Chiral NMR analysis reveals the environmental dependence of areolal scalemization in Piptothrix areolare.

The occurrence of racemic and enantiomerically enriched (scalemic) mixtures of secondary metabolites in their natural sources is a rare phenomenon. The unprecedent case of enantiomeric variations from levorotatory to dextrorotatory, and back to levorotatory, passing through an almost racemic mixture, was recently documented for areolal, the major epoxythymol of Piptothrix areolare. In an attempt to shed some light to understand the reasons for such an unusual behavior, herein, we evaluated this phenomenon by correlating the areolal enantiomeric purity with several environmental variables, including temperature, humidity, rain precipitation, wind speed, and radiation during over 1 year of the plant life cycle. The specific rotation and enantiomeric excess determined by 1 H-NMR-BINOL measurements provided the scalemic variations of areolal samples isolated from the roots collected from the same location along a 427-day period. The 1 H-NMR-BINOL methodology provided better sensitivity to enantiomeric variations than specific rotation measurements. Statistical data, including matrix correlation analysis, exploratory analysis by heatmap plotting, and the principal component analysis (PCA), suggested direct correlation of the scalemic variation with humidity, rain precipitation, and radiation variables with the best PCA explanation (78.4%) and noncritical or poor correlations in PCA explained in 60.2% and 48.4%, respectively. When variations in the optical activity parameter of any metabolite are observed, the search for scalemic mixtures along their host plant life cycle should be undertaken. Herein, this phenomenon could be associated with interactions with soil microorganisms and with evolutionary aspects of Piptothrix areolare which belongs to Asteraceae, one of the most successfully adaptable plant families.

Novel Sesquiterpenoid Skeletons by Wagner-Meerwein Rearrangements of Longipinane-9,13-diol-1-one Derivatives.

The partial or total hydrolysis of (3R,4S,5S,6S,9R,10R,11R)-9,13-diangeloyloxylongipinan-1-one (1), isolated from the roots of Stevia viscida, gave alcohols 2 or 3, respectively, which were subjected to molecular rearrangements with boron trifluoride etherate. Compound 2 afforded (3R,4R,5R,6S,9R,10S,11S)-11,13-oxyneomorelian-1-one (10) and (4S,5R,6S,8S,10R)-10,13-oxyneojiquilp-2-en-1-one (11), both possessing novel sesquiterpenoid skeletons. In turn, 3 provided (3R,4R,5S,6S,9R,11R)-13-hydroxymoreli-10(14)-en-1-one (7) and 10. Acetylation of 3 gave 4, thus allowing reduction of the C-1 carbonyl group to yield 5, which was rearranged to (1S,3R,4S,5S,6S,9R,10R,11R)-13-acetoxy-9,11-epoxyjiquilpane (6), while an attempt to mesylate 3 directly gave rearranged (3R,4R,5S,6S,9R,11R)-13-mesyloxymoreli-10(14)-en-1-one (8) through expulsion of the C-9 mesylate group by the antiperiplanar C-4-C-10 bond migration to C-4-C-9. In addition, treatment of 1 with boron trifluoride etherate generated (3R,4R,5S,6S,9R,11R)-13-angeloyloxymoreli-10(14)-en-1-one (9). The structures of 2-11 were elucidated by 1D and 2D NMR experiments and those of 2, 3, 8, 10, and 11 were confirmed by single-crystal X-ray diffraction analysis.

Methodology for the Absolute Configuration Determination of Epoxythymols Using the Constituents of Piptothrix areolare.

