Enhanced xyloglucan-specific endo-ß-1,4-glucanase efficiency in an engineered CBM44-XegA chimera.

Xyloglucan-specific endo-ß-1,4-glucanases (Xegs, EC 3.2.1.151) exhibit high catalytic specificity for ß-1,4 linkages of xyloglucan, a branched hemicellulosic polysaccharide abundant in dicot primary cell walls and present in many monocot species. In nature, GH12 Xegs are not associated with carbohydrate-binding modules (CBMs), and here, we have investigated the effect of the fusion of the xyloglucan-specific CBM44 on the structure and function of a GH12 Xeg from Aspergillus niveus (XegA). This fusion presented enhanced catalytic properties and conferred superior thermal stability on the XegA. An increased k cat (chimera, 177.03 s(-1); XegA, 144.31 s(-1)) and reduced KM (chimera, 1.30 mg mL(-1); XegA, 1.50 mg mL(-1)) resulted in a 1.3-fold increase in catalytic efficiency of the chimera over the parental XegA. Although both parental and chimeric enzymes presented catalytic optima at pH 5.5 and 60 °C, the thermostabilitiy of the chimera at 60 °C was greater than the parental XegA. Moreover, the crystallographic structure of XegA together with small-angle X-ray scattering (SAXS) and molecular dynamics simulations revealed that the spatial arrangement of the domains in the chimeric enzyme resulted in the formation of an extended binding cleft that may explain the improved kinetic properties of the CBM44-XegA chimera.

In vitro metabolism of monensin A: microbial and human liver microsomes models.

1. Monensin A, an important antibiotic ionophore that is primarily employed to treat coccidiosis, selectively complexes and transports sodium cations across lipid membranes and displays a variety of biological properties. 2. In this study, we evaluated the fungi Cunninghamella echinulata var. elegans ATCC 8688A, Cunninghamella elegans NRRL 1393 ATCC 10028B and human hepatic microsomes as CYP-P450 models to investigate the in vitro metabolism of monensin A and compare the products with the metabolites produced in vivo. 3. Mass spectrometry analysis of the products from these model systems revealed the formation of three metabolites: 3-O-demethyl monensin A, 12-hydroxy monensin A and 12-hydroxy-3-O-demethyl monensin A. We identified these products by tandem mass spectrometry and through comparison with the in vivo metabolites. 4. This analysis demonstrated that the model systems produce the same metabolites found in in vivo studies, thus they could be used to predict the metabolism of monensin A. Furthermore, we verified that liquid chromatography coupled to mass spectrometry is a powerful tool to study the in vitro metabolism of drugs, because it allows the successful identifications of several derivatives from different metabolic models.

Pradimicin-IRD from Amycolatopsis sp. IRD-009 and its antimicrobial and cytotoxic activities.

A new polycyclic antibiotic, pradimicin-IRD, was isolated from actinobacteria Amycolatopsis sp. IRD-009 recovered from soil of Brazilian rainforest undergoing restoration area. This molecule is the major compound produced in solid culture media. The new compound was detected by a focused method of precursor ion (high-performance liquid chromatography coupled to tandem mass spectrometer) developed previously to identify unusual aminoglycosyl sugar moieties. The compound was isolated and its structure was, therefore, elucidated by high-resolution mass spectrometry, and 1D and 2D nuclear magnetic resonance experiments. Pradimicin-IRD displayed potential antimicrobial activity against Streptococcus agalactiae (MIC 3.1 µg/mL), Pseudomonas aeruginosa (MIC 3.1 µg/mL) and Staphylococcus aureus (MIC 3.1 µg/mL), and also cytotoxicity against tumour and non-tumour cell lines with IC50 values ranging from 0.8 µM in HCT-116 colon carcinoma cells to 2.7 µM in MM 200 melanoma cells. Particularly, these biological properties are described for the first time for this chemical class.

Anthocyanidins structural study using positive electrospray ionization triple quadrupole mass spectrometry and H/D exchange.

We report herein a detailed structural study by collision-induced dissociation (CID) of nonglycosylated anthocyanins (anthocyanidins) using electrospray ionization triple quadrupole mass spectrometry (ESI-QqQ) and isotope labeling experiments to understand the fragmentation process often used in mass spectrometry analysis of this class of compounds. Tandem mass spectrometric product ion spectra for three anthocyanidins (cyanidin, delphynidin, and pelargonin) were evaluated to propose fragmentation mechanisms to this natural colorant class of organic compounds. The proposed rearrangements, retro Diels-Alder reaction, water loss, CO losses, and stable acylium ion formation, were evaluated based on tandem mass spectrometric experiments of normal and labeled precursor ions together to computational thermochemistry. B3LYP/6-311 + G** ab initio calculations studies were carried out to obtain energy diagrams to show the viability of the proposed mechanisms. The CO losses fragmentation channels have lower energies when compared with water losses and the other proposed fragmentations. The isotope labeling experiments indicate the H/D exchange of the hydroxyl protons and corroborate the proposed general fragmentation mechanism for anthocyanidins.

