The key step in the chemoenzymatic synthesis of apremilast was to produce the chiral alcohol (R)-1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethanol, (R)-3. Two enzymatic approaches were evaluated to obtain (R)-3, one using ketoreductases and the other lipases. Bioreduction of 1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethanone (2), using ketoreductase KRED-P2-D12, led to (R)-3 with 48% conversion and 93% enantiomeric excess (ee). Kinetic resolution of rac-1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethyl acetate (rac-4), via hydrolysis reaction, with 20% of n-butanol, catalyzed by lipase from Aspergillus niger yielded (R)-3 with > 99% ee, 50% conversion and E-value (enantiomeric ratio) > 200. The reaction between enantiomerically pure (R)-3 and 4-acetylamino-isoindol-1,3-dione (8) afforded apremilast in 65% yield and 67% ee.
Abstract The apolar fractions components of fruit latex of twelve species of Clusia belonging to four different taxonomic sections were examined by GC-MS. The latex of Clusia is characterised by large amounts of sesquiterpene hydrocarbons as major constituents like germacrene D: C. paralicola (44.28 %), C. criuva subsp. criuva (29.03 %); β-caryophyllene: C. fluminensis (35.61 %), C. lanceolata (36.39 %), C. hilariana (58.10 %); α-trans-bergamontene: C. spirictus-sanctensis (36.30 %); α-bulnesene: C. weddelliana (25.61 %); bicyclogermacrene: C. panapanari (25.93 %) and trans-β-farnesene: C. nemorosa (24.63 %), while C. grandiflora is composed of 42.16 % monoterpene hydrocarbons. Verbenone (31.91 %) was the major component. In contrast, C. rosea, C. grandiflora, C. lanceolata and C. criuva subsp. parviflora are rich in 3-methylcyclohexanone (19.56 %), hexadecanol (22.72 %), p-anisaldehyde (23.39 %) and octadecanol (26.81 %), respectively. This study suggests considerable chemical variation among the non-polar fractions of fruit latex of the twelve Clusia species.
We propose a practical, low-cost and selective fluorescence-based protocol adapted to identify polyethylene terephthalate (PET) degrading microorganisms. The microbial hydrolysis of PET nanoparticles was monitored by 2-hydroxyterephthalate, a fluorophore produced in situ after radical hydroxylation of terephthalic acid (TPA), the final hydrolysis product, by the Fenton reaction. Seven fungi presenting promising PET hydrolytic potential using the proposed microscale screening assay were identified. The strains evaluated presented a substantial increase of up to 18-fold in PET nanoparticles conversion, such as obtained by the fungus Trichoderma sp. C70, after their cultivation in a PET-enriched medium. The formation of other hydrolysis products, along with TPA, was observed using matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS).
The simultaneous screening of multiple enzyme activities in a single assay has numerous advantages over the traditional format, since it decreases sampling errors, allows savings in reagents and consumables and reduces the time and labor required to conduct the assays. In the present study, a direct and sensitive assay for the simultaneous detection of epoxide hydrolase and esterase (or lipase) activities was developed. Signal overlap is avoided by synthesizing fluorogenic probes with enzyme-specific alkyl linkers, connected to different fluorophores (resorfurin and umbelliferone), which exhibit emission spectra at different wavelengths. The simultaneous assays were conducted in microplate format with the fluorogenic probes monitored in the same well that uses microorganisms as enzyme source. Our results show that the fluorescent signal from each of the probes used here can be discriminated, allowing multiple enzyme activity detection and quantitation.
Piperidine alkaloids are predominant in ant venom glands of S. invicta and S. saevissima. These ants are broadly sympatric in Southeastern Brazil, and potentially diagnostic compounds could assist in ant identification. We determined the absolute configuration of 2,6-dialkyl-piperidines of S. invicta and S. saevissima workers and used these data to group Southeastern Brazil species. The monitoring of venom samples by chiral gas chromatography coupled to a flame ionization detector (GC-FID) revealed that S. saevissima produces the four stereoisomers of 2-methyl-6-undecyl-piperidines, 2-methyl-6-tridecyl-piperidines and 2-methyl-6-tridecenyl-piperidines, while S. invicta venom consisted primarily of a single enantiomer cis and trans from each piperidine alkaloid. Our findings showed that the absolute configuration of piperidine alkaloids of fire ant venom may be a potential chemical tool to distinguish S. invicta and S. saevissima sympatric species in Southeastern Brazil.