Since epoxythymols occur in Nature either as scalemic mixtures or as pure enantiomers, the knowledge of their chiral composition and of the absolute configuration (AC) of the dominant enantiomer turns out to be mandatory. This task has already been faced using 1,1-bis-2-naphthol (BINOL), as a chiral solvating agent in accurate 1H NMR quantifications to determine the enantiomeric ratio, and vibrational circular dichroism (VCD) to evidence the AC of the dominant enantiomer. We now explore the use of electronic circular dichroism (ECD) to determine the AC of an epoxythymol for which time-expensive DFT calculations would be required unless the AC of a related molecule is already known, from either VCD studies or single-crystal X-ray diffraction analysis, since one could correlate the ECD Cotton effect with the AC because in ECD only chromophores and their neighborhoods are evidenced. This method is now applied by using the epoxythymols from Piptothrix areolare. Known areolal (1) and 10-cinnamoyloxy-8,9-epoxythymol isobutyrate (2) were isolated from the roots, while known 7-acetoxy-10-cinnamoyloxy-8,9-epoxythymol isobutyrate (3) and 10-cinnamoyloxy-7-hydroxy-8,9-epoxythymol isobutyrate (4), as well as the new enantiopure 7-acetoxy-10-cinnamoyloxy-6-hydroxy-8,9-epoxythymol isobutyrate (5) and 10-cinnamoyloxy-8,9-epoxy-6-hydroxy-7-northymol isobutyrate (6), were obtained from the extract of the flowers. Chemical correlation of epoxythymols 1 and 3 was achieved. Compounds 1-4 were obtained as scalemic mixtures, and 5 and 6 as the pure (8S) enantiomers. In addition, the new 10-cinnamoyloxy-7-oxo-8,9-dehydrothymol isobutyrate (7) was isolated from the roots. The structures of 5-7 followed from NMR and HRMS data, while enantiomeric compositions of 1-6 were determined by 1H NMR-BINOL measurements. The AC determination for 2-6 was done by ECD using a sample of 1 to reference the ECD Cotton effect. In turn, the AC of 1 was determined by VCD and extensive DFT calculations. The ECD-BINOL methodology turned out to be some 500 times more sensitive than that combining VCD and 1H NMR-BINOL.

Tubulin Inhibitors: A Chemoinformatic Analysis Using Cell-Based Data.

Inhibiting the tubulin-microtubules (Tub-Mts) system is a classic and rational approach for treating different types of cancers. A large amount of data on inhibitors in the clinic supports Tub-Mts as a validated target. However, most of the inhibitors reported thus far have been developed around common chemical scaffolds covering a narrow region of the chemical space with limited innovation. This manuscript aims to discuss the first activity landscape and scaffold content analysis of an assembled and curated cell-based database of 851 Tub-Mts inhibitors with reported activity against five cancer cell lines and the Tub-Mts system. The structure-bioactivity relationships of the Tub-Mts system inhibitors were further explored using constellations plots. This recently developed methodology enables the rapid but quantitative assessment of analog series enriched with active compounds. The constellations plots identified promising analog series with high average biological activity that could be the starting points of new and more potent Tub-Mts inhibitors.

Parvifoline Derivatives as Tubulin Polymerization Inhibitors.

A series of functionalized sesquiterpenoids derived from benzocyclooctene, including natural parvifoline (1), isoparvifoline (3), epoxyparvifoline (5), epoxyisoparvifoline (7), 8,12-oxyparfivoline (9), 8,14-oxyparvifoline (11), and the respective benzoyl derivatives 2, 4, 6, 8, 10, and 12, were prepared and tested for their inhibitory effect on the in vitro α,ß-tubulin polymerization process. The structural analysis and characterization of the new compounds 5-7 and 9-12 were achieved by 1D and 2D NMR spectroscopy, mass spectrometry, and X-ray diffraction analysis of 6, 7, and 9. Preparation of 9 and 12 involved molecular rearrangements of the epoxide group with transannular 1,5-hydride shifts. At 10 µM compounds 1, 5, and 8 inhibited the polymerization of the α,ß-tubulin heterodimer by 24%, 49%, and 90% as compared to colchicine. These compounds were subjected to docking analysis that supported their interactions in a colchicine binding site located in the α-tubulin subunit, in the pocket formed by Phe296, Pro298, Pro307, His309, Tyr312, Lys338, Thr340, Ile341, and Gln342. Competitive inhibition assays with colchicine were also performed for the three compounds, which supported their binding at the colchicine secondary site in α-tubulin. Also, evaluations of their cytotoxicity on MCF7 breast carcinoma, HeLa cervix carcinoma, and HCT 116 colon carcinoma cell lines were carried out and showed that 8 is active against the HeLa and HCT 116 cell lines with IC50 3.3 ± 0.2 and 5.0 ± 0.5 µM, respectively.

