Vesselin Petrov

UV-Vis spectroscopic and chemometric study on the aggregation of ionic dyes in water

The monomer-dimer equilibrium in several ionic dyes (Methylene Blue, Acridine Orange, Nile Blue A, Neutral Red, Rhodamine 6G and Safranine O) has been investigated by means of UV-Vis spectroscopy. The data have been processed by a recently developed method for quantitative analysis of undefined mixtures, based on simultaneous resolution of the overlapping bands in the whole set of absorption spectra. In the cases of Acridine Orange a second chemometric approach has been used as a reference. It is based on a decomposition of the recorded spectra into a product of target and projection matrices using non iterative partial least squares (NIPALS). The matrices are then rotated to give the correct concentrations, spectral profiles of the components and the equilibrium constant. The dimeric constants determined by the two methods were in excellent agreement, evidencing the accuracy of the analysis. From the calculated dimeric constant and monomer and dimer spectra, the structures of the dimeric forms of the studied dyes are estimated.

The effect of self-aggregation on the determination of the kinetic and thermodynamic constants of the network of chemical reactions in 3-glucoside anthocyanins.

The six most common 3-glucoside anthocyanins, pelargonidin-3-glucoside, peonidin-3-glucoside, delphinidin-3-glucoside, malvidin-3-glucoside, cyanidin-3-glucoside and petunidin-3-glucoside were studied in great detail by NMR, UV-vis absorption and stopped flow. For each anthocyanin, the thermodynamic and kinetic constants of the network of chemical reactions were calculated at different anthocyanin concentration, from 6 × 10⁻⁶ M up to 8 × 10⁻⁴ M; an increasing of the flavylium cation acidity constant to give quinoidal base and a decreasing of the flavylium cation hydration constant to give hemiketal were observed by increasing the anthocyanin concentration. These effects are attributed to the self-aggregation of the flavylium cation and quinoidal base, which is stronger in the last case. The UV-vis and ¹H NMR spectral variations resulting from the increasing of the anthocyanin concentration were discussed in terms of two aggregation models; monomer-dimer and isodesmic, the last one considering the formation of higher order aggregates possessing the same aggregation constant of the dimer. The self-aggregation constant of flavylium cation at pH=1.0, calculated by both models increases by increasing the number of methoxy (-OCH₃) or hydroxy (-OH) substituents following the order: myrtillin (2 -OH), oenin (2 -OCH₃), 3-OGl-petunidin (1 -OH, 1 -OCH₃), kuromanin (1 -OH), 3-OGl-peonidin (1 -OCH₃) and callistephin (none). Evidence for flavylium aggregates possessing a shape between J and H was achieved, as well as for the formation of higher order aggregates.

Thermodynamic and kinetic properties of a red wine pigment: catechin-(4,8)-malvidin-3-O-glucoside.

Catechin-(4,8)-malvidin-3-glucoside, a red pigment adduct (at acid pH) found in red wine and resulting from the reaction between malvidin-3-glucoside and flavan-3-ols during wine aging, was synthesized. The thermodynamic and kinetic constants of the network of chemical reactions were fully determined by stopped flow: (i) Direct pH jumps, from thermal equilibrated solutions at pH = 1.0 (flavylium cation, AH(+)), show three kinetic processes. The first one occurs within the mixing time of the stopped flow and leads to the formation of quinoidal bases A and/or A(-) depending on the final pH; the second one takes place with a rate constant equal to 0.075 + 33[H(+)] and was attributed to the hydration reaction that forms the pseudobases (hemiketals), B/B(-). The third process is much slower, 2 × 10(-4) s(-1), and is due to the cis-trans isomerization giving rise to a small fraction of trans-chalcones, Ct/Ct(-). (ii) Reverse pH jumps from the thermally equilibrated solution at moderate to neutral pH values back to a sufficiently acidic medium clearly distinguish three kinetic processes: the first one takes place within the dead time and is due to the protonation of the bases; the second process occurs with the same rate constant of the hydration reaction monitored by direct pH jumps and is attributed to the formation of flavylium cation from the B; the last process occurs with a rate constant of 1.8 s(-1), and results from the formation of AH(+) from Ct through B, reflecting the rate of the ring closure (tautomerization). The separation of the hydration from the tautomerization upon a reverse pH jump is only possible because at pH < 1 the former reaction is faster than the last. An identical situation was observed for malvidin-3-glucoside (oenin) for pH < 2.

