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The experimental procedure above reported was used to rationalize the multistate of heavenly blue anthocyanin and two derivatives (Scheme 1.6).

Heavenly blue anthocyanin, HBA1, has attracted the attention of the scientific community due to its peculiar properties, specifically the fact that the same anthocyanin is used by the plant to confer purplish color to the buds and blue color to the petals (Yoshida et al. 1995; Goto and Kondo 1991; Kondo et al. 1992). Moreover, in vitro the blue color is persistent in neutral and moderately basic solutions (Kondo et al. 1992; Yoshida et al. 2009). Structural information regarding HBA1 fully supports the intramolecular stacking shown in Scheme 1.7.

The system was studied up to the mono‐anionic forms because at higher pH values a slow decomposition takes place and the data does not have sufficient accuracy. In spite of equilibrium being reached in one to two weeks, the neutral and mono‐anionic species are relatively stable. Table 1.1 summarizes the data.

Scheme 1.6 Heavenly blue anthocyanin HBA1 and their derivatives bis‐deacyl‐HBA2 and tris‐deacyl‐HBA3.

Source: Mendoza et al. 2018.

Scheme 1.7 Sketch representing the intramolecular copigmentation in polyacylated anthocyanins; CPK models of heavenly blue anthocyanin.

Source: Mendoza et al. 2018.

Table 1.1 Equilibrium constants of heavenly blue anthocyanin and their derivatives.

Source: Mendoza et al. 2018.

	pK’a	pK”a	pK^a	pKa	pKh	K t
HBA1	3.5	7.3	3.6	3.8	4.6	1.1
HBA2	—	—	2.92	4.23	3.1	0.35
HBA3	—	—	1.95	4.19	2.1	0.37
	K i	pK ^^a	pK A/A‐	pK B/B‐	pK Cc/Cc‐	pK Ct/Ct‐
HBA1	4.0	7.35	7.35	7.5	7.25	7.36

Estimated error 10%.

In Table 1.1 the equilibrium constants of the non‐acylated, di‐acylated and tri‐acylated derivatives of heavenly blue anthocyanin are also reported (Scheme 1.8). HBA2 and HBA3 behave as common anthocyanins, being relatively stable only in acidic medium, preventing the calculation of the data regarding the anionic species at equilibrium.

The mole fraction distribution for HBA1 of the several species is represented in Figure 1.5. This distribution is in line with the previous observation (Yoshida et al. 1995) that the buds of heavenly blue anthocyanin are purple while the petals are blue. In fact the pH of the vacuoles in buds is around 6.6, while in petals pH=7.7 (Yoshida et al. 1995). In that pH region a small pH change gives different contributions of the quinoidal base and anionic quinoidal base and significant color changes can be observed.

Scheme 1.8 Energy level diagrams of HBA1 (black), HBA2 (blue), and HBA3 (red). In HBA1 there is an inversion of the relative stability between the quinoidal base and hemiketal. The energy levels of Ct cannot be measured with the necessary accuracy due to some decomposition in HBA2 and HBA3 for longer reaction times.

Source: Mendoza et al. 2018.

Figure 1.5 Mole fraction distribution of heavenly blue anthocyanin.

Source: Mendoza et al. 2018.

The rate constants of the kinetic steps of HBA1 and derivatives were also determined and are reported in Table 1.2, together with the data for HBA2 and HBA3. The isomerization is very slow and some decomposition was observed, preventing the determination of the isomerization constants with accuracy.

Inspection of Table 1.2 shows that the hydration constant of HBA1 decreases 35‐fold when compared with HBA3, while the dehydration increases c. nine‐fold. This kinetic effect is compatible with the previous explanation considering a π‐π stacking effect of the caffeoyl residues that protect positions C2 and C4 from the water attack. This kinetic effect also has thermodynamic consequences, because the equilibrium constant K_h increases 306‐fold. In addition, the equilibrium constant of the quinoidal base does not change significantly. The most interesting feature in HBA1 is the inversion of the energy levels of the quinoidal base relative to the hemiketal (Scheme 1.8).

Table 1.2 Rate constants between AH+ and CB (estimated error 10%). Reproduced from Mendoza et al. (2018), with permission.

Source: Mendoza et al. 2018

	k h / s‐1	k ‐h / M‐1 s‐1	k t / s‐1	k ‐t / s‐1	k i / s‐1	k ‐i / s‐1
HBA1	0.01	377	0.09	0.086	2x10‐6	5x10‐7
HBA2	0.12	145	0.08	0.23	—	—
HBA3	0.35	43	0.12	0.33	—	—

Scheme 1.9 (Left) The polyacylated anthocyanins (Dangles et al. 1993); (Right) representation of the relative energy levels at equilibrium using the hydration and acidity constants reported by the authors. The construction of this type of energy level diagram is shown in Scheme 1.2. It is worth noting that two acylated units are not enough to invert the energy levels between the hemiketal (B) and the quinoidal base (A).

Source: Dangles et al. 1993.

The protection effect of the acylated sugars for the hydration reaction was studied previously (Fernandes et al. 2015; Dangles et al. 1993), but according to our knowledge no other study of the physical chemistry of three acylated sugars has been reported. The most complete study previously published, regarding this type of molecules, was reported by Dangles et al. (1993) (Scheme 1.9). In this work, the proton transfer as well as the hydration constants were determined, allowing the construction of an energy level diagram as in Scheme 1.8.

The same type of effect reported for HBA1 was observed: raising of the energy level of the hemiketal and a more or less constant energy level of the quinoidal base as the acylated units increase.⁵ However, no inversion of the energy levels of the hemiketal compared with quinoidal base was observed, suggesting that three acylated units are necessary.⁶

The energy level diagram of the HBA1 was extended to the basic region; Scheme 1.10. Details can be found elsewhere (Mendoza et al. 2019).

After a pH jump to pH=5.5, for example, the system equilibrates between A, B, Cc, and Ct, but the most stable species is the quinoidal base A. In the case of a pH jump to the region of the mono‐anionic species, pH=8.5, the anionic quinoidal base becomes the most stable species.

Scheme 1.10 Energy level diagram of the compound HBA1 extended to the mono‐anionic species. In the case of moderately acidic pHs the purple color of the quinoidal base is observed; at higher pH values it is the blue anionic quinoidal base that becomes more stable.

Source: Mendoza et al. 2018.