Discovery of Prenyltransferase-Guided Hydroxyphenylacetic Acid Derivatives from Marine Fungus Penicillium sp. W21C371.

Traditional isolation methods often lead to the rediscovery of known natural products. In contrast, genome mining strategies are considered effective for the continual discovery of new natural products. In this study, we discovered a unique prenyltransferase (PT) through genome mining, capable of catalyzing the transfer of a prenyl group to an aromatic nucleus to form C-C or C-O bonds. A pair of new hydroxyphenylacetic acid derivative enantiomers with prenyl units, (±)-peniprenydiol A (1), along with 16 known compounds (2-17), were isolated from a marine fungus, Penicillium sp. W21C371. The separation of 1 using chiral HPLC led to the isolation of the enantiomers 1a and 1b. Their structures were established on the basis of extensive spectroscopic analysis, including 1D, 2D NMR and HRESIMS. The absolute configurations of the new compounds were determined by a modified Mosher method. A plausible biosynthetic pathway for 1 was deduced, facilitated by PT catalysis. In the in vitro assay, 2 and 3 showed promising inhibitory activity against Escherichia coli ß-glucuronidase (EcGUS), with IC50 values of 44.60 ± 0.84 µM and 21.60 ± 0.76 µM, respectively, compared to the positive control, D-saccharic acid 1,4-lactone hydrate (DSL). This study demonstrates the advantages of genome mining in the rational acquisition of new natural products.

New Hydroxyphenylacetic Acids and α-Pyrone Derivative from the Deep-Sea Cold Seep Sediment-Derived Fungus Penicillium corylophilum CS-682.

Two pairs of new enantiomeric hydroxyphenylacetic acid derivatives, (±)-corylophenols A and B ((±)-1 and (±)-2), a new α-pyrone analogue, corylopyrone A (3), and six andrastin-type meroterpenoids (4-9) were isolated and identified from the deep-sea cold-seep sediment-derived fungus Penicillium corylophilum CS-682. Their structures and stereo configurations were determined by detailed spectroscopic analysis of NMR and MS data, chiral HPLC analysis, J-based configuration analysis, and quantum chemical calculations of ECD, specific rotation, and NMR (with DP4+ probability analysis). Compound 3 showed inhibitory activity against some strains of pathogenic bacteria.

Secondary Metabolites from the Leather Coral-Derived Fungal Strain Xylaria sp. FM1005 and Their Glycoprotein IIb/IIIa Inhibitory Activity.

Five new tyrosine derivatives (1-5), one new phenylacetic acid derivative (6), two new quinazolinone analogues (7 and 8), one new naphthalenedicarboxylic acid (9), and one new 3,4-dihydroisocoumarin derivative (10), together with seven known compounds, were isolated from the fungus Xylaria sp. FM1005, which was isolated from Sinularia densa (leather coral) collected in the offshore region of the Big Island, Hawaii. The structures of compounds 1-10 were elucidated by extensive analysis of NMR spectroscopy, HRESIMS, and ECD data. Due to their structure similarity to the antiplatelet drug tirofiban, compounds 1-5 together with 6 were investigated for their antithrombotic activities. Compounds 1 and 2 strongly inhibited the binding of fibrinogen to purified integrin IIIb/IIa in a dose-dependent manner with the IC50 values of 0.89 and 0.61 µM, respectively, and compounds 1 and 2 did not show any cytotoxicity against A2780 and HEK 293 at 40 µM.

Larvicidal and ovicidal activities of phenyl acetic acid isolated from Streptomyces collinus against Culex quinquefasciatus Say and Aedes aegypti L. (Diptera: Culicidae).