Pressurized liquid extraction of flavanols and alkaloids from cocoa bean shell using ethanol as solvent.

Cocoa shell (CS) is a co-product of the cocoa industry used mainly as fuel for boilers but with secondary applications as fertilizer and in animal feed. Although it is known that this material is rich in flavanols and alkaloids, to date, a study has not been conducted that has quantitatively identified these compounds in CS. Thus, the aim of this work was to characterize CS in terms of its composition, regarding catechin, epicatechin, procyanidin B2, caffeine and theobromine, and to evaluate the extraction kinetics of the total flavanols using pressurized liquid extraction (PLE) with absolute ethanol. For the determination of the extraction kinetic data, the DMAC method was used, while each compound was quantified using a UPLC-MS/MS analysis. The major compounds found were theobromine and epicatechin (mean values of 9.89 and 3.5 mg/g CS, respectively). PLE proved to be quite effective; the flavanols extraction yield was enhanced by increasing the temperature and extraction time however, high extraction times and temperatures degraded the procyanidins B2. Peleg's model applied to extraction data description provided a reasonable agreement with the experimental results, which allows their application in modeling and optimization of solid-liquid extraction of the total flavanols from cocoa bean shell.

Toxicological and behavioral responses as a tool to assess the effects of natural and synthetic dyes on zebrafish early life.

Organic dyes extracted from natural sources have been widely used to develop safety and eco-friendly dyes as an alternative to synthetic ones, since the latter are usually precursors of mutagenic compounds. Thereby, toxicity tests to non-target organisms are critical step to develop harmless dyes to environment and in this context, zebrafish early life stages are becoming an important alternative model. We aimed to assess the toxic effects of the synthetic dye Basic Red 51 (BR51, used in cosmetic industry), the natural dye erythrostominone (ERY, a potential commercial dye extracted from fungi) and its photodegradation product (DERY), using zebrafish early life assays. Developmental malformations on embryos and behavioral impairment on larvae were explored. Our results showed that embryos exposed to BR51 and ERY exhibited a large yolk sac (LOEC = 7.5 mg L-1), possibly due to a deformity or delayed resorption. ERY also induced pericardial and yolk sac edemas at high concentrations (LOEC = 15 and 30 mg L-1, respectively). Moreover, larvae swan less distance and time when exposed to ERY (LOEC = 7.5 mg L-1) and BR51 (LOEC = 1.875 mg L-1). The lowest larvae locomotion have been associated with impairment of the yolk sac, important tissue of the energy source. Interestingly, DERY did not affect neither development nor behavior of zebrafish, showing that ERY photodegradation is sufficient to prevent its toxic effects. In conclusion, both natural and synthetic dyes impaired development and behavior of zebrafish early life, therefore, a simple treatment of the natural dye can prevent the aquatic life impact.

Apigenin-7-O-glucoside oxidation catalyzed by P450-bioinspired systems.

Apigenin-7-O-glucoside (A7G) is the main flavonoid of Bidens gardneri Bak., a Brazilian plant with wide application in folk medicine. Despite the popular use of this plant, its biological effects are not completely known. This work tested the 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin iron(III) and manganese(III) chloride (Fe(TFPP)Cl and Mn(TFPP)Cl), and Jacobsen's catalyst as P450-bioinspired catalysts for A7G oxidation by different oxidants (PhIO, H2O2, m-CPBA, and t-BuOOH). Up to nine different products were detected by HPLC analysis; Reactions with metalloporphyrin/PhIO systems afforded high catalytic conversions (58-89%). In spite of providing smaller product yields, the metalloporphyrin/H2O2 systems led to superior product distribution. Fe(TFPP)Cl yielded the highest A7G conversion rates (79-93%) with the four different oxidants tested herein. In the presence of PhIO, the oxidative profile of the manganese catalysts was very close to the oxidative profile of Fe(TFPP)Cl. However, in medium containing peroxide, the reactivity of the manganese catalysts was lower as compared to the reactivity of Fe(TFPP)Cl. Reactions with Fe(TFPP)Cl/oxidant systems were analyzed by UPLC-MS; up to thirteen compounds were detected. A7G oxidation catalyzed by Fe(TFPP)Cl yielded seven compounds. Three other compounds had m/z profile compatible with the profile of the A7G metabolites. The A7G oxidation assays performed in the presence of P450-bioinspired catalysts demonstrated their great catalytic potential toward A7G. The present results may be useful to many areas of knowledge and to the research and development of numerous chemical and phamarcological processes, especially in terms of drug design, biological assays, and applications in medicinal chemistry.

