Manipulation of the Reactivity of a Synthetic Anthocyanin Analogue in

Quı´mica e Biolo´gica, ITQB/UNL, Oeiras, Portugal, and Departamento de Quı´mica, IST/UTL,. Lisbon, Portugal. Received August 1, 2002. In Final Fo...
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Langmuir 2002, 18, 10109-10115

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Manipulation of the Reactivity of a Synthetic Anthocyanin Analogue in Aqueous Micellar Media Carolina Vautier-Giongo,† Chang Yihwa,† Paulo F. Moreira, Jr.,† Joa˜o C. Lima,‡,| Adilson A. Freitas,† Marilene Alves,† Frank H. Quina,† and Antonio L. Mac¸ anita*,‡,§ Instituto de Quı´mica, Universidade de Sa˜ o Paulo, Sa˜ o Paulo, Brasil, Instituto de Tecnologia Quı´mica e Biolo´ gica, ITQB/UNL, Oeiras, Portugal, and Departamento de Quı´mica, IST/UTL, Lisbon, Portugal Received August 1, 2002. In Final Form: October 11, 2002 The effects of anionic, cationic, and nonionic micelles on the dynamics of proton transfer in the ground and excited state of anthocyanins have been examined employing the 4-methyl-7-hydroxyflavylium ion (HMF) as a model compound. Unlike other anthocyanins and anthocyanin analogues, HMF does not hydrate in water, allowing study of the acid-base (AH+/A) equilibrium in the absence of competing hydration or tautomerism reactions. Anionic sodium dodecyl sulfate (SDS) micelles are found to stabilize the acid form of HMF, AH+, relative to A, while both cationic hexadecyltrimethylammonium chloride and nonionic Triton X-100 and Brij 35 micelles stabilize A more than AH+. Stabilization in SDS is reflected in the decrease of the deprotonation rate constant (ca. 40-fold relative to water), while destabilization in cationic and nonionic micelles is evidenced by a decrease in the protonation rate of A. Surprisingly, HMF is no longer resistant to hydration in cationic and nonionic micelles. Thus, one can manipulate the reactivity of anthocyanins by changing the detergent type in such a way as to selectively stabilize or destabilize AH+ relative to the neutral forms. In the case of HMF, destabilization of AH+ by cationic and nonionic micelles is sufficient to elicit chemistry that does not occur in the absence of the detergent.

Introduction Anthocyanins are interesting compounds for many reasons including their omnipresence in our diet, their unusual chemical and photochemical properties,1-8 and their potential for application as food dyes9,10 and antioxidant additives.11,12 Rationalization of the chemical and photochemical properties of anthocyanins is quite complex, in part because anthocyanins can exist in aqueous solution in at least five different forms coupled via pH-dependent equilibria (Scheme 1).5 At pH < 3, the dominant form is the flavylium cation (AH+), which in fact is an electron acceptor, that is, a pro-oxidant with a much greater reduction potential than the neutral forms.13-15 At physi†

Instituto de Quı´mica, Universidade de Sa˜o Paulo. Instituto de Tecnologia Quı´mica e Biolo´gica, ITQB/UNL. § Departamento de Quı´mica, IST/UTL. | Present address: Centro de Quı`mica Fina e Biotecnologia, Dep. Quı´mica, FCT/UNL, Portugal. ‡

