Systematic UPLC-ESI-MS/MS Study on the Occurrence of Staurosporine and Derivatives in Associated Marine Microorganisms from Eudistoma vannamei

Former bioactivity-guided analysis of the marine invertebrate Eudistoma vannamei led to the isolation of staurosporine derivatives, which revealed strong cytotoxic activity against several human cancer cell lines. The occurrence of such alkaloids in E. vannamei may be correlated to the presence of associated biota, such as Streptomyces bacteria. In agreement to this hypothesis, marine microorganisms associated with E. vannamei were recovered and cultured, leading to a total of 84 isolated bacterial strains. Gas phase fragmentation reactions of staurosporine and derivatives were systematically studied and the analyzed results further supported by computational chemistry studies. The resulting fragment patterns were used to search for the presence of different derivatives in extracts of isolated microorganisms, thereby using LC-MS/MS analysis in MRM mode. These results evidenced that one isolated Streptomyces sp. was able to generate staurosporine, while none of the hydroxy-7-oxo derivatives were detected. Finally, significant cytotoxic activity against human cancer lines was observed for one of the extracts.

Efficiency of extrinsic and intrinsic charge-carrier photogeneration processes obtained from the steady-state photocurrent action spectra of poly(p-phenylene vinylene) derivatives

The efficiency of the charge-carrier photogeneration processes in poly(2,5-bis(3',7'-dimethyl-octyloxy)-1,4-phenylene vinylene) (OC(1)OC10-PPV) has been analyzed by the spectral response of the photocurrent of devices in ITO/polymer/Al structures. The symbatic response of the photocurrent action spectra of the OC1OC10-PPV devices, obtained for light-excitation through the ITO electrode and for forward bias, has been fitted using a phenomenological model which considers that the predominant transport mechanism under external applied electric field is the drift of photogenerated charge-carriers, neglecting charge-carrier diffusion. The proposed model takes into account that charge-carrier photogeneration occurs via intermediate stages of bounded pairs (excitonic states), followed by dissociation processes. Such processes result in two different contributions to the photoconductivity: The first one, associated to direct creation of unbound polaron pairs due to intrinsic photoionization; and the second one is associated to secondary processes like extrinsic photoinjection at the metallic electrodes. The results obtained from the model have shown that the intrinsic component of the photoconductivity at higher excitation energies has a considerably higher efficiency than the extrinsic one, suggesting a dependence on the photon energy for the efficiency of the photogeneration process.

Two series of new semisynthetic triterpene derivatives: differences in anti-malarial activity, cytotoxicity and mechanism of action

Background The discovery and development of anti-malarial compounds of plant origin and semisynthetic derivatives thereof, such as quinine (QN) and chloroquine (CQ), has highlighted the importance of these compounds in the treatment of malaria. Ursolic acid analogues bearing an acetyl group at C-3 have demonstrated significant anti-malarial activity. With this in mind, two new series of betulinic acid (BA) and ursolic acid (UA) derivatives with ester groups at C-3 were synthesized in an attempt to improve anti-malarial activity, reduce cytotoxicity, and search for new targets. In vitro activity against CQ-sensitive Plasmodium falciparum 3D7 and an evaluation of cytotoxicity in a mammalian cell line (HEK293T) are reported. Furthermore, two possible mechanisms of action of anti-malarial compounds have been evaluated: effects on mitochondrial membrane potential (ΔΨm) and inhibition of β-haematin formation. Results Among the 18 derivatives synthesized, those having shorter side chains were most effective against CQ-sensitive P. falciparum 3D7, and were non-cytotoxic. These derivatives were three to five times more active than BA and UA. A DiOC6(3) ΔΨm assay showed that mitochondria are not involved in their mechanism of action. Inhibition of β-haematin formation by the active derivatives was weaker than with CQ. Compounds of the BA series were generally more active against P. falciparum 3D7 than those of the UA series. Conclusions Three new anti-malarial prototypes were obtained from natural sources through an easy and relatively inexpensive synthesis. They represent an alternative for new lead compounds for anti-malarial chemotherapy.

