Pushing the boundaries of VCD spectroscopy in natural product chemistry.

Vibrational circular dichroism (VCD) is one of the most powerful techniques to assess the stereochemistry of chiral molecules in solution state. The need for quantum chemical calculations to interpret experimental data, however, has precluded its widespread use by non-experts. Herein, we propose the search and validation of IR and VCD spectral markers to circumvent the requirement of DFT calculations allowing for absolute configuration assignments even in complex mixtures. To that end, a combination of visual inspection and machine learning based methods is used. Monoterpene mixtures are selected for this proof-of-concept study.

Revisiting the structure of Heliannuol L: A computational approach.

Recently, structural elucidation of natural products has undergone a revolution. The combined use of different modern spectroscopic methods has allowed obtaining a complete structural assignment of natural products using small amounts of sample. However, despite the extraordinary ongoing advances in spectroscopy, the mischaracterization of natural products has been and remains a recurrent problem, especially when the substance presents several stereogenic centers. The misinterpretation of nuclear magnetic resonance (NMR) data has resulted in frequent reports addressing structural reassignment. In this context, a great effort has been devoted to developing quantum chemical calculations that simulate NMR parameters accurately, allowing to achieve a more precise spectral interpretation. In this work, we employed a protocol for theoretical calculations of 1 H NMR chemical shifts and coupling constants using density functional theory (DFT), followed by the application of the DP4+ method to revisit the structure of Heliannuol L, a member of the Heliannuol class, isolated from Helianthus annuus. Our results indicate that the originally proposed structure of Heliannuol L needs a stereochemical reassignment, placing the hydroxyl bonded to C10 in the opposite side of the methyl and hydroxyl groups bonded to C7 and C8, respectively.

New Law of Brazilian Biodiversity: Legal Aspects and Impact in the Field of Biotechnology.

Access to genetic resources (GR) and/or traditional knowledge associated with genetic resources (ATK) has been regulated in Brazil since 2001. The law 13,123 / 2015 determined a significant change in the theme, mainly on the rules of distribution of benefits obtained for conservation and sustainable use of biodiversity, the access to technology and technology transfer, the exploitation of products or reproductive material from the GR or ATK and consignment to the outside of part or all the living or dead organism shipped for GR. The implementation of international treaties on GR and ATK for research, biotechnological development and bioprospecting have been causing difficulties for Brazilian researchers, mainly due to the lack of information and dissemination available for compliance with the legislation. In this work, the members of the Committee for Access to Genetic Resources and Associated Traditional Knowledge of the Federal Fluminense University (UFFGEN) - Brazil, and collaborators performed a critical reflection on the new law, helping Brazilian researchers with information necessary to understand the changes made by the new legislation, especially in the field of Biotechnology associated with Brazilian Biodiversity.

The Brazilian octocoral Phyllogorgia dilatata as a source of cytotoxic compounds.

The extensive marine biodiversity has proved to be a promising source of substances with biomedical potential. In this study, the cytotoxicity of the Brazilian octocoral Phyllogorgia dilatata (Gorgoniidae) was evaluated against two tumor cell lines and three bacterial strains. The methanol/dichloromethane crude extract presented no antibacterial activity up to the highest concentration tested (512 µg/mL), however it revealed a noteworthy antiproliferative effect against HCT-116 (80%) and MCF-7 (54%) cell lines at 50 µg/mL. Therefore, guided by the cytotoxic activity, a multistep chemical fractionation of the extract provided the subfraction 5 (PDPH2-5) with IC50 values of 3.18 and 17.80 µg/mL against HCT-116 and MCF-7, respectively. The LC-HRMS/MS analysis of PDPH2-5 showed ions of m/z 219.1742 and 219.1743, characterized as (E,E) and (Z,E) germacrone, after a LC-DAD-SPE/NMR analysis of the hexanic fraction and comparisons of NMR data with the literature. Previously reported assessments to the cytotoxic activity of the (E,E)-diastereoisomer disclosed higher IC50 values than that obtained for the PDPH2-5 fraction, suggesting, herein, a potentiated effect of the diastereoisomeric mixture. Such remark encourage further bioactivity studies with stereoisomer mixtures and reduce the urge for compound isolation.

Integrated analytical workflow for chromatographic profiling and metabolite annotation of a cytotoxic Phorbas amaranthus extract.