Biomimetic Transformation of p-Menthene Glucosides into p-Cymenes and Carvotanacetone.

A biomimetic transformation of p-menthene glucosides into aromatic monoterpenoids that alluded to mechanisms for essential oil metabolism, which lines up with the precepts of molecular economy, is described. Acid treatment of (-)-(3 S,4 S,6 R)-3,6-dihydroxy-1-menthene 3- O-ß-d-glucopyranoside (1) and (-)-(3 S,4 R,5 R,6 S)-3,5,6-trihydroxy-1-menthene 3- O-ß-d-glucopyranoside (2), from Ageratina glabrata, yielded p-cymene (7) and carvacrol (9). The stable oxidized intermediates (+)-(3 S,4 S,6 R)-3,6-dihydroxy-1-menthene (3), (+)-(1 S,4 S,6 R)-1,6-dihydroxy-2-menthene (4), (+)-(1 R,4 S,6 R)-1,6-dihydroxy-2-menthene (5), (+)-(4 S,6 R)-yabunikkeol (6), (+)-(4 S)-carvotanacetone (8), (+)-(1 S,4 S,5 R,6 R)-1,5,6-trihydroxy-2-menthene (15), (+)-(1 R,4 S,5 R,6 R)-1,5,6-trihydroxy-2-menthene (16), and the new (+)-(4 S,5 R,6 S)-1(7),2-menthadiene (17) permitted establishment of the reaction mechanisms. The reactivity of the hydroxy groups of 4 and 5, as well as those of 15 and 16, was compared by acetylation reactions and supported by DFT calculations, revealing diminished reactivity in 4 and 15 due to the cis configuration of their hydroxy groups at C-1 and C-6. In addition, p-cymene (7) was detected as one of the major constituents of the essential oil of A. glabrata, which matches well with the biomimetic study.

Structure Elucidation, Conformation, and Configuration of Cytotoxic 6-Heptyl-5,6-dihydro-2 H-pyran-2-ones from Hyptis Species and Their Molecular Docking to α-Tubulin.

Cytotoxic 6-heptyl-5,6-dihydro-2 H-pyran-2-ones are chemical markers of Hyptis (Lamiaceae) and are responsible for some of the therapeutic properties of species with relevance to traditional medicine. The present investigation describes the isolation of known pectinolides A-C (1-3), in addition to the new pectinolides I-M (4-8), from two Mexican collections of H. pectinata by HPLC. The novel biosynthetically related monticolides A (9) and B (10) were also isolated by high-speed countercurrent chromatography from H. monticola, an endemic species of the Brazilian southeastern high-altitude regions. A combination of chemical correlations, chiroptical measurements, and Mosher ester NMR analysis was used to confirm their absolute configuration. The utility of DFT-NMR chemical shifts and JH-H calculations was assessed for epimer differentiation. Molecular docking studies indicated that 6-heptyl-5,6-dihydro-2 H-pyran-2-ones have a high affinity for the pironetin-binding site of α-tubulin, which may be a possible mechanism contributing to the cytotoxic potential of these small and flexible molecules.

Novel Sesquiterpene Skeletons by Multiple Wagner-Meerwein Rearrangements of a Longipinane-1,9-diol Derivative.

The tricyclic sesquiterpene (1R,3R,4S,5S,7S,8S,9S,10R,11R)-7,8-diangeloyloxylongipinan-1,9-diol, or rasteviol (7), underwent multiple Wagner-Meerwein molecular rearrangements and several hydride shifts when treated with Et2O-BF3 to generate the six new compounds (1R,3R,4S,5R,7S,8S,9S,10R,11S)-7,8-diangeloyloxy-1,9-epoxyjiquilpane (8), (1R,3R,4S,5R,7R,8S,9S,11S)-8-angeloyloxy-1,7-epoxyzamor-10(14)-ene (11), (2S,3R,4R,5R,6R,7R,8S,9S,10S)-7,8-diangeloyloxy-6,9-epoxyjanitziane (14), (4R,5R,7S,8S,9S,10S,11S)-7,8-diangeloyloxy-9-hydroxyjiquilp-3(15)-ene (16), (2S,3S,5R,7S,8R,10S,11R)-7,8-diangeloyloxyiratzian-9-one (18), and (2S,3S,5R,10S,11R)-8-angeloyloxyiratzi-7-en-9-one (22), of which 8, 11, 14, and 18 possess new hydrocarbon skeletons. Their structures were determined by 1D and 2D NMR in combination with single-crystal X-ray diffraction analyses of derivatives 10, 15, 20, and 21, which allowed confirmation of their absolute configurations by means of the Flack and Hooft parameters. In addition, some reaction mechanism information was gained from deuterium labeling experiments.