The bio-efficacy of crude ethyl acetate extract, fractions and a compound phenyl acetic acid from the ethyl acetate extract of Streptomyces collinus was evaluated on Culex quinquefasciatus Say and Aedes aegypti L. mosquitoes (Diptera: Culicidae). The larvae were exposed to concentrations of 2.5, 5.0, 7.5 and 10.0 ppm for fractions and 0.5, 1.0, 1.5 and 2.0 ppm for compound. After 24 h, the larval mortality was assessed and the LC50 and LC90 values were calculated. Similarly, per cent ovicidal activity was calculated for eggs after 120 h post treatment for phenyl acetic acid. Among the eleven fractions screened, fraction 7 from the ethyl acetate extract of Streptomyces collinus exhibited good larvicidal activity against both mosquito species. The LC50 and LC90 values of fraction 7 were 4.42, 6.23 ppm against Cx. quinquefasciatus larvae and 5.13, 14.51 ppm against Ae. aegypti larvae, respectively. Further, the isolated compound, phenyl acetic acid from fraction 7 recorded 100% larvicidal activity at 2 ppm concentration with LC50 and LC90 values of 2.07, 4.87 ppm on Cx. quinquefasciatus larvae and 3.81, 9.87 ppm on Ae. aegypti larvae, respectively. Phenyl acetic acid presented 50.3% and 42.0% ovicidal activity against Cx. quinquefasciatus and Ae. aegypti eggs at 2 ppm concentration after 120 h post treatment. The compound, phenyl acetic acid could be used in mosquito control programme.

Three New Hydroxyphenylacetic Acid Derivatives and A New Alkaloid from Endophytic Fungus Mortierella sp. in Epimedium acuminatum Franch. and Their Antibacterial Activity.

Three new hydroxyphenylacetic acid derivatives, stachylines E-G (1-3), and a new alkaloid, mortieridinone (4), along with six known compounds (5-10), were isolated from endophytic fungus Mortierella sp. in Epimedium acuminatum Franch. Their structures were determined by their spectroscopic analyses and by comparison with the literature data. Compounds 7 and 10 showed selective antibacterial activity against tested multidrug-resistant bacteria with minimum inhibitory concentration (MIC) values ranging from 25 to 3.13 µg/mL.

Stability study of α-bromophenylacetic acid: Does it represent an appropriate model analyte for chiral separations?

The stability of α-bromophenylacetic acid (BPAA) in 50% aqueous methanol solution has been tested. CE in different running buffers was used to separate BPAA from the decomposition reaction products α-hydroxyphenylacetic (mandelic) acid and α-methoxyphenylacetic acid. Suitable CE separation of all three compounds and other product, bromide, was achieved in 60 mmol/L formate buffer (pH 3.0) at -30 kV in 50 µm (i.d.) poly(vinyl alcohol)-coated fused silica capillary (30 cm/24.5 cm) with UV detection at 200 nm. The CE method was applied to determine the reaction order of the decomposition of BPAA (0.47 mmol/L) via nucleophilic substitution in 50% aqueous methanol. The first-order reaction kinetics was confirmed by linear and non-linear regression, giving the rate constants 1.52 × 10-4 ± 2.76 × 10-5 s-1 and 7.89 × 10-5 ± 5.02 × 10-6 s-1, respectively. Additionally, the degradation products were identified by CE coupled to mass spectrometric (MS) detection. The CE-MS experiments carried out in 60 mmol/L formate buffer (pH 3.0) and in 60 mmol/L acetate buffer (pH 5.0) confirmed the results obtained by CE-UV. Furthermore, the stability of BPAA in polar solvents was tested by 1H NMR experiments. Our results provide strong evidence of the instability and fast degradation of BPAA in 50% aqueous methanol indicating that BPAA is not suitable as the model analyte for chiral separations.

The characteristic smell emitted from two scale insects, Ceroplastes japonicus and Ceroplastes rubens.