(1) Brouillard, R.; Dubois, J. E. J. Am. Chem. Soc. 1977, 99, 1359. (2) Brouillard, R.; Delaporte R. J. Am. Chem. Soc. 1977, 99, 8461. (3) McClelland, R. A.; Gedge, S. J. J. Am. Chem. Soc. 1980, 102, 5838. (4) Santos, H.; Turner, D. L.; Lima, J. C.; Figueiredo, P.; Pina, F.; Mac¸ anita, A. L. Phytochemistry 1993, 33, 1227. (5) Houbiers, C.; Lima, J. C.; Mac¸ anita, A. L.; Santos, H. J. Phys. Chem. B 1998, 102, 3578. (6) Lima, J. C.; Abreu, I.; Brouillard, R.; Mac¸ anita, A. L. Chem. Phys. Lett. 1998, 298, 189. (7) Figueiredo, P.; Lima, J. C.; Santos, H.; Wigand, M.-C.; Brouillard, R.; Pina, F.; Mac¸ anita, A. L. J. Am. Chem. Soc. 1994, 116, 1249. (8) Pina, F.; Benedito, L.; Melo, M. J.; Bernardo, M. A.; Parola, A. J. J. Chem. Soc., Faraday Trans. 1996, 92, 1693. (9) Harborne, J. B.; Williams, C. A. Phytochemistry 2000, 55, 481 (review). (10) Brouillard, R. In Anthocyanins as Food Colors; Markakis, P., Ed.; Academic Press: New York, 1982; Chapter 9. (11) Wang, H.; Cao, G.; Prior, R. L. J. Agric. Food Chem. 1997, 45, 304. (12) Satue´-Gracia, M. T.; Heinonen, M.; Frankel, E. N. J. Agric. Food Chem. 1997, 45, 304. (13) Harper, K. A.; Chandler, B. V. Aust. J. Chem. 1967, 20, 745. (14) Harper, K. A. Aust. J. Chem. 1968, 21, 221.

ological pH values, where the antioxidant properties would be most likely to manifest themselves, the dominant form of anthocyanins is typically the hemiacetal (B), in equilibrium with minor amounts of the isomeric chalcones (CE and CZ).1 This intrinsic complexity is increased in microheterogeneous media such as micelles, which have been shown to modify significantly the position and dynamics of the equilibria involved in anthocyanin chemistry.16 Thus, for example, sodium dodecyl sulfate (SDS) micelles shift the range of stability of the flavylium cation form of oenin to higher pH by about 2.5 units and invert the relative stability of the hemiacetal (B) and the Z-chalcone (CZ) at pH 7 (CZ becomes the major form and B a minor form in micellar SDS).16 Consequently, the dominant forms of an anthocyanin present in a living organism (blood or cells) might be substantially different from those in water at the same pH value. Elucidation of the physical chemical basis of these effects of microheterogeneous media on the multiequilibria of anthocyanins is thus crucial to an understanding of the chemistry and the antioxidant properties of anthocyanins. Specifically, we must know how and why the medium affects the four key reactions involved in these multiequilibria: proton transfer, hydration, tautomerization, and isomerization. Compared to aqueous solution, incorporation of oenin into SDS micelles results in large (25- to 50-fold) decreases in the rate constants for deprotonation (kd, Scheme 1) and hydration of AH+ (kh).16 Consequently, the kinetics of ground-state proton transfer and of hydration show similar sensitivity to environmental effects on the stability of AH+ relative to that of the neutral species A and B. From a practical standpoint, however, determination of the rate constants for proton transfer of anthocyanins by laser flash (15) Harper, K. A.; Chandler, B. V. Aust. J. Chem. 1967, 20, 731. (16) Lima, J. C.; Vautier-Giongo, C.; Lopes, A.; Melo, E.; Quina, F. H.; Mac¸ anita, A. L. J. Phys. Chem. A 2002, 106, 5851.

10.1021/la026336z CCC: $22.00 © 2002 American Chemical Society Published on Web 12/17/2002

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Vautier-Giongo et al. Scheme 1

perturbation of the ground-state acid-base equilibrium17 is much simpler than determination of the hydration rate constants. In the present work, we have employed the protontransfer dynamics of the 4-methyl-7-hydroxyflavylium ion (HMF) to probe the effects of anionic, cationic, and nonionic

