Chemoenzymatic preparation of optically active 4-aryl-5-carboxy-6-methyl-3,4-dihydro-2(1H)-pyridone derivatives

A series of racemic 4-aryl-5-(tert-butoxycarbonyl)-6-methyl-3,4-dihydro-2(1H)-pyridones have been prepared by means of a modified Hantzsch reaction using commercially available starting materials. An easy removal of the tert-butyl group of these pyridones and subsequent reaction with cesium carbonate and chloromethyl 2-methylpropanoate provided us suitable substrates (±)-5 to be used in lipase-catalyzed hydrolysis reactions. Lipase B from Candida antarctica (CAL-B) was the most adequate lipase in the hydrolysis of (±)-5. Despite the low enantioselectivity values obtained (E≤12), several optically active pyridone derivatives were finally isolated with high enantiomeric excesses (ee≥91%) and moderate yields.     

Total synthesis of (±)-brazilin and formal synthesis of (±)-brazilein, (±)-brazilide A using m-CPBA

Total synthesis of (±)-brazilin has been accomplished. m-CPBA epoxidation of allyl alcohol 10 and epoxy opening reaction mediated by m-chlorobenzoic acid, formed in situ as a byproduct, gave advanced intermediate diol 14. O-alkylation and cyclization gave phenol 6 which enabled the formal synthesis of (±)-brazilein and (±)-brazilide A.     

Daphnauranols A–C,new antifeedant sesquiterpenoids with a 5/6/7 ring system from Daphne aurantiaca

Daphnauranols A–C (1–3), three new sesquiterpenoids with an unprecedented 5/6/7 ring system, were isolated from the stems of Daphne aurantiaca Diels. Their structures were elucidated on the basis of comprehensive spectroscopic methods including MS and NMR. The absolute configuration of daphnauranol A was further confirmed by single crystal X-ray diffraction (Cu Kα). Their anti-feedant activity was tested, and the fruit fly antifeedant index (AI) were 39.8 ± 7.2%, 29.4 ± 7.2%, and 26.3 ± 6.4%, respectively.     

Intramolecular ortho-arylation of phenols utilized in the synthesis of the aporphine alkaloids (±)-lirinidine and (±)-nuciferine

A palladium-mediated intramolecular phenol ortho-arylation reaction applied to the construction of aporphine alkaloids is reported. Most significantly, the efficiency of this transformation was enhanced by the utilization of trialkylphosphine (i.e. tricyclohexylphosphine) or trialkylphosphonium salts (i.e. di-tert-butylmethyl-phosphonium tetrafluoroborate) as co-catalysts in the presence of cesium carbonate. This methodology was employed in the syntheses of the aporphine alkaloids (±)-lirinidine and (±)-nuciferine.     

Luminaolide, a novel metamorphosis-enhancing macrodiolide for scleractinian coral larvae from crustose coralline algae

A new metamorphosis-enhancing macrodiolide, luminaolide (1), was isolated from the crustose coralline algae (CCA) Hydrolithonreinboldii. Its structure was determined by spectroscopic analysis. A fraction (1.30 μg/mL) eluted with 80% aqueous MeOH by ODS gel column chromatography of the same CCA extract induced larval metamorphosis (25.9 ± 7.4%) against Leptastrea purpurea, and its metamorphosis-inducing activity was further enhanced to 92.6 ± 2.9% with the addition of 1 (25.6 ng/mL).     

The use of fast protein liquid chromatography with ICP-OES and ES-MS-MS detection for the determination of various forms of aluminium in the roots of Chinese cabbage

The distribution of aluminium (Al) species was investigated in the roots of Al-tolerant Chinese cabbage (Brassica rapa L. ssp. pekinensis) by employing fast protein liquid chromatography (FPLC) with inductively coupled plasma optical emission spectrometry (ICP-OES) and electrospray tandem mass spectrometry (ES-MS-MS) detection. The cabbage was exposed to a nutrient solution that contained 10 μg cm⁻³ of Al³⁺. The results demonstrated that after 24 h of exposure, Al was quantitatively taken up by the cabbage and was distributed in different parts of the plant. 36 ± 6% of total Al was located in the roots, while the remaining 64 ± 10% was transferred to the leaves. It was found that in the roots Al was partially present in the root sap (15.5%), while the majority (84.5%) was accumulated in its apoplasmic compartments. It was further demonstrated that the proportion of Al that entered the symplasm formed a complex with organic acid. Speciation analysis by FPLC with ICP-OES detection and ES-MS-MS identification of the binding ligand indicated that Al-citrate complex was the prevailing species in the root sap.The results of the present study showed that both immobilization of Al in the apoplasmic compartments of the roots and transformation of Al³⁺ to Al-citrate are most likely responsible for the tolerance of Chinese cabbage (B. rapa L. ssp. pekinensis) to the toxic effects of Al³⁺.     

