This review deals with two-dimensional liquid chromatography (2D-LC) separations encompassing target heart-cut (LC-LC), multiple heart-cut (mLC-LC), non-targeted comprehensive (LC × LC), and selective comprehensive (sLC × LC) analysis. It presents an overview of basic concepts and emphasizes the versatility of the applications gained by going from one-(1D) to two-dimensional (2D) separations. This review also discusses target analysis of achiral and chiral drugs for different applications and the use of 2D-LC in zonal bioaffinity chromatography. Advances in instrumental and column technologies have widened the application of LC × LC and sLC × LC separations, and we will discuss some of them.

Croton grewioides Baill. popularly known as “canelinha” or “canelinha-de-cheiro” has been used for the treatment of influenza, antitussive, febrifuge and headache; however, the study of its phytochemical composition is limited. The aim of this study was to investigate the effect of the extraction method and seasonality through leaf extracts of four accessions of Croton grewioides by fingerprint chromatograms aided by principal component analysis to analyze the differences and similarities among the samples. We aimed also to provide chemical characterization of isolated secondary metabolites using semi-preparative liquid chromatography. The results showed that only the chemical profile of the methanolic extracts of accessions 101 and 113 were influenced by the seasonality. For the first time, four flavonoids were isolated through semi-preparative chromatography in this species, characterized as quercetin 3-O-β-D-galactopyranosyl-(1→2)-α-L-rhamnopyranoside-(1→6)-α-L-rhamnopyranoside (1), quercetin 3-O-β-D-galactopyranosyl-(1→2)-α-apiopyranoside-(1→6)-α-L-rhamnopyranoside (2), quercetin 3-O-glucopyranoside (3) and 3-O-methyl-quercetin (4), the flavonoid (2) has been recognized as a new triglycosylated derivative.

Abstract 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.

Qualea grandiflora Mart. (“pau-terra”) is a medicinal plant symbolic from Cerrado biome. Qualea species reportedly have antiparasitic properties, but their active compounds have yet to be identified. In this study, we investigated the antiplasmodial activity of Q. grandiflora fruit and stem extracts against Plasmodium falciparum. The polar extracts of both fruits and stems showed promising in vitro antiplasmodial activity against sensitive and resistant P. falciparum strains (half-maximal inhibitory concentration (IC50) = 1.2 and 4.2 ng mL-1, respectively), low cytotoxicity against human hepatic cells (IC50HepG2 ≥ 400 ng mL-1), and a noteworthy selectivity index (SI ≥ 322 and ≥ 96, respectively). The fruit ethanolic extract revealed significant oral efficacy at 100 mg kg-1 in a mouse model of P. berghei malaria (100% reduction in parasitemia on day 5 post-infection). To investigate the chemical composition of the extracts, we developed a method involving liquid chromatography-high resolution quadrupole-time of flight mass spectrometer. The chemical profiling of active extracts indicated 32 compounds, including gallotannins, ellagic acid derivatives and flavonoids. The common component in the active polar extracts was found to be ellagic acid (EA), which may contribute favorably to the in vitro and in vivo inhibitory activity observed in this study.

In recent years, great advances have been observed in the proteomic area regarding the different approaches adopted as top-down, middle-down and bottom-up. Regarding middle-down and bottom-up approaches, a previous step for protein digestion is necessary for further analysis of the generated peptides. Classical digestion protocols employing proteases are usually carried out in solution, which can lead to a number of disadvantages such as high digestion time, enzymatic autolysis and impossibility to reuse the enzymes. In view of these drawbacks, different approaches have been studied and applied for improving enzymatic efficiency, inhibition of autolysis, reduction of digestion time, and to minimize the number of missed cleavages. In the literature, great efforts have been made in order to achieve maximum efficiency in enzymatic digestion, with emphasis on proteases immobilization. In this review, concepts related to protein digestion, immobilization techniques, as well as the main support used for enzyme immobilization are discussed considering new tools for proteomic analysis. years topdown, topdown top down, down top-down middledown middle bottomup. bottomup bottom up. up peptides solution time enzymes drawbacks cleavages literature review techniques

Phosphoenolpyruvate carboxykinase (PEPCK) is a ubiquitous enzyme found in all known groups of organisms, acting in the reversible conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) in the presence of divalent metal ion, and dependent of adenosine 5’-triphosphate (ATP) or guanosine-5’-triphosphate (GTP). PEPCK is an important enzyme in the metabolism of some organisms, such as Trypanosoma cruzi, being suggested as a potential drug target to treat Chagas’ disease. Its catalytic activity is, classically, measured by coupled assays. Herein, a direct assay by liquid chromatography-tandem mass spectrometry (LC-MS/MS) capable of quantifying PEP, in co-elution with OAA by the differentiation obtained by the mass spectra, is reported. The developed assay was used throughout the purification protocol in order to measure the activity of PEPCK of T. cruzi, which was expressed in Escherichia coli. The purified enzyme was kinetically characterized by the developed method with Michaelis-Menten constant (KMapp) values of 96 ± 4 and 275 ± 18 µmol L-1 to OAA and ATP as substrates, respectively. The developed assay was also used for ligand screening and proved to be able to identify very low inhibitions for small molecules (50 µmol L-1).