micelles on the relative stabilities of the cationic and neutral forms of anthocyanins. The choice of HMF stemmed from the fact that it is the only known anthocyanin analogue that is resistant to hydration in water, which was expected to eliminate interference from the other forms (B and C).6 The ground-state proton-transfer rate constants show that anionic micelles stabilize the acid form of HMF, AH+, relative to A, while both cationic and nonionic micelles stabilize A more than AH+. In contrast to the ground state, proton transfer in the excited state of HMF is only slightly sensitive to micellar type. An unexpected outcome of the present study was the finding that in cationic and nonionic micelles HMF is no longer resistant to hydration in the ground state. Thus, by changing the detergent, one can selectively stabilize or destabilize AH+ relative to the neutral forms and manipulate the reactivity of anthocyanins. In the case of HMF, destabilization of AH+ by cationic and nonionic micelles is sufficient to elicit chemistry that does not occur in the absence of the detergent or in SDS micelles. Experimental Section Materials. The synthesis of 4-methyl-7-hydroxyflavylium chloride was previously described.17 SDS, polyoxyethylene[10]dodecyl ether (C12E10), Triton X-100 (TX100), and sodium phosphate and chloride, p.a., from Sigma were used without further purification in the equilibrium measurements. Ultrapure bioreagent grade SDS (Mallinckrodt Baker, Inc.), sodium phosphate and borate (Merck), and bis-tris (Aldrich) were employed in the kinetic measurements. Brij 35 (polyoxyethylene[23]dodecyl ether) and purified hexadecyltrimethylammonium chloride (CTAC) were available from previous studies.18 Hexadecyltri(17) Macanita, A. L.; Moreira, P. F.; Lima, J. C.; Quina, F. H.; Yihwa, C.; Vautier-Giongo, C. J. Phys. Chem. A 2002, 106, 1248-1255. (18) Ranganathan, R.; Okano, L. T.; Yihwa, C.; Alonso, E. O.; Quina, F. H. J. Phys. Chem. B 1999, 103, 1977-1981.

methylammonium bromide (CTAB, Aldrich, 99%) and dodecyltrimethylammonium chloride (DTAC, ChemionAB Acro, 98%) were used as received. Sample Preparation. Aqueous solutions were prepared in Millipore Milli-Q quality water. An appropriate buffer (typically 10 mM; sodium phosphate for SDS; bis-tris chloride for CTAC; sodium borate or phosphate or bis-tris chloride for Brij 35) was employed to maintain the required pH and ionic strength in the aqueous phase of the micellar solutions. The final pH was measured using an ORION 720A pH meter with a combined RedRod electrode. The concentration of HMF was ca. 10-5 M. “Detergent-Concentration Jump” Experiments. Detergent-concentration jumps were typically performed by rapidly mixing an aliquot of a concentrated solution of HMF (5 µL of 6.6 × 10-3 M HMF in methanol) into an aqueous detergent solution (1.80 mL) of the desired final pH. The moment of mixing was taken as time zero, and the initial absorption spectrum of the resultant mixture was measured at 10 s after mixing with a Hewlett-Packard 8452A diode array spectrometer. The spectra obtained at subsequent time intervals were analyzed by subtraction of the initial spectrum of A after normalization at ca. 477 nm. Incorporation Coefficients. Micelle-water incorporation coefficients are defined as KS ) [Smic]/([Saq]CD), where [Smic] and [Saq] are the concentrations of HMF in the micellar and aqueous phases, respectively, and CD ) CT - cmc is the concentration of micellized detergent, equal to the total detergent concentration (CT) minus the critical micelle concentration (cmc). The KS values in CTAB and the nonionic detergents were determined at 30 °C by micellar liquid chromatography employing the liquid chromatograph and the procedure detailed previously.19 The micellar mobile phases were acidified to approximately pH 2 in all cases to ensure that the predominant species present was the flavylium cation form AH+. The solutes were detected by absorbance (422 nm). Retention times on a 15 cm × 4.6 mm reverse phase Hypersil ODS column (Sigma-Aldrich) were determined at a mobile phase flow rate of 1 mL/min after saturation of the column with detergent (concentration range, 0.040-0.008 M). The KS values for incorporation of the acid (AH+) and base (A) forms of HMF into SDS micelles were determined in 10 mM sodium citrate buffer, pH 3.0, or 10 mM sodium borate buffer, pH 9.1, respectively, from the change in absorbance of HMF+ as a function of added detergent concentration, by the method previously described.20 Reproducibility of the KS values is estimated to be ca. (20%. Time-Resolved Fluorescence. Fluorescence decays were measured with picosecond resolution using the time-correlated single photon counting technique, as previously described.6,21 The excitation wavelength was 420 nm. Automatic alternate (19) Rodrigues, M. A.; Alonso, E. O.; Yihwa, C.; Farah, J. P. S.; Quina, F. H. Langmuir 1999, 15, 6770-6774. (20) Quina, F. H.; Toscano, V. T. J. Phys Chem. 1977, 81, 1750. (21) Giestas, L.; Yihwa, C.; Lima, J. C.; Vautier-Giongo, C.; Lopes, A.; Quina, F. H.; Mac¸ anita, A. L. The Dynamics of Ultrafast ExcitedState Proton Transfer in Anionic Micelles. J. Phys. Chem., submitted.