Establishment of the Chemical Equilibria of Different Types of Pyranoanthocyanins in Aqueous Solutions: Evidence for the Formation of Aggregation in Pyranomalvidin-3-O-coumaroylglucoside-(+)-catechin

The chemical equilibria of the pyranomalvidin-3-glucosides linked to (+)-catechin, (-)-epicatechin, and catechol moieties (and the respective coumaroylglucoside compounds) were established by means of UV-vis spectroscopy. The conjugated double bonds among pyranic rings C and D provide a higher electronic delocalization that prevents the nucleophilic attack of water at position 2. Consequently, besides flavylium cation (AH(+)), the bases A, A(-), and A(2-) have been identified by increasing pH, and the respective acidity constants were determined by spectrophotometry. The formation of dimers at higher concentration was observed for pyranomalvidin-3-O-coumaroylglucoside-(+)-catechin, and the respective data treated by the exciton model suggests the formation of a dimer where the monomers form J-type aggregates with the dipolar moments in opposite directions and rotated by 174° at a distance of 5.2 Å (from the center).

Step by step filter based program for calculations of highly informative derivative curves

We have developed a microcomputer program, based on a recently described approach called step by step filter (SBSF), for calculation of derivative curves directly from spectra recorded as a function of wavelength. This program avoids the long wavelength attenuation featured at conventional method for derivative curves calculation, and in this extent could be very helpful for daily spectroscopy practice. The features of the SBSF program include: easy treatment of data through a windowed environment, calculating of both conventional and step by step filter derivatives, possibilities for selection of the mathematical conditions for smoothing and differentiation simultaneous plotting of the original curve and its derivative and a mouse pointer. Several examples from different branches of the molecular spectroscopy (absorption UV-VIS, CD and fluorescence) are provided and discussed in the terms of advantages of SBSF.

Chemical Behavior of Methylpyranomalvidin-3-O-glucoside in Aqueous Solution Studied by NMR and UV-Visible Spectroscopy

In the present work, the proton-transfer reactions of the methylpyranomalvidin-3-O-glucoside pigment in water with different pH values was studied by NMR and UV-visible spectroscopies. The results showed four equilibrium forms: the methylpyranomalvidin-3-O-glucoside cation, the neutral quinoidal base, the respective anionic quinoidal base, and a dianionic base unprotonated at the methyl group. According to the NMR data, it seems that for methylpyranomalvidin-3-O-glucoside besides the acid-base equilibrium between the pyranoflavylium cation and the neutral quinoidal base, a new species is formed at pD 4.88-6.10. This is corroborated by the appearance of a new set of signals in the NMR spectrum that may be assigned to the formation of hemiketal/cis-chalcone species to a small extent. The two ionization constants (pK(a1) and pK(a2)) obtained by both methods (NMR and UV-visible) for methylpyranomalvidin-3-O-glucoside are in agreement (pK(a1) = 5.17 ± 0.03; pK(a2) = 8.85 ± 0.08; and pK(a1) = 4.57 ± 0.07; pK(a2) = 8.23 ± 0.04 obtained by NMR and UV-visible spectroscopies, respectively). Moreover, the fully dianionic unprotonated form (at the methyl group) of the methylpyranomalvidin-3-O-glucoside is converted slowly into a new structure that displays a yellow color at basic pH. On the basis of the results obtained through LC-MS and NMR, the proposed structure was found to correspond to the flavonol syringetin-3-glucoside.

Circular dichroism of anthocyanidin 3-glucoside self-aggregates

Self-association constants for the flavylium cations of the six most common anthocyanidin 3-glucosides were determined by circular dichroism (CD) and UV-Vis spectroscopy. Along with previous (1)H NMR results, all measurements were consistent with a monomer-dimer model. The CD spectra of the anthocyanidin 3-glucosides were similar to the analogues 3,5-diglucosides. All dimers of the anthocyanidin 3-glucosides exhibited left-handed CD signals, with petunidin-3-glucoside and myrtillin having the most intense signals. In addition, the magnitude of the molar ellipticity, [θ], was generally higher for the 3-glucosides than for the 3,5-diglucosides. For all six anthocyanins studied, the CD absorption spectra of their dimers showed evidence of the splitting of the monomer absorption into lower (J aggregates) and higher (H aggregates) energy bands. The angle and the distance between the dipolar moments of the two monomers comprising the dimer were obtained from the lower energy absorption band. While the angle was more or less similar in all six dimers, the separation distance between the monomer dipole moments differed dramatically. The intensity of the CD signal displayed a linear dependence with the inverse square of the dipole moment distances.