The continuous search for biologically active compound candidates pushes the pursuit of new substances produced by diverse organisms. Endophytic fungi are known as a promising source of metabolites with several biological activities. Based on the rich Amazonian biodiversity and the chemical potential of microbial sources, three Talaromyces strains, all major endophytes from their respective host plants, were studied aiming at the isolation of biologically active secondary metabolites. Through classical chromatographic approaches, 13 compounds were isolated from the antimicrobial extracts of the studied strains. From these, polyketides, steroids, anhydrides, and phenolic compounds were identified. Among them, two previously undescribed compounds were elucidated based on spectroscopic and spectrometric methods, a homodimer chromanone and a maleic anhydride methyl ester. The antimicrobial activity against a panel of pathogenic microorganisms from extracts, fractions, and isolated compounds was evaluated.

The genus Lippia comprises approximately 200 species of herbs, shrubs and small trees, distributed throughout the South and Central America and tropical Africa. The species Lippia gracilis Schauer is an endemic aromatic plant of the Brazilian Northeast normally found in the states of Bahia, Sergipe and Piauí. The traditional communities of Caatinga, a semi-arid region of the Brazilian Northeast, use its leaves to treat throat and mouth infections, cutaneous diseases, ulcers, vagina disorders, acne, Pityriasis alba, dandruff, burns, wounds, sinusitis, bronchitis, nasal congestion, headache, jaundice and paralysis. Considering that the main investigations into the L. gracilis Schauer species are focused on its volatile constituents, in this report, we describe the isolation, by liquid chromatography with diode array detector-solid phase extraction/nuclear magnetic resonance (LC-DAD-SPE/NMR), of five flavonoids besides free and glycosidically bound carvacrol from an infusion of the leaves of a genotype of this species.

Coumarins are a large class of compounds that display a range of interesting biological properties, being considered privileged structures because of the ability of their 2H-chromen-2-one nuclei to bind to multiple pharmacological targets. We hypothesized that the linkage of a second pharmacophore nucleus to the 2H-chromen-2-one core, the 1,2,3-triazole moiety, would entail more selective and pharmacologically active coumarins. Therefore, we describe the synthesis of fourteen 4-methylcoumarins/1,4-substituted 1,2,3-triazole conjugates, which were predicted by in silico methods to inhibit acetylcholinesterase (AChE) and proved to be moderate in vitro inhibitors of this enzyme. Molecular docking simulations suggest that the most active of these compounds has a putative binding mode similar to donepezil, both occupying the peripheral anionic site of AChE, which is associated with the secondary noncholinergic functions of the enzyme. This highlights the potential of this series for further optimization in the search of new coumarins for the treatment of Alzheimer's disease.

A new series of Mannich bases derived from 2-hydroxy-1,4-naphthoquinone (lawsone), substituted benzaldehydes and two primary amines, and their Cu2+ complexes were synthesized and evaluated for their potential as selective cholinesterase inhibitors (ChEIs). Immobilized capillary enzyme reactors (ICERs) bearing butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) were used not only for the on-flow screening assay but also for determining the inhibitory potency and equilibrium binding constants of the lead inhibitors. Eight copper complexes were identified and characterized as potent reversible and selective ChEIs with inhibitory potencies (IC50) and constants of inhibition (Ki) ranging from 1.24 to 11.5 µmol L-1. One of the compounds was particularly promising, showing IC50 and Ki values of 1.24 ± 0.01 and 1.06 ± 0.01 µmol L-1, respectively, for huAChE. These values were lower than those for the standard inhibitor galanthamine (IC50 = 206 ± 30.0 and Ki = 126 ± 18.0 µmol L-1). Even though, it is showing noncompetitive inhibition of huAChE and linear mixed-type inhibition of eeAChE. These complexes showed a promising cholinesterase inhibitory activity and can be used as model inhibitors.

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.

