Proanthocyanidin precursors

Flavan-3,4-diols FIavan-3,4-diols, also known as leucoanthocyanidins, are not particularly prevalent in the plant kingdom, instead being themselves precursors of flavan-3-ols (catechins), anthocyanidins, and condensed tannins (proanthocyanidins) (see Fig. 5.4). Flavan-3,4-diols are synthesized from dihydroflavonol precursors by the enzyme dihydroflavonol 4-reductase (DFR), through an NADPH-dependent reaction (Anderson and Markham 2006). The substrate binding affinity of DFR is paramount in determining which types of downstream anthocyanins are synthesized, with many fruits and flowers unable to synthesize pelargonidin type anthocyanins, because their particular DFR enzymes cannot accept dihydrokaempferol as a substrate (Anderson and Markham 2006). 

Finally, reactions of flavonoid and nonflavonoid precursors are affected by other parameters like pH, temperature, presence of metal catalysts, etc. In particular, pH values determine the relative nucleophilic and electrophilic characters of both anthocyanins and flavanols. Studies performed in model solutions showed that acetaldehyde-mediated condensation is faster at pH 2.2 than at pH 4 and limited by the rate of aldehyde protonation. The formation of flavanol-anthocyanin adducts was also limited by the rate of proanthocyanidin cleavage, which was shown to take place at pH 3.2, but not at pH 3.8. Nucleophilic addition of anthocyanins was faster at pH 3.4 than at pH 1.7, but still took place at pH values much lower than those encountered in wine, as evidenced by the formation of anthocyanin-caffeoyltartaric acid adducts, methylmethine anthocyanin-flavanol adducts,and flavanol-anthocyanin adducts. The formation of pyranoanthocyanins requiring the flavylium cation was faster under more acidic conditions, as expected, but took place in the whole wine pH range. Thus, the availability of either the flavylium or the hemiketal form does not seem to limit any of the anthocyanin reactions. 

Proanthocyanidins and Procyanidins - In a classical study Bate-Smith ( ) used the patterns of distribution of the three principal classes of phenolic metabolites, which are found in the leaves of plants, as a basis for classification. The biosynthesis of these phenols - (i) proanthocyanidins (ii) glycosylated flavonols and (iii) hydroxycinnamoyl esters - is believed to be associated with the development in plants of the capacity to synthesise the structural polymer lignin by the diversion from protein synthesis of the amino-acids L-phenylalanine and L-tyro-sine. Vascular plants thus employ one or more of the p-hydroxy-cinnarayl alcohols (2,3, and 4), which are derived by enzymic reduction (NADH) of the coenzyme A esters of the corresponding hydroxycinnamic acids, as precursors to lignin. The same coenzyme A esters also form the points of biosynthetic departure for the three groups of phenolic metabolites (i, ii, iii), Figure 1. 

The branch pathway for anthocyanin biosynthesis starts with the enzymatic reduction of dihydrofiavonols to their corresponding flavan 3,4-diols (leucoanthocyanidins) by substrate-specific dihydroflavonol 4-reductases (DFR). Flavan 3,4-diols are the immediate precursors for the synthesis of catechins and proanthocyanidins. Catechins are formed by enzymatic reduction of the flavan 3,4-diols in the presence of NADPH to leucoanthocyanidins, which are subsequently converted to anthocyanidins by the 2-oxoglutarate-dependant dioxygenase, anthocyanidin synthase. Further glycosylation, methylation, and/or acylation of the latter lead to the formation of the more stable, colored anthocyanins (Scheme 1.1). The details of the individual steps involved in flavonoid and isoflavonoid biosynthesis, including the biochemistry and molecular biology of the enzymes involved, have recently appeared in two excellent reviews.7,8... 

The precursors of these reactions are, on one hand, proanthocyanidins and, on the other hand, any kind of flavonoid that can act as a nucleophile. The latter include flavonols, dihydroflavonols, flavanol monomers, proanthocyanidins, and anthocyanins under their hemiketal form (for anthocyanin reactivity, see Chapter 9A). 

The biosynthetic pathways of epicatechin (55), cyanidin (51) and proan-thocyanidin (PA) (44) had the same intermediates to leucocyanidin (48). Next, leucoanthocyanidin reductase (LAR) converts the leucocyanidin (48) to epicathechin (55), whereas leucoanthocyanidin dioxygenase (LDOX) converted leucocyanidin (48) to cyanidin (51). Two cyanidin (51) and epicathechin (55) are a precursor of proanthocyanidins (PA) (44). 

The predominant feature of the flavan-3,4-diols as far as the chemistry of the proanthocyanidins is concerned is their role as precursors to flavan-4-yl carhocations and/or A-ring quinone methide electrophiles. As such they represent the extension units of the polymeric chain of the naturally occurring proanthocyanidins. The physico-chemical properties regulating their role and function in the synthesis of the proanthocyanidins were extensively covered in CONAP-I. ... 

Jannie P. J. Marais studied at the University of the Free State, Bloemfontein, South Africa where he obtained his Ph.D. in organic chemistry in 2002. His research focused on characterization of the free phenolic profile of South African red wine, and the stereoselective synthesis of flavonoids, for example, flavan-3,4-diols and flavanones. He joined the National Center for Natural Products Research at the University of Mississippi as a postdoctoral associate in July of 2002, where he worked with Dr. Ferreira on the stereoselective synthesis of flavonoid precursors, the semi-synthesis of proanthocyanidin oligomers, characterization of proanthocyanidin profiles of selected transgenic plants, and the synthesis of radioactive antimalarial 8-aminoquinolines. He was promoted to associate research scientist in 2005, where his main area of research still remains the synthesis of A- and B-type proanthocyanidins, starting at the monomeric level and continuing through the tri- and tetra-meric, to the deca-mer level. 