Phloea subquadrata and Phloeophana longirostris are subsocial species that differ from each other in their host plants and degrees of aggregation. Until recently, only ten low-molecular weight compounds, a sesquiterpene, and dimers of (E)-4-oxo-2-hexenal had been reported from the contents of P. subquadrata glands. Here, the main goal was to distinguish the volatile compounds released by adult males, adult females and nymphs of the two species. The analyses revealed 103 compounds, of which 44 were exclusive to Phloeophana longirostris and 15 were exclusive to Phloea subquadrata. The data were analyzed by chemometric methods, which discriminated the phloeid species by developmental stage and sex. (E)-2-Hexenal, (E)-4-oxo-2-hexenal and (E)-2-octenal were found in both species. Adult individuals were characterized by the presence of methyl 2-methylbutanoate, (E)-2-hexenyl acetate and 2-hexenyl butanoate, and nymphs were characterized by 5-ethyl-2(3H)-furanone and (E)-2-octenal. There was chemical distinction between volatiles of adults and nymphs of the two species studied.
A set of eight tar sand samples with identical source, from sedimentary facies of the Pirambóia Formation, Paraná Basin, provides an interesting opportunity to assess the biodegradation extent of bitumens using high-resolution mass spectrometry with Orbitrap mass analyzer. The results suggest that acidic compounds have been degraded by microorganisms, since the O2 and O3 classes were lower to bitumens from dry interdune facies, the most biodegraded. It was also proposed a new parameter to evaluate biodegradation, DBE ratio (ratio between acidic species with the same number of double bond equivalent), that was used together with the already proposed A/C ratio (acyclic to cyclic naphthenic acids), both obtained from O2 class distribution. The bitumens from dry interdune facies presented the highest DBE ratios and lowest A/C ratios, which suggested that these parameters can be used to assess the extent of bitumen biodegradation in which the DBE ratios increase and the A/C ratios decrease with increasing biodegradation.
Abstract Copper mine drainages are restricted environments that have been overlooked as sources of new biocatalysts for bioremediation and organic syntheses. Therefore, this study aimed to determine the enzymatic activities (esterase, epoxide hydrolase and monooxygenase) of 56 heterotrophic bacteria isolated from a neutral copper mine drainage (Sossego Mine, Canaã dos Carajás, Brazil). Hydrolase and monooxygenase activities were detected in 75% and 20% of the evaluated bacteria, respectively. Bacterial strains with good oxidative performance were also evaluated for biotransformation of organic sulfides. Fourteen strains with good enzymatic activity were identified by 16S rRNA gene sequencing, revealing the presence of three genera: Bacillus, Pseudomonas and Stenotrophomonas. The bacterial strains B. megaterium (SO5-4 and SO6-2) and Pseudomonas sp. (SO5-9) efficiently oxidized three different organic sulfides to their corresponding sulfoxides. In conclusion, this study revealed that neutral copper mine drainages are a promising source of biocatalysts for ester hydrolysis and sulfide oxidation/bioremediation. Furthermore, this is a novel biotechnological overview of the heterotrophic bacteria from a copper mine drainage, and this report may support further microbiological monitoring of this type of mine environment.
Bark bugs belonging to the family Phloeidae are known for their camouflage on tree trunks. The nymphs store in dorsal abdominal glands defensive secretions with a pungent odor mainly constituted of (E)-2-hexenal, (E)-2-octenal, and (E)-4-oxo-2-hexenal. The metathoracic glands of adults (male and female) store (E)-2-hexen-1-ol and (E)-2-hexenyl acetate, which are less irritating than their corresponding aldehydes. Additional compounds of m/z 224 were detected in the scent glands of these insects and were previously suggested to be dimers of the (E)-4-oxo-2-hexenal. Thus, the aim of this study was to elucidate the details of the chemical structure of the dimers found in the scent glands of Phloea subquadrata. These dimers were obtained by synthesis and were compared with the natural products, confirming the dimeric structures of the latter. These (E)-4-oxo-2-hexenal dimeric compounds are novel and have not been reported before.
Indigenous bacteria isolated from a crude oil sample from a deep water reservoir in the Pampo Sul Oilfield (Campos Basin-RJ, Brazil) were previously classified as strains of B. pumilus. However, their enzymatic activities with fluorogenic probes and rates of petroleum biodegradation were completely different. Some of the bacteria depleted n-alkanes, whereas others did not. Aromatic compounds reported to be recalcitrant were also biodegraded by some of these Bacillus strains, revealing their outstanding ability to deplete petroleum. Further classification using matrixassisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) followed by statistical analysis revealed that these strains could be clustered into three different groups, consistent with their enzymatic activity evaluation. A more accurate phylogenetic analysis using gyrB gene sequences confirmed the MALDI-TOF MS classification of three groups of strains and identified them as Bacillus safensis, B. cereus and B. thuringiensis.
