Leucoanthocyanidins flavan-3,4-diols

Biosynthesis of Anthocyanins (39) via Leucoanthocyanidins (Flavan-3,4-diols, 37) from Dihydroflavonols (3-OH-Flavanones, 31, 32) 

Dihydroflavonol 4-reductase (DFR) converted dihydroflavonols (3-OH-flava-nones, 32) to leucoanthocyanidins (flavan-3,4-diols, 37). The leucoanthocyanidins (flavan-3,4-diols, 37) were converted by leucoanthocyanidin dioxygenase (LDOX) to 3-hydroxy-anthocyanidins (38). Finally, 3-hydroxy-antho-cyanidins (38) were converted by three enzymes of O-methyltransferase (OMT), UDPG-flavonoid glucosyl transferase (UFGT) and rhamnosyl transferase (UFGT) to anthocyanins (39) (Fig. 8) [23,24], 

Leucoanthocyanidin reductase (LAR) converted leucoanthocyanidins (flavan-3,4-diols, 37) to flavan-3-ols (40). The flavan-3-ols (40) were converted to condensed tannins (proanthocyanidins, PA, 44) by condensing enzyme (CE). Following this condensed tannins (proanthocyanidins, PA, 44) finally yielded their oxidized tannins (oxidized proanthocyanins, 45) by proanthocyanidine oxidase (PRO) (Fig. 9) [23,24]. 

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Fig. 9 Biosynthesis of flavan-3-ols (40), condensed tannins (proanthocyanidins, 44) and oxidized tannins (oxidized proanthocyanidines, 45) from leucoanthocyanidins (flavan-3,4-diols, 37)...

Anthocyanidin synthase (ANS), the key enzyme in the biosynthesis of anthocyanins, catalyzes oxidation of leucoanthocyanidin (flavan-3,4-diol) to a 2-flaven-3,4-diol that spontaneously isomerizes to 3-flaven-2,3-diol (anthocyanidin) (Fig. 5). This is subsequently glycosylated at C-3, transported to the vacuole, and finally converted to the colored flavilium cation at the acidic... 

The natural absence of leucoanthocyanidins (flavan-3,4-diols) of the delphinidin, cyanidin and pelargonidin series may be rationalized by the knowledge of their excessively high reactivity. 

From extensive analysis of recombinant proteins, and the crystal structure of A. thaliana protein, detailed reaction mechanisms have been proposed. The ANS reaction likely proceeds via stereospecific hydroxylation of the leucoanthocyanidin (flavan-3,4-cA-diol) at the C-3 to give a flavan-3,3,4-triol, which spontaneously 2,3-dehydrates and isomerizes to 2-flaven-3,4-diol, which then spontaneously isomerizes to a thermodynamically more stable anthocyanidin pseudobase, 3-flaven-2,3-diol (Figure 3.2). The formation of 3-flaven-2,3-diol via the 2-flaven-3,4-diol was previously hypothesized by Heller and Forkmann. The reaction sequence, and the subsequent formation of the anthocyanidin 3-D-glycoside, does not require activity of a separate dehydratase, which was once postulated. Recombinant ANS and uridine diphosphate (UDP)-glucose flavonoid 3-D-glucosyltransferase (F3GT, sometimes... 

Among the hypotheses formulated, the most likely mechanism calls for the formation of a covalent bond between carbon 4 of the 3,4-flavan-diol (leucoanthocyanidin) and carbons 6 or 8 of another flavan molecule. Benzylic alcohol is a reactive electrophile (loss of OH"), and it donates readily in acid media leucoanthocyanin (25) functions similarly at position 4. The phenolic group is a mesomeric structure which displays negatively charged nucleophilic centers in the ortho and para positions analogous centers may be found at positions 6 and 8 of flavan molecules. This would allow the possibility of covalent bond formation between carbon 4 of 25 and carbons 6 or 8 of 26a or 26b. This bond is attributable to the elimination of a water molecule. 

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. 

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). 

Dihydroflavonol 4-reductase (DFR EC 1.1.1.219) is a member of the short-chain dehydrogenase/reductase family and catalyzes the stereospecific conversion of (+)-(2R,3R)-dihydroflavonols to the corresponding (2R,3S,4S) flavan-3,4-cw-diols (leucoanthocyanidins), with NADPH as a required cofactor. The enzyme activity was first identified in cell suspension cultures of Douglas fir (Pseudotsuga menziesii) and was shown to be related to the accumulation of flavan-3-ols and proanthocyanidins [96]. Leucoanthocyanidins and DFR were later shown to be required for anthocyanidin formation by complementation of Matthiola incana mutants blocked between dihydroflavonol and anthocyanidin biosynthesis [97, 98], DFR has been purified to apparent homogeneity and biochemically analyzed from flower buds of Dahlia variabilis [99]. DFR was shown to accept different substrates depending on the plant species from which it was isolated (reviewed in 100). 

