Leucoanthocyanidins

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. 

Because of their completely saturated heterocycle, leucoanthocyanidins, together with flavan-3-ols are referred to as flavans. Examples of flavan-3-ols are catechin (1.39) and gallocatechin (1.40). The gallo in the latter compound refers to the vic-tri-hydroxy substitution pattern on the B-ring. Unlike most other flavonoids, the flavans are present as free aglycones or as polymers of aglycones, i.e. they are not glycosylated. 

Catechins (1.41) can also be found as gallic acid esters that are esterified at the 3 hydroxyl group. Note the difference between the gallic acid ester of catechin (1.41) and gallocatechin (1.40). 

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Xie and Dixon [27] underlined that although some models of proanthocyanidin biosynthesis show the extension units arising from condensation of an leucoanthocyanidin-derived electrophile with the nucleophilic 8 or 6 position of the starter unit, this scheme fails because of stereochemistry of leucoanthocyanidin is most likely 2,3-trans, whereas, in many cases, the extension units are 2,3-cis. One possible solution for this stereochemical para-... 

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]... 

Figure 5.4. Abbreviated scheme for biosynthesis of major flavonoid subclasses, showing the primary enzymes and substrates leading to different subclasses. Bold-faced, uppercase abbreviations refer to enzyme names, whereas substrate names are presented in lowercase letters. PAL, phenylalanine ammonia lyase C4H, cinnamate 4-hydroxylase 4CL, 4-coumarate CoA ligase CHS, chalcone synthase CHI, chalcone isomerase CHR, chalcone reductase IPS, isoflavone synthase F3H, flavonone 3-hydroxylase F3 H, flavonoid 3 -hydroxylase F3 5 H, flavonoid 3 5 -hydroxylase FNSI/II, flavone synthase DFR, dihydroflavonol 4-reductase FLS, flavonol synthase ANS, anthocyanidin synthase LAR, leucoanthocyanidin reductase ANR, anthocyanidin reductase UFGT, UDP-glucose flavonoid 3-O-glucosyltransferase. R3 = H or OH. R5 = H or OH. Glc = glucose. Please refer to text for more information.

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). 

Figure 6.1 Major branch pathways of flavonoid biosynthesis in Arabidopsis. Branch pathways, enzymes, and end products present in other plants but not Arabidopsis are shown in light gray. Abbreviations cinnamate-4-hydroxylase (C4H), chalcone isomerase (CHI), chalcone synthase (CHS), 4-coumarate CoA-ligase (4CL), dihydroflavonol 4-reductase (DFR), flavanone 3-hydroxylase (F3H), flavonoid 3 or 3 5 hydroxylase (F3 H, F3 5 H), leucoanthocyanidin dioxygenase (LDOX), leucoanthocyanidin reductase (LCR), O-methyltransferase (OMT), phenylalanine ammonia-lyase (PAL), rhamnosyl transferase (RT), and UDP flavonoid glucosyl transferase (UFGT).

Table 6.1 Abbreviations BAN, BANYULS bHLH, basic helix-loop-helix CHS, chalcone synthase CHI, chalcone isomerase DFR, dihydroflavonol reductase F3H, flavonol 3-hydroxylase F3 H, flavonoid 3 -hydroxylase FLS, flavonol synthase icx, increased chalcone synthase expression LDOX, leucoanthocyanidin dioxygenase LCR, leucoanthocyanidin reductase MATE, multidrug and toxic compound extrusion NR, not yet reported tt, transparent testa ttg, transparent testa glabrous the WD40 and WRKY transcription factors are named for conserved amino acid sequences within these proteins. PC = personal communication.

PELLETIER, M.K., MURRELL, J., SHIRLEY, B.W., Arabidopsis flavonol synthase and leucoanthocyanidin dioxygenase further evidence for distinct regulation of "early" and "late" flavonoid biosynthetic genes, Plant Physiol., 1997,113, 1437-1445. 

The R locus determines the presence (R) or absence (r) of anthocyanins in the seed coat. R is required (with i and T) to produce black seed [10]. However the identity of the gene product encoded by this locus has not been reported. Todd and Vodkin [25] have demonstrated that brown seed coats (r) contain proanthocyanidin (PAs) and black seed coats (R) contain anthocyanins in addition to PAs and suggested that R acts subsequent to the formation of leucoanthocyanidin but previous to the formation of anthocyanins. UDP-glucose flavonoid 3-0-glucosyltransferase (UF3GT) should be considered a candidate gene of the R locus but its identiflcation has not yet been reported. 

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). 

PeUetier MK, MurreU JR, Shirley BW (1997) Characterization of flavonol synthase and leucoanthocyanidin dioxygenase genes in arabidopsis. Further evidence for differential regulation of early and late genes. Plant Physiol 113(4) 1437-1445... 