Configurational Variation of a Natural Compound within Its Source Species. The Unprecedented Case of Areolal in Piptothrix areolare.

The exceptional case of a natural compound that shows drastic absolute configuration variations within the same species was examined. Sequential samples of areolal (1) isolated from Piptothrix areolare showed dextrorotatory (ee 32%), almost racemic (ee 4%), levorotatory (ee 82%), and again dextrorotatory (ee 10%) values. Enantiomeric compositions of this epoxythymol derivative were determined from individual plant specimens collected from the same geographical location over a 46-day period, which were processed using the same extraction and isolation methods. Detection of this unusual phenomenon was possible by analysis of NMR data recorded in the presence of BINOL as a chiral solvating agent. The absolute configuration of (-)-(8S)-areolal followed from vibrational circular dichroism data of an enantiomerically enriched sample, while single-crystal X-ray diffraction and supramolecular analyses revealed interactions that diminish the crystal entropy in rac-1. These results might be related with environmental factors and biochemical processes, suggesting the need of strict evaluations of enantiomeric composition of natural products that could be considered for human applications.

Conformational, configurational, and supramolecular studies of podocephalol acetate from Lasianthaea aurea.

Although podocephalol (1) and its derived acetate 2 were found in Lasianthaea podocephala four decades ago, and 1 was later detected in the essential oils of several vegetal species, its absolute configuration (AC) and conformational preferences remained to be established. The structures of ar-himachalene 1, now isolated from Lasianthaea aurea, and its derived acetate 2, were herein confirmed by extensive 1D and 2D nuclear magnetic resonance (NMR) studies, while the conformational preferences of the cycloheptene was established by density functional theory (DFT) calculations, which in combination with vibrational circular dichroism measurements provided the (R) absolute configuration of the molecules. The structure and AC were further verified through the Flack and Hooft parameters calculations derived from single crystal X-ray diffraction data of 2. In addition, careful evaluation of the crystal data allowed observing supramolecular layers cell package, an uncommon property in natural terpenes that might have potential applications. A transmission electron microscopy analysis of crystal of 2 was also possible, providing its physical characteristics at the micrometric scale.

Absolute configuration of phomactatriene diterpenoids obtained by Wagner-Meerwein rearrangement of epimeric verticillols.

The epimeric diterpenes (+)-(1S,3E,7E,11S,12S)-verticilla-3,7-dien-12-ol (1), isolated from Bursera suntui, and (+)-(1S,3E,7E,11S,12R)-verticilla-3,7-dien-12-ol (2), isolated from Bursera kerberi, gave the same Wagner-Meerwein rearrangement product (-)-(1E,4Z,8Z,11S,12R)-phomacta-1,(15)4,8-triene (3). The Et2 O:BF3 -induced transformations evidence that verticillenes and phomactanes, both containing the bicyclo[9.3.1]pentadecane skeleton, are biogenetically related through the verticillen-12-yl cation (A+ ), which also is a key intermediate in the biosynthetic pathways to generate antitumor taxanes. Molecular modeling using the Monte Carlo protocol, followed by density functional theory (DFT) geometry optimization employing the hybrid functionals B3LYP and B3PW91, both with the DGDZVP basis set, secured the configuration of 3 as followed from the good agreement between the calculated and experimental vibrational circular dichroism spectra. Similar DFT calculations allowed determining the absolute configuration of (+)-(1R,4R,5R,8S,9S,11S,12R,15R)-1,15:4,5:8,9-triepoxyphomactane (9), which surprisingly derives from epoxidation of the second minimum energy conformer of 3.