In vitro trypanocidal evaluation of pinane derivatives from essential oils of ripe fruits from Schinus terebinthifolius Raddi (Anacardiaceae)

Essential oils of ripe fruits from Schinus terebinthifolius (Anacardiaceae), obtained using a pilot extractor and a Clevenger apparatus were chemically characterized. Due the high amount of (-)- α-pinene in both oils, this monoterpene was tested against the protozoan parasite Trypanosoma cruzi, showing a moderate potential (IC50 63.56 µg/mL) when compared to benznidazole (IC50 43.14 µg/mL). Otherwise, (-)- α-pinene oxide did not showed anti-trypanosomal activity (IC50 > 400 µg/mL) while (-)-pinane showed an IC50 of 56.50 µg/mL. The obtained results indicated that the epoxydation of α-pinene results to the loss of the anti-parasitic activity while its hydrogenation product, contributed slightly to the increased activity.

Systematic UPLC-ESI-MS/MS study on the occurrence of staurosporine and derivatives in associated marine microorganisms from Eudistoma vannamei

Former bioactivity-guided analysis of the marine invertebrate Eudistoma vannamei led to the isolation of staurosporine derivatives, which revealed strong cytotoxic activity against several human cancer cell lines. The occurrence of such alkaloids in E. vannamei may be correlated to the presence of associated biota, such as Streptomyces bacteria. In agreement to this hypothesis, marine microorganisms associated with E. vannamei were recovered and cultured, leading to a total of 84 isolated bacterial strains. Gas phase fragmentation reactions of staurosporine and derivatives were systematically studied and the analyzed results further supported by computational chemistry studies. The resulting fragment patterns were used to search for the presence of different derivatives in extracts of isolated microorganisms, thereby using LC-MS/MS analysis in MRM mode. These results evidenced that one isolated Streptomyces sp. was able to generate staurosporine, while none of the hydroxy-7-oxo derivatives were detected. Finally, significant cytotoxic activity against human cancer lines was observed for one of the extracts.

Self-assembled films from chitosan and poly(vinyl sulfonic acid) on Nafion® for direct methanol fuel cell

Chitosan/poly(vinyl sulfonic acid) (PVS) films have been prepared on Nafion® membranes by the layer-by-layer (LbL) method for use in direct methanol fuel cell (DMFC). Computational methods and Fourier transform infrared (FTIR) spectra suggest that an ionic pair is formed between the sulfonic group of PVS and the protonated amine group of chitosan, thereby promoting the growth of LbL films on the Nafion® membrane as well as partial blocking of methanol. Chronopotentiometry and potential linear scanning experiments have been carried out for investigation of methanol crossover through the Nafion® and chitosan/PVS/Nafion® membranes in a diaphragm diffusion cell. On the basis of electrical impedance measurements, the values of proton resistance of the Nafion® and chitosan/PVS/Nafion® membranes are close due to the small thickness of the LbL film. Thus, it is expected an improved DMFC performance once the additional resistance of the self-assembled film is negligible compared to the result associated with the decrease in the crossover effect.

Chitosan: effect of a new chelating agent on the microhardness of root dentin

The effect of solutions of 0.2% chitosan, 15% EDTA and 10% citric acid on the microhardness of root dentin was evaluated comparatively in this study. Thirteen sound human maxillary central incisors were selected and decoronated at the cementoenamel junction. Ten roots were set into rapid polymerization acrylic resin and the root/resin block was fitted to the cutting machine to obtain slices from the cervical third. The first slice was discarded and the second slice was divided into four quadrants. Each quadrant was used to construct a sample, so that 4 specimens were obtained from each root slice, being one for each chelating solution to be tested: 15% EDTA, 10% citric acid, 0.2% chitosan and distilled water (control). The specimens were exposed to 50 μL of the solution for 5 min, and then washed in distilled water. A microhardness tester (Knoop hardness) with a 10 g load was used for 15 s. Data were analyzed statistically by one-way ANOVA and Tukey-Kramer test (α=0.05). The other 3 roots had the canals instrumented and irrigated at the end of the biomechanical preparation with the test solutions, and then examined by scanning electron microscopy (SEM) for qualitative analysis. All solutions reduced the microhardness of root dentin in a way that was statistically similar to each other (p>0.05) but significantly different from the control (p>0.05). The SEM micrographs showed that the three solutions removed smear layer from the middle third of the root canal. In conclusion, 0.2% chitosan, 15% EDTA and 10% citric acid showed similar effects in reducing dentin microhardness.

