B-ring hydroxylation

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. 

In a few species, the B-ring hydroxylation pattern is thought to be fully or partially determined through the HCA-CoA ester used by CHS (see Section 3.4.1). However, most commonly hydroxylation at the C-3 and C-5 positions is determined at the C15 level by the activity of two P450s, the F3 H and F3, 5 H. Genes and cDNAs for both enzymes, sometimes informally referred to as the red and blue genes because of their impact on flower color, were first cloned and characterized from Petunia and subsequently from several other species (listed in Ref. 5). Based on sequence analysis, the F3 H and F3, 5 H proteins are 56 to 58 kDa in size. 

This enzyme was first reported in the microsomal fractions of Haplopappus gracilis cell cultures,33 studied in more detail in parsley,34 and later shown to occur in the flowers of several species.7 F3 H catalyzes the hydroxylation of naringenin and dihydrokaempferol (DHK), as well as of apigenin or kaempferol to their respective 3 -hydroxy derivatives, eriodictyol, dihydroquercetin (DHQ), luteolin, and quercetin, but does not accept the flavan 3,4-diols or anthocyanidins as substrates,34 indicating that B-ring hydroxylation of the latter is determined at the dihydroflavonol level. It was more than two decades later that the first cDNA clone encoding F3 H was isolated and characterized from the flowers of Petunia hybridal Arabidopsis thaliana,36 and Perilla frutescens.37 Their recombinant proteins were shown to... 

Hydroxyflavanone represents the simplest compound of this class and is found in many legumes.9 2-Hydroxyflavanones, which are expected to be quite labile and converted to their corresponding flavones, have also been identified. These include the 2,5,7-trihydroxyflavanone 7-0-glucoside (8) in Malus spp.,80 2,5-dihydroxy-7-methoxyflavanone in Populus nigra, 1 6,7,8-trihydroxy-5-methoxyflavanone (9) in Isodon oresbius,82 and 2,5-dihydroxy-7-methoxyflavanone and its 6-C- and 8-C-methyl isomers (10) in Friesodielsia enghiana.83 All of these exhibit a lack of B-ring hydroxylation. 

Figure 4.5 Sites of tissue modification of isoflavones. In the A-ring, chlorination and hydroxy-lation occur at the 6- and 8-positions. In the B-ring, hydroxylation and nitration occur at the 3 -position.

The condensation reaction may take place at position 6 or 8. Position 6 of the flavan skeleton is favored because it is usually less sterically hindered than position 8. In contrast to the proanthocyanidin assay the condensation reaction runs without depolymerization of the proanthocyanidins [135]. Consequently monomers also react [116] and a multitude of different dyes are produced depending on the complexity of the sample. The maxima of the chromophores of the resulting dyes are not influenced by the B-ring hydroxylation pattern (i.e. procyanidin-prodelphinidin ratio) like in the proanthocyanidin assay [116]. Absorbances are however influenced by the substitution pattern on the ring at which condensation takes place. Condensation products of phloroglucinol nucleis show absorbance maxima at about 500 nm, whereas resorcinol and pyrogallol nucleis exhibit absorbance maxima around 520 nm [77]. 

The flavonols, kaempferol (6), quercetin (7), and myri-cetin (8), have increasing B-ring hydroxylation. These compounds occur as copigments in flowers and fruits, but also they are found commonly in leaves, stems, and roots. In a sample of 1000 plant species, over 60% had 1 or more of these compounds as glycosides. Myricetin tends to be found in association with tannins in woody plants. 

The most important members, however, are proanthocyanidin polymers, also called nonhydrolyzable or condensed tannins. Proanthocyanidin polymers exist as chains of C-4-C-8 (or C-6) linked flavan-3-ol units (Fig. 318). The monomer unit may be based on either of two stereochemistries designated as cis and trans and on either of two B-ring hydroxylation patterns, the procyanidin (PC) and the prodelphinidin (PD) units. Thus the polymer chains are characterized by the average stereochemistry and the PC PD ratios (Table 56). 

The structural requirements for 8-hydroxyflavone activation are distinct from those for flavonols such as quercetin or kaempferol. Whereas B-ring hydroxylation, 2,3-unsaturation, and 3-hy-droxylation appear critical for flavonol mutagenicity, the B-ring and 2,3-positions do not appear to be involved in the activation of 8-substituted flavones. The 5,7,8-substitution pattern on the A-ring is particularly effective at conferring high activity in the flavone series. The seven most active compounds known at present all have -OH or -OCH substituents at these positions. 

NADH-oxidase activity was based on the O2 uptake in the presence of complex 1 substrates (e.g., NADH) and is dependent on complexes 1, 111, and IV. Hydroxy-lation of the flavonoid B-ring was an important determinant of inhibitory potency toward NADH-oxidase [2]. The absence of B-ring hydroxyl substituents or their... 

The occurrence of flavones lacking B-ring hydroxylation - e.g. of chrysin - is noteworthy. A wider range of flavones occur in Prunus than in Pinus, with luteolin being repordes in Prunus ssiori. It is interesting that the barks of Prunus species appear to contain some of the 5- or 7-glucosides of those flavones that occur free in the heartwood. 

It may be noted from Table 7.7.2 that a number of proanthocyanidin polymers contain units with the same B-ring oxidation but differing stereochemistry (e.g., catechin-4 and epicatechin-4), and others with common stereochemistry but differing B-ring oxidation (e.g., epicatechin-4 and epigallocatechin-4), whereas still others may have both stereochemistry and B-ring hydroxylation mixed. These ob-... 

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