Dihydroflavonol 4-reductase (DFR) catalyzes the stereospecific conversion of 2R,3R)-trans-DHFs to the respective (2R,35, 45)-flavan-2,3-traKi-3,4-cA-diols (leucoanthocyanidins) through a NADPH-dependent reduction at the 4-carbonyl. DNA sequences for DFR were first identified from A. majus and Z. mays, and the identity of the Z. mays sequence confirmed by in vitro transcription and translation of the cDNA and assay of the resultant protein. DNA sequences have now been cloned from many species, with the size of the predicted protein averaging about 38kDa. Stereospecificity to (2R,3R)-dihydroquercetin (DHQ) has been shown for some recombinant DFR proteins. ... 

Owing to the purported role of the flavans and flavan-3-ols as nucleophilic chain-terminating units, and of the flavan-4-ols and flavan-3,4-diols (leucoanthocyanidins) as electrophilic chain-extension units in the biosynthesis of the proanthocyanidins," the chemistry of these four classes of compounds is intimately linked to that of the proanthocyanidins. 

Leucoanthocyanidins are also referred to as flavan-3.4-c/.s-diols. They are synthesized from flavanonols via a reduction of the ketone moiety on C4. Examples are leucocyanidin (1.37) and leucodelphinidin (1.38). These compounds are often present in wood and play a role in the formation of condensed tannins. 

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... 

This enzyme catalyzes the conversion of flavan 3,4-diols (leucoanthocyanidins) to their corresponding anthocyanidins.60 A cDNA encoding ANS was recently isolated from Perilla frutescens,61 and its recombinant protein catalyzed the oxidation of both leucocyanidin and leucopelargonidin to their corresponding anthocyanidins, most likely via their 2-flaven-3,4-diols upon subsequent acidification. The enzyme exhibited a 3-fold higher affinity for leucocyanidin over leucopelargonidin.61 Leucodelphinidin was not tested as a substrate. 

Flavanol oligomers and polymers are also called condensed tannins or proan-thocyanidins. The term tannin refers to their capacity to interact or react with proteins and precipitate them out. When heated under acidic conditions, these molecules release red anthocyanidin pigments, hence the term proanthocyanidins. The term leucoanthocyanidin, also referring to this particular property, is sometimes encountered in the literature. However, this should be restricted to another group of compounds, flavan 3,4-diols, which are intermediates in the biosynthetic pathway leading to flavanols and anthocyanins (Stafford and Lester 1984 Nakajima et al. 2001 Abrahams et al. 2003) but have never been isolated from grapes, presumably due to their instability. 

Metabolically, anthocyanins are built up from the dihydroflavonols by means of a reduction of C4, catalyzed by the dihydroflavonol reductase, which leads to the flavan-2,3-trans-3,4-cis-diols, which are intermediates of proanthocyanidins and anthocyanidins. However, despite all the data in this direction, it has not been possible to obtain in vitro the transformation of leucoanthocyanidins in anthocyanins [33],... 

Subsequent reduction of dihydroflavonols, catalyzed by dihydroflavonol 4-reductase, leads to flavan-3,4-diols (Fig. 11.15) (leucoanthocyanidins), which are intermediates in an-... 

The condensed tannins, also referred to as procyanidins (Weinges et al., 1969), and formerly as leucoanthocyanidins (Rosenheim, 1920) because many form cyanidin upon acid hydrolysis, are mostly flavolans or pol3nners of flavan-3-ols (catechins) and/or flavan 3 4-diols (leucoanthocyanidins) (Fig. 3). Both catechins and leucoanthocyanidins are readily converted by dehydrogenating enzymes or even by very dilute mineral acids at room temperature into flavonoid tannins (Weinges, 1968). Heating in acid solution converts leucoanthocyanidins to the corresponding anthocyanidins and brown phlobaphene-like" polymers (Swain and Hillis, 1959). 

The condensed tannins are synthesized by the condensation of two or more molecules of flavonoids such as flavan-3-ols or flavan-3 4-diols, or a mixture of both. The flavan-3-ols exist as two series of stereoisomers the compounds in which the 2,3-hydrogen atoms are trans are known as catechins when these hydrogen atoms are cis, the epi-catechins result. Flavan-3 4-diols are also known as leucoanthocyanidins because, when treated with acid, they give the corresponding anthocyanidin. In plants, however, the latter compounds are not biosynthesized from their leuco counterparts. 

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