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.     

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.     

8-O-methyl- and the first 3-O-methylflavan-3,4-diols from Acacia saxatilis

The light purple heartwood of Acacia saxatilis contains (+)-2,3-trans-3,4-trans- and (+)-2,3-trans-3,4-cis-diastereoisomers of 8-methoxy-7,j',4'-trihydroxy- and 7,3′,4′-trihydroxyflavan-3,4-diols as major components. Evidence was also obtained of the first 3-methyl ether of metabolites: of this type, notably of (+)-8-methoxy-7,3′,4′-trihydroxy-2,3-trans-flavan-3,4-cis-diol. Flavonol, dihydroflavonol and flavanone analogies accompany these. The correlation between colour of Acacia heartwoods and structure, phenolic substitution, stereochemistry and composition of their flavonoid components is discussed.     

Flavonoids and tannins of Acacia species

The heartwoods of Acacia giraffae and A. galpinii were selected from South African Acacias as representative of those with abnormally high and minimal tannin contents respectively. A. galpinii contains amongst other analogues, the first natural (+)-2,3-trans-3,4-trans-teracacidin (7,8,4′-trihydroxy-flavan-3,4-diol and novel 3-O-methyl-, 7,8-di-O-methyl- and 7,8,4′-tri-O-methylflavonol analogues. (−)-2,3-cis-3,4-cis-Melacacidin (7,8,3′,4′-tetrahydroxyflavan-3,4-diol) is also present, but tannins are absent. By contrast, from the large excess of leueofisetinidin tannins which characterizes the wood of A. giraffae, only (+)-catechin, (+)-2,3-trans-3,4-trans-leucofisetinidin (7,3′,4′,trihydroxyflavan-3,4-diol and all-trans-(+)-leueofisetinidin-(+)-catechin could be isolated.     

Biflavanoid proguibourtinidin carboxylic acids and their biflavanoid homologues from Acacia luederitzii

[4,6]- and [4,8]-Proguibourtinidin carboxylic acids (3,7,4′-trihydroxyl functionality) of 2,3-trans-3,4-trans: 2,3-cis- and 2,3-trans-3,4-trans: 2,3-trans-configuration based on (?)-epicatechin or (+)-catechin as constituent units, and their associated biflavanoid homologues, predominate in the heartwood of Acacia luederitzii. They are accompanied by stereochemical and functional analogues and by their putative flavan-3,4-diol and flavan-3-ol precursors.     

Chemical and enzymatic synthesis of monomeric procyanidins (leucocyanidins or 3′,4′,5,7-tetrahydroxyflavan-3,4-diols) from (2R,3R)-dihydroquercetin

The major product from the reduction of (2R,3R)-dihydroquercetin with sodium borohydride is the 2,3-trans-3,4-trans isomer of leucocyanidin [(2R,3S,4R-3,3′,4,4′,5,7-hexahydroxyflavan] whereas the enzymatic reduction product is the 2,3-trans-3,4-cis isomer [(2R,3S,4S)-3,3′,4,4′,5,7-hexahydroxyflavan]. The 3,4-trans isomer may be partly converted to the 3,4-cis isomer under mild acid conditions. The 3,4-cis isomer is more acid-labile, and more reactive both chemically with thiols and enzymatically with a diol reductase, than the 3,4-trans isomer.     

The first 2,3-trans-3,4-cis procyanidin

Condensation of (+)-leucocyanidin with (+)-catechin under acidic conditions afforded the novel 2,3-trans-3,4-cis: 2,3-trans [4,8]-bi-[(+)-catechin] as the first representative of a 3,4-cis procyanidin unit.     

Degradation of Polychlorinated Biphenyl Metabolites by Naphthalene-Catabolizing Enzymes

The ability of the dehydrogenase and ring cleavage dioxygenase of the naphthalene degradation pathway to transform 3,4-dihydroxylated biphenyl metabolites was investigated. 1,2-Dihydro-1,2-dihydroxynaphthalene dehydrogenase was expressed as a histidine-tagged protein. The purified enzyme transformed 2,3-dihydro-2,3-dihydroxybiphenyl, 3,4-dihydro-3,4-dihydroxybiphenyl, and 3,4-dihydro-3,4-dihydroxy-2,2′,5,5′-tetrachlorobiphenyl to 2,3-dihydroxybiphenyl, 3,4-dihydroxybiphenyl (3,4-DHB), and 3,4-dihydroxy-2,2′,5,5′-tetrachlorobiphenyl (3,4-DH-2,2′,5,5′-TCB), respectively. Our data also suggested that purified 1,2-dihydroxynaphthalene dioxygenase catalyzed the meta cleavage of 3,4-DHB in both the 2,3 and 4,5 positions. This enzyme cleaved 3,4-DH-2,2′,5,5′-TCB and 3,4-DHB at similar rates. These results demonstrate the utility of the naphthalene catabolic enzymes in expanding the ability of the bph pathway to degrade polychlorinated biphenyls.     