Metalloporphyrins as biomimetic models for cytochrome p-450 in the oxidation of atrazine.

The aim of this work was to evaluate whether metalloporphyrin models could mimic the action of cytochrome P-450 in the oxidation of atrazine, a herbicide. The commercially available second-generation metalloporphyrins 5,10,15,20-tetrakis(2,6-dichlorophenyl)porphyrin metal(III) chloride [M(TDCPP)Cl] and 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin metal(III) chloride [M(TFPP)Cl] (metal = Fe or Mn) and the oxidants iodosylbenzene and metachloroperbenzoic acid were employed in this study. Results showed that the metalloporphyrins used here can oxidize atrazine. Yields as high as 32% were obtained for the Mn(TFPP)Cl/PhIO system, which shows that these catalysts can mimic both the in vivo and the in vitro action of cytochrome P-450, with formation of the metabolites DEA and DIA. The formation of five other unknown products was also detected, but only one of them could be identified, since the other four were present in very low concentrations. The compound COA, identified by mass spectrometry, was the main product in most of the oxidation reactions.

Direct Analysis of Amphetamine Stimulants in a Whole Urine Sample by Atmospheric Solids Analysis Probe Tandem Mass Spectrometry.

Amphetamine-type stimulants (ATS) are among illicit stimulant drugs that are most often used worldwide. A major challenge is to develop a fast and efficient methodology involving minimal sample preparation to analyze ATS in biological fluids. In this study, a urine pool solution containing amphetamine, methamphetamine, ephedrine, sibutramine, and fenfluramine at concentrations ranging from 0.5 pg/mL to 100 ng/mL was prepared and analyzed by atmospheric solids analysis probe tandem mass spectrometry (ASAP-MS/MS) and multiple reaction monitoring (MRM). A urine sample and saliva collected from a volunteer contributor (V1) were also analyzed. The limit of detection of the tested compounds ranged between 0.002 and 0.4 ng/mL in urine samples; the signal-to-noise ratio was 5. These results demonstrated that the ASAP-MS/MS methodology is applicable for the fast detection of ATS in urine samples with great sensitivity and specificity, without the need for cleanup, preconcentration, or chromatographic separation. Thus ASAP-MS/MS could potentially be used in clinical and forensic toxicology applications.

Versatility of tandem mass spectrometry for focused analysis of oxylipids.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the multiple reaction monitoring (MRM) scan mode has been the primary MS method applied for the target identification of specific and minor oxylipids in complex matrices, such as eicosanoids and docosanoids, which are potent lipid mediators derived from polyunsaturated fatty acid oxygenation. However, the high specificity of MRM can limit the detection of species with m/z MRM transitions not covered by the method. In addition to MRM, tandem-quadrupole mass analyzers enable other experiments to be conducted, by fragmenting ions via collision-induced dissociation process (CID). This paper presents the potential of tandem mass spectrometry for the focused analysis of oxylipids. We have successfully developed an LC-MS/MS method for the identification of precursor ions of m/z 115, a diagnostic product ion of 5-hydroxy- and 5-epoxy-fatty acids. As a proof of concept, the developed method was used to discover several oxylipids oxidized at C5 derived from arachidonic acid (C20 : 4) oxygenation in a hypothalamus rat extract that were not identified using the target MRM methodology. The proposed focused MS/MS-based approach in a tandem mass analyzer has proven to be a powerful strategy to accelerate the identification of oxylipids with structural similarities and assist the field of lipidomic research.

Dereplication of Streptomyces sp. AMC 23 polyether ionophore antibiotics by accurate-mass electrospray tandem mass spectrometry.

Actinomycetes, especially those belonging to the genus Streptomyces, are economically important from a biotechnological standpoint: they produce antibiotics, anticancer compounds and a variety of bioactive substances that are potentially applicable in the agrochemical and pharmaceutical industries. This paper combined accurate-mass electrospray tandem mass spectrometry in the full scan and product ion scan modes with compounds library data to identify the major compounds in the crude extract produced by Streptomyces sp. AMC 23; it also investigated how sodiated nonactin ([M + Na](+)) fragmented. Most product ions resulted from elimination of 184 mass units due to consecutive McLafferty-type rearrangements. The data allowed identification of four macrotetrolides homologous to nonactin (monactin, isodinactin, isotrinactin/trinactin and tetranactin) as well as three related linear dimer compounds (nonactyl nonactoate, nonactyl homononactoate and homononactyl homononactoate). The major product ions of the sodiated molecules of these compounds also originated from elimination of 184 and 198 mass units. UPLC-MS/MS in the neutral loss scan mode helped to identify these compounds on the basis of the elimination of 184 and 198 mass units. This method aided monitoring of the relative production of these compounds for 32 days and revealed that the biosynthetic process began with increased production of linear dimers as compared with macrotetrolides. These data could facilitate dereplication and identification of these compounds in other microbial crude extracts.

