Lipase-Catalyzed Kinetic Resolution of (±)-cis-Flavan-4-ol and Its Acetate: Synthesis of Chiral 3-Hydroxyflavanones

Lipase-catalyzed kinetic resolution of (±)-cis-flavan-4-ol and its acetate led to enantiomerically enriched flavan-4-ol and its acetate. These chiral compounds were converted to (2R, 3R)- and (2S, 3S)-3-hydroxyflavanones.     

Synthesis and characterization of procyanidin dimers as their peracetates and octamethyl ether diacetates

Condensation of (2R,3S,4R or S)-leucocyanidin or the 5,7,3′,4′-tetramethyl ether of (2R,3^R,4S)-leucocyanidin with flavan-3-ols yielded dimeric flavanoids which were converted to their octamethyl ether diacetates, or the deca-acetates for the 2,3-trans-procyanadin series. Comparison is made of the ¹H NMR spectra of the deca-acetate and octamethyl ether diacetate derivatives which lead to useful diagnostic shift parameters characteristic of their structures. Condensations afforded a novel biflavanoid with a 3,4-cis-configuration and a triflavanoid of ‘mixed’ stereochemistry.     

Anthocyanins in the epacridaceae

An examination of 73 species of the family Epacridaceae resulted in the identification of the following anthocyanins: cyanidin 3-galactoside, cyanidin 3-glucoside, cyanidin 3-arabinoside, cyanidin 3-rhamnoside, cyanidin 3-rhamnosylgalactoside, cyanidin 3-rhamnosylglucoside, cyanidin 3-xylosylgalactoside, cyanidin 3-xylosylarabinoside, delphinidin 3-galactoside, delphinidin 3-arabinoside, delphinidin 3-rhamnosylgalactoside, delphinidin 3-rhamnosylglucoside and pelargonidin 3-rhamnosylglucoside. No acylated or 5-substituted anthocyanins were detected in any of the species examined. Evidence of methylated anthocyanidin was found only in one species, Woollsia pungens. The occurrence of cyanidin 3-galactoside and cyanidin 3-arabinoside forms a chemical link between this family and the related Ericaceae.     

Anthocyanidin reductases from Medicago truncatula and Arabidopsis thaliana

Anthocyanidin reductase (ANR), encoded by the BANYULS gene, is a newly discovered enzyme of the flavonoid pathway involved in the biosynthesis of condensed tannins. ANR functions immediately downstream of anthocyanidin synthase to convert anthocyanidins into the corresponding 2,3-cis-flavan-3-ols. We report the biochemical properties of ANRs from the model legume Medicago truncatula (MtANR) and the model crucifer Arabidopsis thaliana (AtANR). Both enzymes have high temperature optima. MtANR uses both NADPH and NADH as reductant with slight preference for NADPH over NADH. In contrast, AtANR only uses NADPH and exhibits positive cooperativity for the co-substrate. MtANR shows preference for potential anthocyanidin substrates in the order cyanidin>pelargonidin>delphinidin, with typical Michaelis-Menten kinetics for each substrate. In contrast, AtANR exhibits the reverse preference, with substrate inhibition at high concentrations of cyanidin and pelargonidin. (+)-Catechin and (+/-)-dihydroquercetin inhibit AtANR but not MtANR, whereas quercetin inhibits both enzymes. Possible catalytic reaction sequences for ANRs are discussed.     

An integrated approach to demonstrating the ANR pathway of proanthocyanidin biosynthesis in plants

Proanthocyanidins (PAs) are oligomers or polymers of plant flavan-3-ols and are important to plant adaptation in extreme environmental conditions. The characterization of anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) has demonstrated the different biogenesis of four stereo-configurations of flavan-3-ols. It is important to understand whether ANR and the ANR pathway widely occur in the plant kingdom. Here, we report an integrated approach to demonstrate the ANR pathway in plants. This includes different methods to extract native ANR from different tissues of eight angiosperm plants (Lotus corniculatus, Desmodium uncinatum, Medicago sativa, Hordeum vulgare, Vitis vinifera, Vitis bellula, Parthenocissus heterophylla, and Cerasus serrulata) and one fern plant (Dryopteris pycnopteroides), a general enzymatic analysis approach to demonstrate the ANR activity, high-performance liquid chromatography-based fingerprinting to demonstrate (-)-epicatechin and other flavan-3-ol molecules, and phytochemical analysis of PAs. Results demonstrate that in addition to leaves of M. sativa, tissues of other eight plants contain an active ANR pathway. Particularly, the leaves, flowers and pods of D. uncinatum, which is a model plant to study LAR and the LAR pathways, are demonstrated to express an active ANR pathway. This finding suggests that the ANR pathway involves PA biosynthesis in D. uncinatum. In addition, a sequence BLAST analysis reveals that ANR homologs have been sequenced in plants from both gymnosperms and angiosperms. These data show that the ANR pathway to PA biosynthesis occurs in both seed and seedless vascular plants.     