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measurements (103 counts at the maximum per cycle) of the excitation pulse profile and sample emissions were made until a typical value of 5 × 103 total counts had been accumulated at the maximum. Fluorescence decays were deconvoluted from the excitation pulse (ca. 38 ps) using G. Striker’s Sand program, which allows individual and global analysis of the decays with individual shift optimization.22 Laser Flash Photolysis. The laser flash photolysis experiments were carried out with an Edinburgh Analytical Instruments LP900 laser flash photolysis system as previously described,17 employing the third harmonic (355 nm) of the Surelite I-10 Nd:YAG laser for excitation. The transients exhibited singleexponential decay in all cases and were analyzed using the standard software routines of the LP900. Lifetimes shorter than 30 ns were deconvoluted using the pulse shape of the laser (obtained by monitoring the Raman scattering peak of water at 407 nm in the fluorescence mode of the LP900, i.e., monitoring beam shutter closed). Transient absorption spectra were determined point by point, and in all cases, the lifetime of the transient showed no significant variation with wavelength.

Results pH-Dependent Equilibrium Spectra and Detergent-Concentration Jump Experiments. Figure 1 shows the absorption spectra of HMF in equilibrated 0.10 M aqueous micellar solutions of the anionic detergent SDS, the cationic detergent CTAC, and the nonionic detergent Triton X-100, as a function of the pH of the intermicellar aqueous phase (pHw). Addition of detergent induces a red shift of the absorption maxima of the acid form, AH+, relative to that of water (λmax ) 416.5 nm) that ranges from slight in CTAC (λmax ) 417 nm) to moderate in Brij 35 (λmax ) 420 nm) and substantial in SDS (λmax ) 427 nm). The increase in the magnitude of the shift upon going from micellar CTAC to Triton X-100 (or Brij 35) to SDS follows the micellar charge dependence of the incorporation coefficient of the cationic form (AH+) of HMF into the micellar phase (SDS, KS ) 1200 M-1; Brij 35, KS ) 100 M-1; Triton X-100, KS ) 36 M-1; CTAB, KS ) 3.2 M-1). In contrast, the detergent-induced red shift of the absorption maximum of the base form, A, is similar and quite substantial with respect to that of water (λmax ) 464 nm) for all three detergents (CTAC, λmax ) 478.5 nm; Triton X-100, λmax ) 476.5 nm; SDS, λmax ) 476 nm, KS > 5000 M-1). This is consistent with the expected complete incorporation of the neutral species A into the micelle in all three cases under our conditions. Figure 2 shows the effect of rapid mixing with detergent on the absorption spectra of equilibrated aqueous solutions of HMF at pH values where only the base form, A, exists in water (pH > 7). Mixing of HMF with 0.10 M SDS (final concentration) at pH ) 10.0 results in an immediate (less than 10 s) shift of the absorption maximum of the base (spectrum at 10 s after the initial addition of detergent in Figure 2a). The slight decrease in maximal absorbance is mostly due to broadening of the absorption band; that is, the integrated extinction coefficient does not change appreciably. In micellar SDS, the absorption spectrum does not undergo any further change with time. In contrast, mixing with 0.10 M CTAC or Brij 35 under similar conditions causes both an initial red shift of the absorption maximum of A and subsequent profound, timedependent changes in the absorption spectrum of the solution (Figure 2b,c). In both cases, new absorption bands gradually appear at 356 and 276 nm as the absorbance due to the base form, A, decreases. Acidification of an equilibrated (64 h old) solution of HMF in CTAC from pH 7.27 to pH 2.2 with HCl induces immediate (5 s), the disappearance of A is first order. Thus, we observe only a single decay time at all pHs, as opposed to the biexponential decay that can normally be observed for other anthocyanins both in water and in micellar SDS solution.16 Despite this limitation of our present data, the magnitudes and pH dependence of the observed first-order rate constants are consistent with an increase of the hydration rate constant and a decrease of the dehydration rate constant in the presence of CTAC. A detailed kinetic study of the cationic and nonionic detergent-induced hydration, tautomerization, and isomerization reactions of HMF, with better time resolution, is currently in progress in our laboratories.

