Proanthocyanidins and anthocyanidins

Evidence for the presence of covalent adducts involving proanthocyanidins and anthocyanidins was also observed by infusion mass spectrometry. These minor ions appeared in two series, one series starting at m/z 781 and one at m/z 783. Each of these series is observable up to m/z 2509/2511, with ions separated by a mass of 288 (Figure 4). Based upon previous studies and speculation, these ions were consistent with two types of anthocyanin-proanthocyanidin adducts. Specifically, these ions agree with the direct condensation products of... 

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],... 

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). 

Bogs J, Downey MO, Harvey JS, Ashton AR, Tanner GJ, Robinson SP. 2005 Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves. Plant Physiol 139 652-663. 

Paolocci F, Robbins MP, Madeo L, Arcioni S, Martens S, Damiani F. 2007. Ectopic expression of a basic helix-loop-helix gene transactivates parallel pathways of proanthocyanidin biosynthesis. Structure, expression, analysis, and genetic control of leucoanthocyanidin 4-reductase and anthocyanidin reductase genes in Lotus corniculatus. Plant Physiol 143 504-516. 

The proanthocyanidins share a biosynthesis sequence with the anthocyanins, a class of flavanoids well understood in biochemical and genetic terms. Three enzymes, leucoanthocyanidin reductase (LAR), anthocyanidin synthase (ANS), and anthocyanidin reductase (ANR), function at branches between the anthocyanin and proanthocyanidin biosynthesis pathways (Scheme 1). LAR is a member of the reductase—eptmerase—dehydrogenase enzyme... 

Figure 12.12. Biosynthetic pathways of flavonoid compounds from phenylalanine into isoflavonoids, flavones, flavonols, anthocyanidins, leucoanthocyanidins, proanthocyanidins, and phlobaphenes. The figure was kindly provided by Winkel-Shirley, B. (2003) as modified from Winkel-Shirley (2001). Reprinted with permission from Winkel-Shirley B., Grotewold E., Martins C., Hirsch A. M., and Quattrocchio E...

Flavonoids are the largest and best studied class of naturally occurring polyphenols, comprising different subclasses such as anthocyanidins, proanthocyanidins and isoflavones. 

Fig. 5 Scheme of the flavonoid pathway leading to synthesis of proanthocyanidins. The enzymes involved in the pathway are shown as follows CHS = chalcone synthase CHI = chalcone isomerase F3H = flavanone-3B-hydroxylase DFR = dihydroflavonol-4-reductase LDOX = leucoanthocynidin dioxygenase LAR = leucoanthocyanidin reductase ANR = anthocyanidin reductase adapted from [27] and [28]... 

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). 

The two principal classes of proanthocyanidins found (10) in plant tissues are the procyanidins (1, R e H) and the prodeTphin-idins (1, R s OH). Proanthocyanidins of mixed anthocyanidin character (1, R = H or OH) have been noted. In any tissue where proanthocyan din synthesis occurs there is invariably found a range of molecular species - from the monomeric flavan-3-ols (catechins, gallocatechins) to the polymeric forms (1) and biosynthetic work (11) suggests a very close relationship between the metabolism of the parent f1avan-3-o1 and the synthesis of proanthocyanidins, Figure 4. 

Proanthocyanidins are so named because under oxidative and acidic conditions they are converted to anthocyanidins, a subclass of flavonoid (for general structure, see Fig. 11.3.4). Historically, they have also been referred to as leucoanthocyanidins, condensed tannins, or simply tannins because of their ability to fix or tan leather hides. 

Condensed tannins are also referred to as proanthocyanidins. They are oligomeric or polymeric flavonoids consisting of flavan-3-ol (catechin) units. Hydrolysis under harsh conditions, such as heating in acid, yields anthocyanidins. An example of a condensed tannin is procyanidin B2 (epicatechin-(4 3—>8 )-epicatechin 1.90). In this case the interflavanyl linkage is between C4 of the lower unit, and C8 of the upper unit. The linkage can also be between C4 of one unit and C6 of the second unit. 

Xie DY, Sharma SB, Wright E, Wang ZY, Dixon RA. 2006. Metabolic engineering of proanthocyanidins through co-expression of anthocyanidin reductase and the PAP1 MYB transcription factor. Plant J 45 895-907. 

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... 

Nonhydrolyzable or condensed tannins are also named proanthocyanidins. These are polymers of flavan-3-ols, with the flavan bonds most commonly between C4 and C8 or C6 (Figure 6-23) (Macheix et al. 1990). Many plants contain tannins that are polymers of (+)-catechin or (-)-epicatechin. These are hydrogenated forms of flavonoids or anthocyanidins. Other monomers occupying places in condensed fruit tannins have trihydroxylation in the B-ring (+)-gallocat-echin and (-)-epigallocatechin. Oligomeric and polymeric procyanidins are formed by addition of more flavan-3-ol units and result in the formation of helical structures. These structures can form bonds with proteins. 

Condensed tannins, on the other hand, occur in the bark of all conifers and hardwoods examined to date, and they are frequently present in the wood. They are primarily responsible for the tan to brown color of wood after it is exposed to air. In their purest form, condensed tannins are colorless, but they become colored very readily once isolated because of their propensity to oxidize to quinones. The primary characteristic of the water-soluble condensed tannins (4) is dehydration/oxidation to intensely colored anthocyanidin pigments (5) when refluxed in butanol and hydrochloric acid (Figure 2). For this reason, there has been a tendency to refer to these compounds as proanthocyanidins in the last few years. Prior to that, they were referred to as leucoanthocyanidins (i.e., the colorless chemical form of anthocyanidins). All references earlier than the late 1950 s, when the structure of these substances was just beginning to be understood, used the term condensed tannin. 

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. 

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