Condensed tannins. Optically active diastereoisomers of (+)-mollisacacidin by epimerization

1. (+)-Mollisacacidin [(+)-3′,4′,7-trihydroxy-2,3-trans-flavan-3,4-trans- diol] is converted by autoclaving into the optically active free phenolic 2,3-trans-3-4-cis (12% yield), 2,3-cis-3,4-trans (11%) and 2,3-cis-3,4-cis (2·8%) diastereoisomers through epimerization at C-2 and C-4. 2. The relative configurations of the epimeric forms were determined by nuclear-magnetic-resonance spectrometry and paper ionophoresis in comparison with synthetic reference compounds, and was confirmed by chemical interconversions. 3. From this a scheme of epimerization is inferred and their absolute configurations are assigned as (2R:3S:4S), (2S:3S:4R) and (2S:3S:4S) respectively from the known absolute configuration (2R:3S:4R) of (+)-mollisacacidin.     

Anthocyanin content of Tulipa species and cultivars and its impact on tepal colours

Flowers of tulips (17 species and 25 cultivars) were subjected to qualitative and relative quantitative examination for anthocyanins. Altogether five anthocyanins were identified as the 3-O-(6″-O-α-rhamnopyranosyl-β-glucopyranoside) of delphinidin (1), cyanidin (2) and pelargonidin (3), and the 3-O-[6″-O-(2‴-O-acetyl-α-rhamnopyranosyl)-β-glucopyranoside] of cyanidin (4) and pelargonidin (5). The pigments 1–5 represented 7%, 43%, 12%, 2% and 31%, respectively, of the total anthocyanin amount in the tepals of the Tulipa species, and 20%, 37%, 30%, 6% and 4%, respectively, in the cultivar tepals. Nearly 50% of the samples contained acetylated anthocyanins. The colours of the freeze-dried tepals described by the CIELab coordinates, hue angle (h_ab), saturation (C*), and lightness (L*) together with the anthocyanin content were subjected to multivariate analysis. All tepals classified with hue angles described as “blue nuances” were from cultivars. They contained 1 as the major anthocyanin, and no or just traces of pelargonidin derivatives. The species and cultivars having “magenta nuances” showed similar anthocyanin content with increased relative proportions of 2 at the expense of 1. Orange coloured tepals were to a large extent correlated with high relative proportions of the pelargonidin derivatives, 3 and 5. Acetylation of anthocyanins furnished a weak colour effect opposite to the bluing effect previously reported for anthocyanins with aromatic acyl groups. All six species belonging to the section Eichleres (subgenus Tulipa) were after principal component analysis grouped closely together. They were characterized by high concentrations of the pelargonidin derivatives 3 and 5, and orange petal nuances. However, within section Tulipa (subgenus Tulipa), considerable anthocyanin variation was observed. Species in the subgenus Eriostemones were generally characterized by the two anthocyanins 1 and 2, and no pelargonidin derivatives.     

Fractionation and chemistry of ethyl acetate-soluble thearubigins from black tea

Sephadex LH-20 chromatography was used to fractionate purified ethyl acetate-soluble thearubigins, prepared from an aqueous ethanolic extract of black tea. Three subfractions were so produced, each having a MW of about 1500 and each being degradable into cyanidin, delphinidin, gallic acid, the same two flavan-3-ols, and the same two flavan-3-ol gallates, though in different yield. Some evidence for the presence of benzotropolone moieties in at least one of the subfractions was obtained. Overall the ethyl acetate-soluble thearubigins are viewed as pentameric flavan-3-ols/flavan-3-ol gallates, containing both hydrolysable and non-hydrolysable interflavanoid links, as well as benzotropolone units, rather than as polymeric proanthocyanidins, a term previously used for all thearubigin subgroups.     

Crystal Structure and Catalytic Mechanism of Leucoanthocyanidin Reductase from Vitis vinifera