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Figure 5. Decay of the base form, A, at 470 nm, and recovery of the acid form, AH+, at 420 nm, of HMF in 0.10 M Triton X-100 at pH 4.0.

Ground-State Proton-Transfer Kinetics. Nanosecond laser flash photolysis (5 ns, 355 nm) shifts the groundstate acid-base equilibrium of HMF toward the base form A.17 This is due to fast adiabatic deprotonation of (AH+)* to form the excited base A*, which in turn decays to the ground state of A on a subnanosecond time scale.6 The relaxation back to equilibrium (decay of A and recovery of AH+) follows single-exponential kinetics, both in water and in micellar solutions of SDS, CTAC, and Triton X-10. The observed rate constant, kobs, equal to the reciprocal of the transient lifetime, τ, is the sum of the rates of deprotonation of ground-state AH+ (kd) and protonation of ground-state A (kp[H+]w), as given by24

kobs ) kd + kp[H+]w

(1)

As shown in Figure 6, kobs is a linear function of the hydronium ion concentration in the intermicellar aqueous phase, [H+]w, for all three surfactants.24 From the slope of the plot in Figure 6a, the value of the protonation rate constant of A in SDS, kp ) (1.82 ( 0.05) × 1011 L mol-1 (24) The applicability of eq 1 to nonheterogeneous media and, in particular, the interpretation given to kp perhaps require some justification. The important consideration here is that the transient lifetimes of A (Table 2) are of the order of tens to hundreds of nanoseconds in our experimental pH range. Typical probe-quencher intramicellar encounter rates in SDS micelles are of the order of 3 × 107 s-1. Given the greater mobility of the proton, we estimate the probe-proton encounter rate at the surface of an SDS micelle to be of the order of 108 s-1.21 Consequently, any A that is generated in a SDS micelle containing one or more protons should have a lifetime of 10 ns or less. Thus, our laser flash perturbation method only detects A molecules that, at the end of the laser pulse, are incorporated into proton-free SDS micelles. The same is, of course, true for nonionic Triton X-100 and Brij 35 micelles that efficiently incorporate AH+ and A but show little or no tendency to concentrate protons. Micellar CTAC is a special case, since incorporation of HMF is only partial (ca. 25%) under our conditions. Here it is instructive to compare the obseved lifetimes of A in the presence of CTAC to those in its absence at the same pH. For A in water, τaq ) 1/(kp[H+]) ) 2.8 × 10-2/[H+] (in ns), where we have used the value of kp ) 3.6 × 1010 M-1 s-1 determined previously.17 Thus, for the range of H+ concentrations of Table 2, the corresponding lifetimes of the fraction of A that is formed in the aqueous phase would be 90 ns at [H+] ) 0.32 mM, 28 ns at [H+] ) 1.0 mM, 5 ns at [H+] ) 5.6 mM, and 95% incorporated into the micellar pseudophase (based on the value of KS), the deprotonation and protonation rate constants of HMF are kd ) (3.4 ( 0.3) × 106 s-1 and kp ) (8.4 ( 0.7) × 109 L mol-1 s-1. These values lead to a calculated value of pKa ) 3.39 (similar to the experimental value of the apparent pKa ) 3.51 in Brij 35, Figure 4). Thus, deprotonation is modestly favored (2fold increase of kd) and protonation slightly disfavored (4-fold decrease of kp, Table 3). The important observation is that, even though the effect is small, AH+ is destabilized in neutral micelles where electrostatic effects are absent. This confirms that nonionic micelles do indeed destabilize the ionic AH+ form of HMF with respect to the neutral form A. Possible origins of this micellar medium effect could be the lower dielectric constant of the micelle surface and/or the somewhat lower effective concentration of water (the proton acceptor) near the micelle surface.26 Comparison with Excited-State Proton Transfer. In contrast to the ground state, the rate of deprotonation of (AH+)* is slowed relative to that of water in all types of micelles examined, whether anionic, cationic, or nonionic (Table 3). This is consistent with the fact that the excited flavylium cation of HMF is a superphotoacid, that is, deprotonation has a negligible energetic barrier with a rate controlled by proton solvation,25 which would appear to be slightly slower at the micelle surface than in water. (25) Lima, J. C.; Moreira, P. F.; Quina, F. H.; Yihwa, C.; VautierGiongo, C.; Mac¸ anita, A. L. To be published. (26) (a) Mac¸ anita, A. L.; Costa, F. P.; Costa, S. M. B.; Melo, E. C.; Santos, H. J. Phys. Chem. 1989, 93, 336. (b) Melo, E. C.; Costa, S. M. B.; Mac¸ anita, A. L.; Santos, H. J. Colloid Interface Sci. 1991, 141, 439.

