Chemically engineered essential oils prepared through thiocyanation under solvent-free conditions: chemical and bioactivity alteration.

The generation of chemically engineered essential oils (CEEOs) prepared from bi-heteroatomic reactions using ammonium thiocyanate as a source of bioactive compounds is described. The impact of the reaction on the chemical composition of the mixtures was qualitatively demonstrated through GC-MS, utilizing univariate and multivariate analysis. The reaction transformed most of the components in the natural mixtures, thereby expanding the chemical diversity of the mixtures. Changes in inhibition properties between natural and CEEOs were demonstrated through acetylcholinesterase TLC autography, resulting in a threefold increase in the number of positive events due to the modification process. The chemically engineered Origanum vulgare L. essential oil was subjected to bioguided fractionation, leading to the discovery of four new active compounds with similar or higher potency than eserine against the enzyme. The results suggest that the directed chemical transformation of essential oils can be a valuable strategy for discovering new acetylcholinesterase (AChE) inhibitors.

Effect-directed analysis in food by thin-layer chromatography assays.

Thin-layer chromatography (TLC) is widely used for food analysis and quality control. As an open chromatographic system, TLC is compatible with microbial-, biochemical-, and chemical-based derivatization methods. This compatibility makes it possible to run in situ bioassays directly on the plate to obtain activity-profile chromatograms, i.e., the effect-directed analysis of the sample. Many of the properties that can be currently measured using this assay format are related to either desired or undesired features for food related products. The TLC assays can detect compounds related to the stability of foods (antioxidant, antimicrobial, antibrowning, etc.), contaminants (antibiotics, pesticides, estrogenic compounds, etc.), and compounds that affect the absorption, metabolism or excretion of nutrients and metabolites or could improve the consumers health (enzyme inhibitors). In this article, different food related TLC-assays are reviewed. The different detection systems used, the way in which they are applied as well as selected examples are discussed.

Modification of PapA5 acyltransferase substrate selectivity for optimization of short-chain alcohol-derived multimethyl-branched ester production in Escherichia coli.

Plant waxes are interesting substitutes of fossil-derived compounds; however, their limited sources and narrow structural diversity prompted the development of microbial platforms to produce esters with novel chemical structures and properties. One successful strategy was the heterologous expression of the mycocerosic polyketide synthase-based biosynthetic pathway (MAS-PKS, PapA5 and FadD28 enzymes) from Mycobacterium tuberculosis in Escherichia coli. This recombinant strain has the ability to produce a broad spectrum of multimethyl-branched long-chain esters (MBE) with novel chemical structures and high oxidation stability. However, one limitation of this microbial platform was the low yields obtained for MBE derived of short-chain alcohols. In an attempt to improve the titers of the short-chain alcohol-derived MBE, we focused on the PapA5 acyltransferase-enzyme that catalyzes the ester formation reaction. Specific amino acid residues located in the two-substrate recognition channels of this enzyme were identified, rationally mutated, and the corresponding mutants characterized both in vivo and in vitro. The phenylalanine located at 331 position in PapA5 (F331) was found to be a key residue that when substituted by other bulky and aromatic or bulky and polar amino acid residues (F331W, F331Y or F331H), gave rise to PapA5 mutants with improved bioconversion efficiency; showing in average, 2.5 higher yields of short-chain alcohol-derived MBE compared with the wild-type enzyme. Furthermore, two alternative pathways for synthetizing ethanol were engineered into the MBE producer microorganism, allowing de novo production of ethanol-derived MBE at levels comparable with those obtained by the external supply of this alcohol. KEY POINTS: • Mutation in channel 2 changes PapA5 acyltransferase bioconversion efficiency. • Improved production of short-chain alcohol derived multimethyl-branched esters. • Establishing ethanologenic pathways for de novo production of ethanol derived MBE. • Characterization of a novel phenylethanol-derived MBE.

Enzymatic Bioautographic Methods.

Enzymatic bioautography enables the detection of enzyme inhibitors absorbed on a thin-layer chromatography plate. Therefore, it is an assay format that is particularly useful for the detection of inhibitors present in complex mixtures. The inhibition properties of compounds separated by thin-layer chromatography can be directly analyzed to produce an inhibition profile. Here, we describe the conditions to detect inhibitor of the enzymes xanthine oxidase and ß-glucosidase immobilized on agar gel.

A Tyrosinase Inhibitor from a Nitrogen-Enriched Chemically Engineered Extract.

The enzyme tyrosinase is involved in the biosynthesis of melanin and the enzymatic browning of fruits and vegetables, and therefore, its inhibitors have potential to treat hyperpigmentary disorders or to function as food antibrowning agents. The use of hydrazine monohydrate as a reagent to prepare chemically engineered extracts can lead to semisynthetic compounds that contain the portion N-N, a fragment rarely found in natural products and present in some tyrosinase inhibitors. Here, we report the tyrosinase inhibition screening of a series of chemically engineered extracts that are diversified by reaction with hydrazine. LC-MS was used to evaluate the change in composition produced by the reaction. Bioguided fractionation of the most active chemically engineered extract, prepared from Matricaria recutita L., led to the discovery of a pyrazole that inhibits tyrosinase with an IC50 value of 28.20 ± 1.13 µM. This compound was produced by a one-pot double chemical transformation of its natural precursor, which includes an unexpected selective removal of one -OH group.

