Flavonoid variation in some North Americna Saxifraga species

Eight species of Saxifraga representing sections Micranthes, Hirculus, Dactyloides and Xanthizoon were studied for their flavonoids (S. california, S. integrifolia, S. michauxii, S. ferruginea, S. eschscholtzii, S. hirculus, S. caespitosa and S. aizoides). The major compounds present in most species were kaempferol and quercetin monogluocosides and galactosides. Glucosides of kaempferol and quercetin predominate in the first four species listed, while galactosides of quercetin and myricetin are dominant in the lastthree. 3-O-Methyl- and 3,3′-di-O-methylquercetin were identified from S. californica and S. integrifolia. Saxifraga caespitosa synthesizes a complex mixture of O-methylated flavonols as well as a novel O-methylated dihydrokaempferol.Species pairs S. michauxii/S. ferruginea and S. californica/S.integrifolia exhibit close flavonoid similarity which may reflect their morphological similarities.     

Molecular cloning and biochemical characterization of the UDP-glucose: Flavonoid 3-O-glucosyltransferase from Concord grape (Vitis labrusca)

Glucosylation of anthocyanidin substrates at the 3-O-position is crucial for the red pigmentation of grape berries and wine. The gene that encodes the enzyme involved in this reaction has been cloned from Vitis labrusca cv. Concord, heterologously expressed, and the recombinant enzyme (rVL3GT) was characterized. VL3GT has 96% amino acid sequence identity with Vitis vinifera VV3GT and groups phylogenetically with several other flavonoid 3-O-glycosyltransferases. In vitro substrate specificity studies and kinetic analyses of rVL3GT indicate that this enzyme preferentially glucosylates cyanidin as compared with quercetin. Crude protein extracts from several Concord grape tissues were assayed for glucosyltransferase activity with cyanidin and quercetin as acceptor substrates. A comparison of the VL3GT activities toward with these substrates showed that the 3GT enzyme activity is consistent with the expression of VL3GT in these tissues and is coincident with the biosynthesis of anthocyanins in both location and developmental stages. Enzyme activities in grape mesocarp, pre-veraison exocarp, leaf, flower bud, and flower tissues glucosylated quercetin but not cyanidin at high rates, suggesting the presence of additional enzymes which are able to glucosylate the 3-O-position of flavonols with higher specificity than anthocyanidins.     

Non-polar flavonoids of perityle vaseyi (asteraceae)

Ten flavonoids have been isolated from a dichloromethane leafwash of Perityle vaseyi. Trace amounts of 7,4′-dimethylnaringenin were observed along with nine O-methylated flavonols. Two kaempferol, four 6-hydroxykaempferol and three 6-hydroxyquercetin O-methylated derivatives were identified. 5,7,4-Trihydroxy-3,6-dimethoxyflavone and 5,4′-dihydroxy-3,6,7-trimethoxyflavone were the major components.     

Flavonoids of Chrysosplenium tetrandrum

Chrysosplenium tetrandrum, from northern British Columbia, accumulates a variety of flavonoid glycosides. Several kaempferol and quercetin mono- and diglycosides were identified. The major flavonoid fraction consisted of O-methylated compounds having an hydroxyl or methoxyl substituent at position-6. Aglycones identified were 5,4′-dihydroxy-3,6,7-trimethoxyflavone, 5,6,7,3′,4′-pentahydroxy-3-methoxyflavone, 5,6,3′,4′-tetrahydroxy-3,7-dimethoxyflavone, 5,6,4′-trihydroxy-3,7,3′-trimethoxyflavone, 5,3′,4′-trihydroxy-3,6,7-trimethoxyflavone, and 5,4′-dihydroxy-3,6,7,3′-tetramethoxyflavone. All occurred as glucosides. The occurrence of 6-substitution and the preponderance of O-methylated flavonoids supports removal of Chrysosplenium from Engler's Saxifraginae.     

O-methylation of flavonoids by cell-free extracts of calamondin orange

Cell-free extracts of calamondin orange (Citrus mitis) catalysed the O-methylation of almost all hydroxyls of a number of flavonoids, indicating the existence in citrus tissues of ortho, meta, para and 3-O-methyltransferases. The latter, hitherto unreported enzyme, catalysed the formation of 3-O-methyl ethers of galangin and quercetin. The stepwise O-methylation of a number of compounds, especially quercetin and quercetagetin, tends to suggest a coordinated sequence of O-methylations on the surface of a multienzyme complex. The methyl acceptor abilities of the flavonoid substrates used are discussed in relation to their hydroxyl substitution patterns and their negative electron density distribution.     

