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Achieving Complexity at the Bottom Through the Flavylium Cation-Based Multistate : A Comprehensive Kinetic and Thermodynamic Study

Johan Mendoza and Fernando Pina

Department of Chemistry, Nova School of Science and Technology, Caparica, Portugal

1.1 Introduction

Complexity is ubiquitous in biological systems. The main strategy to study complexity has been carried out using a top-down approach. Though the top-down approach the simpler components of the complex systems are identified, and whenever possible, up to the molecular level. In contrast, supramolecular chemistry, a concept well established and recognized after the 1987 Nobel Prize awarded to Donald J. Cram, Jean-Marie Lehn, and Charles J. Pedersen, is a bottom-up approach (Figure 1.1). Supramolecular chemistry studies how molecules interact to form higher-dimension entities and tends to fill the gap between "classical chemistry" and biology (Lehn, 1995).

A beautiful example of supramolecular chemistry is the structure of the metalloanthocyanin that gives color to Commelina communis (Kondo et al. 1992; Yoshida et al. 2009). An anthocyanin, a flavone, and a metal ion in a ratio 6:6:2 are organized into two parallel plans, each one containing three anthocyanins, three flavones, and one metal ion that organizes the space Figure 1.1.

There is an alternative to achieve complexity that we coin metamorphosis (Petrov et al. 2012). When a molecule (generator) is able to be transformed into other molecules by means of successive conversions and as a response to external stimuli, new molecules are formed. The complexity results from the number of the species and everything takes place at the bottom.

The pH-dependent multistate of species of anthocyanins and related compounds is a paradigm of the metamorphosis concept; see Scheme 1.1.

1.2 Flavylium Cation as a Metamorphosis Generator

The flavylium cation, AH+, is the most stable species at very low pH values, in anthocyanins generally for pH<1. The system is conveniently studied by direct pH jumps when base is added to the flavylium cation, and reverse pH jumps, defined as addition of acid to equilibrated solutions at higher pH values. After a direct pH jump to moderately acidic pHs, the flavylium cation equilibrates in microseconds with quinoidal base, A eq. (1). The next step is the formation of the hemiketal, B, through the hydration of AH+ (min) eq. (2), followed by the ring opening to form cis-chalcone, Cc, (ms) eq. (3). The fact that the quinoidal base does not open in acidic medium is a breakthrough discovery (Brouillard and Dubois 1977) crucial for the comprehension of anthocyanins and related compounds systems. The Cc isomerization to trans-chalcone, Ct, in anthocyanins takes place in several hours eq. (4). When the system is equilibrated in moderately acidic pH values, a reverse pH jumps restores the flavylium cation. The following set of equilibrium reactions accounts for the system:

Figure 1.1 Sketch of the metalloanthocyanin responsible for the color in Cummelina communis. The building blocks self-associate to create the supramolecule in a bottom-up approach.

Source: Courtesy of Prof. Kumi Yoshida.

Scheme 1.1 The metamorphosis concept in biology and in chemistry applied to anthocyanins and related compounds in acidic medium.

Source: Reproduced from Mendoza et al. (2018), with permission.

A few years ago we introduced an energy level diagram that accounts for the thermodynamic of the anthocyanin system in acidic medium (Pina et al. 1997; Pina 2014a). This diagram can be straightforwardly constructed provided that the equilibrium constants, eq. (1) to eq. (4), of the system have been determined, see Scheme 1.2.

Scheme 1.2 Energy level diagram for anthocyanins and related compounds in acidic medium.

Source: Adapted from Pina 2014a. © 2014 John Wiley & Sons.

1.3 Extending the Multistate of Anthocyanins and Related Compounds to the Basic Region

In many flavylium derivatives from natural or synthetic origin, including anthocyanins, it is indispensable to extend the multistate study to basic medium.

In order to account for these new species, eight equilibrium equations should be added to eq. (1) through eq. (4).

For the formation of the mono-anionic species1

And for the formation of the di-anionic species

The system can be generalized for higher charged anionic species.

In spite of the complexity of this system, the set of eqs. 1 through 12 can be simplified considering a triprotic acid, eq. (13) through eq. (15), with constants K'a, eq. (19) K"a, eq. (20), and K"'a, eq. (21). The complete mathematical development of the system above was previously reported (supplementary information, Mendoza et al. 2019) and is straightforwardly obtained from a mass balance and representation of all species as a function of AH+.

Where

and

The mole fraction distribution XR of all species can be expressed in terms of the 12 linearly independent constants reported in Scheme 1.3. Since the flavylium cation and the quinoidal bases are in very fast equilibrium (microseconds scale), it is convenient to consider them altogether. The same is valid for the other species related through the proton transfer reaction.

where

Scheme 1.3 Extension to the basic medium of Pelargonidin-3-glucoside.

Figure 1.2 Absorption spectrum of heavenly blue anthocyanin, a peonidin derivative, black full line, flavylium cation; black pointed line, quinoidal base; black traced line, ionized quinoidal base. pK'a=3.47; pK"a=7.05; pK"'a=8.30.

Source: Mendoza et al. 2018.

Since the complex system shown in Scheme 1.3 behaves as a simple triprotic acid, the respective apparent equilibrium constants K'a, K"a, and K"'a are experimentally obtained from the inflection points of the absorbance representation as a function of the pH. Consequently, the term D is a parameter obtained experimentally. In Figure 1.2 the example of the heavenly blue anthocyanin is shown (Mendoza et al. 2018).

The question now is to define the experimental strategy to calculate the equilibrium constants of the system.

1.3.1 Reverse pH Jumps from Pseudo-equilibrium Followed by Stopped Flow UV-visible Spectroscopy

Recently we have reported a new experimental procedure that allows the experimental determination of all equilibrium constants (as shown in Scheme 1.3) of the flavylium-based multistates including anthocyanins (Mendoza et al. 2019; Mendoza et al. 2018; Slavcheva et al. 2018). It is based on the reverse pH jumps defined above, followed by stopped flow. In Figure 1.3 the stopped flow traces of the model compound 4'-hydroxyflavylium are shown. The initial solutions should be equilibrated or pseudo-equilibrated. The reverse pH jumps consist of the addition of acid to make the solutions with pH=1, where flavylium cation is the sole species. In both cases of Figure 1.3 the initial absorbance is due to the quinoidal bases (independently on their protonation state) that give flavylium cation (absorption at 450 nm) during the mixing time of the stopped flow together with some flavylium cation present at the initial equilibrium (at lower pH values) prior to the jump; see also Scheme 1.3. This is the reason why the mole fraction distribution of the flavylium cation and quinoidal bases are represented together in eq. (22). At the final very low pH jump (pH=1) the hydration reaction becomes faster than the tautomerization because it is directly proportional to the proton concentration (Pina 2014b). Therefore, the faster trace is due to the conversion of B into AH+. The slower trace is the formation of more flavylium cation from Cc via B (Scheme 1.4) (Mendoza et al. 2019).

In anthocyanins and most flavylium derivatives the cis-trans isomerization is much...