Stracke R, De Vos RC, Bartelniewoehner L, Ishihara H, Sagasser M, Martens S, Weisshaar B (2009) Metabolomic and genetic analyses of flavonol synthesis in Arabidopsis thaliana support the in vivo involvement of leucoanthocyanidin dioxygenase. Planta 229(2) 427-445... 

HeUer W, Britsch L, Eorkmann G, Grisebach H (1985) Leucoanthocyanidins as intermediates in anthocyanin biosynthesis in flowers of Matthiola incana. R. Br. Planta 163(2) 191-196... 

FIGURE 3.2 General phenylpropanoid and flavonoid bios5mthetic pathways. The B-ring hydroxylation steps are not shown. For formation of anthocyanins from leucoanthocyanidins two routes are represented a simplified scheme via the anthocyanidin (pelargonidin) and the likely in vivo route via the pseudobase. Enzyme abbreviations are defined in the text and in Table 3.1. 

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. ... 

DFR belongs to the single-domain-reductase/epimerase/dehydrogenase (RED) protein family, which has also been termed the short chain dehydrogenase/reductase (SDR) superfamily. This contains other flavonoid biosynthetic enzymes, in particular the anthocyanidin reductase (ANR), leucoanthocyanidin reductase (EAR), isoflavone reductase (IFR), and vestitone reductase (VR), as well as mammalian, bacterial, and other plant enzymes. ... 

The role of anthocyanidin synthase (ANS) in the biosynthetic pathway is to catalyze reduction of the leucoanthocyanidins to the corresponding anthocyanidins. However, in vivo it is anthocyanidins in pseudobase form that are formed, as is described below. In this chapter, use of anthocyanidin should be taken to include the pseudobase form. Furthermore, although the name ANS is commonly used, the enzyme is also referred to in the literature as leucoantho-cyanidin dioxygenase (LDOX), reflecting the reaction type. 

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... 

LAR removes the 4-hydroxyl from leucoanthocyanidins to produce the corresponding 2,3-tran5-flavan-3-ols, e.g., catechin from leucocyanidin. Despite early biochemical characterization, it is only recently that a LAR cDNA was isolated and the encoded activity characterized in detail. Tanner et al. purified LAR to homogeneity from Desmodium uncinatum (silverleaf desmodium), and used a partial amino acid sequence to isolate a LAR cDNA. The cDNA was expressed in E. coli, N. tabacum, and Trifolium repens (white clover), with the transgenic plants showing significantly higher levels of LAR activity than nontransformed plants. 

Nakajima, J.-I. et al.. Reaction mechanism from leucoanthocyanidin to anthocyanidin 3-glucoside, a key reaction for coloring in anthocyanin biosynthesis. J. Biol. Chem., Tib, 25797, 2001. 

Abrahams, S. et al., The Arabidopsis TDS4 gene encodes leucoanthocyanidin dioxygenase (LDOX) and is essential for proanthocyanidin synthesis and vacuole development. Plant Cell, 35, 624, 2003. 

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. 

In three of the volumes of The Flavonoids, Advances in Research, published between 1975 and 1993, flavanones and dihydroflavonols were part of the chapter on Minor Flavonoids, expertly written by Professor Bruce Bohm. These Minor Flavonoid chapters also included chalcones, dihydrochalcones, and aurones. The term Minor Flavonoids was first used by Harborne in 1967 to encompass not only flavanones, chalcones, and aurones, but also isoflavonoids, biflavonyls, and leucoanthocyanidins, because so few compounds belonging to each of these flavonoid classes were known at that time. For example, only about 30 flavanone and dihydroflavonol aglycones, 19 chalcones, and 7 aurones were known in 1967. The number of known minor flavonoids increased considerably in the next two decades, so that when the checklist for The Flavonoids, Advances in Research Since 1980 was published in 1988, 429 known flavanones and dihydroflavonols (including glycosides) were listed, 268 chalcones and dihydrochalcones, and 29 aurones. In the last 15 years, the total number of known compounds in these flavonoid classes has more than doubled, so that the term minor flavonoids is no longer appropriate. Consequently, it has been decided that separate chapters should be devoted to the flavanones and dihydroflavonols (this chapter), and chalcones, dihydrochalcones, and aurones (Chapter 16). 

Condensed Tannins. The tannins found in grapes and wines are condensed polymers from 3-flavanols (catechins) (17, 18, 19) and from 3,4-flavandiols (leucoanthocyanidins) (20, 21, 22). The monomeric leucoanthocyanidins, like their polymerized forms, display the characteristic, which differentiates them from the catechins, of trans-... 

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. 

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