Bioassay-guided isolation of a low molecular weight PHB from Burkholderia sp. with phytotoxic activity.

This work reports on the bioassay-guided isolation and identification of the macrocyclic pentolide 1, a cyclic polyhydroxybutyrate (PHB) with low molecular weight. This metabolite is produced by Burkholderia sp. and it exhibited phytotoxic activity in a Lemna minor bioassay. Its structure was determined by (1)H and (13)C NMR, heteronuclear multiple quantum correlation, heteronuclear multiple bond correlation, IR, and electrospray ionization tandem mass spectrometry analyses. The period for maximum production of the pentolide was optimized and determined on the basis of multiple reaction monitoring experiments at 15 days. The potential of Burkholderia sp. as a producer of higher biopolymers of PHB was also investigated. The methodology employed here accelerated the isolation and characterization of a phytotoxic metabolite whose structure can serve as a model for the synthesis of new classes of herbicides.

Cyclam kappa4 to kappa3 ligand denticity change upon mono-n-substitution with a carboxypropyl pendant arm in a ruthenium nitrosyl complex.

The complex fac-[Ru(NO)Cl2(kappa(3)N(4),N(8),N(11)(1-carboxypropyl)cyclam)]Cl.H2O (1-carboxypropyl)cyclam=3-(1,4,8,11-tetraazacyclotetradecan-1-yl)propionic acid) was prepared in a one pot reaction by mixing equimolar amounts of RuNOCl 3 and (1-carboxypropyl)cyclam and was characterized by X-ray crystallography, electrospray ionization tandem mass spectrometry (ESI-MS/MS), elemental analysis, NMR, and electronic and vibrational (IR) spectroscopies. fac-[Ru(NO)Cl 2(kappa(3)N(4),N(8),N(11)(1-carboxypropyl)cyclam)]Cl.H2O crystallizes in the triclinic, space group P1, No. 2, with unit cell parameters of a=8.501(1) A, b=9.157(1) A, c=14.200(1) A, alpha=72.564(5) degrees , beta=82.512(5) degrees , gamma=80.308(5) degrees , and Z=2. The Ru-N interatomic distance and bond angle in the [Ru-NO] unit are 1.739(2) A and 167.7(2) degrees , respectively. ESI-MS/MS shows characteristic dissociation chemistry that initiates by HCl or NO loss. The IR spectrum displays a nu(NO) at 1881 cm(-1) indicating a nitrosonium character. The electronic spectrum shows absorptions bands at 264 nm (log epsilon=3.27), 404 nm (log epsilon=2.53), and 532 nm (log epsilon=1.88). (1)H and (13)C NMR are in agreement with the proposed molecular structure, which shows a very singular architecture where the cyclam ring N (with the carboxypropyl pendant arm) is not coordinated to the ruthenium resulting in a kappa(3) instead of the expected kappa(4) denticity.

Biomimetic oxidation studies of monensin A catalyzed by metalloporphyrins: identification of hydroxyl derivative product by electrospray tandem mass spectrometry

Monensin A is an important commercially available natural product isolated from Streptomyces cinnamonensins that shows antibiotic and anti-parasitic activities. This molecule has a significant influence in the antibiotic market, but until now there are no studies on putative metabolite formations. Bioorganic catalysts applying metalloporphyrins and mono-oxygen donors are able to mimic the cytochrome P450 reactions. This model has been employed for natural product metabolism studies affording several new putative metabolites and in vivo experiments confirming the relevance of this procedure. In this work we evaluated the potential of 10,15,20-tetrakis (pentafluorophenyl) porphyrin metal(III) chloride [Fe(TFPP)Cl] catalyst models to afford a putative monensin A metabolite. Oxidation agents such as meta-chloroperoxy benzoic acid, iodosylbenzene, hydrogen peroxide 30 wt.% and tert-butyl hydroperoxide 70 wt.%, were used to investigate different reaction conditions, in addition to the analysis of the influence of the solvent. The quantification of total monensin A conversion and the structure of the new hydroxylated putative metabolite were proposed based on electrospray ionization tandem mass spectrometry analysis. The porphyrin tested, afforded moderate conversions of monensin A in all reaction conditions and the selectivity was found to be dependent on the oxidation/medium employed.