Procyanidins are the most widely distributed members of the class of polyphenolic compounds known as proanthocyanidins (synonyms oligoflavanoids, condensed polyphenols, condensed tannins). Proanthocyanidins are colorless compounds, occurring predominantly in woody or herbaceous plants. They got their name from the characteristic hydrolyzation reaction they undergo in acidic medium which yields colored anthocyanidins. Proanthocyanidins consist of flavonoid precursors, which are commonly linked by carbon-carbon bonds at C4—>C8 or C4—>C6. A variety of different classes are known, depending on the substitution pattern of the monomer units (see Fig. (1)). More details on structures, distribution and general features of this class of compounds can be found in numerous reviews [1-6]. 

Flavan-3-ols are derived from flavanones, via 3-hydroxy-flavanones (or dihydroflavonols). Naiingenin (31) gives rise to 5,7-dihydroxyproanthocyanidins, whereas liquiritigenin (32) (Fig. 12.11) appears to be the precursor for 5-deoxy-proanthocyanidins, both via the intermediacy of flavan-3-ols and the same basic pathway as in Fig. 11.16 (Heller and Forkmann, 1988 Lewis and Yamamoto, 1989 Stafford, 1989). 

Incorporation of labeled phenylalanine and cinnamic acid into dimeric proanthocyanidins occurs asymmetrically, with differences in the amount of precursors incorporated into the two portions of the molecule (Stafford, 1989). Further, the pool of free flavan-3-ol monomers including (+ )-catechin (3) and (— )-epicatechin (30) contained much higher levels of radioactive label than the comparable initiating (or terminating) unit. Tritium label at the C-2 position is retained, whereas that at the C-3 position is lost in the dimers (Stafford, 1989). 

Condensed tannins or proanthocyanidins are high-molecular-weight polymers. The monomeric unit is a flavan-3-ol (e.g., catechin and epicatechin), with a flavan-3,4-diol as its precursor (Figure 14.1). Oxidative condensation occurs between carbon C-4 in the heterocycle and carbons C-6 or C-8 of adjacent units [14]. However, most of the literature on the condensed tannin contents refers only to oligomeric proanthocyanidins (dimers, trimers, and tetramers) because of the difficulty of analyzing highly polymerized molecules. Proanthocyanidins, however, can occur as polymers with a degree of polymerization of 50 and more. 

Havanols are a wide group of polyphenols that include flavan-3-ols (e.g., catechin and proanthocyanidins), flavan-4-ols, and flavan-3,4-diols. They arise from plant secondary metabolism through condensation of phenylalanine derived from the shikimate pathway with malonyl-CoA obtained from citrate that is produced by the tricarboxylic acid cycle, leading to the formation of the key precursor in the flavonoids biosynthesis the naringenin chalcone. The exact nature of the molecular species that undergo polymerization and the mechanism of assembly in proanthocyanidins are still unknown. From a structural point of view, flavanols... 

The significance of flavan-3,4-diols in plants rests primarily on their probable role as precursors of the polymeric proanthocyanidins. Co-occurrence of the 5-deoxy compounds - i.e., quibourtacacidins, mollisacacidins, and robinetinidins - with the related proanthocyanidins in Acacia species and the ready synthesis of naturally occurring proanthocyanidins from reactions of these flavan-3,4-diols with catechin under mild acidic conditions constitutes heavy but not definitive evidence for this thesis (31, 315-317). 

The vast majority of recent works on the reactions of flavan-3,4-diols has centered on their role as precursors to flavan-4-carbocations or quinone methide electrophiles in proanthocyanidin syntheses. The regioselectivity of their condensation with flavan-3-ols or oligomeric proanthocyanidins is discussed in Sect. 7.6.3.1.2. Our concern here is with the factors governing the stability of the electrophile and the stereospecificity or stereoselectivity of its reaction with phenolic nucleophiles. Roux and coworkers have made extensive studies of these reactions and have prepared reviews of their recent results (316, 317). The intent here is to summarize some of the more important principles that have been developed. 

Stafford H A, Lester H H 1981 Proanthocyanidins and potential precursors in needles of Douglas-fir and in cell suspension cultures derived from seedling shoot tissues. Plant Physiol 68 1035-1040... 

Dihydroflavonol 4-reductase (DFR) is involved in the biosynthesis of anthocyanins and proanthocyanidins (PAs). DFRs catalyze the stereospecific reduction of (2R,3R)-dihydrofla-vonols to (2R,3R,45)-leucoanthocyanidins [77] (Fig. 5). Petunia possesses three different DFR genes (dfrA-C), but only dfrA is transcribed in floral tissues. DFR-A does not accept dihy-drokaempferol, the precursor for the synthesis of pelar-gonidin-type anthocyanins. Consequently, no orange-colored petunia flowers are foimd in nature [89]. Dihydroquercetin and (hhydramyrice-tin are also substrates for DFRs and provide leu-cocyanidin and leucodelphinidin, respectively. 

The name proanthocyanidins, previously called leucoanthocyanidins, implies that these are colorless precursors of anthocyanidins. On heating in acidic solution, the C—C bond made during formation is cleaved and terminal flavan units are released from the oligomers as carbocations, which are then oxidized to colored anthocyanidins (cf. 18.1.2.5.3) by atmospheric oxygen (Formula 18.21). Base-catalyzed cleavage via the quinone methode is also possible. 

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