Rebaudioside A administration prevents experimental liver fibrosis: an in vivo and in vitro study of the mechanisms of action involved.

Rebaudioside A (Reb A) is a diterpenoid isolated from the leaves of Stevia rebaudiana (Bertoni) that has been shown to possess pharmacological activity, including anti-inflammatory and antioxidant properties. However, the ability of Reb A to prevent liver injury has not been evaluated. Therefore, we aimed to study the potential of Reb A (20 mg/kg; two times daily intraperitoneally) to prevent liver injury induced by thioacetamide (TAA) administration (200 mg/kg; three times per week intraperitoneally). In addition, cocultures were incubated with either lipopolysaccharide or ethanol. Antifibrotic, antioxidant and immunological responses were evaluated. Chronic TAA administration produced considerable liver damage and distorted the liver parenchyma with the presence of prominent thick bands of collagen. In addition, TAA upregulated the expression of α-smooth muscle actin, transforming growth factor-ß1, metalloproteinases 9, 2 and 13, and nuclear factor kappaB and downregulated nuclear erythroid factor 2. Reb A administration prevented all of these changes. In cocultured cells, Reb A prevented the upregulation of genes implicated in fibrotic and inflammatory processes when cells were exposed to ethanol and lipopolysaccharide. Altogether, our results suggest that Reb A prevents liver damage by blocking oxidative processes via upregulation of nuclear erythroid factor 2, exerts immunomodulatory effects by downregulating the nuclear factor-κB system and acts as an antifibrotic agent by maintaining collagen content.

Methodology for the Absolute Configuration Determination of Epoxythymols Using the Constituents of Ageratina glabrata.

A methodology to determine the enantiomeric excess and the absolute configuration (AC) of natural epoxythymols was developed and tested using five constituents of Ageratina glabrata. The methodology is based on enantiomeric purity determination employing 1,1'-bi-2-naphthol (BINOL) as a chiral solvating agent combined with vibrational circular dichroism (VCD) measurements and calculations. The conformational searching included an extensive Monte Carlo protocol that considered the rotational barriers to cover the whole conformational spaces. (+)-(8S)-10-Benzoyloxy-6-hydroxy-8,9-epoxythymol isobutyrate (1), (+)-(8S)-10-acetoxy-6-methoxy-8,9-epoxythymol isobutyrate (4), and (+)-(8S)-10-benzoyloxy-6-methoxy-8,9-epoxythymol isobutyrate (5) were isolated as enantiomerically pure constituents, while 10-isobutyryloxy-8,9-epoxythymol isobutyrate (2) was obtained as a 75:25 (8S)/(8R) scalemic mixture. In the case of 10-benzoyloxy-8,9-epoxythymol isobutyrate (3), the BINOL methodology revealed a 56:44 scalemic mixture and the VCD measurement was beyond the limit of sensitivity since the enantiomeric excess is only 12%. The racemization process of epoxythymol derivatives was studied using compound 1 and allowed the clarification of some stereochemical aspects of epoxythymol derivatives since their ACs have been scarcely analyzed and a particular behavior in their specific rotations was detected. In more than 30 oxygenated thymol derivatives, including some epoxythymols, the reported specific rotation values fluctuate from -1.6 to +1.4 passing through zero, suggesting the presence of scalemic and close to racemic mixtures, since enantiomerically pure natural constituents showed positive or negative specific rotations greater than 10 units.

Complementarity of DFT Calculations, NMR Anisotropy, and ECD for the Configurational Analysis of Brevipolides K-O from Hyptis brevipes.