Phorbas is a widely studied genus of marine sponge and produce structurally rich cytotoxic metabolites. Still, only few studies have assessed metabolites present in Brazilian species. To circumvent redundancy, in this work, we applied and herein report the use of a scouting liquid chromatographic system associate to the design of experiment produced by the DryLab® software to obtain a fast and efficient chromatographic separation of the active hexane fraction, further enabling untargeted high-resolution mass spectrometry (HRMS) data. To this end, a crude hydroalcoholic extract of the sponge Phorbas amaranthus collected in Brazilian coast was prepared and partitioned. The cytotoxicity of the crude extract and the fractions was evaluated using tumor cell culture models. Fragmentation pathways assembled from HRMS data allowed the annotation of 18 known Phorbas metabolites, while 17 metabolites were inferred based on Global Natural Product Social Molecular Networking (GNPS), matching with a further 29 metabolites annotated through molecular subnetwork. The workflow employed demonstrates that chromatographic method development can be accelerated by the use of automated scouting systems and DryLab®, which is useful for profiling natural product libraries, as well as data curation by molecular clusters and should be incorporated to the tools of natural product chemists.

Phosphoenolpyruvate carboxykinase from T. cruzi magnetic beads affinity-based screening assays on crude plant extracts from Brazilian Cerrado.

In T. cruzi, a causative agent of Chagas disease, phosphoenolpyruvate carboxykinase (TcPEPCK) is associated with carbohydrate catabolism. Due to its importance in the metabolism of the parasite, it has become a promising target for the development of new drugs against Chagas disease. Aiming to investigate different approaches for ligands screening, TcPEPCK was immobilized on amine-terminated magnetic beads (TcPEPCK-MB) and kinetically characterized by liquid chromatography tandem mass spectrometry activity assay with a KMapp value of 10 ± 1 µM to oxaloacetate as substrate. Natural products library affords highly diverse molecular frameworks through their secondary metabolites, herein a ligand fishing TcPEPCK-MB assay is described for prospecting ligands in four ethanolic extracts of Brazilian Cerrado plants: Qualea grandiflora (Vochysiaceae), Diospyros burchellii (Ebenaceae), Anadenanthera falcata (Fabaceae) and Byrsonima coccolobifolia (Malpighiaceae). The chemical characterization of eleven identified ligands was carried out by liquid chromatography tandem high-resolution mass spectrometry experiments. Senecic acid, syneilesinolide A, phytosphingosine and vanillic acid 4-glucopyranoside are herein reported for the first time for Q. grandiflora, D. burchellii, A. falcata, respectively. In addition, the specificity of the assay was observed since only catechin was fished out from the ethanolic extract of B. coccolobifolia leaves, despite the presence of epicatechin epimer.

Artemisia annua L. and photoresponse: from artemisinin accumulation, volatile profile and anatomical modifications to gene expression.

KEY MESSAGE: Blue and yellow light affected metabolism and the morphology. Blue and red promote the DOXP/MEP pathway. ADS gene expression was increased in plants cultivated under blue, promoting artemisinin content. Artemisinin-based combination therapies are the most effective treatment for highly lethal malaria. Artemisinin is produced in small quantities in the glandular trichomes of Artemisia annua L. Our aim was to evaluate the effect of light quality in A. annua cultivated in vitro under different light qualities, considering anatomical and morphological changes, the volatile composition, artemisinin content and the expression of two key enzymes for artemisinin biosynthesis. Yellow light is related to the increase in the number of glandular trichomes and this seemed to positively affect the molecular diversity in A. annua. Yellow light-stimulated glandular trichome frequency without triggered area enhancement, whereas blue light stimulated both parameters. Blue light enhanced the thickness of the leaf epidermis. The B-promoting effect was due to increased cell size and not to increased cell numbers. Green and yellow light positively influenced the volatile diversity in the plantlets. Nevertheless, blue and red light seemed to promote the DOXP/MEP pathway, while red light stimulates MVA pathway. Amorpha-4,11-diene synthase gene expression was significantly increased in plants cultivated under blue light, and not red light, promoting artemisinin content. Our results showed that light quality, more specifically blue and yellow light, positively affected secondary metabolism and the morphology of plantlets. It seemed that steps prior to the last one in the artemisinin biosynthesis pathway could be strongly influenced by blue light. Our work provides an alternative method to increase the amount of artemisinin production in A. annua without the use of transgenic plants, by the employment of blue light.

Stereochemistry of spongosoritins: beyond optical rotation.