In the case of SDS, the decrease of the excited-state deprotonation rate constant, kd, is somewhat more pronounced (ca. 5-fold) than for the other detergents, indicating the presence of a small energy barrier to deprotonation reflecting an electrostatic stabilization of (AH+)* in the SDS micelle. Conclusions Microheterogeneous media such as micelles can profoundly modify the reactivity of anthocyanins in general and HMF in particular. Anionic SDS micelles preferentially stabilize the cationic form AH+ with respect to the base form A. On the other hand, both cationic and nonionic micelles destabilize the cationic form AH+ with respect to the neutral forms (A, B, and C). Moreover, this destabilization is large enough to make HMF, which normally does not hydrate or tautomerize in the absence of micelles or in SDS, spontaneously hydrate and tautomerize in the presence of cationic or nonionic micelles! Presumably, a lower water activity at the micelle surface should reduce the hydration of the cationic form AH+ and improve color stability of anthocyanins (a long standing aim in the field of food dyes). At the same time, however, the lower dielectric constant at the micelle surface should increase the rate of hydration and decrease that of dehydration, resulting in destabilization of AH+. Apparently, the latter effect predominates in nonionic micelles, suggesting that incorporation of an anthocyanin into a lipidlike medium at physiological pH should favor the hemiacetal and chalcone forms of the anthocyanin, which, though colorless or pale yellow, may be the species responsible for the antioxidant properties of anthocyanins. Acknowledgment. This work was partially supported by research grants from PRAXIS/PCEX/C/QUI/56/96, ICCTI/CAPES/423, POCTI/33679/QUI/2000, and FAPESP. A.L.M. acknowledges the Programa CIENCIA for partial funding in the acquisition of the picosecond laser system. J.C.L. is grateful to FCT-Fundac¸ a˜o para a Cieˆncia e Tecnologia for a postdoctoral grant (PRAXIS 4/4.1/BPD/ 3410). P.F.M. and F.H.Q. thank the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) for fellowship support. The laser flash photolysis system was acquired with a multiuser equipment grant from the Fundac¸ a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) to F.H.Q. C.Y. and A.A.F. acknowledge doctoral fellowship support and M.A. postdoctoral fellowship support from FAPESP. LA026336Z