Application of a new hybrid organic-inorganic monolithic column for efficient deoxyribonucleic acid purification

A new hybrid organic-inorganic monolithic column for efficient deoxyribonucleic acid (DNA) extraction was prepared in situ by polymerization of N-(β-aminoethyl)-γ-aminopropyltriethoxysilane (AEAPTES) and tetraethoxysilane (TEOS). The main extraction mechanism was based on the Coulombic force between DNA and the amino silica hybrid monolithic column. DNA extraction conditions, such as pH, ion concentration and type, and loading capacity, were optimized online by capillary electrophoresis with laser-induced fluorescence detection. Under optimal condition, a 6.0-cm monolithic column provided a capacity of 48 ng DNA with an extraction efficiency of 74 ± 6.3% (X ± RSD). The DNA extraction process on this monolithic column was carried out in a totally aqueous system for the successful purification of DNA and removal of proteins. The PBE2 plasmid could be extracted from Bacillus subtilis (B. subtilis) crude lysate within 25 min, and the purified DNA was suitable for the amplification of a target fragment by polymerase chain reaction. This study demonstrates a new attractive solid-phase support for DNA extraction to meet the increasingly miniaturized and automated trends of genetic analyses.     

Stereoselective total synthesis of (±)-7-deoxy-trans-dihydronarciclasine, a potent antineoplastic phenanthridone alkaloid

A short and efficient stereoselective total synthesis of (±)-7-deoxy-trans-dihydronarciclasine, a highly potent antineoplastic agent and constituent of the Amaryllidaceae alkaloids, is described. Starting from a known arylcyclohexylamine-type precursor 6, the C-ring with the required stereochemistry is constructed using a chemo- and stereoselective enone reduction (NaBH₄/CaCl₂ system) and a Mitsunobu reaction. For the B-ring closure, the Banwell modification of the Bischler-Napieralski reaction was applied.     

Novel sesquiterpenes and norergosterol from the soft corals Nephthea erecta and Nephtheachabroli

Chemical investigations on the acetone extract of the Formosan soft coral Nephthea erecta have afforded a new calamenene-type sesquiterpene with a mercaptan group at C-15, erectathiol (1), and a previously reported sesquiterpenoid, (+)-trans-calamenene (2). A novel sec-germacrane sesquiterpene (3), along with a novel norergosterol, chabrosterol (4), possessing a 19-norergostane skeleton, was isolated from the other soft coral Nephthea chabroli. The structures of these metabolites were elucidated through extensive spectroscopic analyses. In vitro anti-inflammatory activity of 1 and 4 (10 μM) significantly reduced the levels of the iNOS protein (58.0 ± 6.5% and 12.4 ± 2.9%) and COX-2 protein (108.7 ± 4.5% and 45.2 ± 5.4%). In addition, metabolite 1 (166 μg/disk) exhibited antimicrobial activities against a small panel of bacterial strains.     

The use of Agrobacterium tumefacients immobilized on Amberlite XAD-4 as a new biosorbent for the column preconcentration of iron(III), cobalt(II), manganese(II) and chromium(III)

A microorganism Agrobacterium tumefacients as an immobilized cell on a solid support was presented as a new biosorbent for the enrichment of Fe(III), Co(II), Mn(II) and Cr(III) prior to flame atomic absorption spectrometric analysis. Amberlite XAD-4 was used as a support material for column preconcentration. Various parameters such as pH, amount of adsorbent, eluent type and volume, flow rate of sample solution, volume of sample solution and matrix interference effect on the retention of the metal ions have been studied. The optimum pH for the sorption of above mentioned metal ions were about 6, 8, 8 and 6, respectively. The loading capacity of adsorbent for Co(II) and Mn(II) were found to be 29 and 22 μmol g⁻¹, respectively. The recoveries of Fe(III), Co(II), Mn(II) and Cr(III), under the optimum conditions were found to be 99 ± 3, 99 ± 2, 98 ± 3 and 98 ± 3%, respectively, at the 95% confidence level. The limit of detection was 3.6, 3.0, 2.8 and 3.6 ng ml⁻¹ for Fe(III), Co(II), Mn(II) and Cr (III), respectively, by applying a preconcentration factor of 25. The proposed enrichment method was applied for metal ion determination from water samples, alloy samples, infant foods and certified samples such as whey powder (IAEA-155) and aluminum alloy (NBS SRM 85b). The analytes were determined with a relative error lower than 10% in all samples.     

