Hector H.F. Koolen

Determination of the phenolic composition from Brazilian tropical fruits by UHPLC–MS/MS

Although Brazil is the third largest fruit producer in the world, several specimens consumed are not well studied from the chemical viewpoint, especially for quantitative analysis. For this reason and the crescent employment of mass spectrometry (MS) techniques in food science we selected twenty-two phenolic compounds with important biological activities and developed an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method using electrospray (ESI) in negative ion mode aiming their quantification in largely consumed Brazilian fruits (açaí-do-Amazonas, acerola, cashew apple, camu-camu, pineapple and taperebá). Multiple reaction monitoring (MRM) was applied and the selection of proper product ions for each transition assured high selectivity. Linearity (0.995<r(2)<0.999), limit of detection (28.85-333.3 pg/mL), limit of quantification (96.15-1111 pg/mL), inter- and intraday accuracy (>80%), precision (CV<20%) and extraction recovery rate (>80%) were satisfactory and showed that the method provides an efficient protocol to analyze phenolic compounds in fruit pulp extracts.

Phytochemical Study of the Alkaloidal Fractions of Unonopsis duckei R. E. Fr. Guided by Electrospray Ionisation Ion-trap Tandem Mass Spectrometry

INTRODUCTION The Unonopsis genus is a promising source of aporphinoid alkaloids, substances with great biological potential. These alkaloids have a well-defined mass spectrometry fragmentation pattern that, together with previous phytochemical knowledge, can guide the isolation of alkaloids not yet described for the genus. OBJECTIVE Isolate substances not yet described in the Unonopsis genus, guided by alkaloidal profile analyses of stem barks, twigs and leaves of Unonopsis duckei using electrospray ionisation ion-trap tandem mass spectrometry (ESI-IT/MS(n) ). METHODS Methanolic extracts from stem barks, twigs and leaves were submitted to a liquid-liquid, acid-base partitioning treatment to obtain the alkaloidal fractions. These fractions were analysed by direct infusion into an ESI-IT/MS(n) system. The major alkaloids observed for each fraction were submitted to fragmentation analysis. RESULTS The MS fragmentation patterns revealed the presence of alkaloids previously reported for Annonaceae, including nornuciferine, anonaine, asimilobine, liriodenine and lysicamine, known for the Unonopsis genus, as well as others that were not yet described for this genus. In this way, the proaporphine alkaloid glaziovine was isolated, as well as a mixture of the aporphine alkaloids glaucine and norglaucine, all described for the first time in the Unonopsis genus. CONCLUSION Mass spectrometry monitoring was fundamental to prioritise the isolation of substances not yet identified for the Unonopsis genus, dismissing known compounds and simplifying the phytochemical study.

Endophytic fungi from Myrcia guianensis at the Brazilian Amazon: distribution and bioactivity

Beneficial interactions between plants and microorganisms have been investigated under different ecological, physiological, biochemical, and genetic aspects. However, the systematic exploration of biomolecules with potential for biotechnological products from this interaction still is relatively scarce. Therefore, this study aimed the evaluation of the diversity and antimicrobial activity of the endophytic fungi obtained from roots, stems and leafs of Myrcia guianensis (Myrtaceae) from the Brazilian Amazon. 156 endophytic fungi were isolated and above 80% were identified by morphological examination as belonging to the genera Pestalotiopsis, Phomopsis, Aspergillus, Xylaria, Nectria, Penicillium and Fusarium. Fermented broth of those fungi were assayed for antimicrobial activity and four inhibited the growth of Staphylococcus aureus, Enterococcus faecalis, Candida albicans and Penicillium avellaneum. As the strain named MgRe2.2.3B (Nectria haematococca) had shown the most promising results against those pathogenic strains, its fermented broth was fractioned and only its two low polar fractions demonstrated to be active. Both fractions exhibited a minimum bactericidal concentration of 50 μg.mL−1 against S. aureus and a minimum fungicidal concentration of 100 μg.mL−1 against P. avellaneum. These results demonstrate the diversity of fungal genera in M. guianensis and the potential of these endophytic fungi for the production of new antibiotics.

