A new experimental procedure to calculate all equilibrium constants of the multistate of species of anthocyanins and related compounds, including those in basic medium, is reported. The procedure is based on a series of pH jumps monitored by stopped flow from an extended pH range of solutions at pseudo-equilibrium (when there is no significant formation of trans -chalcones) or at equilibrium to pH = 1.0. The experimental procedure is described for the anthocyanin model compound 4′-hydroxyflavylium, which exhibits a peculiar behavior in moderately acidic medium, because the quinoidal base, hemiketal, and cis -chalcone have similar mole fractions at pseudo-equilibrium, permitting good discrimination among these species. The experimental procedure can be extended to the copigmentation phenomenon and allow the calculation of the 1:1 copigmentation constants of the flavylium cation, quinoidal base, hemiketal, and cis - and trans -chalcones (this last from the equilibrium) and their respective ionized forms. The method was applied to calculate the copigmentation constants of the model compound 4′-hydroxyflavylium as well as malvidin-3-glucoside with caffeine. In the last compound, the strongest interaction takes place with the quinoidal base ( K = 303 M –1 ) and flavylium cation ( K = 134 M –1 ) and, to a lesser extent, with the ionized quinoidal base ( K = 43 M –1 ) and cis -chalcone ( K = 17 M –1 ). The caffeine interaction with the hemiketal and the other ionized species is negligible.

All equilibrium and rate constants of heavenly blue anthocyanin (HBA 1) as well as the derivatives with two (HBA 2) or none (HBA 3) acylated units were determined. The three acylated units of the sugar in position 3 of the peonidin chromophore of HBA 1 are essential to confer the peculiar stability of its purple and blue colors. The sugars generate an efficient protective environment around position 2 (and 4) of the flavylium cation, through an intramolecular sandwich-type stacking that retards 35-fold the hydration reaction ( k) and increases 8.8-fold the dehydration reaction ( k), when compared with the peonidin chromophore HBA 3. The conjugation of these two rates lowers 308-fold the hydration equilibrium constant ( K), corresponding to a raise of the energy level of the hemiketal by 14.2 kJ mol. Conversely, the p K of the quinoidal base in HBA 1 is only slightly stabilized in comparison with that of HBA 2 and HBA 3. The energy level of hemiketal increases with the number of acylated units, but the inversion of energies between hemiketal and quinoidal base takes place exclusively for HBA 1 (three acylated units), permitting in moderately acidic solutions the stabilization of the purple quinoidal base. Identical inversion of energy was observed for the corresponding ionized species, allowing the stabilization of the blue ionized quinoidal base in slightly basic solutions. At pH values higher than 8, the hydroxyl groups of the hydroxycinnamic acid units start to deprotonate disrupting the intramolecular sandwich-type stacking and the more or less slow degradation of the anthocyanin is observed.

The interaction of two anthocyaninsw ith aw atersoluble polyanionic dendrimerw as studied through UV/Vis, stopped-flow,a nd NMR spectroscopy. Cyanidin-3-glucoside (cy3glc)r evealed as tronger interactiont han malvidin-3-glucoside (mv3glc) at pH 1a ccording to their apparent association constants. Ah igher color increased was also obtained for cy3glca tp H3.5 as ar esult of this strongeri nteraction.A high-frequency chemical shift of the cy3glc aromatic protons suggest the formation of ionic pairs. The interaction parame-ters (K % 700 m À1 , n % 295) indicated the binding of approximately two anthocyanin molecules by each sulfate group. The equilibriuma nd ratec onstantso fc y3glc in the presence of dendrimer showed an increased stability of the flavylium cationand ahigher protectiono fthis speciesf rom hydration (pK' a and pK h increased almost one pH unit).T he tuning and color stabilizationo fa nthocyanins by using this dendrimer allow novel applications as colorimetric sensors for food packaging.[a] L. Cruz, N. Mateus,V .d eFreitas Supporting Information and the ORCID identification number(s) for the author(s) of this articlecan be found under: https://doi.