Stereochemical determinations based solely on the comparison of optical rotation (OR) measured at a single wavelength may commonly result in misassignments. Herein, we use vibrational and electronic CD, NMR, OR, and DFT calculations, to confirm the absolute configuration (AC) of the cytotoxic marine polyketides spongosoritin A (1) and 9,10-dihydrospongosoritin A (2) as (-)-(6R,8R) and (-)-(6R,8S), respectively. The AC of natural 1 and 2 has so far relied upon comparisons of OR values only. Besides showing the dependence of OR on achiral structural features, such as E/Z double bond geometries, an IR spectral marker is provided to help distinguish the geometric isomers of related molecules.

Cytotoxicity and selectiveness of Brazilian Piper species towards oral carcinoma cells.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is one of the ten most common types of cancer worldwide. Plants of the genusPiper are used in traditional medicine to treat cancer, and they have a vast diversity of phytochemicals with cytotoxic potential. Purpose and Study Design: In this work, we analyzed the cytotoxic and selective potential of extracts and semipurified fractions of Piper mollicomum (PM), Piper truncatum (PT), Piper cernuum (PC), Piper arboreum (PA), and Piper cabralanum (PCa) using three different OSCC cell lines (SCC4, SCC9 and SCC25), and we measured their in vivo toxicities and conducted chemical analyses of their active fractions. RESULTS: The dichloromethane fractions of the crude methanolic extracts of the leaves of PM(-L-D), PC(-L-D) and PCa(-L-D) exhibited notable IC50 values of 94.2, 47.2 and 47.5 µg/mL, respectively, and all three of these extracts were more active than carboplatin (172.3 µg/mL). The most selective fraction was PC-L-D, which exhibited SI > 4.5; less than 5% hemolysis; and no significant alterations in in vivo acute toxicology. The major constituents in active fractions were lignans (PC-L-D and PCa-L-D) and chromenes (PM-L-D). CONCLUSION: PC-L-D demonstrated great potential for further development as an anticancer drug and could be the key to developing more effective and less toxic therapies against oral cancer.

Clusia criuva Cambess. (Clusiaceae): anatomical characterization, chemical prospecting and antioxidant activity

ABSTRACT This study aims the anatomical description and chemical characterization of aerial parts of Clusia criuva Cambess., Clusiaceae in addition to the evaluation of the antioxidant activity of crude extracts, correlated to the flavonoid content. The morphological characterization was performed using traditional techniques of plant anatomy. For phytochemical studies, crude extracts were obtained by static maceration and analyzed by thin layer chromatography. The antioxidant activity and the flavonoids content were determined by colorimetric methods involving, respectively, 2,2-diphenyl-1-picrylhydrazyl free radical and aluminum chloride. C. criuva has uniseriate epidermis, paracytic stomata, hypostomatic leaves, cuticular flanges and cordiform vascular cylinder with accessory bundles. Chemical prospecting confirmed the abundant presence of terpenes and phenols in the extracts of leaves and of fruits. The methanolic extract of seeds showed the lowest EC50 value, but the methanolic extract of pericarps exhibited the highest maximum antioxidant activity. The results suggested a high percentage of flavonoids in the hexanic extract of pericarps, however, this could represent, in fact, the presence of benzophenones. Secretory ducts and the shape of the midrib are diagnostic for C. criuva. The antioxidant activity is not directly related to the flavonoids. The results indicate the importance of future studies with C. criuva chemical constituents.

Clusia criuva Cambess. (Clusiaceae): anatomical characterization, chemical prospecting and antioxidant activity.

This study aims the anatomical description and chemical characterization of aerial parts of Clusia criuva Cambess., Clusiaceae in addition to the evaluation of the antioxidant activity of crude extracts, correlated to the flavonoid content. The morphological characterization was performed using traditional techniques of plant anatomy. For phytochemical studies, crude extracts were obtained by static maceration and analyzed by thin layer chromatography. The antioxidant activity and the flavonoids content were determined by colorimetric methods involving, respectively, 2,2-diphenyl-1-picrylhydrazyl free radical and aluminum chloride. C. criuva has uniseriate epidermis, paracytic stomata, hypostomatic leaves, cuticular flanges and cordiform vascular cylinder with accessory bundles. Chemical prospecting confirmed the abundant presence of terpenes and phenols in the extracts of leaves and of fruits. The methanolic extract of seeds showed the lowest EC50 value, but the methanolic extract of pericarps exhibited the highest maximum antioxidant activity. The results suggested a high percentage of flavonoids in the hexanic extract of pericarps, however, this could represent, in fact, the presence of benzophenones. Secretory ducts and the shape of the midrib are diagnostic for C. criuva. The antioxidant activity is not directly related to the flavonoids. The results indicate the importance of future studies with C. criuva chemical constituents.