Abscisic (ABA)-Aldehyde Is a Precursor to, and 1',4'-trans-ABA-Diol a Catabolite of, ABA in Apple

Previous ¹⁸O labeling studies of abscisic acid (ABA) have shown that apple (Malus domestica Borkh. cv Granny Smith) fruits synthesize a majority of [¹⁸O]ABA with the label incorporated in the 1′-hydroxyl position and unlabeled in the carboxyl group (JAD Zeevaart, TG Heath, DA Gage [1989] Plant Physiol 91: 1594-1601). It was proposed that exchange of ¹⁸O in the side chain with the medium occurred at an aldehyde intermediate stage of ABA biosynthesis. We have isolated ABA-aldehyde and 1′-4′-trans-ABA-diol (ABA-trans-diol) from ¹⁸O-labeled apple fruit tissue and measured the extent and position of ¹⁸O incorporation by tandem mass spectrometry. ¹⁸O-Labeling patterns of ABA-aldehyde, ABA-trans-diol, and ABA indicate that ABA-aldehyde is a precursor to, and ABA-trans-diol a catabolite of, ABA. Exchange of ¹⁸O in the carbonyl of ABA-aldehyde can be the cause of loss of ¹⁸O from the side chain of [¹⁸O]ABA. Results of feeding experiments with deuterated substrates provide further support for the precursor-product relationship of ABA-aldehyde → ABA → ABA-trans-diol. The ABA-aldehyde and ABA-trans-diol contents of fruits and leaves were low, approximately 1 and 0.02 nanograms per gram fresh weight for ABA-aldehyde and ABA-trans-diol, respectively, while ABA levels in fruits ranged from 10 to 200 nanograms per gram fresh weight. ABA biosynthesis was about 10-fold lower in fruits than in leaves. In fruits, the majority of ABA was conjugated to β-d-glucopyranosyl abscisate, whereas in leaves ABA was mainly hydroxylated to phaseic acid. Parallel pathways for ABA and trans-ABA biosynthesis and conjugation in fruits and leaves are proposed.     

Solution structure of the DNA decamer duplex containing a 3'-T x T basepair of the cis-syn cyclobutane pyrimidine dimer: implication for the mutagenic property of the cis-syn dimer

The cis–syn dimer is the major DNA photoproduct produced by UV irradiation. In order to determine the origin of the mutagenic property of the cis–syn dimer, we used NMR restraints and molecular dynamics to determine the solution structure of a DNA decamer duplex containing a wobble pair between the 3′-T of the cis–syn dimer and the opposite T residue (CS/TA duplex). The solution structure of the CS/TA duplex revealed that the 3′-T·T base pair of the cis–syn dimer had base pair geometry that was significantly different from the canonical Watson–Crick base pair and caused destabilization and conformational distortion of its 3′-region. However, a 3′-T·A base pair at the cis–syn dimer within this related DNA decamer maintains the normal Watson–Crick base pair geometry and causes little distortion in the conformation of its 3′-side. Our results show that in spite of its stable hydrogen bonding, the insertion of a T residue opposite the 3′-T of the cis–syn dimer is inhibited by structural distortion caused by the 3′-T·T base pair. This may explain why the frequency of the 3′-T→A transversion, which is the major mutation produced by the cis–syn dimer, is only 4%.     

Structure-Activity Relationships of Abscisic Acid Analogs Based on the Induction of Freezing Tolerance in Bromegrass (Bromus inermis Leyss) Cell Cultures

The induction of freezing tolerance in bromegrass (Bromus inermis Leyss) cell culture was used to investigate the activity of absisic acid (ABA) analogs. Analogs were either part of an array of 32 derived from systematic alterations to four regions of the ABA molecule or related, pure optical isomers. Alterations were made to the functional group at C-1 (acid replaced with methyl ester, aldehyde, or alcohol), the configuration at C-2, C-3 (cis double bond replaced with trans double bond), the bond order at C-4, C-5 (trans double bond replaced with a triple bond), and ring saturation (C-2′, C-3′ double bond replaced with a single bond so that the C-2′ methyl and side chain were cis). All deviations in structure from ABA reduced activity. A cis C-2, C-3 double bond was the only substituent absolutely required for activity. Overall, acids and esters were more active than aldehydes and alcohols, cyclohexenones were more active than cyclohexanones, and dienoic and acetylenic analogs were equally active. The activity associated with any one substituent was, however, markedly influenced by the presence of other substituents. cis, trans analogs were more active than their corresponding acetylenic analogs unless the C-1 was an ester. Cyclohexenones were more active than cyclohexanones regardless of oxidation level at C-1. An acetylenic side chain decreased the activity of cyclohexenones but increased the activity of cyclohexanones relative to their cis, trans counterparts. Trends suggested that for activity the configuration at C-1′ has to be the same as in (S)-ABA, in dihydro analogs the C-2′-methyl and the side chain must be cis, small positional changes of the 7′-methyl are tolerable, and the C-1 has to be at the acid oxidation level.     