Time-dependent effects of chitosan on dentin structures

Complete debridement with smear layer removal are essential measures for achieving a successful outcome of root canal treatment. The aim of this study was to evaluate the effects of chitosan at different concentrations on the removal of the smear layer and on dentin structure after 3 and 5 min of application. Twelve recently extracted maxillary canine teeth were instrumented using the crown-down technique and irrigated with 1% sodium hypochlorite. The specimens were distributed according to the time and concentration of the final irrigating solution: G1: 0.1% chitosan for 3 min; G2: 0.2% chitosan for 3 min; G3: 0.37% chitosan for 3 min; G4: 0.1% chitosan for 5 min; G5: 0.2% chitosan for 5 min; G6: 0.37% chitosan for 5 min. All samples were prepared for SEM analysis. G1 exhibited removal of the smear layer, but not the smear plugs. G2 showed visible and open tubules with slight erosion of the peritubular dentin. Cleaning in G3 was similar to that in G2, however, the erosive effect was greater. There was expansion of the diameter of the tubules in G4; and in G5 and G6, there was severe erosion with deterioration of dentin surface. In conclusion, 0.2% chitosan for 3 min appeared to be efficient for removing the smear layer, causing little erosion of dentin.

Cytotoxicity tests for nanostructured chitosan/PEO membranes using the agar diffusion method

Electrospinning is used to produce fibers in the nanometer range by stretching a polymeric jet using electric fields of high magnitude. Chitosan is an abundant natural polymer that can be used to obtain biocompatible nanostructured membranes. The objectives of this work were to obtain nanostructured membranes based on blends of chitosan and polyoxyethylene (PEO), and evaluate their thermal and morphological properties, as well as their in vitro biocompatibility by agar diffusion cytotoxicity tests for three different cell lines. A nanostructured fibrous membrane with fiber diameters in the order of 200 nm was obtained, which presented a rough surface and thickness ranging from one to two millimeters. The results of the cytotoxicity tests evidenced that the chitosan/PEO membranes are non-toxic to the cells studied in this work. Further, the electrospinning technique was effective in obtaining nanostructured chitosan/PEO membranes, which showed biocompatibility according to in vitro preliminary tests using the cell lines.

Chitosan-based biomaterials used in critical-size bone defects: radiographic study in rat's calvaria

OBJETIVO: Este estudo avaliou através de imagens radiográficas digitais, a ação de biomateriais de quitosana e de cloridrato de quitosana, com baixo e alto peso molecular, utilizados na correção de defeitos ósseos de tamanho crítico (DOTC)em calvária de ratos. MATERIAL E MÉTODO: DOTCs com 8 mm de diâmetro foram criados cirurgicamente na calvária de 50 ratos Holtzman. Em 10 animais o defeito foi preenchido foram preenchidos com coágulo sanguíneo (controle negativo). Os 40 animais restantes foram divididos de acordo com o biomaterial utilizado no preenchimento do defeito (quitosana de baixo peso e de alto peso molecular, e cloridrato de quitosana de baixo e de alto peso molecular), e foram avaliados em dois períodos experimentais (15 e 60 dias), totalizando 5 animais/biomaterial/período de avaliação. RESULTADO: A avaliação radiográfica foi feita utilizando duas radiografias digitais do crânio do animal: uma tomada logo após o defeito ósseo ser criado e a outra no momento do sacrifício. Nessas imagens, foi avaliada a densidade óssea radiográfica inicial e a final na área do defeito, que foram comparadas. As análises na densidade óssea radiográfica indicaram aumento da densidade óssea radiográfica dos DOTCs tratados para todos os biomateriais testados, em ambos os períodos. Resultados semelhantes foram encontrados no grupo controle. CONCLUSÃO: Conclui-se que os biomateriais de quitosana testados não foram capazes de aumentar a densidade radiográfica em DOTC realizados em calvária de ratos.