Leucoanthocyanidin reductase (LAR) catalyzes the NADPH-dependent reduction of 2R,3S,4S-flavan-3,4-diols into 2R,3S-flavan-3-ols, a subfamily of flavonoids that is important for plant survival and for human nutrition. LAR1 from Vitis vinifera has been co-crystallized with or without NADPH and one of its natural products, (+)-catechin. Crystals diffract to a resolution between 1.75 and 2.72 Å. The coenzyme and substrate binding pocket is preformed in the apoprotein and not markedly altered upon NADPH binding. The structure of the abortive ternary complex, determined at a resolution of 2.28 Å, indicates the ordering of a short 3₁₀ helix associated with substrate binding and suggests that His122 and Lys140 act as acid-base catalysts. Based on our 3D structures, a two-step catalytic mechanism is proposed, in which a concerted dehydration precedes an NADPH-mediated hydride transfer at C4. The dehydration step involves a Lys-catalyzed deprotonation of the phenolic OH7 through a bridging water molecule and a His-catalyzed protonation of the benzylic hydroxyl at C4. The resulting quinone methide serves as an electrophilic target for hydride transfer at C4. LAR belongs to the short-chain dehydrogenase/reductase superfamily and to the PIP (pinoresinol-lariciresinol reductase, isoflavone reductase, and phenylcoumaran benzylic ether reductase) family. Our data support the concept that all PIP enzymes reduce a quinone methide intermediate and that the major role of the only residue that has been conserved from the short-chain dehydrogenase/reductase catalytic triad (Ser…TyrXXXLys), that is, lysine, is to promote the formation of this intermediate by catalyzing the deprotonation of a phenolic hydroxyl. For some PIP enzymes, this lysine-catalyzed proton abstraction may be sufficient to trigger the extrusion of the leaving group, whereas in LAR, the extrusion of a hydroxide group requires a more sophisticated mechanism of concerted acid-base catalysis that involves histidine and takes advantage of the OH4, OH5, and OH7 substituents of leucoanthocyanidins.     

Anthocyanins in the genus Erica

3-Glucosides, 3-galactosides and 3-arabinosides of cyanidin, delphinidin, malvidin, peonidin and pelargonidin have been identified as major floral pigments in Erica (Ericaceae). Unidentified 3-biosides are present as minor pigments in some species. A comparison is made with floral anthocyanins occurring in the related family Epacridaceae.     

A survey of anthocyanins in fruits of some angiosperms,I

The anthocyanin pigments in the fruits of fifty-two species belonging to seventeen families of angiosperms were investigated paper-chromatographicallly. They were identified as cyanidin 3-monoglucoside, pelargonidin 3-monoglucoside, cyanidin 3-rutinoside, pelargonidin 3-rutinoside, cyanidin 3-xylosylglucoside, cyanidin 3-xylosylgalactoside, delphinidin 3-xylosylglucoside and delphinidin 3-sophorosido-5-monoglucoside. Of those anthocyanins detected, the most common was cyanidin 3-monoglucoside. In general, the plants belonging to a certain genus contained the same anthocyanin.     

Correlations between anthocyanin chemistry and pollination ecology in the polemoniaceae

A study of the anthocyanins in a representative sample (34 species from 14 genera) of Polemoniaceae has shown that the pigment type in the flowers is broadly correlated with pollination ecology. Thus, hummingbird pollinated species such as Ipomopsis aggregata generally contain pelargonidin sometimes with cyanidin, while bee and beefly pollinated species (e.g. Gilia latiflora) contain mainly delphinidin. On the other hand, lepidopteran species such as Leptodactylon californicum have cyanidin or mixtures of cyanidin with delphinidin. The above three anthocyanidins occur usually as the 3-glucoside, 3,5-diglucoside, $\text{3-(p-}\text{coumarylglucoside}\text{)}$ and $\text{3-(p-}\text{coumarylglucoside}\text{)-5-}\text{glucoside}$, although other types are occasionally found. The distribution of glycosidic types and of acylation, unlike that of the anthocyanidins, is more closely correlated with systematic position than with pollinating vectors. In autogamous species where animal pollination is absent or unimportant, anthocyanin pigmentation in the flowers retains the complexity present in related animal-pollinated taxa. Anthocyanins were also identified in hummingbird pollinated plants from two related families and pelargonidin derivatives were detected. In Fouquieria splendens (Fouquieriaceae), the glycosidic pattern was different from that in Polemoniaceae in being 3-galactoside. In Penstemon (Scrophulariaceae) a study of flower anthocyanins was consistent with Straw's hypothesis that the wasp-pollinated P. spectabilis originated by hybridization between the hummingbird-pollinated P. centranthifolius and the bee-pollinated P. grinnellii.     

Isorhamnetin 3,7-disulphate from Flaveria bidentis

3-Glucosides and 3,5-diglucosides of pelargonidin, cyanidin, peonidin, delphinidin, petunidin and malvidin have been identified as flower pigments in Fuchsia species. These pigments in varying admixture appear to be solely responsible for different flower colours in this genus. Their production and inheritance seems to be under a complex system of genetic control.     