Os etenoditioacetais polarizados são conhecidos como importantes intermediários em muitos processos de síntese. Neste trabalho, reporta-se a anelação livre de metal de transição de 1,1-bis(tiometil)-2-nitroetileno com hidroxilalquilaminas e alquildiaminas. Esta metodologia permite a síntese direta de N-heterociclos tais como imidazolidinas, oxazolidinas e benzoxazóis sob irradiação de micro-ondas. Estes compostos foram avaliados como inibidores da acetilcolinesterase usando um reator capilar contendo a enzima imobilizada acoplado a um espectrômetro de massas.

Polarized ketene dithioketals have been recognized as useful building blocks in many synthetic operations. In this work, a transition-metal-free annulations of 1,1-bis(thiomethyl)-2-nitroethylene with hydroxylalkylamines or alkyldiamines have been reported. This methodology provides a directed approach to N-heterocycles, e.g., imidazolidines, oxazolidines and benzoxazoles under microwave conditions. These compounds were evaluated as acetylcholinesterase inhibitors by using an enzyme immobilized capillary reactor-tandem mass spectrometry.

Esta revisão versa sobre o uso de novas tecnologias no preparo de amostras biológicas, com ênfase nos trabalhos reportados por pesquisadores brasileiros. A miniaturização, automação e injeção direta de amostra com o intuito de atender a demanda por métodos mais eficientes e com menor geração de resíduos, apresentam-se criticamente discutidos.

This paper reviews the new technologies used for bioanalytical sample preparation as developed and explored by Brazilian researchers. Off-line, at-line and on-line sample clean-up approaches, as well microextraction systems, have been critically discussed. The main gains in these applications are enhanced sensitivity with cleaner samples and/or high-throughput capabilities.

The development of Chiral Stationary Phases (CSPs) for high performance liquid chromatography has been studied by various researches around the world, especially, since 1980. This simple interest has been transformed into a tool of great technological value for the industrial community and scholars in general providing the existence of several CSPs, which act through different mechanisms of chiral discrimination. This paper describes the main types of CSPs that are used for the resolution of the majority of chiral compounds.

O desenvolvimento de métodos rápidos e eficazes para a identificação de novas moléculas bioativas é fundamental para o processo de descoberta e planejamento de fármacos. A integração de um sistema de cromatografia liquida de alta eficiência (CLAE), com biorreatores como fase estacionária (IMER) é uma estratégia atrativa e versátil para a triagem de coleções de compostos visando à identificação de novos agentes terapêuticos. Os parâmetros cinéticos da enzima imobilizada gliceraldeído-3-fosfato desidrogenase (GAPDH) de Trypanosoma cruzi e humana foram determinados (T. cruzi: K M G3P = 0.50 mmol L-1; K M NAD+ = 0.67 mmol L-1; humana: K M G3P = 3.7 mmol L-1; K M NAD+ = 0.75 mmol L-1) e comparados com aqueles observados em solução (T. cruzi: K M G3P = 0.42 mmol L-1; K M NAD+ = 0.26 mmol L-1; humana: K M G3P = 0.16 mmol L-1; K M NAD+ = 0.18 mmol L-1). Os resultados indicaram uma diminuição na afinidade das enzimas imobilizadas, entretanto, os elementos estruturais necessários para o processo de reconhecimento molecular e atividade biológica permaneceram inalterados, aumentando a estabilidade das enzimas. Além disso, a análise estrutural forneceu dados moleculares importantes envolvidos nos efeitos da imobilização sobre as interações entre ligante e receptor e, consequentemente, sobre a atividade enzimática e parâmetros cinéticos.

The development of fast and reliable methods for the identification of new bioactive compounds is of utmost importance to boost the process of drug discovery and development. Immobilized enzyme reactors (IMERs), integrated with high performance liquid chromatography (HPLC), are attractive and versatile tools for screening collections consisting of natural products and synthetic small molecules. Standard kinetic parameters of the immobilized enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from both Trypanosoma cruzi de and human have been determined (T. cruzi: K M G3P = 0.50 mmol L-1; K M NAD+ = 0.67 mmol L-1; humana: K M G3P = 3.7 mmol L-1; K M NAD+ = 0.75 mmol L-1), and comparisons of these values with those of the parasite and human free enzymes indicate a decrease in the affinity for the immobilized system (T. cruzi: K M G3P = 0.42 mmol L-1; K M NAD+ = 0.26 mmol L-1; humana: K M G3P = 0.16 mmol L-1; K M NAD+ = 0.18 mmol L-1). Interestingly, despite the kinetic differences between the two systems, the immobilized GAPDHs retained the required structural requirements for molecular recognition and biological activity, increasing the stability the enzyme. In the present work, we described an integrated structural analysis which has provided important insights into the molecular basis underlying the effects of immobilization on the ligand-receptor interactions and consequent enzymatic activity and kinetics parameters.