Parallel kinetic resolution of propargyl ketols: formal synthesis of (+)-bakkenolide A

We describe an intriguing new example of a parallel kinetic resolution; an asymmetric cyclization-carbonylation of propargyl ketols catalyzed by palladium(II) with chiral bisoxazoline (box) ligands. The 2S,3S enantiomer of (±)-6 was preferentially converted to 13 (45-49% yields, 37-46% ee), and the 2R,3R enantiomer of (±)-6 was preferentially converted to 14 (21-23% yields, 92-97% ee). As an application of this reaction, formal synthesis of (+)-bakkenolide A was achieved.     

Dihydroisocoumarin derivatives with antifouling activities from a gorgonian-derived Eurotium sp. fungus

Five pairs of new dihydroisocoumarin enantiomers, (±)-eurotiumides A–E, and two related racemates, (±)-eurotiumides F and G, were isolated from a gorgonian-derived fungus, Eurotium sp. XS-200900E6. The enantiomeric separations for (±)-eurotiumides A–E were achieved by chiral-HPLC, and their absolute configurations were determined by ECD spectra. All of the isolated compounds are rare dihydroisocoumarin derivatives with a methoxy at C-4. (+)- And (−)-eurotiumides B and D with cis configurations of H-3/H-4 exhibited potent antifouling activities against the larval settlement of the barnacle Balanus amphitrite with the EC₅₀ values ranging from 0.7 to 2.3 μg/mL, and displayed high therapeutic ratios (LC₅₀/EC₅₀ >15). The tested compounds also showed extensive antibacterial activities. It was the first report of antifouling activities for dihydroisocoumarin derivatives.     

Regio- and stereoselective 12-O-demethylation of schizandrin into gomisin T, an important intermediate to gomisin A, by Mortierella sp. (TM-I1104)

A strain TM-I1104 identified as Mortierella sp. was discovered from soil as the most efficient fungus, which converted schizandrin into gomisin T in 91% regioselectivity by microbial 12-O-demethylation. Under optimum conditions, the yield of gomisin T reached around 80%. The faculty of 12-O-demethylation was specific on (+)-schizandrin (natural form) and the optical purity of gomisin T converted from (±)-schizandrin was 96% ee.     

Highly sensitive method for determination of N-nitrosamines using high-performance liquid chromatography with online UV irradiation and luminol chemiluminescence detection

A new method for the measurement of N-nitrosamines in part-per-trillion concentrations from water samples without preconcentration steps has been developed. This method is based on online UV irradiation after high-performance liquid chromatographic separation and subsequent luminol chemiluminescence detection without addition of an oxidant. It was confirmed that N-nitrosamines in basic aqueous solution were transformed to peroxynitrite by UV irradiation. The detection limits for this method were 1.5 ng/L, 2.9 ng/L, 3.0 ng/L, and 2.7 ng/L for N-nitrosodimethylamine, N-nitrosomorpholine, N-nitrosomethylethylamine, and N-nitrosopyrrolidine, respectively, at a signal-to-noise ratio of 3. The calibration graphs were linear in the range of 5–1000 ng/L for these N-nitrosamines. This method was used for the determination of N-nitrosamines in tap water, river water, and industrial plant effluent samples. The recoveries of N-nitrosodimethylamine, N-nitrosomorpholine, N-nitrosomethylethylamine, and N-nitrosopyrrolidine present in tap water sample at a concentration of 10 ng/L (mean ± standard deviation, n = 4) were (94.8 ± 2.7)%, (102.0 ± 6.9)%, (99.3 ± 3.9)%, and (102.8 ± 2.5)%, respectively. These results indicate that our proposed method can be applied satisfactorily to the determination of N-nitrosamines in water samples.     

First synthesis of naturally occurring (±)-epi-conocarpan

(±)-epi-Conocarpan 1 was synthesized via the key intermediate 5-bromo-cis-2-(4-methoxyphenyl)-3-methyl-2,3-dihydrobenzofuran 6 which was synthesized by a ruthenium(II) porphyrin-catalyzed intramolecular C-H insertion reaction using aryl tosylhydrazone salt 5 as the carbene source, starting from the commercially available 5-bromo-2-hydroxyacetophenone.     