Effect of a polymeric protective coating on optical and electrical properties of poly (p-phenylene vinylene) derivatives

The resistance to photodegradation of poly [(2-methoxy-5-n-hexyloxy)-p-phenylene vinylene] (OC1OC6-PPV) films was significantly enhanced by the use of poly(vinyl alcohol) 99% hydrolyzed as protective coating. The deposition of poly(vinyl alcohol) onto OC1OC6-PPV films did not affect the absorption and the emission spectra of the luminescent polymer. The protected film showed 5% drop on the absorbance at 500nm after 270 hours of light exposure while the unprotected film completely degraded in the same conditions. The conductivity of the protected film remained stable (around 7 × 10-10 S/m) while the value for the unprotected one dropped around two orders of magnitude after 100 hours of light exposure.

Effects of Betuletol derivatives in human normal cells and human leukaemia cells lines

Estudio de la citotoxicidad de varios derivados del Betuletol y sus efectos sobre células humanas de leucemia mieloide (U937)comparando sus efectos con celulas humanas normales.

Organocatalytic asymmetric mannich-type reactions: an easy approach to optically active amine derivatives

The topics I came across during the period I spent as a Ph.D. student are mainly two. The first concerns new organocatalytic protocols for Mannich-type reactions mediated by Cinchona alkaloids derivatives (Scheme I, left); the second topic, instead, regards the study of a new approach towards the enantioselective total synthesis of Aspirochlorine, a potent gliotoxin that recent studies indicate as a highly selective and active agent against fungi (Scheme I, right). At the beginning of 2005 I had the chance to join the group of Prof. Alfredo Ricci at the Department of Organic Chemistry of the University of Bologna, starting my PhD studies. During the first period I started to study a new homogeneous organocatalytic aza-Henry reaction by means of Cinchona alkaloid derivatives as chiral base catalysts with good results. Soon after we introduced a new protocol which allowed the in situ synthesis of N-carbamoyl imines, scarcely stable, moisture sensitive compounds. For this purpose we used α-amido sulfones, bench stable white crystalline solids, as imine precursors (Scheme II). In particular we were able to obtain the aza-Henry adducts, by using chiral phase transfer catalysis, with a broad range of substituents as R-group and excellent results, unprecedented for Mannich-type transformations (Scheme II). With the optimised protocol in hand we have extended the methodology to the other Mannich-type reactions. We applied the new method to the Mannich, Strecker and Pudovik (hydrophosphonylation of imines) reactions with very good results in terms of enantioselections and yields, broadening the usefulness of this novel protocol. The Mannich reaction was certainly the most extensively studied work in this thesis (Scheme III). Initially we developed the reaction with α-amido sulfones as imine precursors and non-commercially available malonates with excellent results in terms of yields and enantioselections.3 In this particular case we recorded 1 mol% of catalyst loading, very low for organocatalytic processes. Then we thought to develop a new Mannich reaction by using simpler malonates, such as dimethyl malonate.4 With new optimised condition the reaction provided slightly lower enantioselections than the previous protocol, but the Mannich adducts were very versatile for the obtainment of β3-amino acids. Furthermore we performed the first addition of cyclic β-ketoester to α-amido sulfones obtaining the corresponding products in good yield with high level of diastereomeric and enantiomeric excess (Scheme III). Further studies were done about the Strecker reaction mediated by Cinchona alkaloid phase-transfer quaternary ammonium salt derivatives, using acetone cyanohydrin, a relatively harmless cyanide source (Scheme IV). The reaction proceeded very well providing the corresponding α-amino nitriles in good yields and enantiomeric excesses. Finally, we developed two new complementary methodologies for the hydrophosphonylation of imines (Scheme V). As a result of the low stability of the products derived from aromatic imines, we performed the reactions in mild homogeneous basic condition by using quinine as a chiral base catalyst giving the α-aryl-α-amido phosphonic acid esters as products (Scheme V, top).6 On the other hand, we performed the addition of dialkyl phosphite to aliphatic imines by using chiral Cinchona alkaloid phase transfer quaternary ammonium salt derivatives using our methodology based on α-amido sulfones (Scheme V, bottom). The results were good for both procedures covering a broad range of α-amino phosphonic acid ester. During the second year Ph.D. studies, I spent six months in the group of Prof. Steven V. Ley, at the Department of Chemistry of the University of Cambridge, in United Kingdom. During this fruitful period I have been involved in a project concerning the enantioselective synthesis of Aspirochlorine. We provided a new route for the synthesis of a key intermediate, reducing the number of steps and increasing the overall yield. Then we introduced a new enantioselective spirocyclisation for the synthesis of a chiral building block for the completion of the synthesis (Scheme VI).

