloride with p-methoxybenzaldehyde in ethanol solution employing the general procedures described by Chassaing et al. [17]. The structure of the compound was confirmed by comparing the absorption and ¹H-NMR spectral data with those reported by Chassaing et al. [17] and by determination of the high-resolution molecular mass of the cation (Calc. for C₂₄H₁₇O₄: 369.1127; Found: 369.1125 g mol⁻¹, IQ-USP Analytical Center). Spectroscopic grade acetonitrile, absolute ethanol and methanol (Merck) and deionized water (Elgastat UHQPS System) were used in the preparation of all solutions. Acidic solutions of I were prepared with either 0.10 mol dm⁻³ aqueous perchloric acid (60% concentrated, p.a., Merck) in the case of ethanol-water or trifluoroacetic acid (TFA, Sigma-Aldrich) in the case of acetonitrile, methanol and methanol-water. The pH values of the 30% methanol-water mixtures were adjusted by addition of TFA. As discussed by Castells et al. [18], up to ca. 50% methanol, the actual pH of a methanol/water mixture differs only slightly from the apparent pH measured in the same methanol/water mixture with a glass electrode calibrated against standard aqueous buffer solutions. The measured pH values can then be corrected via the expression: pH(methanol/water)=pH(measured) + δ, where the correction factor δ is a function of percent methanol and temperature, to obtain the actual pH of the mixture. For 30% methanol/water (v/v), the average correction factor, δ=0.06 at 20°C [18], is relatively insensitive to temperature and the nature of the acid or buffer employed to adjust or control the pH. Basic solutions (pH 12) of I for the fluorescence measurements were prepared using K₂CO₃ (Merck). The acidic ethanol-water solutions of I were filtered through a LiChrolut RP-10 E column (40–63 nm, Merck Millipore) to guarantee to absence of microcrystals in the solution.

Scheme 3:

Preparation of I from the 5,7-dihydroxy-4-methylflavylium cation.

Results and discussion

In contrast to most natural anthocyanins and flavylium cations, the pyranoflavylium cation I was not particularly soluble in acidic aqueous solution. As a result, most spectroscopic measurements were performed in methanol acidified with trifluoroacetic acid (TFA), 30% metanol:water (v:v; X_MeOH=0.16) acidified with TFA or 50% ethanol:0.1 M aqueous HClO₄ (v:v; X_EtOH=0.24), solvents in which I was completely soluble with no indications of aggregation. Over the pH range from ca. 1–8, only the prototropic equilibrium of the hydroxyl group was observed for I, confirming the greater pH stability of the color of pyranoflavylium cations relative to flavylium ions due to the absence of the other multiequilibria of Scheme 1. A pK_a value of 3.7±0.1 was determined for I in 0.01 mol dm⁻³ sodium acetate/acetic acid buffer solutions, similar to the usual range of values for anthocyanins and 7-hydroxyflavylium cations in aqueous solution [1], [2], for other synthetic pyranoflavylium cations [16] and for analogous natural pyranoanthocyanins [14], [16].

In methanol acidified with 0.58 mol dm⁻³ TFA, I exhibits a first absorption band with a maximum at 480 nm and a fluorescence maximum at 500 nm. The fluorescence decay (410 nm excitation, 490 nm emission) was monoexponential within experimental error with a lifetime of 2.4 ns, indicating the absence of appreciable net excited state proton transfer. Fluorescence quantum yields (±10%) of 0.25 and 0.41 were determined in methanol and acetonitrile, respectively, both containing 0.010 mol dm⁻³ TFA. In 30% methanol:water (v:v, X_MeOH=0.16) acidified with TFA to pHs in the range of pH 1.9–2.75, emission from both the excited acid (HPF⁺*) and excited base (PF*) forms of I could be detected (Figs. 1 and 2). Global analysis of the fluorescence decays (410 nm excitation) was performed at 490 nm, dominated by the emission of HPF⁺*, and 640 nm, dominated by the emission of PF*. At pH 2.33, for example (Fig. 2), the fluorescence decay of HPF⁺* at 490 nm was biexponential with decay times of 0.66 and 1.02 ns, while at 640 nm PF* exhibited a rise time of 0.66 ns followed by a decay time of 1.02 ns. Thus, the 0.66 ns decay time clearly corresponds to proton dissociation of the pyranoflavylium excited state. Together with the decay time of 0.95 ns for PF* at pH 12 in 30% metanol:water and the pH dependence of the decay constants, the protonation (k_p) and deprotonation (k_d) rate constants of the excited state can be estimated to be 4.7×10⁹ mol dm⁻³ s⁻¹ and 1.0×10⁹ s⁻¹, respectively, corresponding to an excited state pK_a*=log (k_p/k_d) of 0.67. Compared to anthocyanins and hydroxyflavylium cations, the rate constant for proton dissociation from I is substantially smaller (by almost two orders of magnitude) and the excited state pK_a* higher by about one unit [1], [3], [9], [22].

