Leucoanthocyanidins from

Agrawal S, Misra K 1977 Leucoanthocyanidins from Ficus racemosa bark. Chem Scripta 12 37-39... 

Drewes S E, Roux D G 1965 Absolute configuration of mopanol, a new leucoanthocyanidin from Colophospermum mopane. J Chem Soc Chem Commun 500-501... 

Foo L Y, Wong H 1986 Diastereoisomeric leucoanthocyanidins from the heartwood of Acacia melanoxylon. Phytochemistry 25 1961-1965... 

Keppler H H 1975 The isolation and constitution of mollisacacidin, a new leucoanthocyanidin from the heartwood of Acacia mollismi Willd. J Chem Soc 2721 -2724... 

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]... 

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). 

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. ... 

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. 

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-... 

Finally, Weinges et al. (31, 33) postulated the formation of dimers such as 27 or 28, 29 or 30 directly from catechins without involving 3,4-flavandiols (leucoanthocyanidins). This process has never been demonstrated in fruits directly and specifically not in grapes. Only the proanthocyanin dimers have been positively identified through the formation of catechins and anthocyanidins during acid hydrolysis. Dimer formation proceeds by enzymatic oxidation of two molecules of catechin... 

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. 

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. 

Fig. 8 Biosynthesis of anthocyanins (39) via leucoanthocyanidins (flavan-3,4-diols, 37) from dihydroflavonols (3-hydroxy-flavanones, 31, 32)...

Biosynthesis of Flavan-3-ols (40) and Condensed Tannins (Proanthocyanidins, 44) from Leucoanthocyanidins (Flavan-3,4-diols, 37)... 

Fig. 12 Biosynthesis of epicatechin (55), cyanidin (51) and proanthocyanidins (44) by two enzymes of leucoanthocyanidin dioxygenase (LDOX) and leucoanthocyanidin reductase (LAR) from 4-hydroxycinnamic acid (p-coumaric acid, 3)...

The anthocyanidin reductase enzyme recently described in Arabidopsis and Medicago was shown to be present in tea with very high activity and could produce epicatechin as well as epigallocatechin from the respective anthocyanidins, thus explaining very high contents of llavan-3-ols. Especially, two enzymes, dihydroflavonol 4-reductase and leucoanthocyanidin 4-reductase selectively catalyze key steps in their biosynthesis of catechins (43,62) and gallocatechins (63,64), respectively (Fig. 14) [31]. 

Catechins are also derived from flavones. The leucoanthocyanidins have an additional... 

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