Perezone, from the gorgonian Pseudopterogorgia rigida, induces oxidative stress in human leukemia cells

Abstract Four bisabolanes 1–4, including perezone (1) and triacetyl perezone (2), were isolated through a bioassay-guided fractionation of the extract obtained from the Caribbean gorgonian coral Pseudopterogorgia rigida collected during an expedition cruise to the Bahamas. All isolated compounds showed to be cytotoxic toward panel of four human tumor cell lines, as quantified by the MTT assay after 72 h incubation. Perezone (1), the most active one, was further analyzed, showing to be cytotoxic, but not selective, in a 12-cell line panel comprising tumor and non-tumor, as well as human and murine cells. Additionally, 1 was assayed for cytotoxicity against HL-60 leukemic cells. Pre-treatment with an acute free radical scavenger (L-NAC) before exposure of cells to perezone virtually eliminated the generation of intracellular ROS and lessened its severe cytotoxicity. The protective effect delivered by L-NAC evidences that the mechanism of perezone-induced cytotoxicity is partially associated to production of ROS and a consequent induction of oxidative stress.

New trifluoromethyl triazolopyrimidines as anti-Plasmodium falciparum agents.

According to the World Health Organization, half of the World's population, approximately 3.3 billion people, is at risk for developing malaria. Nearly 700,000 deaths each year are associated with the disease. Control of the disease in humans still relies on chemotherapy. Drug resistance is a limiting factor, and the search for new drugs is important. We have designed and synthesized new 2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine derivatives based on bioisosteric replacement of functional groups on the anti-malarial compounds mefloquine and amodiaquine. This approach enabled us to investigate the impact of: (i) ring bioisosteric replacement; (ii) a CF3 group substituted at the 2-position of the [1,2,4]triazolo[1,5-a]pyrimidine scaffold and (iii) a range of amines as substituents at the 7-position of the of heterocyclic ring; on in vitro activity against Plasmodium falciparum. P. falciparum dihydroorotate dehydrogenase (PfDHODH) through strong hydrogen bonds. The presence of a trifluoromethyl group at the 2-position of the [1,2,4]triazolo[1,5-a]pyrimidine ring led to increased drug activity. Thirteen compounds were found to be active, with IC50 values ranging from 0.023 to 20 µM in the anti-HRP2 and hypoxanthine assays. The selectivity index (SI) of the most active derivatives 5, 8, 11 and 16 was found to vary from 1,003 to 18,478.

Atividade moluscicida de plantas: uma alternativa profilática / Plant molluscicidal activity: a prophylactic alternative

Uma das formas de combate a helmintos que possuem moluscos em seu ciclo biológico é o controle da população malacológica. A tentativa de combater os criadouros naturais de hospedeiros intermediários, através de moluscicidas, tem sido uma das formas almejadas para a redução da incidência de enfermidades, como as esquistossomoses e fascioloses. Essa medida epidemiológica tem se concretizado através da utilização dos moluscicidas sintéticos que, dentre outras desvantagens, trazem danos ao ecossistema. Visando a obtenção de novos compostos, os estudos sobre a potencialidade de produtos naturais moluscicidas têm crescido consideravelmente. Alguns pesquisadores apontam os moluscicidas vegetais como fontes alternativas para o tratamento profilático de parasitoses. Tendo em vista a abrangência, endemicidade e relevância, este trabalho revela um maior enfoque à atividade moluscicida de plantas relacionada às esquistossomíases.

An effective way to combat helminthes having mollusk in their life cycle is by controlling their population. The attempt to battle the natural breeding of intermediary hosts through molluscicides has been the targeted approach to reduce the incidence of such diseases as schistosomiasis and fascioloses. This epidemiological measure has been performed using synthetic molluscicides which, among other drawbacks, cause damages to the ecosystem. In order to obtain new compounds, studies on the potential of natural molluscicides products are increasing. Researchers draw attention to molluscicides plants as alternative sources for the prophylactic management of parasitoses. In view of the scope, significance and endemism, the present work highlights plant activities related to schistosomiasis.