Leishmanicidal activity of fractions rich in aporphine alkaloids from Amazonian Unonopsis species

In vitro evaluation of alkaloidal fractions of twigs, barks and leaves from two Unonopsis species, Unonopsis guatterioides R.E. Fr. and Unonopsis duckei R.E. Fr., Annonaceae, against promastigote forms of Leishmania amazonensis revealed these species as sources of substances with promising leishmanicidal potential. All alkaloidal fractions from twigs, barks and leaves of U. guatterioides were classified as highly active, with IC50 1.07, 1.90, and 2.79 mg/mL, respectively. Only the alkaloidal fraction from the twigs of U. duckei was classified as inactive.

7,7-Dimethylaporphine and Other Alkaloids from the Bark of Guatteria friesiana

Phytochemical investigation of the bark of Guatteria friesiana afforded 12 new aporphines (1-12), along with nine known alkaloids (13-21). The structures of the new alkaloids were determined on the basis of spectroscopic data interpretation. The cytotoxic activity of the isolated compounds against a small panel of tumor cell lines was assessed using the Alamar blue assay.

Mauritic acid: a new dammarane triterpene from the roots of Mauritia flexuosa L.f. (Arecaceae)

A new dammarane triterpene named mauritic acid (1) was isolated from the roots of Mauritia flexuosa L.f. The complete structural assignment of this new compound was elucidated from spectroscopic methods. Moreover, this compound was evaluated for its cytotoxicity against human cancer cell lines (OVCAR-8, PCM3, NCIH358M and different leukaemia cell strains). The mauritic acid presented significant cytotoxicity against OVCAR-8, PCM3 and NCIH358M cell lines with IC50 3.02, 2.39 and 6.19 μM, respectively. The triterpenes 1 and 2 were also tested for their antimicrobial activity against 15 strains of microorganisms, including fungi and bacteria, with the best minimal inhibitory concentration values ranging from 50.8 to 203.5 μM.

Imidate-Based Cross-Linkers for Structural Proteomics: Increased Charge of Protein and Peptide Ions and CID and ECD Fragmentation Studies

AbstractChemical cross-linking is an attractive low-resolution technique for structural studies of protein complexes. Distance constraints obtained from cross-linked peptides identified by mass spectrometry (MS) are used to construct and validate protein models. Amidinating cross-linkers such as diethyl suberthioimidate (DEST) have been used successfully in chemical cross-linking experiments. In this work, the application of a commercial diimidate cross-linking reagent, dimethyl suberimidate (DMS), was evaluated with model peptides and proteins. The peptides were designed with acetylated N-termini followed by random sequences containing two Lys residues separated by an Arg residue. After cross-linking reactions, intra- and intermolecular cross-linked species were submitted to CID and ECD dissociations to study their fragmentation features in the gas phase. Fragmentation of intramolecular peptides by collision induced dissociation (CID) demonstrates a unique two-step fragmentation pathway involving formation of a ketimine as intermediate. Electron capture and electron transfer dissociation (ECD and ETD) experiments demonstrated that the cyclic moiety is not dissociated. Intermolecular species demonstrated previously described fragmentation behavior in both CID and ECD experiments. The charge state distributions (CSD) obtained after reaction with DMS were compared with those obtained with disuccinimidyl suberate (DSS). CSDs for peptides and proteins were increased after their reaction with DMS, owing to the higher basicity of DMS modified species. These features were also observed in LC-MS experiments with bovine carbonic anhydrase II (BCA) after cross-linking with DMS and tryptic proteolysis. Cross-linked peptides derived from this protein were identified at high confidence and those species were in agreement with the crystal structure of BCA. Figureᅟ

Antiprotozoal and antioxidant alkaloids from Alternanthera littoralis

Five alkaloids, in addition to hydroxytyrosol and uridine, were isolated from aerial parts of Alternanthera littoralis P. Beauv. Among the isolated compounds, alternamide A was an unusual tricyclic alkaloid with a bridged benzoazepine core. All isolated alkaloids have a catechol moiety, indicating a possible common biosynthetic route. Their structures were established by 1D and 2D NMR spectroscopy in combination with extensive tandem MS experiments by collisional induced dissociation (CID). The antiprotozoal activity of the isolated compounds was assayed against trypomastigote forms of Trypanosoma cruzi and amastigotes of Leishmania amazonensis. Alternamine A was the most active compound, reducing markedly the viability of both parasites. Antioxidant capacities evaluated by ORACFL assay showed that the isolated alkaloids (mainly alternamide B) contributed to the high activity recorded for the ethanolic crude extract; possibly, the catechol moiety present in all structures plays a central role in this result.