Flavonoids of Balsamorhiza deltoidea and Wyethia mollis

Eleven O-methylated derivatives of kaempferol, quercetin and quercetagetin were isolated from the dichloromethane leaf-wash of Balsamorhiza deltoidea. Four of these compounds represent new reports from either Balsamorhiza or Wyethia: 6-hydroxykaempferol 7-O-methyl ether, quercetin 3′,4′-O-dimethylether, quercetagetin 7-O-methyl ether, and quercetagetin 3,6,7-O-trimethyl ether. We also confirmed the presence of two isoflavones, santal and orobol 3′-O-methyl ether, in W. mollis. The 8-C-prenylated derivatives of naringenin, eriodictyol, and dihydroisorhamnetin were also identified as constituents of W. mollis. The vacuolar flavonoid fraction of Balsamorhiza deltoidea and Wyethia helenioides was shown to consist of simple mono and diglycosides of kaempferol and quercetin.     

Rare 6- and 8-O-methylated epicuticular flavonols from two Cistus species

Leaves of Cistus albanicus and C. parviflorus secrete a resin which contains several rare 6- and 8-O-methylated flavonols. In addition, some common kaempferol, quercetin methyl ethers and one myricetin methyl ether were identified. Some typical colour reactions of the 6- and 8-methoxy derivatives on polyamide TLC plates are reported. The systematic implications concerning the flavonoids found are discussed briefly.     

Flavonoids from the leaves and flowers of the Himalayan Cathcartia villosa (Papaveraceae)

Five flavonols, four flavones and one C-glycosylflavone were isolated from the leaves of Cathcartia villosa which is growing in the Himalayan Mountains. They were characterized as quercetin 3-O-vicianoside (1), quercetin 7,4′-di-O-glucoside (3), quercetin 3-O-rutinoside (4), quercetin 3-O-glucoside (5), quercetin 3-O-arabinosylarabinosylglucoside (6) (flavonols), luteolin (7), luteolin 7-O-glucoside (8), apigenin (9), chrysoeriol (10) (flavones), and vicenin-2 (11) (C-glycosylflavone) by UV, LC-MS, acid hydrolysis, NMR and/or HPLC and TLC comparisons with authentic samples. On the other hand, two flavonols 1 and kaempferol 3-O-vicianoside (2) were isolated and identified from the flowers of the species. Flavonoids were reported from the genus Cathcartia in this survey for the first time. Their chemical characters were chemotaxonomically compared with those of related Papaveraceous genera, Meconopsis and Papaver.     

Biochemical and structural analysis of substrate promiscuity in plant Mg2+-dependent O-methyltransferases

Plant S-adenosyl-l-methionine-dependent class I natural product O-methyltransferases (OMTs), related to animal catechol OMTs, are dependent on bivalent cations and strictly specific for the meta position of aromatic vicinal dihydroxy groups. While the primary activity of these class I enzymes is methylation of caffeoyl coenzyme A OMTs, a distinct subset is able to methylate a wider range of substrates, characterized by the promiscuous phenylpropanoid and flavonoid OMT. The observed broad substrate specificity resides in two regions: the N-terminus and a variable insertion loop near the C-terminus, which displays the lowest degree of sequence conservation between the two subfamilies. Structural and biochemical data, based on site-directed mutagenesis and domain exchange between the two enzyme types, present evidence that only small topological changes among otherwise highly conserved 3-D structures are sufficient to differentiate between an enzymatic generalist and an enzymatic specialist in plant natural product methylation.     

Flavonoids of four species of Parthenium (compositae)

Kaempferol and quercetin 3-O-glycosides were found in the closely related species, Parthenium hysterophorus, P. bipinnatifidum and P. glomeratum; the major aglycone flavonols in P. hypterophorus are quercetagetin 3,7-dimethyl ether and a new flavonoid, 6-hydroxykaempferol 3,7-dimethyl ether. The North-South American species-pair P. glomeratum (Argentina) and P. bipinnatifidum (Mexico) yielded quercetagetin 3,7,3′-trimethyl ether as the major aglycone. The desert species P. rollinsianum yielded five methylated flavonols: quercetin 3,3′-dimethyl ether, penduletin, quercetagetin 3,6,7-trimethyl ether, polycladin and artemetin.     

Determination of minor flavonol-glycosides and sugar-free flavonols in the tepals of several species of cereoideae (Cactaceae)

Eight kinds of flavonoids were isolated by crystallization or paper-chromatography from the tepals of several cactaceous plants, i.e.,Astrophytum ornatum Web.,Notocactus apricus A. Berg.,Echinopsis huotii Lab.,Aylostera pseudodeminuta Backbg. andNeochilenia napina Backbg. The structures of six flavonols were determined by UV spectral means and co-PC comparison as quercetin and its 7-O-galactoside (coptiside II), kaempferol and its 3-O-rhamnosylglucoside (nicotiflorin), and isorhamnetin and its 3-O-rhamnosylglucoside (narcissin). The remaining two flavonoids were partially characterized as kaempferol 3, 7-O-diglycoside and 5-hydroxy-3,4′-oxygenated flavonol derivative.     