High-yield anthocyanin biosynthesis in engineered Escherichia coli

Anthocyanins are red, purple, or blue plant water-soluble pigments. In the past two decades, anthocyanins have received extensive studies for their anti-oxidative, anti-inflammatory, anti-cancer, anti-obesity, anti-diabetic, and cardioprotective properties. In the present study, anthocyanin biosynthetic enzymes from different plant species were characterized and employed for pathway construction leading from inexpensive precursors such as flavanones and flavan-3-ols to anthocyanins in Escherichia coli. The recombinant E. coli cells successfully achieved milligram level production of two anthocyanins, pelargonidin 3-O-glucoside (0.98 mg/L) and cyanidin 3-O-gluside (2.07 mg/L) from their respective flavanone precursors naringenin and eriodictyol. Cyanidin 3-O-glucoside was produced at even higher yields (16.1 mg/L) from its flavan-3-ol, (+)-catechin precursor. Further studies demonstrated that availability of the glucosyl donor, UDP-glucose, was the key metabolic limitation, while product instability at normal pH was also identified as a barrier for production improvement. Therefore, various optimization strategies were employed for enhancing the homogenous synthesis of UDP-glucose in the host cells while at the same time stabilizing the final anthocyanin product. Such optimizations included culture medium pH adjustment, the creation of fusion proteins and the rational manipulation of E. coli metabolic network for improving the intracellular UDP-glucose metabolic pool. As a result, production of pelargonidin 3-O-glucoside at 78.9 mg/L and cyanidin 3-O-glucoside at 70.7 mg/L was achieved from their precursor flavan-3-ols without supplementation with extracellular UDP-glucose. These results demonstrate the efficient production of the core anthocyanins for the first time and open the possibility for their commercialization for pharmaceutical and nutraceutical applications.     

Comparison of Proanthocyanidins and Related Compounds in Leaves and Leaf-Derived Cell Cultures of Ginkgo bioloba L., Pseudotsuga menziesii Franco, and Ribes sanguineum Pursh

Proanthocyanidins, flavan-3-ols, and their flavanoid precursors in leaves and leaf-derived callus and cell suspension cultures have been isolated and analyzed by high performance liquid chromatography with C₁₈ columns, paper chromatography, and by chemical and spectrophotometric methods. Cultures of Ginkgo biloba and Pseudotsuga menziesii (Douglas-fir) produced much greater amounts of proanthocyanidins than leaves per milligram dry weight. In cultures, however, the prodelphinidin component relative to that of procyanidins decreased; this was most pronounced in Pseudotsuga. In contrast, callus cultures of Ribes sanguineum accumulated proanthocyanidins in amounts about equal to those in intact leaves per milligram dry weight and the prodelphinidin content remained high. Although Ginkgo and Ribes leaves contained major amounts of flavan-3-ols and dimers with the 2,3-cis-stereochemistry, their cultures tended to synthesize 2,3-trans-isomers instead. Glycosides of flavanone and 3-hydroxyflavanone precursors accumulated in medium to high amounts on a dry weight basis in leaves and cultures of Ribes and Pseudotsuga, and the 3′-glycosidic linkage predominated when the latter species was cultured with 2,4-dichlorophenoxyacetic acid rather than naphthaleneacetic acid.     

Dimalonated anthocyanins from the flowers of Salvia splendens and S. coccinea

The pigments of Salvia splendens flowers (scarlet cvs) have been identified as pelargonidin 3-caffeoylglucoside-5-dimalonylglucoside and pelargonidin 3-p-coumaroylglucoside-5-dimalonylglucoside. The flowers of S. coccinea contain the same pigments in the corolla but the calyx contains, in addition, the cyanidin analogues.     

Structure des caboxines: Alcaloïdes oxindoliques du Cabucala fasciculata

The structures of three new 11-monomethoxy pentacyclic oxindole alkaloids have been elucidated by chemical correlations with reserpinine: caboxine-A was assigned to the allo C₁₉-méthyl α series: 3S, 4R, 7S, 19S; isocaboxine-A and B to the epi-allo C₁₉-methyl α series and have, respectively, the following configurations 3R, 4S, 7S, 19S and 3R, 4S, 7R, 19S.     

Natural and synthetic diastereoisomeric (?)-3′,4′,7-trihydroxyflavan-3,4-diols

1. Rhodesian copalwood (Guibourtia coleosperma) contains three diastereo-isomeric leuco-fisetinidins. These consist of the (−)-2,3-cis–3,4-cis (2R,3R,4R) and (−)-2,3-cis–3,4-trans (2R,3R,4S) 3′,4′,7-trihydroxyflavan-3,4-diols, and the third was shown to be a 2,3-trans–3,4-cis isomer by means of paper ionophoresis. 2. There occurrence in similar proportions as tannin precursors also in the tropical hardwoods G. tessmannii and G. demeusii implies a close taxonomic relationship between these, and with G. coleosperma. 3. Epimerization of the natural (−)-3′,4′,7- trihydroxy-2,3-trans-flavan-3,4-trans-diol affords a mixture from which the (−)-2,3-cis–3,4-cis isomer was separated readily, but the (−)-2,3-trans–3,4-cis isomer was obtained with difficulty. These were formed by epimerization of the (−)-2,3-trans–3,4-trans isomer at C-2 and C-4, and at C-4, respectively.