Procyanidins polymerization reactions

Flavanols and procyanidins Flavanols, or flavan-3-ols, are synthesized via two routes, with (+) catechins formed from flavan-3,4-diols via leucoanthocyanidin reductase (LAR), and (—) epicatechins from anthocyanidins via anthocyanidin reductase (ANR) (see Fig. 5.4). These flavan-3-ol molecules are then polymerized to condensed tannins (proanthocyanidins or procyanidins), widely varying in the number and nature of their component monomers and linkages (Aron and Kennedy 2008 Deluc and others 2008). It is still not known whether these polymerization reactions happen spontaneously, are enzyme catalyzed, or result from a mixture of both. 

The procyanidins polymerized during the various reactions oxidize other components in the medium, especially ethanol into ethanal. 

The last decade has seen quite remarkable advances in our knowledge of the structure and properties of the proanthocyanidins. Viscosity measurements were made of solutions of procyanidins isolated from Theobroma cacao and Chaenomeles speciosa with number-average degrees of polymerization of 6.1 and 11.8, respectively, in water and 1% sodium hydroxide at 25 °C. Procyanidins are apparently completely crosslinked by formaldehyde up to a chain length of 6 units, but few units are crosslinked in polymeric procyanidins. The second order rate constants observed for the formaldehyde reaction with catechin or epicatechin are approximately six times higher than that observed for the C. speciosa polymer. 

If polymeric procyanidins extractable from conifer tree barks are to be used in adhesive formulations requiring condensation with phenol-formaldehyde prepolymers, these reactions must be performed at acidic pH conditions, and because of solubility limitations, this will probably require the use of sulfonate derivatives. 

A major drawback of all functional group assays is that a satisfactory standard does not exist. For a given sample the most appropriate standard is a purified procyanidin fraction prepared from the same matrix. The isolation and characterization of such purified fractions are laborious. Added to that procyanidins undergo oxidation, complexation and self-polymerization very easily, rendering such purified fractions only reproducible to a limited degree. At least in the proanthocyanidin assay the color reaction depends not only on the polyphenols themselves, but also on the matrix. The use of specified proanthocyanidins as a standard in a suitable blank matrix is an attempt to correct for such effects [67],... 

Some derivatization reactions which are frequently used for the structural elucidation of procyanidins have been adopted for HPLC analysis. Koupai-Abyazani et al. [231] developed a qualitative HPLC procedure to separate flavan-3-ol monomers and phloroglucinol adducts. The reaction is based on the acid degradation of procyanidins in the presence of phloroglucinol. The procedure has been used for quantitation of polymeric proanthocyanidins from sainfoin leaves [63]. HPLC analysis of benzylthioethers after acid degradation of procyanidins in the presence of toluene-a-thiol has so far only been used for qualitative analysis [250-251],... 

Acid solutions of dimeric, oligomeric and polymerized procyanidins are unstable. Even under nitrogen with sulfur dioxide in the absence of light, the color yellows, then browns and, after a short time, a precipitate is observed. At pH 3.2, the reaction takes about ten months at 5°C, a few months at 20°C and one to two months at 30°C. In the presence of oxygen from the air, and especially at high temperatures, conversion of the solution is more intense and the precipitates look different. It is impossible to dissolve them in any solvents other than formic acid. They can only be studied after acetylation. Results obtained by molecular screening (TSK), NMR and mass spectrometry show that these complex polymers have molecular weights above 3000. 

Fig. 6.26. Example of tannin polymerization (a) organized polymerization of tannins, an example of a tetrameric procyanidin (b) disorganized polymerization of tannins (reactions with free radicals) (Glavin, 1993)...

In wine, this type of reaction occurs at the same time as the heterogeneous polymerization of the procyanidins (Section 6.3.7), as a result of the controlled oxidation during barrel aging, when traces of ethanal are produced by the oxidation of ethanol. The color of the wine becomes more intense and changes tone, becoming darker after a few months in the barrel. 