O-diphenol O-methyltransferases of healthy and tobacco-mosaic-virus-infected hypersensitive tobacco

Three distinct o-diphenol O-methyltransferases (OMTs) were found in leaves of Nicotiana tabacum, variety Samsun NN. They could be clearly distinguished by differences in elution pattern upon chromatography on DEAE-cellulose and in specificity towards 16 diphenolic substrates. The phenylpropanoids caffeic acid and 5-hydroxyferulic acid, whose importance as lignin precursors is well known, were the best substrates of OMT I, but they were also efficiently methylated by the two other OMTs that showed a broader substrate specificity. The highest rates of methylation were observed by assaying these latter enzymes with catechol, homocatechol and protocatechuic aldehyde. The flavonoid quercetin, the major o-diphenol of tobacco leaves, was a good substrate for OMTs II and III, but was also methylated significantly by OMT I. The tobacco OMTs showed both para-and meta-directing activities with protocatechuic acid, protocatechuic aldehyde and esculetin as substrates. Para-O-methylation of the former substrate arose almost exclusively from OMT I whereas that of the two latter substrates from all three enzymes. In healthy leaves the total O-methylating activity varied very much with the batch of plants whereas the relative contributions of the three enzymes were rather constant. On an average, OMTs I, II and III acounted towards caffeic acid, respectively. In tobacco mosaic virus-infected leaves carrying local necrotic lesions we found the same three OMTs with the same substrate specificities, but with increased activities. The degree of stimulation of both OMTs II and III was 2–3 times greater than that of OMT I when the leaves had a moderate number of lesions, and 3–5 times greater with large number of lesions. It is very likely that the changes in both the pattern of the O-methylating enzymes and the concentrations of the naturally occuring o-diphenolic substrates are related to an increased biosynthesis of lignins and of lignin-like compounds. These aromatic polymers could be involved in the cell wall thickening associated with the hypersensitive reaction and with the resistance to virus spread that occur in the cells surrounding the local lesions.Abbreviations OMT O-methyltransferase - TMV tobacco mosaic virus - SAM S-adenosyl-L-methionine     

Enzymatic synthesis of sulphated flavonols in Flaveria

Cell-free extracts of Flaveria bidentis and F. chloraefolia catalysed the transfer of sulphate groups from 3′-phosphoadenosine-5′-phosphosulphate to the hydroxyl groups of a variety of hydroxylated and O-methylated flavonols, but not to flavones or phenylpropanoids. Enzymatic sulphation was more predominant at the 3-hydroxyl group, but not to the exclusion of other hydroxyl substituents on the flavonoid ring. Quercetin was sulphated to yield its mono-, di-, tri- and tetrasulphate esters. This, together with the differences observed in the sulphation of different flavonols by extracts of both Flaveria species, suggests the existence of a number of distinct, position-specific sulphotransferases (EC 2.8.2.-). The sulphation reaction was catalysed at an optimum pH of 7.5 in Tris-HCl buffer, required SH groups for activity and was stimulated in the presence of divalent cations.     

Evolution of Novel O-methyltransferases from the Vanilla planifolia Caffeic Acid O-methyltransferase

The biosynthesis of many plant secondary compounds involves the methylation of one or more hydroxyl groups, catalyzed by O-methyltransferases (OMTs). Here, we report the characterization of two OMTs, Van OMT-2 and Van OMT-3, from the orchid Vanilla planifolia Andrews. These enzymes catalyze the methylation of a single outer hydroxyl group in substrates possessing a 1,2,3-trihydroxybenzene moiety, such as methyl gallate and myricetin. This is a substrate requirement not previously reported for any OMTs. Based on sequence analysis these enzymes are most similar to caffeic acid O-methyltransferases (COMTs), but they have negligible activity with typical COMT substrates. Seven of 12 conserved substrate-binding residues in COMTs are altered in Van OMT-2 and Van OMT-3. Phylogenetic analysis of the sequences suggests that Van OMT-2 and Van OMT-3 evolved from the V. planifolia COMT. These V. planifolia OMTs are new instances of COMT-like enzymes with novel substrate preferences.     

Methylated flavonols from Wyethia bolanderi and Balsamorhiza macrophylla

A leaf wash of Wyethia bolanderi afforded eight known methylated flavonols: santin, ermanin, jaceidin, 3,6-dimethoxyapigenin, kaempferide, isokaempferide, axillarin and quercetin 3-methyl ether. A leaf wash of Balsamorhiza macrophylla afforded six known methylated flavonols: centaureidin, quercetin 3,4′-dimethyl ether, axillarin, spinacetin, tamarexetin and quercetin 3-methyl ether. The chemotaxonomy of the two genera is discussed briefly.     