Thermodynamics, kinetics, and photochromism of oaklins: a recent family of deoxyanthocyanidins.

Two oaklins guaiacylcatechinpyrylium (GCP) and syringylcatechinpyrylium (SCP) and a model compound deoxypeonidin (DOP) were synthesized, and the rate and equilibrium constants of the respective pH dependent network of chemical reactions were calculated. In contrast to anthocyanins, the three compounds possess a small cis-trans isomerization barrier and hence the rate of the trans-chalcone formation follows a bell-shaped curve as a function of pH. The three compounds exhibit photochromism obtained by irradiation of the trans-chalcone, which, depending on pH, leads to the colored species flavylium cation and quinoidal base. The flash photolysis together with pH jumps followed by UV-vis absorption and stopped flow is a very useful tool to achieve the rate and equilibrium constants of the network of chemical reactions followed by these molecules. Oaklin compounds which are formed in wine aged in oak barrels present physical-chemical properties more similar to simpler deoxyanthocyanidins rather than anthocyanins and may play a significant role in color changes observed in wine aging. Given their higher stability, they may be regarded as potential food colorants.

Isomerization between 2-(2,4-dihydroxystyryl)-1-benzopyrylium and 7-hydroxy-2-(4-hydroxystyryl)-1-benzopyrylium.

2-Phenyl-1-benzopyrylium (flavylium) and 2-styryl-1-benzopyrylium (styrylflavylium) cations establish in aqueous solution a series of equilibria defining chemical reaction networks responsive to several stimuli (pH, light, redox potential). Control over the mole fraction distribution of species by applying the appropiate stimuli defines a horizontal approach to supramolecular chemistry, in agreement with the customary bottom-up approach toward complex systems. In this work, we designed an asymmetric styrylchalcone able to cyclize in two different ways, producing two isomeric styrylflavylium cations whose chemical reaction networks are thus interconnected. The chemical reaction networks of 2-(2,4-dihydroxystyryl)-1-benzopyrylium (AH(+)) and 7-hydroxy-2-(4-hydroxystyryl)-1-benzopyrylium (AH(+)(iso)) comprise the usual species observed in flavylium-derived networks, in this case, the styryl derivatives of quinoidal bases, hemiketals, and chalcones. The thermodynamics and kinetics of the crossed networks were characterized by the use of UV-vis absorption and NMR spectroscopy as well as time-resolved pH jumps followed by stopped-flow. The two styrylflavylium cations are connected (isomerize) through two alternative intermediates, the asymmetric trans-styrylchalcone (Ct) and a spiropyran-type intermediate (SP). At pH = 1, AH(+) slowly evolves (k(obs) ≈ 10(-5) s(-1)) to a mixture containing 62% AH(+)(iso) through the Ct intermediate, while at pH = 5, the SP intermediate is involved. The observed rate constants for the conversion of the styrylflavylim cations into equilibrium mixtures containing essentially Ct follow a pH-dependent bell-shaped curve in both networks. While at pH = 1 in the dark, AH(+) evolves to an equilibrium mixture containing predominantly AH(+)(iso), irradiation at λ > 435 nm induces the opposite conversion.

Unidirectional switching between two flavylium reaction networks by the action of alternate stimuli of acid and base.

The introduction of an ester group in the flavylium core allowed the reversible conversion between two different flavylium compounds each one exhibiting its own reaction network. An unidirectional switching cycle between 7-diethylamino-2-(4-(methoxycarbonyl)phenyl)-1-benzopyrylium and 2-(4-carboxyphenyl)-7-diethylamino-1-benzopyrylium was achieved by means of alternate acid and base stimuli. Addition of base to a methanolic solution of the ester derivative gives rise to the trans-chalcone of the parent carboxylic acid, which upon acidification of the solution forms the respective flavylium cation. This species esterifies under very acidic conditions to restore the original methyl ester derivative. The chemical reaction networks of both compounds were fully characterized from their thermodynamic and kinetic aspects, by a series of pH jumps followed by UV-vis absorption and emission spectroscopy, stopped flow and (1)H NMR. The crystal structure of the trans-chalcone of the ester derivative was unveiled showing a supramolecular structure involving hydrogen bonding.