Metabolic Footprinting of a Clear Cell Renal Cell Carcinoma in Vitro Model for Human Kidney Cancer Detection.

A protocol for harvesting and extracting extracellular metabolites from an in vitro model of human renal cell lines was developed to profile the exometabolome by means of a discovery-based metabolomics approach using ultraperformance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry. Metabolic footprints provided by conditioned media (CM) samples ( n = 66) of two clear cell Renal Cell Carcinoma (ccRCC) cell lines with different genetic backgrounds and a nontumor renal cell line, were compared with the human serum metabolic profile of a pilot cohort ( n = 10) comprised of stage IV ccRCC patients and healthy individuals. Using a cross-validated orthogonal projection to latent structures-discriminant analysis model, a panel of 21 discriminant features selected by iterative multivariate classification, allowed differentiating control from tumor cell lines with 100% specificity, sensitivity, and accuracy. Isoleucine/leucine, phenylalanine, N-lactoyl-leucine, and N-acetyl-phenylalanine, and cysteinegluthatione disulfide (CYSSG) were identified by chemical standards, and hydroxyprolyl-valine was identified with MS and MS/MS experiments. A subset of 9 discriminant features, including the identified metabolites except for CYSSG, produced a fingerprint of classification value that enabled discerning ccRCC patients from healthy individuals. To our knowledge, this is the first time that N-lactoyl-leucine is associated with ccRCC. Results from this study provide a proof of concept that CM can be used as a serum proxy to obtain disease-related metabolic signatures.

A New Fluorinated Tyrosinase Inhibitor from a Chemically Engineered Essential Oil.

The generation of fluorinated essential oils as a source of bioactive compounds is described. Most of the components of the natural mixtures were altered, leading to the discovery of a new fluorinated tyrosinase inhibitor.

Thin Layer Chromatography-Autography-High Resolution Mass Spectrometry Analysis: Accelerating the Identification of Acetylcholinesterase Inhibitors.

INTRODUCTION: The prevailing treatment for Alzheimer's disease is the use of acetylcholinesterase (AChE) inhibitors. Natural extracts are the principal source of AChE's inhibitors. However, their chemical complexity demands for simple, selective and rapid assays. OBJECTIVE: To develop a strategy for identification of AChE inhibitors present in mixtures employing high resolution mass spectrometry (HRMS) and thin layer chromatography (TLC)-biological staining. METHODOLOGY: The strategy uses an autographic assay based on the α-naphthyl acetate - fast blue B system for the detection of AChE activity. The immobilisation of AChE in agar allowed the extraction of the compounds for analysis by HRMS. Three TLC experiments employing different solvent systems were used in parallel and the mass spectra of the compounds extracted from the inhibition halos, were compared. The analysis was performed under MatLab environment. RESULTS: The strategy was used to detect the presence of physostigmine in an extract of Brassica rapa L. spiked with the inhibitor. Similarly, caffeine was straightforwardly spotted as responsible for the inhibitory properties of an extract of Ilex paraguariensis Saint-Hilaire. Comparison of the HRMS profiles lead to the facile identification of the [M+H](+) and [M+Na](+) of the compounds responsible for the inhibition. CONCLUSION: The proposed methodology, coupling TLC-AChE autography-HRMS, illustrates the feasibility of assigning molecular formulas of active compounds present in complex mixtures directly from autography. The new AChE agar-immobilised assay presented a more homogenous colour and a better definition than direct spraying methods, reducing the cost of the assay and improving its sensitivity.

A thin-layer chromatography autographic method for the detection of inhibitors of the Salmonella PhoP-PhoQ regulatory system.

INTRODUCTION: The PhoP-PhoQ system from Salmonella enterica serovar Typhimurium controls the expression of factors that are critical for the bacterial entry into host cells and the bacterial intramacrophage survival. Therefore it constitutes an interesting target to search for compounds that would control Salmonella virulence. Localisation of such compounds in complex matrixes could be facilitated by thin-layer chromatography (TLC) bioautography. OBJECTIVE: To develop a TLC bioautography to detect inhibitors of the PhoP-PhoQ regulatory system in complex matrixes. METHODS: The TLC plates were covered by a staining solution containing agar, Luria-Bertani medium, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal), kanamycin and a S. typhimurium strain that harbours a reporter transcriptional lacZ-fusion to an archetypal PhoP-activated gene virK. After solidification, the plate was incubated at 37°C for 16 h. RESULTS: A bioautographic assay suitable for the localisation of inhibitors of the PhoP-PhoQ system activity in S. enterica serovar Typhimurium present in a complex matrix is described. The assay was used to analyse a series of hydrolysed extracts prepared by alkaline treatment of crude plant extracts. Bioassay-guided analysis of the fractions by NMR spectroscopy and MS led to the identification of linolenic and linoleic acids as inhibitory input signals of the PhoP-PhoQ system. CONCLUSION: A practical tool is introduced that facilitates detection of inhibitors of the Salmonella PhoP-PhoQ regulatory system. The assay convenience is illustrated with the identification of the first naturally occurring organic compounds that down-regulate a PhoP-PhoQ regulatory system from a hydrolysed extract.