The volatile components emitted from two scale insects, Ceroplastes japonicus and Ceroplastes rubens, were identified using GC-MS analysis. The major volatile components of the solvent extract from C. japonicus were α-humulene (35.8%) and δ-cadinene (17.0%), while those of C. rubens were ß-selinene (10.3%) and ß-elemene (5.1%). In GC/olfactometry, linalool, butyric acid, 3-methylbutyric acid, 2-methylbutyric acid, and vanillin were identified as the odor-active components of the extract from C. japonicus, in addition to trace amounts of trans-4,5-epoxy-(2E)-decenal, 4-methyl-(3E)-hexenoic acid, and phenylacetic acid. With regard to C. rubens, trans-4,5-epoxy-(2E)-decenal, 3-methylbutyric acid, and phenylacetic acid were identified as the odor-active components. Besides, decan-1,4-olide (γ-decalactone) with milky cherry-like note and 3-hydroxy-4,5-dimethylfuran-2(5H)-one (sotolone) with brown sugar-like note were also detected as the characteristic cherry-like sweet-and-sour note of these two scale insects. ABBREVIATIONS: GC: Gas chromatography; GC/O: gas chromatography/olfactometry.

Retention Modelling of Phenoxy Acid Herbicides in Reversed-Phase HPLC under Gradient Elution.

Phenoxy acid herbicides are used worldwide and are potential contaminants of drinking water. Reversed phase high-performance liquid chromatography (RP-HPLC) is commonly used to monitor phenoxy acid herbicides in water samples. RP-HPLC retention of phenoxy acids is affected by both mobile phase composition and pH, but the synergic effect of these two factors, which is also dependent on the structure and pKa of solutes, cannot be easily predicted. In this paper, to support the setup of RP-HPLC analysis of phenoxy acids under application of linear mobile phase gradients we modelled the simultaneous effect of the molecular structure and the elution conditions (pH, initial acetonitrile content in the eluent and gradient slope) on the retention of the solutes. In particular, the chromatographic conditions and the molecular descriptors collected on the analyzed compounds were used to estimate the retention factor k by Partial Least Squares (PLS) regression. Eventually, a variable selection approach, Genetic Algorithms, was used to reduce the model complexity and allow an easier interpretation. The PLS model calibrated on the retention data of 15 solutes and successively tested on three external analytes provided satisfying and reliable results.

Comparative Studies on Polysaccharides, Triterpenoids, and Essential Oil from Fermented Mycelia and Cultivated Sclerotium of a Medicinal and Edible Mushroom, Poria Cocos.

Poria cocos, an important medicinal and edible fungus, is well known in East Asia. The main active components are water-soluble polysaccharides (WPS) and triterpenoids. Due to the growing market demand, long cultivation period, and consumption of pine trunk during cultivation, alternative methods for producing P. cocos or its active components should be investigated. In this study, WPS, triterpenoids, monosaccharide composition, and essential oil in fermented mycelia and cultivated sclerotium were analyzed using UV spectrophotometry, HPLC, pre-column derivatization, and HS-GC/MS, respectively. Our results showed that the WPS and triterpenoids in mycelia are several times higher than those in sclerotium. Among the 62 compounds identified by HS-GC/MS analysis from the essential oil obtained from the fermentation media and a fresh external layer, the two main fragrances in common were linalool and methyl phenylacetate. Our results suggested that it is applicable to produce polysaccharides and triterpenoids by the fermentation of P. cocos, and a strategy to improve triterpenoid production in the fermentation process was proposed.

Why chartreuse? The pigment vulpinic acid screens blue light in the lichen Letharia vulpina.