Sample preparation focusing on plant proteomics: extraction, evaluation and identification of proteins from sunflower seeds

In the present work 17 different procedures for extraction of proteins from sunflower (Helianthus annuus L.) seeds in natura were evaluated regarding extraction time and temperature, type of solvent, and analytical procedure. After each extraction procedure, total protein was determined, ranging from 260 ± 5 to 2727 ± 11 μg g−1. Then, a 2-D PAGE was used for P4, P5, P13 and P17, which were considered the most efficient extraction protocols, to calculate the match between them. The highest (77 ± 3%) and the lowest (48 ± 1%) values were achieved when P13 × P17 and P4 × P17 protocols were compared, respectively. Additionally, the P4 and P5 protocols presented the highest number of spots (196 ± 11 and 194 ± 20) after 2-D PAGE protein separation. From both protocols (P4 and P5), 66 protein spots were visualized in both extraction protocols and also analyzed by the 2-D PAGE technique for verifying possible changes in protein expression. Thirty-six spots were differentially found at 90% (or 1.8 times) and related to their relative volume and/or intensity. From these protein spots, 24 (ca. 67%) were successfully identified, 17 being proteins from sunflower seeds. The others are homologous proteins from other plant organisms. All of these analyses contributed to choosing the protocol P4 as the best for protein extraction from sunflower seeds.

Fullerene separation and identification by traveling wave ion mobility mass spectrometry in laser desorption processes during asphaltene analysis.

* Correspondence to: Hector Henrique Ferreira Koolen, Institute of Chemistry, University of Campinas, Campinas, SP 13083-970, Brazil. E-mail: hectorkoolen@gmail.com; hkoolen@uea.edu.br Asphaltenes constitute the heaviest polar fraction in crude petroleum, and the structural elucidation of its components has been a challenge for many analytical techniques. The structurally related components of asphaltenes normally associate in solution to form complex colloidal structures, and this behavior is responsible for many of its phenomenological properties, including some of practical relevance associated with oil transportation and storage, or with the behavior of the oil/water emulsions formed in marine spills and several problems such as rapid deactivation of catalysts, coking within the reactor or fractionator, and formation of sludge in the products. Ion mobility spectrometry, originally known as plasma chromatography, was introduced in the 1960s and consists of a gas-phase ion separation technique. Species are separated based on their charge states and sizes/shapes, the latter being more rigorously described as their collision cross sections (CCS). Resolution and sensitivity have significantly improved in the last years, and it has been explored to analyse carbon clusters, peptides and proteins, nucleotides, synthetic polymers, and petroleum samples. Traveling wave ion mobility (TWIM) has been introduced more recently as a new mode of ion propulsion and separation for ion mobility spectrometry experiments. Briefly, in TWIM, ions are accumulated and periodically released into a stacked-ring ion guide (T-wave cell), where they drift under the action of a continuous train of transient voltage pulses (traveling waves) applied to pairs of stacked ring electrodes, encompassing a very compact device (18.5 cm long) but with advantages in the ion transmission and resolution. Herein, we report the formation of fullerene compounds during the ionization process of asphaltene, its separation through TWIM, and its identification by an approach of CCS measurements and theoretical calculations. Asphaltene fractions were obtained from Brazilian crude oils, by precipitation with n-heptane according to the standard procedure IP143/96. A Brazilian coal sample was purchased from Copelmi Mineração Ltda. (Butiá, RS, Brazil) for comparison purposes. The samples were solubilized in toluene and spotted in steel plates suitable for laser desorption ionization (LDI) analysis. Thin layers of the carbonaceous material were obtained after application of 1μL of the samples and drying under atmospheric conditions. Mass spectrometric experiments were performed with a Waters Synapt G1 HDMS. Gas-phase ions were generated by a LDI probe operating in positive mode. LDI acquisitions were carried out using a 200-Hz solid state laser (Nd:YAG, UV 355nm) (Waters). LDIspecific instrumental parameters were as follows: pulse energy 400, focal length 60mm. The separations were carried out in the