The second method for calculating the tannin concentration is based on examining the visible spectrum of the reaction (LA). The following equations apply, whatever the degree of polymerization and concentration of the procyanidins. AOD 520, AOD 470 and AOD 570 represent the difference in OD, with or without heating, for the three corresponding wavelengths ... 

According to Lea (1992) the reaction between tannins and proteins depends on the degree of polymerization of the procyanidins. Astringency increases up to heptamer level and then decreases, as the molecules are too bulky. Maximum bitterness occurs with tetrameric procyanidins. These findings were confirmed by Mirabel (2000), showing that the difference between bitterness and astringency varied widely from one taster to another and that the distinction was not clear. 

The procyanidin molecules from the grapes tend to polymerize, condense with anthocyanins and combine with plant polymers such as proteins and polysaccharides. Several reactions are involved (Section 6.3) ... 

The temperature depends on the winery. Low temperatures are useful for precipitating unstable colloids. On the one hand, temperatures above 20°C promote the formation of carbocations from procyanidins, and therefore the TA complex (red, orange), as well as homogeneous polymerization. On the other hand, they also facilitate combinations with polysaccharides as well as color breakdown reactions. Furthermore, it promotes the thermal degradation of some anthocyanins, particularly malvidin. Alternating a low temperature with a temperature around 20°C promotes development, while maintaining it within certain limits. 

Laks, RE., and R.W. Hemingway Condensed Tannins Base-catalyzed Reactions of Polymeric Procyanidins with Toluene-a-thiol. Lability of the Interflavonoid Bond and Pyran Ring. J. Chem. Soc., Perkin Trans. 1, 465 (1987). 

The quantification of the procyanidins with a degree of polymerization higher than 2 also varied according to the cocoa source analyzed. For instance, cocoa powder showed the lowest content compared with the other cocoa sources studied, which could be related to the manufacturing process comprising oxidation steps and condensation reactions. Lacueva et al. [58] suggested a 60% loss of the flavonoid content during the process of alkalinization to obtain the cocoa powder. 

Two other approaches to determination of a kinetic control ratio for substitution at the C-6 and C-8 positions of procyanidins have been attempted (139,148). Determination of the relative rate of cleavage of C-8 and C-6 regio-isomers (2.6-1) and a determination of their relative yields at the thermodynamic equilibrium (1.3-1) permitted an estimation of a kinetic control ratio of 3.3 1 for the C-8 and C-6 linked isomers (148). The other approach was to determine the ratio of the C-8 and C-6 linked dimers after synthesis by reaction of epicatechin-(4)ff)-phenylsulfide and catechin at pH 9.0 and ambient temperature through quinone methide intermediates (139). Here the C-8 and C-6 linked isomers were obtained in an approximate ratio of 3.5-4 1. These later results, together with evidence for interflavanoid linkage isomerism in trimeric and polymeric procyanidins (141,143) (Sect. 7.6.3.3), show that substitution is not as heavily favored at the C-8 position in procyanidins as had been thought. 

An explanation for the formation of 2jR,3jR-(2,3-c/5)-proanthocyanidins from the 2i ,3S-(2,3-/ra/Z5 )-flavan-3,4-diols could lie in a tautomeric rearrangement of quinone methide intermediates to flav-3-en-3-ols, which could then be stereospecifically converted back to either 2,3-trans or 2,3-cis quinone methides (145). Chemical evidence supporting this thesis has been obtained by the formation of diarylprqpanone derivatives from the reaction of polymeric procyanidins with phenylmethanethiol under alkaline conditions (223). Enzymes controlling the quinone methide to flav-3-en-3-ol rearrangements rather than C-3 inversion of dihydroflavonols may be involved. In either case, evidence continues to mount that the flavan-3,4-diols are indeed central intermediates in the biogenesis of proanthocyanidins and that this conversion is under enzymic (genetic) control (219, 341, 342). 

Until recently, little attention has been given to the reactions of polymeric proanthocyanidins under alkaline conditions. It was known that solution of polymeric procyanidins in base caused a marked decrease in the reactivity of these polymers with aldehyde and that both carbonyl and relatively acidic functions were generated analogous to the rearrangement of catechin to catechinic acid (152, 335). 

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