MAIN CONCLUSION: Chlorophyll fluorescence, infrared gas exchange and photoinhibition data consistently show that vulpinic acid in L. vulpina functions as a strong blue light screening compound. The cortical lichen compounds, parietin, atranorin, usnic acid and melanins are known to screen photosynthetically active radiation (PAR), thereby protecting the underlying photobionts. The role of the toxic UV-/blue light-absorbing vulpinic acid in lichen cortices is poorly documented. By comparing controls with acetone-rinsed Letharia vulpina thalli (75% reduced vulpinic acid concentration), we aimed to test PAR screening by vulpinic acid. We exposed such thalli to blue, green and red irradiance, respectively, and recorded light quality-specific light saturation curves of CO2 uptake, quantum yields of CO2 uptake (QYCO2) and effective quantum yields of PSII (ΦPSII). We also quantified light quality-dependent photoinhibition after 4-h exposure to 400 µmol photons m-2 s-1. In controls, the greatest high light-induced reductions in CO2 uptake and ΦPSII, as well as the strongest photoinhibition [lowered maximal quantum yield of PSII (Fv/Fm)], occurred in red light, followed by green, and was low in blue light. Removal of vulpinic acid significantly exacerbated photoinhibition, reduced ΦPSII, and increased QYCO2 in blue light. By contrast, acetone rinsing had no or weak effects in green and red lights. Comparing control with acetone-rinsed thalli, blue light screening was estimated at 69% using ΦPSII data and 49% using QYCO2. To compensate for the 25% residual vulpinic acid left after rinsing, we repeated the screening estimation by comparing responses in blue and red lights. This resulted in 88% screening using ΦPSII data and 77% using QYCO2. The consistent responses in all photosynthetic parameters support the hypothesis that vulpinic acid functions as a blue light screen in L. vulpina.

Cablinosides A and B, Two Glycosidic Phenylacetic Acid Derivatives from the Leaves of Pogostemon cablin.

A pair of new glycosidic epimers, cablinosides A (1a) and B (1b) were isolated from the leaves of Pogostemon cablin. The structures with absolute configurations of 1a and 1b were elucidated by extensive NMR investigation, and quantum chemical CD calculations. The epimer mixture 1 showed moderate α-glucosidase inhibitory activity and no significant cytotoxic activity against HepG2 cells.

Specialized Metabolites of the Lichen Vulpicida pinastri Act as Photoprotective Agents.

The extreme resiliency of lichens to UV radiations makes them an interesting model to find new photoprotective agents acting as UV-blockers and antioxidant. In this research, using a new in vitro method designed to overcome the shortage of material associated to many studies dealing with natural products, we show that the three major compounds isolated from the lichen Vulpicida pinastri, vulpinic acid, pinastric acid and usnic acid, were UV blocker agents. Antioxidant assays evidenced superoxide anion scavenging activity. Combination of the most promising compounds against UVB and UVB radiations, usnic acid, vulpinic acid and pinastric acid, increased the photoprotective activity. At the same time, they were found not cytotoxic on keratinocyte cell lines and photostable in the UVA and UVB ranges. Thus, lichens represent an attractive source to find good candidate ingredients as photoprotective agents. Additionally, the uncommon scalemic usnic acid mixture in this Vulpicida species was proven through electronic circular dichroism calculation.

Letharia vulpina, a vulpinic acid containing lichen, targets cell membrane and cell division processes in methicillin-resistant Staphylococcus aureus.

CONTEXT: Antibiotic resistance in humans is a major concern. Drugs that target traditional sites and pathways are becoming obsolete; thus, compounds affecting novel targets are needed. Screening lichen metabolites for antimicrobials has yielded promising antimicrobial compounds, yet their mode of action is poorly understood. Letharia vulpina (L.) Hue (Parmeliaceae) has traditionally been used to poison predators, and treat stomach disorders; more recently L. vulpina extracts have demonstrated promising antimicrobial properties. OBJECTIVE: This study investigates the mode of action of L. vulpina acetone extract against a methicillin-resistant Staphylococcus aureus (MRSA). MATERIAL AND METHODS: We treated MRSA with L. vulpina extracts at 1×, 5×, and 10 × MIC values (MIC = 31.25 µg/ml) for 24 h and optical density (OD660) was measured over time to determine bacteriolytic activity; counted colony forming units (CFUs) to determine time kill dynamics; the propidium iodide (PI) assay and transmission electron microscopy were used to assess membrane-damage potential, and thin-layer chromatography was used to identify secondary compounds. RESULTS: Bacteriolytic assays showed that L. vulpina extracts, containing only vulpinic acid, do not cause cell lysis, even at 10 × MIC values but there was 92% reduction in bacterial CFUs when treated with increased concentrations of lichen extracts over 24 h at 4 h intervals. Our data indicate that the L. vulpina extract compromises membrane integrity of the MRSA isolate and disrupts cell division processes. DISCUSSION AND CONCLUSION: Based on this study, detailed examination of acetone extracts of L. vulpina as well as pure extracts of vulpinic acid as potential antibacterial compounds merit further study.