A quantitative method to determine the points of O-(2-hydroxypropyl) substitution in O-(2-hydroxypropyl)-guar

A method is described for locating the O-(2-hydroxypropyl) groups in O-(2-hydroxypropyl)-substituted guar. Per-O-methylation of the O-(2-hydroxypropyl)guar yielded guar that was partially O-methylated and partially O-(2-methoxypropyl)ated. This polymer was hydrolyzed, to afford a mixture of partially O-methylated monosaccharides and partially O-(2-methoxypropylated), partially O-methylated monosaccharides. These monosaccharide derivatives were reduced, and the alditols acetylated, to give a mixture of partially O-acetylated, partially O-methylated alditols with partially O-acetylated, partially O-(2-methoxypropyl)ated, partially O-methylated alditols. These alditol derivatives were identified by gas-liquid chromatography-mass spectrometry, and quantitated by gas-liquid chromatography.     

Structural characterization of oligosaccharides isolated from the pectic polysaccharide rhamnogalacturonan II

Rhamnogalacturonan II (RG-II) is a structurally complex pectic (d-galactosyl-uronic acid-rich) polysaccharide that is present in the primary (growing) cell-walls of higher plants. RG-II is composed of ∼60 glycosyl residues. The isolation and structural characterization of 23 oligosaccharide fragments of the residue of RG-II that remained after removal of hepta- and di-saccharides by partial hydrolysis with acid are reported. In order to obtain the oligosaccharide fragments characterized herein, the carboxyl groups of RG-II were dideuterio-reduced, and the carboxyl-reduced polysaccharide was per-O-methylated. The per-O-methylated polysaccharide was fragmented by partial hydrolysis with acid, producing partially O-methylated oligosaccharides. These derivatized oligosaccharides were reduced, to afford a mixture of partially O-methylated oligoglycosyl-alditols, which was then per-O-methylated. The structures of the resulting per-O-methylated oligoglycosylalditols were determined by chemical-ionization mass spectrometry, electron-impact mass spectrometry, fast-atom-bombardment mass spectrometry, ¹H-n.m.r. spectroscopy, and analysis of corresponding, partially O-acetylated, partially O-methylated alditols. Seventeen of the oligosaccharides isolated from RG-II were parts of a single heptasaccharide, namely.     

The flavonoids of Stenosiphon (Onagraceae)

Stenosiphon linifolius is a monotypic genus of the tribe Onagreae of the Onagraceae. The species is widespread in, but restricted to, the Great Plains of the United States. Three flavonol glycosides, kaempferol 3-O-rhamnoside, quercetin 3-O-rhamnoside and myricetin 3-O-rhamnoside, were found to occur in methanolic extracts of Stenosiphon leaves. Similar compounds are found in the leaves of such related genera as Oenothera and Gaura, but in the latter genera, additional flavonols exhibiting greater substitutional variation also are found.     

Further studies of flavonols of Clibadium (compositae)

The flavonoids of an additional eight species of Clibadium have been determined. The compounds are derivatives of kaempferol, quercetin and quercetagetin. O-Methylated quercetagetin derivatives were found in several taxa with the possibility that 6-methoxykaempferol may also exist in one collection. Kaempferol and quercetin exist as 3-O-glucosides, galactosides, rhamnosides, rutinosides and diglucosides although not all glycosides occur in each taxon. Quercetagetin derivatives occur as 7-O-glucosides. Observations on these newly investigated species confirm previous work in the genus that three types of flavonoid profiles exist: (1) kaempferol and quercetin 3-glycosides; (2) kaempferol and quercetin 3-glycosides plus quercetagetin 7-glucoside; and (3) kaempferol and quercetin 3-glycosides plus quercetagetin 7-glucoside and O-methylated derivatives of quercetagetin.     

Plant Flavonoid <Emphasis Type="Italic">O</Emphasis>-Methyltransferases: Substrate Specificity and Application

Flavonoids consist of a large family of compounds, which has been estimated to be more than 10,000 compounds. The structural diversity of these compounds comes from different modification reactions. The O-methylation reaction is one of the most important modification reactions of flavonoids and the resulting O-methylated flavonoids have been shown to display new biological activities. The regioselective and substrate specific O-methylation is mediated by O-methyltranferases (OMTs). To date, 30 flavonoid OMTs (FOMTs) have been biochemically characterized from various plants. FOMTs utilize common reaction mechanisms to transfer a methyl group to the hydroxyl group of the flavonoid. Phylogenetic tree analysis along with biochemical characterization of FOMTs provides clues about their substrate specificity and regioselectivity. FOMTs can be used for the production of O-methylated flavonoids that have a particular biological activity.