Brevipolides K-O (1-5), five new cytotoxic 6-(6'-cinnamoyloxy-2',5'-epoxy-1'-hydroxyheptyl)-5,6-dihydro-2H-pyran-2-ones (IC50 values against six cancer cell lines, 1.7-10 µM), were purified by recycling HPLC from Hyptis brevipes. The structures, containing a distinctive tetrahydrofuran ring, were established by comprehensive quantum mechanical calculations and experimental spectroscopic analysis of their NMR and ECD data. Detailed analysis of the experimental NMR 1H-1H vicinal coupling constants in comparison with the corresponding DFT-calculated values at the B3LYP/DGDZVP level confirmed the absolute configuration of 3 and revealed its conformational preferences, which were further strengthened by NOESY correlations. NMR anisotropy experiments by the application of Mosher's ester methodology and chemical correlations were also used to conclude that this novel brevipolide series (1-5) share the same absolute configuration corresponding to C-6(R), C-1'(S), C-2'(R), C-5'(S), and C-6'(S).

Complete 1 H NMR assignment of cedranolides.

Complete and unambiguous 1 H NMR chemical shift assignment of α-cedrene (2) and cedrol (9), as well as for α-pipitzol (1), isocedrol (10), and the six related compounds 3-8 has been established by iterative full spin analysis using the PERCH NMR software (PERCH Solutions Ltd., Kuopio, Finland). The total sets of coupling constants are described and correlated with the conformational equilibria of the five-membered ring of 1-10, which were calculated using the complete basis set method. Copyright © 2015 John Wiley & Sons, Ltd.

Absolute Configuration of Menthene Derivatives by Vibrational Circular Dichroism.

The aerial parts of Ageratina glabrata afforded (-)-(3S,4R,5R,6S)-3,5,6-trihydroxy-1-menthene 3-O-ß-d-glucopyranoside (1) and (-)-(3S,4S,6R)-3,6-dihydroxy-1-menthene 3-O-ß-d-glucopyranoside (3). Acid hydrolysis of 1 yielded (+)-(1R,4S,5R,6R)-1,5,6-trihydroxy-2-menthene (5) and (+)-(1S,4S,5R,6R)-1,5,6-trihydroxy-2-menthene (6), while hydrolysis of 3 yielded (+)-(3S,4S,6R)-3,6-dihydroxy-1-menthene (10), (+)-(1R,4S,6R)-1,6-dihydroxy-2-menthene (11), and (+)-(1S,4S,6R)-1,6-dihydroxy-2-menthene (12). The structures of the new compounds 1, 2, 5-9, and 11 were defined by 1D and 2D NMR experiments, while the absolute configurations of the series of compounds were determined by comparison of the experimental vibrational circular dichroism (VCD) spectra of the 1,6-acetonide 5-acetate derived from 6 and of the 1,6-acetonide derived from 12 with their DFT-calculated spectra. In addition, Flack and Hooft X-ray parameters of 10 permitted the same conclusion. The results further led to the absolute configuration reassignment of 10 isolated from Brickellia rosmarinifolia, Mikania saltensis, Ligularia muliensis, L. sagitta, and Lindera strychnifolia, as well as of 11 from Cacalia tangutica, as ent-11.

Carotenoid production and gene expression in an astaxanthin-overproducing Xanthophyllomyces dendrorhous mutant strain.

The primary carotenoid synthesized by Xanthophyllomyces dendrorhous is astaxanthin, which is used as a feed additive in aquaculture. Cell growth kinetics and carotenoid production were correlated with the mRNA levels of the idi, crtE, crtYB, crtI, crtS and crtR genes, and the changes in gene sequence between the wild-type and a carotenoid overproducer XR4 mutant strain were identified. At the late stationary phase, the total carotenoid content in XR4 was fivefold higher than that of the wild-type strain. Additionally, the mRNA levels of crtE and crtS increased during the XR4 growth and were three times higher than the wild-type strain in the late stationary phase. Moreover, the nucleotide sequences of crtYB, crtI and crtR exhibited differences between the strains. Both the higher crtE and crtS transcript levels and the crtYB, crtI and crtR mutations can, at least in part, act to up-regulate the carotenoid biosynthesis pathway in the XR4 strain.

Long-term response on growth, antioxidant enzymes, and secondary metabolites in salicylic acid pre-treated Uncaria tomentosa microplants.