A new antibacterial octaketide and cytotoxic phenylethanoid glycosides from Pogostemon cablin (Blanco) Benth.

A new octaketide, named cytosporone V (1), and two other known phenylethanoid glycosides (2-3), were isolated from the aerial parts of Pogostemon cablin (Blanco) Benth. The structure of 1 was elucidated by a combination of extensive spectroscopic analyses, including extensive 2D NMR and HR-MS. Compounds 1-3 displayed weak antibacterial activity against two gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus. All isolates were also evaluated for their antiproliferative activities against four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15). Compounds 2 and 3 showed significant cytotoxicity against A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines with IC50 values ranging from 2.73 to 9.52 µM.

Anti-angiogenic and antiproliferative properties of the lichen substances (-)-usnic acid and vulpinic acid.

The anti-proliferative activities of the lichen substances (-)-usnic acid and vulpinic acid on the viability of HepG2 hepatocarcinoma cells, NS20Y neuroblastoma cells and HUVEC endothelial cells were studied by the MTT assay. The anti-angiogenic potential of the substances was determined by the endothelial tube formation assay. Both lichen substances exhibited strong anti-angiogenic activity and were more cytotoxic to the cancer cell lines than to the normal cell line, but vulpinic acid has more potential as an anti-angiogenic substance because of its low cytotoxicity and stronger anti-angiogenic activity on the HUVEC cell line.

Identification of the 5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivatives as highly selective PDE4B inhibitors.

A PDE4B subtype selective inhibitor is expected to have a wider therapeutic window than non-selective PDE4 inhibitors. In this Letter, two series of 7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivatives and 5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivatives were evaluated for their PDE4B subtype selectivity using human PDE4B2 and PDE4D2 full length enzymes. To improve their PDE4B selectivity over PDE4D, we optimized the substituents on the pyrimidine ring and the side chain phenyl ring, resulting in several derivatives with more than 100-fold selectivity for PDE4B. Consequently, we identified 2-(3-chloro-4-methoxy-phenyl)-5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivative 54 as a highly selective PDE4B inhibitor, which had potent hPDE4B inhibitory activity with an IC50 value of 3.0 nM and 433-fold PDE4B selectivity over PDE4D.

Removal of protein-bound, hydrophobic uremic toxins by a combined fractionated plasma separation and adsorption technique.

Protein-bound uremic toxins, such as phenylacetic acid, indoxyl sulfate, and p-cresyl sulfate, contribute substantially to the progression of chronic kidney disease (CKD) and cardiovascular disease (CVD). However, based on their protein binding, these hydrophobic uremic toxins are poorly cleared during conventional dialysis and thus accumulate in CKD-5D patients. Therefore, we investigated whether hydrophobic and cationic adsorbers are more effective for removal of protein-bound, hydrophobic uremic toxins than conventional high-flux hemodialyzer. Five CKD-5D patients were treated using the fractionated plasma separation, adsorption, and dialysis (FPAD) system for 5 h. A control group of five CKD patients was treated with conventional high-flux hemodialysis. Plasma concentrations of phenylacetic acid, indoxyl sulfate, and p-cresyl sulfate were measured. Removal rates of FPAD treatment in comparison to conventional high-flux hemodialysis were increased by 130% for phenylacetic acid, 187% for indoxyl sulfate, and 127% for p-cresol. FPAD treatment was tolerated well in terms of clinically relevant biochemical parameters. However, patients suffered from mild nausea 2 h after the start of the treatment, which persisted until the end of treatment. Due to the high impact of protein-bound, hydrophobic uremic toxins on progression of CKD and CVD in CKD-5D patients, the use of an adsorber in combination with dialysis membranes may be a new therapeutic option to increase the removal rate of these uremic toxins. However, larger, long-term prospective clinical trials are needed to demonstrate the impact on clinical outcome.