Analysis of HIF-1 inhibition by manassantin A and analogues with modified tetrahydrofuran configurations

We have shown that manassantin A downregulated the HIF-1α expression and inhibited the secretion of VEGF. We have also demonstrated that the 2,3-cis-3,4-trans-4,5-cis-configuration of the tetrahydrofuran is critical to the HIF-1 inhibition of manassantin A.     

Microbiological Hydroxylation of Cinerone to Cinerolone

Cinerone [2-(2′-cis-butenyl)-3-methyl-2-cyclopenten-1-one] is hydroxylated to cinerolone [2-(2′-cis-butenyl)-3-methyl-4-hydroxy-2-cyclopenten-1-one] by a number of streptomycetes, bacteria, and fungi. Aspergillus niger ATCC 9,142 and Streptomyces aureofaciens ATCC 10,762 were found to be the most effective hydroxylators. The optical activity of the product was found to range from [α]_D²⁵ 0° to -8.6°, depending on the organism and conditions of culture. Two additional hydroxylated products of cinerone have been isolated and identified as 2-n-butyl-4-hydroxy-3-methyl-2-cyclopenten-1-one and 2-(2′-cis-butenyl-4′-hydroxy)-3-methyl-2-cyclopenten-1-one, respectively.     

Xanthoxin Metabolism in Cell-free Preparations from Wild Type and Wilty Mutants of Tomato

Extracts prepared from the turgid and water-stressed leaves of wild-type tomato (Lycopersicon esculentum Mill cv Ailsa Craig) and the wilty mutants sitiens, notabilis, and flacca were tested for their ability to metabolize xanthoxin to ABA. Extracts from wild type and notabilis converted xanthoxin at similar rates, while extracts from sitiens and flacca showed little or no activity. We also observed no activity when extracts of sitiens and flacca were mixed. Similar results were obtained when ABA aldehyde was used as a substrate, in that extracts from wild type and notabilis were equally active, but extracts from flacca and sitiens showed little activity. None of the tomato extracts showed significant activity with xanthoxin acid, xanthoxin alcohol, or ABA-1′,4-′Trans-diol as substrates. Extracts from bean leaves (Phaseolus vulgaris L. cv Blue Lake) were similar to the wild-type tomato extracts in their ability to convert the various substrates to ABA, although excised bean leaves did convert ABA-1′,4′-trans-diol and xanthoxin alcohol to ABA when these substances were taken up through the petiole. These results are consistent with a role for xanthoxin as a normal intermediate on the ABA biosynthetic pathway, and they suggest that ABA aldehyde is the final ABA precursor.     

Stereochemical aspects of the metabolism of the isomeric methylcyclohexanols and methylcyclohexanones

1. The seven isomeric optically inactive forms of methylcyclohexanol (i.e. 1-, and cis- and trans-2-, -3- and -4-) are excreted by rabbits mainly as glucuronides of the thermodynamically more stable forms of the alcohols. The eighth isomer, cyclohexylmethanol, however, undergoes aromatization in vivo, giving rise to benzoic acid and hippuric acid. The (±)-2-, (±-3- and 4-methylcyclohexanones are reduced in the rabbit and excreted mainly as the glucuronides of the thermodynamically more stable forms of the corresponding methylcyclohexanols. 2. Racemic cis- and trans-2-methylcyclohexanol and 2-methylcyclohexanone are all excreted as conjugated trans-2-methylcyclohexanol. However, when the (±)-cis-alcohol or the (±)-ketone is fed, (+)-trans-2-methylcyclohexanol is excreted, whereas when the (±)-trans-alcohol is fed it is excreted as the (±)-trans-alcohol. 3. Racemic cis- and trans-3-methylcyclohexanol and 3-methylcyclohexanone are all excreted as conjugated racemic cis-3-methylcyclohexanol. cis- and trans-4-Methylcyclohexanol and 4-methylcyclohexanone are all excreted as conjugated trans-4-methylcyclohexanol. 4. The metabolic differences between the various methylcyclohexanols are explicable in terms of their conformations and of Vennesland's (1958) hypothesis of the role of NADH in dehydrogenation reactions.     