OBJECTIVE: To obtain micro propagated Uncaria tomentosa plantlets with enhanced secondary metabolites production, long-term responses to salicylic acid (SA) pre-treatments at 1 and 100 µM were evaluated after propagation of the plantlets in a SA-free medium. RESULTS: SA pre-treatments of single node cuttings OF U. tomentosa produced long-term responses in microplants grown for 75 days in a SA-free medium. Reduction in survival rate, root formation, and stem elongation were observed only with 100 µM SA pre-treatments with respect to the control (0 + DMSO).Both pre-treatments enhanced H2O2 and inhibited superoxide dismutase and catalase activities, while guaiacol peroxidase was increased only with 1 µM SA. Also, both pre-treatments increased total monoterpenoid oxindole alkaloids by ca. 55 % (16.5 mg g(-1) DW), including isopteropodine, speciophylline, mitraphylline, isomitraphylline, rhynchopylline, and isorhynchopylline; and flavonoids by ca. 21 % (914 µg g(-1) DW), whereas phenolic compounds were increased 80 % (599 µg g(-1) DW) at 1 µM and 8.2 % (359 µg g(-1) DW) at 100 µM SA. CONCLUSION: Pre-treatment with 1 µM SA of U.tomentosa microplants preserved the survival rate and increased oxindole alkaloids, flavonoids, and phenolic compounds in correlation with H2O2 and peroxidase activity enhancements, offering biotechnological advantages over non-treated microplants.

DFT 1H-1H coupling constants in the conformational analysis and stereoisomeric differentiation of 6-heptenyl-2H-pyran-2-ones: configurational reassignment of synargentolide A.

Density functional theory (DFT) (1) H-(1) H NMR coupling constant calculations, including solvation parameters with the polarizable continuum model B3LYP/DGDZVP basis set together with the experimental values measured by spectral simulation, were used to predict the configuration of hydroxylated 6-heptenyl-5,6-dihydro-2H-pyran-2-ones 1, 2, 4, and 7, allowing epimer differentiation. Modeling of these flexible compounds requires the inclusion of solvation models that account for stabilizing interactions derived from intramolecular and intermolecular hydrogen bonds, in contrast with peracetylated derivatives (3, 5, and 6) in which the solvation consideration can be omitted. Using this DFT NMR integrated approach as well as spectral simulation, the configurational reassignment of synargentolide A (8) was accomplished by calculations in the gas phase among four possible diastereoisomers (8-11). Calculated (3) JH,H values established its configuration as 6R-[4'S,5'S,6'S-(triacetyloxy)-2E-heptenyl]-5,6-dihydro-2H-pyran-2-one (8), in contrast with the incorrect 6R,4'R,5'R,6'R-diastereoisomer previously proposed by synthesis (12). Application of this approach increases the probability for successful enantiospecific total syntheses of flexible compounds with multiple chiral centers.

Analysis of proteomic changes in colored mutants of Xanthophyllomyces dendrorhous (Phaffia rhodozyma).

The yeast Xanthophyllomyces dendrorhous synthesizes astaxanthin as its most prevalent xanthophyll derivative. Comparisons between the protein profiles of mutant lines of this yeast can provide insight into the carotenogenic pathway. Differently colored mutants (red, orange, pink, yellow, and white) were obtained from this yeast species, and their protein profiles were determined using two-dimensional polyacrylamide gel electrophoresis (2DE). Individual proteins differentially expressed were identified using mass spectrometry. The red mutants hyperproduced total carotenoids (mainly astaxanthin), while in white and orange mutants, mutagenesis affected the phytoene dehydrogenase activity as indicated by the accumulation of phytoene. Inactivation of astaxanthin synthase after the mutagenic treatment was evident in ß-carotene accumulating mutants. Differences in the proteomic profiles of wild-type X. dendrorhous and its colored mutants were demonstrated using 2DE. Of the total number of spots detected in each gel (297-417), 128 proteins were present in all strains. The red mutant showed the greatest number of matches with respect to the wild type (305 spots), while the white and yellow mutants, which had reduced concentrations of total carotenoids, presented the highest correlation coefficient (0.6) between each other. A number of differentially expressed proteins were sequenced, indicating that tricarboxylic acid cycle and stress response proteins are closely related to the carotenogenic process.