Induced production of methyl bromodihydroxyphenyl acetates by the marine-derived fungus Aspergillus sp.

The addition of metal bromides (NaBr and CaBr2) during fermentation of a marine isolate of the fungus Aspergillus sp. induced production of two new brominated dihydroxyphenylacetic acid derivatives, methyl 2-(6-bromo-3,4-dihydroxyphenyl)acetate (1) and methyl 2-(2,5-dibromo-3,4-dihydroxyphenyl)acetate (2), and a known compound, 2-(3,4-dihydroxyphenyl)acetic acid (3). The structures of the two new compounds (1, 2) were assigned through the combination of spectroscopic data analyses and comparison with the spectral data of compound 3. Compounds 1-3 exhibited potent radical-scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) with IC50 values (14.2, 12.1, 11.0 µm, respectively) demonstrating greater activity than the positive control (l-ascorbic acid; IC50, 20.0 µm).

Two new derivatives of 2, 5-dihydroxyphenylacetic acid from the kernel of Entada phaseoloides.

Two new aromatic compounds, butyl 2,5-dihydroxyphenylacetate (1) and butyl 2-O-ß-D-glucopyranosyloxy-5-dihydroxyphenylacetate (2), together with three known ones, methyl 2,5-dihydroxyphenylacetate (3), ethyl 2,5-dihydroxyphenylacetate (4) and 2-O-ß-D-glucopyranosyloxy-5-hydroxyphenylacetic acid (5), were isolated from the EtOH extract of the kernel of Entada phaseoloides. The structures of the new compounds were elucidated by MS and NMR experiments. Compounds 1, 3 and 4 displayed potent inhibitory activities against HIV-1 replication, with EC(50) values of 9.80 µM, 11.70 µM and 9.93 µM, respectively.

Simultaneous detection and degradation patterns of kresoxim-methyl and trifloxystrobin residues in citrus fruits by HPLC combined with QuEChERS.

This study aimed to investigate the residues, kinetics and dissipation patterns of kresoxim-methyl, (E)-methoxyimino[α-(o-tolyloxy)-o-tolyl]acetate, and trifloxystrobin, methyl(E)-methoxyimino-{(E)-α[1-(α,α,α-trifluoro-m-tolyl)ethylideneaminooxy]-o-tolyl}acetate". A simple and sensitive liquid chromatography-ultraviolet detection (LC-UV) method combined with the 'Quick Easy Cheap Effective Rugged and Safe' (QuEChERS) protocol was developed to quantify the levels of kresoxim-methyl and trifloxystrobin residues in citrus. More than 97% of the kresoxim-methyl and trifloxystrobin deposists gradually dissipated from the citrus peels within 15 days. The half-lives of kresoxim-methyl and trifloxystrobin in the peels were in the ranges of 2.63-2.66 d and 3.12-3.15 d, respectively, and the pattern of decline in the peels followed first-order kinetics. The kresoxim-methyl and trifloxystrobin residues in the pulp dissipated below the detectable level of 0.01 mg kg(-1) after 9 days. Kresoxim-methyl and trifloxystrobin were easily decomposed (T1/2 < 30 d), and the observed dissipation patterns could support the application of these two fungicides in the postharvest storage of citrus fruits.