Evaluation of steviol and its glycosides in Stevia rebaudiana leaves and commercial sweetener by ultra-high-performance liquid chromatography-mass spectrometry

Stevia rebaudiana leaves contain non-cariogenic and non-caloric sweeteners (steviol-glycosides) whose consumption could exert beneficial effects on human health. Steviol-glycosides are considered safe; nonetheless, studies on animals highlighted adverse effects attributed to the aglycone steviol. The aim of the present study was to develop and validate two different ultra-high-performance liquid chromatography methods with electrospray ionization mass spectrometry (UHPLC-MS) to evaluate steviol-glycosides or steviol in Stevia leaves and commercial sweetener (Truvia^®). Steviol-glycosides identity was preliminarily established by UV spectra comparison, molecular ion and product ions evaluation, while routine analyses were carried out in single ion reaction (SIR) monitoring their negative chloride adducts. Samples were sequentially extracted by methanol, cleaned-up by SPE cartridge and the analytes separated by UHPLC HSS C₁₈ column (150 mm × 2.1 mm I.D., 1.8 μm). The use of CH₂Cl₂ added to the mobile phase as source of Cl⁻ enhance sensitivity. The LLOD for stevioside, rebaudioside A, steviolbioside and steviol was 15, 50, 10 and 1 ng ml⁻¹, respectively. Assay validation demonstrated good performances in terms of accuracy (89–103%), precision (<4.3%), repeatability (<5.7%) and linearity (40–180 mg/g). Stevioside (5.8 ± 1.3%), rebaudioside A (1.8 ± 1.2%) and rebaudioside C (1.3 ± 1.4%) were the most abundant steviol-glycosides found in samples of Stevia (n = 10) from southern Italy. Rebaudioside A was the main steviol-glycosides found in Truvia^® (0.84 ± 0.03%). The amounts of steviol-glycosides obtained by the UHPLC-MS method matched those given by the traditional LC-NH₂-UV method. Steviol was found in all the leaves extract (2.7–13.2 mg kg⁻¹) but was not detected in Truvia^® (<1 μg kg⁻¹). The proposed UHPLC-MS methods can be applied for the routine quality control of Stevia leaves and their commercial preparations.     

The characterization of (+/-)-anti-benzo[a]pyrene diolepoxide-DNA adducts and (+/-)-anti-dibenzo[a,l]pyrene diolepoxide--DNA adducts in the same DNA sample using solid-matrix phosphorescence

Solid-matrix phosphorescence (SMP) spectra and lifetimes were used to characterize the (±)-anti-benzo[a]pyrene diolepoxide [(±)-anti-B[a]PDE] and (±)-anti-dibenzo[a,l]pyrene diolepoxide [(±)-anti-DB[a,l]PDE] bonded to the same sample of DNA. SMP spectra and lifetimes were also acquired for two samples of DNA that had only (±)-anti-B[a]PDE or (±)-anti-DB[a,l]PDE bonded to the individual samples of DNA. A detailed comparison of the SMP properties was made among the three samples of DNA. The SMP excitation spectra for the (±)-anti-B[a]PDE-DNA and the (±)-anti-DB[a,l]PDE-DNA adducts were very similar. However, the SMP emission spectra of the two DNA adduct systems were very dissimilar with a major emission band for the (±)-anti-B[a]PDE-DNA adducts appearing at 613 nm and for the (±)-anti-DB[a,l]PDE-DNA adducts a major band was at 558 nm. It was possible to selectively use SMP emission wavelengths and obtain a SMP excitation of spectrum of the (±)-anti-DB[a,l]PDE-DNA adducts in the dual adducted DNA sample without the (±)-anti-B[a]PDE-DNA adducts emitting SMP. In addition, it was shown that the SMP emission spectrum of the dual adducted DNA sample could be used to detect both adduct systems in the modified DNA sample. It was demonstrated that the SMP lifetimes could be effectively employed to characterize the dual adducted DNA sample. For example, the SMP decay curve for the (±)-anti-DB[a,l]PDE-DNA adducts could be acquired without any SMP emission from the (±)-anti-B[a]PDE-DNA adducts. Also, ln(SMP intensity) versus time plots were very useful in characterizing the dual adducted DNA sample.     