High throughput techniques to evaluate enantiomeric excess (ee) and enantiomeric ratio (E) of enzymatic reactions are fast and efficient tools that can be applied large quantities of enzymes, microorganisms and clones. The Quick-ee technique proposed in this study produces the ee and conversion values separately.
Bacteria from the genus Methylobacterium interact symbiotically (endophytically and epiphytically) with different plant species. These interactions can promote plant growth or induce systemic resistance, increasing plant fitness. The plant colonization is guided by molecular communication between bacteria-bacteria and bacteria-plants, where the bacteria recognize specific exuded compounds by other bacteria (e.g. homoserine molecules) and/or by the plant roots (e.g. flavonoids, ethanol and methanol), respectively. In this context, the aim of this study was to evaluate the effect of quorum sensing molecules (N-acyl-homoserine lactones) and plant exudates (including ethanol) in the expression of a series of bacterial genes involved in Methylobacterium-plant interaction. The selected genes are related to bacterial metabolism (mxaF), adaptation to stressful environment (crtI, phoU and sss), to interactions with plant metabolism compounds (acdS) and pathogenicity (patatin and phoU). Under in vitro conditions, our results showed the differential expression of some important genes related to metabolism, stress and pathogenesis, thereby AHL molecules up-regulate all tested genes, except phoU, while plant exudates induce only mxaF gene expression. In the presence of plant exudates there is a lower bacterial density (due the endophytic and epiphytic colonization), which produce less AHL, leading to down regulation of genes when compared to the control. Therefore, bacterial density, more than plant exudate, influences the expression of genes related to plant-bacteria interaction.
Changes in fatty acid profiles have been associated with several pathophysiological processes. Gas chromatography-mass spectrometry (GC-MS) data monitoring of selected ions was used with principal components analysis (PCA), revealing a set of relevant ions for quantification and characterization of fatty acids. This protocol was successfully applied to the analyses of fatty acids in different human blood lipids, allowing the quantification of several fatty acids and revealing their unsaturation numbers. Moreover the presences of contaminants, artifacts and co-eluitions in the chromatogram were also revealed without any additional analyses. Thus, fatty acid constituents of triglycerides, phospholipids and esters of cholesterol present in plasma and erythrocyte membranes were accurately determined, with repeatability, low limits of determination and of quantification.
Mudanças no perfil de ácidos graxos têm sido associadas a vários processos patofisiológicos. Dados obtidos por cromatografia gasosa-espectrometria de massas (GC-MS) usando monitoramento seletivo de íons foram empregados para revelarem, após análise por análise de componentes principais (PCA), um conjunto relevante de íons para quantificação e caracterização de ácidos graxos. Este protocolo foi aplicado com sucesso na análise de ácidos graxos presentes em diversas frações lipídicas de sangue, permitindo a quantificação de diversos ácidos graxos e revelando seus números de insaturações. Adicionalmente, a presença de contaminantes, artefatos e co-eluições no cromatograma também foram reveladas sem análises adicionais. Por fim, ácidos graxos presentes em triacilglicerídeos, fosfolipídios e ésteres de colesterol presentes em plasma e membrana de eritrócitos foram determinados com exatidão adequada, repetibilidade e baixos limites de detecção e de quantificação.
Brazil is renowned for its biodiversity; however, its economy is based on exotic plants, extraction and unsustainable use of natural resources. This issue was addressed in a recent QN review entitled "Chemistry without Borders." In order to explore the potential of Brazilian biodiversity fully, sustainable development is required in key technological areas, such as biotechnology. This research field is consistent with the green chemistry and white technology principles. Therefore, biotechnology is a sustainable alternative to conventional technologies and is expected to account for 20% of global chemicals by 2020. Brazil is the second largest grower of biotech crops and biodiesel, but its main activities rely on the fermentative process. In order to stimulate the national biotechnology development, the Brazilian Federal Government launched a national policy for biotechnology in 2007 and the National Committee of Biotechnology was created. Among the outstanding biotechnological processes, biocatalysis is one of the most important alternatives to conventional processing, and this field has changed dramatically with the advent of recombinant DNA technology in the 1970s, when large quantities of enzymes were accessible. The direct evolution methodology in the 1990s was a breakthrough and allowed tailoring of enzymes possessing high stability and stereoselectivity. However, about 60 years after the first industrial enzymatic biotransformation of steroids, the full potential of biocatalysis is far from being achieved. Future challenges in this field concern the multienzyme cascade reactions associated with optimized chemoenzymatic processes, and some recent industrial application of biocatalysts are also highlighted in this perspective.