Ethyl-flavanol adducts

The anthocyanin-ethyl-flavanol adducts thus formed are purple pigments ( nax 5 0 nm), and much more resistant towards acid-catalyzed cleavage than the equivalent colorless ethyl-linked flavanol dimers (29). This presumably results from the displacement of the hydration equilibrium toward the flavylium cation form, due to self association, since similar synthetic pigments have been shown to be greatly stabilized by sandwich-type stacking (28). 

Thiolysis also proved useful for the analysis of derived tannins. Methylmethine-linked tannin-like compounds resulting from acetaldehyde-mediated condensation of flavanols (see Section 5.5.S.2) yielded several adducts when submitted to acid-catalyzed cleavage in the presence of ethanethiol, providing information on the position of linkages in the original ethyl-linked compounds. " Thiolysis of red wine extracts released benzylthioether derivatives of several anthocyanin-flavanol adducts, indicating that such structures were initially linked to proanthocyanidins. However, some of the flavonoid derivatives present in wine (e.g., flavanol-anthocyanins ) are resistant to thiolysis, while others (e.g., flavanol-ethyl anthocyanins) were only partly cleaved. Thiolysis, thus, appears as a rather simple, sensitive, and powerful tool for quantification and characterization of proanthocyanidins, but provides mostly qualitative data for their reaction products. 

Reactions of anthocyanins and flavanols take place much faster in the presence of acetaldehyde that is present in wine as a result of yeast metabolism and can also be produced through ethanol oxidation, especially in the presence of phenolic compounds, or introduced by addition of spirit in Port wine technology. The third mechanism proposed involves nucleophilic addition of the flavanol onto protonated acetaldehyde, followed by protonation and dehydration of the resulting adduct and nucleophilic addition of a second flavonoid onto the carbocation thus formed. The resulting products are anthocyanin flavanol adducts in which the flavonoid units are linked in C6 or C8 position through a methyl-methine bond, often incorrectly called ethyl-link in the literature. 

Precursors. The precursors for this reaction are anthocyanins, flavanols or flavanols containing a vinyl residue at C-8 (i.e., 8-vinylflavanols). 8-Vinylflavanols could arise from the cleavage of flavanol-ethyl-flavanol oligomers or from the dehydration of the flavanol-ethanol adduct formed after the attack of aldehyde cation to the flavanol (Chapter 9B). Saucier et al. (1997) have supported evidence for this precursor when detecting an ion corresponding to vinyl-catechin from the fragmentation of ethyl-linked catechin dimers under ESI-MS in positive or negative mode. 

MS (38) demonstrated that the reaction starts with protonation of acetaldehyde in acidic medium, followed by nucleophilic addition of the flavanol (C6 or C8 of the A-ring) on the resulting carbocation. Figure. 3, as postulated by Timberlake and Bridle (32). The ethanol adduct then looses a water molecule to give a new carbocation which is in turn attacked by the anthocyanin to give the flavanol-ethyl-anthocyanin adduct. 

HPLC-ESI-MS analysis of the mixture conducted in the positive ion mode showed the presence of both ethyl-linked flavanol dimers, detected at m/z 607 amu, and ethyl-linked adducts involving both (-)-epicatechin and the... 

It must also be noted that the anthocyanin and the flavanol conq ete in the different steps of the reaction. Nevertheless, and due to the low proportion of the hydrated form of the anthocyanin at the pH value of the reaction medium (2.2) which considerably decreases its ability to act as a nucleophile, this competition is in favor of the flavanol which is predominantly attacked first. This was shown by detection by LC/ESI-MS of the flavanol intermediate at m/z 333 amu in the negative ion mode, while the corresponding anthocyanin intermediate was not detected. Isolation of the flavanol intermediate was achieved through HPLC at the semi-preparative scale and further reaction with malvidin 3-0-glucoside was shown to afford the two ethyl-bridged anthocyanin-flavanol adducts. 

Monitoring of the reaction also indicated that ethyl-linked flavanol-anthocyanin adducts are more stable than ethyl-linked flavanol oligomers. 

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