Unsaturated long chain free fatty acids are input signals of the Salmonella enterica PhoP/PhoQ regulatory system.

The Salmonella enterica serovar Typhimurium PhoP/PhoQ system has largely been studied as a paradigmatic two-component regulatory system not only to dissect structural and functional aspects of signal transduction in bacteria but also to gain knowledge about the versatile devices that have evolved allowing a pathogenic bacterium to adjust to or counteract environmental stressful conditions along its life cycle. Mg(2+) limitation, acidic pH, and the presence of cationic antimicrobial peptides have been identified as cues that the sensor protein PhoQ can monitor to reprogram Salmonella gene expression to cope with extra- or intracellular challenging conditions. In this work, we show for the first time that long chain unsaturated free fatty acids (LCUFAs) present in Salmonella growth medium are signals specifically detected by PhoQ. We demonstrate that LCUFAs inhibit PhoQ autokinase activity, turning off the expression of the PhoP-dependent regulon. We also show that LCUFAs exert their action independently of their cellular uptake and metabolic utilization by means of the ß-oxidative pathway. Our findings put forth the complexity of input signals that can converge to finely tune the activity of the PhoP/PhoQ system. In addition, they provide a new potential biochemical platform for the development of antibacterial strategies to fight against Salmonella infections.

Chemically engineered extracts: source of bioactive compounds.

Biological research and drug discovery critically depend on access to libraries of small molecules that have an affinity for biomacromolecules. By virtue of their sustained success as sources of lead compounds, natural products are recognized as "privileged" starting points in structural space for library development. Compared with synthetic compounds, natural products have distinguishing structural properties; indeed, researchers have begun to quantify and catalog the differences between the two classes of molecules. Measurable differences in the number of chiral centers, the degree of saturation, the presence of aromatic rings, and the number of the various heteroatoms are among the chief distinctions between natural and synthetic compounds. Natural products also include a significant proportion of recurring molecular scaffolds that are not present in currently marketed drugs: the bioactivity of these natural substructures has been refined over the long process of evolution. In this Account, we present our research aimed at preparing libraries of semisynthetic compounds, or chemically engineered extracts (CEEs), through chemical diversification of natural products mixtures. The approach relies on the power of numbers, that is, in the chemical alteration of a sizable fraction of the starting complex mixture. Major changes in composition can be achieved through the chemical transformation of reactive molecular fragments that are found in most natural products. If such fragments are common enough, their transformation represents an entry point for chemically altering a high proportion of the components of crude natural extracts. We have searched for common reactive fragments in the Dictionary of Natural Products (CRC Press) and identified several functional groups that are expected to be present in a large fraction of the components of an average natural crude extract. To date, we have used reactions that incorporate (i) nitrogen atoms through carbonyl groups, (ii) sulfur by transformation of -OH and amines, and (iii) bromine through double bonds and aromatic rings. The resulting CEEs had different composition and biomolecular properties than their natural progenitors. We isolated a semisynthetic ß-glucosidase inhibitor from a CEE prepared by reaction with benzenesulfonyl chloride, an antifungal pyrazole from a CEE prepared by reaction with hydrazine, and an acetylcholinesterase inhibitor from a CEE prepared through bromination. Our results illustrate how biological activity can be generated through chemical diversification of natural product mixtures. Moreover, the level of control that can be asserted in the process by judicious design and experimental choices underscores the potential for further development of CEEs in both basic research and drug discovery.

Brominated extracts as source of bioactive compounds.

The chemical composition and the biomolecular properties of a crude plant extract were altered through bromination leading to the discovery of an acetylcholinesterase inhibitor.

Discovery of a ß-glucosidase inhibitor from a chemically engineered extract prepared through sulfonylation.

A semisynthetic ß-glucosidase inhibitor was identified from a chemically engineered extract prepared by reaction with benzenesulfonyl chloride. The structure includes a natural histamine portion and a benzenesulfonyl portion introduced during the diversification step.

Chemically engineered extracts: bioactivity alteration through sulfonylation.

The chemical composition and the biomolecular properties of a series of crude plant extracts were altered without previous knowledge of their detailed chemical composition.

A rapid TLC autographic method for the detection of glucosidase inhibitors.

A new bioautographic assay suitable for the localisation of beta-glucosidase inhibitors present in a complex matrix is described. Enzyme activity was detected using esculin as the substrate to produce esculetin, which reacts with ferric ion to form a brown complex.