Activity and metabolism of C-(+/-)-abscisic Acid derivatives

Several radioactive analogues of abscisic acid have been tested for their growth-inhibitory effects and their metabolism in excised embryonic axes of Phaseolus vulgaris. The compounds tested were the methyl and ethyl esters of 2-¹⁴C-abscisic acid and the cis- and trans-1′,4′-diols of 2-¹⁴C-abscisic acid. All four compounds cause less growth inhibition than abscisic acid, and all four compounds are converted to abscisic acid in the axes at rates which are sufficient to account for most, if not all, of the observed growth-inhibitory activity. None of the four compounds is metabolized to the extent that abscisic acid is metabolized in the axes, suggesting that the structural requirements for growth-inhibitory activity and metabolism may be similar.     

Biotransformation of Natural and Synthetic Isoflavonoids by Two Recombinant Microbial Enzymes

Isolation and synthesis of isoflavonoids has become a frequent endeavor, due to their interesting biological activities. The introduction of hydroxyl groups into isoflavonoids by the use of enzymes represents an attractive alternative to conventional chemical synthesis. In this study, the capabilities of biphenyl-2,3-dioxygenase (BphA) and biphenyl-2,3-dihydrodiol 2,3-dehydrogenase (BphB) of Burkholderia sp. strain LB400 to biotransform 14 isoflavonoids synthesized in the laboratory were investigated by using recombinant Escherichia coli strains containing plasmid vectors expressing the bphA1A2A3A4 or bphA1A2A3A4B genes of strain LB400. The use of BphA and BphB allowed us to biotransform 7-hydroxy-8-methylisoflavone and 7-hydroxyisoflavone into 7,2′,3′-trihydroxy-8-methylisoflavone and 7,3′,4′-trihydroxyisoflavone, respectively. The compound 2′-fluoro-7-hydroxy-8-methylisoflavone was dihydroxylated by BphA at ortho-fluorinated and meta positions of ring B, with concomitant dehalogenation leading to 7,2′,3′,-trihydroxy-8-methylisoflavone. Daidzein (7,4′-dihydroxyisoflavone) was biotransformed by BphA, generating 7,2′,4′-trihydroxyisoflavone after dehydration. Biotransformation products were analyzed by gas chromatography-mass spectrometry and nuclear magnetic resonance techniques.     

Formation of the P1.1 pseudoknot is critical for both the cleavage activity and substrate specificity of an antigenomic trans-acting hepatitis delta ribozyme

Hepatitis delta virus RNAs possess self-cleavage activities that produce 2′,3′-cyclic phosphate and 5′-hydroxyl termini (i.e. cis-acting delta ribozyme). Trans-acting delta ribozymes have been engineered by removing a junction from the cis version, thereby producing one molecule possessing the substrate sequence and the other the catalytic domain. According to the pseudoknot model, the secondary structure of the delta ribozyme includes a pseudoknot (i.e. P1.1 stem) formed by two base pairs from residues of the L3 loop and J1/4 junction. A collection of 48 P1.1 stem mutants was synthesized in order to provide an original characterization of both the importance and the structure of this pseudoknot in a trans-acting version of the ribozyme. Several structural differences were noted compared to the results reported for cis-acting ribozymes. For example, a combination of two stable Watson–Crick base pairs composing the essential P1.1 stem was demonstrated to be crucial for a significant level of activity, while the cis version required only one base pair. In addition, we present the first physical evidences revealing that the composition of the P1.1 stem affects the substrate specificity for ribozyme cleavage. Depending on the residues forming the J1/4 junction, non-productive ribozyme–substrate complexes can be observed. This phenomenon is proposed to be important for further development of a gene-inactivation system based on delta ribozyme.     

3,4-dihydroxy-2-hydroxymethylpyrrolidine from Arachniodes standishii

3,4-Dihydroxy-2-hydroxymethylpyrrolidine, which has not been encountered naturally before, was isolated from the Pteridophyte Arachniodes standishii. Its configuration was determined as 2,3-cis and 3,4-trans from NMR spectra.     

(+)-2,3-Trans-pubeschin,the first catechin analogue of peltogynoids from Peltogyne pubescens and P. venosa

The heartwoods of Peltogyne pubescens and P. venosa contain the predominant pair (+)-peltogynol and (+)-mopanol, their 4-epimers, (+)-peltogynol B and (+)-mopanol B, together with the first catechin analogue of peltogynol, (+)-2,3- trans-pubeschin. These are accompanied by ±-2,3-cis- and ±-2,3-trans-3-O-methylfustins, and by α, 2′,3,4,4′-pentahydroxychalcone. Other minor metabolises are 4′,7-dihydroxy- and 3′,4′,7-trihydroxy-flavanones and 5,6-dihydroxyphthalide. (+)-2,3-Trans-pubeschin trimethyl ether was synthesized by reduction of the corresponding (+)-2,3-trans-peltogynone analogue with NaBH₄/BF₃ in diglyme, and its absolute configuration shown to be 2R: 3S.