From 3,3,4,4-Tetraethoxybut-1-yne to furan derivatives

The starting material for this project was the highly functionalized compound 3,3,4,4- tetraethoxybut-1-yne (TEB) and it can be prepared from ethyl vinyl ether by a 4-steps synthesis. The third and the fourth step in TEB synthesis were sensitive to reaction conditions, so it was developed a strategy to try to optimize the third step and obtain TEB with higher yields. An approach, which tries to optimize also the fourth step, will be developed in further works. Several γ-hydroxy-α,β-unsaturated acetylenic ketones can be prepared from 3,3,4,4- tetraethoxybut-1-yne. TEB and γ-hydroxy-α,β-unsaturated acetylenic ketones have been previously synthesized in good yields using various reaction routes. In this work will be shown the synthesis of 1,1-diethoxy-5-hydroxyhex-3-yn-2-one, 1,1-diethoxy-5-hydroxyundec-3-yn-2-one and 1,1-diethoxy-5-hydroxydodec-3-yn-2-one, which will react with ethyl acetoacetate to give, respectively, ethyl 4-(3,3-diethoxy-2-oxopropyl)-2,5-dimethylfuran-3-carboxylate, ethyl 4-(3,3-diethoxy-2-oxopropyl)-5-hexyl-2-methylfuran-3-carboxylate and ethyl 4-(3,3-diethoxy-2-oxopropyl)- 5-heptyl-2-methylfuran-3-carboxylate furan derivatives. This thesis project was carried out during the year 2011, at the Department of Chemistry of the University of Bergen.