Fig. 1:

Absorption spectra (left) and the steady-state fluorescence spectra (right; excitation wavelength 410 nm) of I in 30% methanol:water (v:v; X_MeOH=0.16) at pH 2.33, where only the acid form (HPF⁺) is present in the ground state, and pH 12, where only the base form (PF) is present.

Fig. 2:

Fluorescence decay of I in 30% methanol:water (v:v; X_MeOH=0.16) at pH 2.33 (left) or pH 12 (right). Excitation at 410 nm, 25 ps laser pulse width (IRF). The fit curves, the chi-squared values and the residuals of the fit are shown at the wavelengths of predominant emission of the excited acid (HPF⁺*; 490 nm) and the excited base (PF*; 640 nm) forms of I.

Finally, in 50% ethanol:0.10 mol dm⁻³ aqueous HClO₄ (v:v; X_EtOH=0.24), global analysis of the fluorescence decay of I from 480 to 700 nm required a tetraexponential fit, with decay times of 12, 70, 340 and 1240 ps. Using the decay of the excited acid form (HPF⁺*) to deconvolute the decay of the excited base [19] showed that the 70 and 1240 ps lifetimes correspond to the biexponential decay of the excited base. The 12 and 340 ps lifetime components could be attributed to the decay of HPF⁺*, with 340 ps corresponding to the rise time of PF* as well. These lifetime components are nicely mirrored in the time-resolved emission spectra (Fig. 3), in particular in the time-resolved area-normalized emission spectra (TRANES), which largely compensate for the overall decay of the excited state population with time [23]. Thus, within about 30 ps, the emission maximum of the excited acid HPF⁺* shifts from 500 nm to almost 520 nm and broadens, suggesting a change in solvation and/or conformation of the excited state (perhaps in the direction of an intramolecular charge transfer state). This is followed by much slower (340 ps lifetime component) proton loss to form the excited base PF*. The long lifetime (1240 ps) component of the excited base PF* corresponds to its decay back to the ground state, while the shorter (70 ps) decay component appears to be a solvent relaxation phenomenon. In ethanol-water mixtures, the time scale of the cooperative rearrangement of the hydrogen-bond network of the mixed solvent itself should be in the range of 30 ps at an ethanol mol fraction of 0.24 (interpolated from the data in [24]). On the other hand, given the limited solubility of I in purely aqueous media, selective solvation of both the ground and excited states of I by ethanol is probably to be expected [25], [26]. Thus, an increase in the local mol fraction of ethanol to ca. 0.5–0.6 in the solvation sphere of the excited base PF* would be consistent with an increase of the time scale for local rearrangement of the hydrogen-bond network to ca. 70 ps [24].

Fig. 3:

Time-resolved emission spectra (TRES, left) and time-resolved area-normalized emission spectra (TRANES, right) of ^I in 50% ethanol:0.10 mol dm⁻³ aqueous HClO₄ (v:v; X_EtOH=0.24) at the indicated times, in ps.

Conclusions

Like 7-hydroxyflavylium cations and naturally-occurring anthocyanins, the p-methoxyphenyl-substituted pyranoflavylium cation I is a weak acid in the ground state. However, I is notably less water-soluble than most flavylium cations and its pH-dependent chemistry is much simpler, limited to the acid-base equilibrium, which eliminates the visible color loss due to hydration at neutral pH typical of natural anthocyanins. In the lowest excited singlet state, anthocyanins and 7-hydroxyflavylium cations are strong photoacids in aqueous solution (excited state pK_a*<0) and deprotonation times to water of the order of 5–20 ps. By comparison, the photoacidity of I is much less pronounced (pK_a*=0.67 in 30% methanol-water) and the rate of proton loss from the excited acid form of I much slower (by a factor of up to 100). In 50% ethanol:0.10 mol dm⁻³ HClO₄, the excited state of the acid form of I, HPF⁺*, undergoes fast (12 ps) initial relaxation (potentially in the direction of a TICT state), followed by much slower (340 ps) adiabatic deprotonation to form the excited base, PF*. The excited base in turn exhibits a moderately fast relaxation (70 ps), consistent with solvent hydrogen-bond reorganization times, followed by slower but efficient decay (1240 ps) back to the ground state. As in uncomplexed anthocyanins and 7-hydroxyflavylium cations, the photophysical behavior of I points to excited state proton transfer as the dominant excited state deactivation pathway of pyranoanthocyanins, consistent with relatively good photostability of natural pyranoanthocyanins.