Application of bench-scale biocalorimetry to photoautotrophic cultures

Bench-scale biocalorimetry (≥1 L) allows for the determination of the metabolic heat flow during bioprocesses under complete control of all process conditions for extended periods of time. It can be combined with a number of on-line and off-line measurement techniques. This combination can significantly improve insight into the metabolism of microorganisms and the optimization of bioprocesses. In this study it is demonstrated that bench-scale biocalorimetry can also be applied to phototrophic microorganisms. The green microalga Chlorella vulgaris CCAP 211/11B was cultivated in a Mettler-Toledo RC1 calorimeter adapted for high-sensitivity biological calorimetry (BioRC1). Heat production was monitored in 1.5 L batch cultures. In the linear phase of growth, inhibitors of photosynthetic electron transport (DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), were used to stop photosynthesis and to monitor the resulting increase in the energy dissipating heat flux. This resulted in a calculated storage of light energy as chemical energy, i.e. biomass, of 141 ± 12.2 mW L⁻¹ (±S.D.). In addition, it was demonstrated that calorimetric determination of the total amount of light energy absorbed within the reactor was accurate by comparing two different calorimetric techniques. Using both the value of the total light input and the quantity stored as chemical energy, the photosynthetic efficiency could be calculated as 10.5% in this example.     

Determination of environmental estrogens in human urine by high performance liquid chromatography after fluorescent derivatization with p-nitrobenzoyl chloride

A high performance liquid chromatographic method (HPLC) for the simultaneous determination of 4-nonylphenol, bisphenol A, 17α-ethinylestradiol and three endogenic estrogens including 17α-estradiol, 17β-estradiol, estriol in urine sample, based on precolumn derivatization with p-nitrobenzoyl chloride, is presented in this paper. The estrogens mentioned above in urine were firstly hydrolyzed with 0.6 mol/l HCl, and then enriched and cleaned-up by ENV-18 C18 solid phase extraction (SPE) column. The estrogens on column were eluted with dichloromethane, and the eluent was evaporated to dryness under gentle nitrogen flow. The residue was allowed to react with p-nitrobenzoyl chloride at 25 °C for 30 min. Separation was performed on a C18 column with gradient elution using acetonitrile and water as mobile phase. A fluorescence detection system was used to detect the fluorescent derivatization products. The detection limit of the method was 2.7 μg/l for bisphenol A and 17β-estradiol, 2.9 μg/l for 4-nonylphenol, 4.6 μg/l for 17α-estradiol and 17α-ethinylestradiol and 8.3 μg/l for estriol, respectively. The relative standard deviations (R.S.D.) ranged from 1.29 to 4.52% and the recoveries ranged from 85.5 to 99.9%. The method was applied to the determination of those six estrogens mentioned above in human urine samples collected from 20 healthy volunteers (aged 21-29). Bisphenol A (BPA) and 4-nonylphenol (NP) were detected with average contents of 1.22 ± 1.38 mg/l and 0.38 ± 0.77 mg/l in 10 male urine samples and 1.29 ± 1.22 mg/l and 0.05 ± 0.05 mg/l in 10 female urine samples, respectively. 17α-ethinylestradiol (α-EE2) was also detected with average contents of 0.13 ± 0.41 mg/l and 0.06 ± 0.15 mg/l in male and female urine samples, respectively.     

New optically active organoantimony (BINASb) and bismuth (BINABi) compounds comprising a 1,1′-binaphthyl core: synthesis and their use in transition metal-catalyzed asymmetric hydrosilylation of ketones

Racemic 2,2′-bis[diarylstibano]-1,1′-binaphthyls [(±)-BINASbs] and 2,2′-bis[di(p-tolyl)bismuthano]-1,1′-binaphthyl [(±)-BINABi], which are the antimony and bismuth congeners of BINAP, have been prepared from 2,2′-dibromo-1,1′-binaphthyl (DBBN) via 2,2′-dilithio-1,1′-binaphthyl intermediate by treatment with the appropriate metal halides [(p-Tol)₂SbBr, Ph₂SbBr and (p-Tol)₂BiCl]. The optical resolution of the (±)-BINASbs could be achieved via the separation of a mixture of the diastereomeric Pd-complexes derived from the reaction of (±)-BINASbs with di-μ-chlorobis{(S)-2-[1-(dimethylamino)-ethyl]phenyl-C¹,N}dipalladium(II). Optically active (R)-BINASb and (R)-BINABi could be also obtained from optically active (R)-DBBN by the same procedure. The enantiopure BINASbs have been shown to be effective chiral ligands for the rhodium-catalyzed asymmetric hydrosilylation of ketones.