Liquid crystalline oligofluorenes and their derivatives

Homo-oligofluorenes (OFn), polyfluorenes (PF2/6) and oligofluorenes with one fluorenenone group in the center (OFnK) were synthesized. They were used as model compounds to understand of the structure-property relationships of polyfluorenes and the origin of the green emission in the photoluminescence (after photooxidation of the PFs) and the electroluminescence (EL) spectra. The electronic, electrochemical properties, thermal behavior, supramolecular self-assembly, and photophysical properties of OFn, PF2/6 and OFnK were investigated. Oligofluorenes with 2-ethylhexyl side chain (OF2-OF7) from the dimer up to the heptamer were prepared by a series of stepwise transition metal mediated Suzuki and Yamamoto coupling reactions. Polyfluorene was synthesized by Yamamoto coupling of 2,7-dibromo-9,9-bis(2-ethylhexyl)fluorene. Oligofluorenes with one fluorenone group in the center (OF3K, OF5K, OF7K) were prepared by Suzuki coupling between the monoboronic fluorenyl monomer, dimer, trimer and 2, 7-dibromofluorenone. The electrochemical and electronic properties of homo-oligofluorenes (OFn) were systematically studied by several combined techniques such as cyclic voltammetry, differential pulse voltammetry, UV-vis absorption spectroscopy, steady and time-resolved fluorescence spectroscopy. It was found that the oligofluorenes behave like classical conjugated oligomers, i.e., with the increase of the chain-length, the corresponding oxidation potential, the absorption and emission maximum, ionization potential, electron affinity, band gap and the photoluminescence lifetime displayed a very good linear relation with the reciprocal number of the fluorene units (1/n). The extrapolation of these linear relations to infinite chain length predicted the electrochemical and electronic properties of the corresponding polyfluorenes. The thermal behavior, single-crystal structure and supramolecular packing, alignment properties, and molecular dynamics of the homo-oligofluorenes (OFn) up to the polymer were studied using techniques such as TGA, DSC, WAXS, POM and DS. The OFn from tetramer to heptamer show a smectic liquid crystalline phase with clearly defined isotropization temperature. The oligomers do show a glass transition which exhibits n-1 dependence and allows extrapolation to a hypothetical glass transition of the polymer at around 64 °C. A smectic packing and helix-like conformation for the oligofluorenes from tetramer to heptamer was supported by WAXS experiments, simulation, and single-crystal structure of some oligofluorene derivatives. Oligofluorenes were aligned more easily than the corresponding polymer, and the alignability increased with the molecular length from tetramer to heptamer. The molecular dynamics in a series of oligofluorenes up to the polymer was studied using dielectric spectroscopy. The photophysical properties of OFn and PF2/6 were investigated by the steady-state spectra (UV-vis absorption and fluorescence spectra) and time-resolved fluorescence spectra both in solution and thin film. The time-resolved fluorescence spectra of the oligofluorenes were measured by streak camera and gate detection technique. The lifetime of the oligofluorenes decreased with the extension of the chain-length. No green emission was observed in CW, prompt and delayed fluorescence for oligofluorenes in m-THF and film at RT and 77K. Phosphorescence was observed for oligofluorenes in frozen dilute m-THF solution at 77K and its lifetime increased with length of oligofluorenes. A linear relation was obtained for triplet energy and singlet energy as a function of the reciprocal degree of polymerization, and the singlet-triplet energy gap (S1-T1) was found to decrease with the increase of degree of polymerization. Oligofluorenes with one fluorenone unit at the center were used as model compounds to understand the origin of the low-energy (“green”) emission band in the photoluminescence and electroluminescence spectra of polyfluorenes. Their electrochemical properties were investigated by CV, and the ionization potential (Ip) and electron affinity (Ea) were calculated from the onset of oxidation and reduction of OFnK. The photophysical properties of OFnK were studied in dilute solution and thin film by steady-state spectra and time-resolved fluorescence spectra. A strong green emission accompanied with a weak blue emission were obtained in solution and only green emission was observed on film. The strong green emission of OFnK suggested that rapid energy transfer takes place from higher energy sites (fluorene segments) to lower energy sites (fluorenone unit) prior to the radiative decay of the excited species. The fluorescence spectra of OFnK also showed solvatochromism. Monoexponential decay behaviour was observed by time-resolved fluorescence measurements. In addition, the site-selective excitation and concentration dependence of the fluorescence spectra were investigated. The ratio of green and blue emission band intensities increases with the increase of the concentration. The observed strong concentration dependence of the green emission band in solution suggests that increased interchain interactions among the fluorenone-containing oligofluorene chain enhanced the emission from the fluorenone defects at higher concentration. On the other hand, the mono-exponential decay behaviour and power dependence were not influenced significantly by the concentration. We have ruled out the possibility that the green emission band originates from aggregates or excimer formation. Energy transfer was further investigated using a model system of a polyfluorene doped by OFnK. Förster-type energy transfer took place from PF2/6 to OFnK, and the energy transfer efficiency increased with increasing of the concentration of OFnK. Efficient funneling of excitation energy from the high-energy fluorene segments to the low-energy fluorenone defects results from energy migration by hopping of excitations along a single polymer chain until they are trapped on the fluorenone defects on that chain or transferred onto neighbouring chains by Förster-type interchain energy transfer process. These results imply that the red-shifted emission in polyfluorenes can originate from (usually undesirable) keto groups at the bridging carbon atoms-especially if the samples have been subject to photo- or electro-oxidation or if fluorenone units are present due to an improper purification of the monomers prior to polymerization.