Mass Spectrometry-Based Systems Approach for Identification of Inhibitors of Plasmodium falciparum Fatty Acid Synthase{triangledown}

The emergence of strains of Plasmodium falciparum resistant to the commonly used antimalarials warrants the development of new antimalarial agents. The discovery of type II fatty acid synthase (FAS) in Plasmodium distinct from the FAS in its human host (type I FAS) opened up new avenues for the development of novel antimalarials. The process of fatty acid synthesis takes place by iterative elongation of butyryl-acyl carrier protein (butyryl-ACP) by two carbon units, with the successive action of four enzymes constituting the elongation module of FAS until the desired acyl length is obtained. The study of the fatty acid synthesis machinery of the parasite inside the red blood cell culture has always been a challenging task. Here, we report the in vitro reconstitution of the elongation module of the FAS of malaria parasite involving all four enzymes, FabB/F (β-ketoacyl-ACP synthase), FabG (β-ketoacyl-ACP reductase), FabZ (β-ketoacyl-ACP dehydratase), and FabI (enoyl-ACP reductase), and its analysis by matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS). That this in vitro systems approach completely mimics the in vivo machinery is confirmed by the distribution of acyl products. Using known inhibitors of the enzymes of the elongation module, cerulenin, triclosan, NAS-21/91, and (–)-catechin gallate, we demonstrate that accumulation of intermediates resulting from the inhibition of any of the enzymes can be unambiguously followed by MALDI-TOF MS. Thus, this work not only offers a powerful tool for easier and faster throughput screening of inhibitors but also allows for the study of the biochemical properties of the FAS pathway of the malaria parasite.

Thermal degradation products of poly(styrenesulfides) investigated by direct pyrolysis�mass spectrometry and flash pyrolysis�gas chromatography/mass spectrometry

The thermal degradation products of two sulfur polymers, poly(styrenedisulfide) (PSD) and poly(styrenetetrasulfide) (PST), were investigated in parallel by direct pyrolysis-mass spectrometry (DPMS) and by flash pyrolysis-GC/MS (Py-GC/MS). The time-scale of the two pyrolysis techniques is quite different, and therefore they were able to detect significantly different products in the pyrolysis of PSD and PST because of the thermal lability of sulfur-containing compounds. However, the results obtained are not contradictory, and satisfactory mechanisms for the thermal degradation of PSD and PST have been derived from the overall evidence available. Pyrolysis compounds containing sulfur, styrene, and a number of cyclic styrene sulfides and diphenyldithianes have been observed by DPMS. However, in flash pyrolysis-GC/MS, styrene, sulfur, only one cyclic styrene sulfide, and two isomers of diphenylthiophene have been detected. These thiophene derivatives were indeed absent among the compounds obtained by DPMS because they were the terminal (most thermally stable) species arising from further decomposition of the cyclic styrene sulfides formed in the primary thermal degradation processes of PSD and PST.

Thermal degradation processes in polysulfide copolymers investigated by direct pyrolysis mass spectrometry and flash pyrolysis-gas chromatography/mass spectrometry

This is the first report on the analysis of random block polysulfide copolymers containing different amounts of repeating units in the copolymer backbone, which has been studied by direct pyrolysis mass spectrometry (DPMS) and by pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS). The homopolymers such as poly(ethylene sulfide) (PES), poly(styrene sulfide) (PSS), and two random copolymers, viz., poly(ethylene sulfide(x)-co-styrene sulfide(y)) [copolymer I (x = y = 0.5) and copolymer II (x = 0.74, y = 0.26)] were investigated by both DPMS and Py-GC/MS (except copolymer II) techniques. In the case of copolymer I, the thermal degradation products of SE1, SE2, S-2, and S2E (S = styrene sulfide, E = ethylene sulfide) were detected in DPMS, whereas the formation of SE1 and SE2 were observed by Py-GC/MS technique. However, for copolymer II, SE3 was also found along with SE1, SE2, S-2, and S2E in DPMS. The formation of additional product (SE3) observed in copolymer II could be due to an increase in the block length formed during copolymerization. Further, a comparative study on thermal degradation of PES, poly(ethylene disulfide) (PEDS), and poly(ethylene tetrasulfide) (PETS) were investigated by Py-GC/MS. The pyrolysis products detected by both DPMS and Py-GC/MS indicates that the thermal decomposition of these polymers yield cyclic sulfides through an intramolecular exchange or by backbiting processes. The linear products with thiol and vinyl groups were also observed by Py-GC/MS along with the cyclic products via carbon hydrogen transfer reaction.

Self-Assembly of Metallamacrocycles Using a Dinuclear Organometallic Acceptor: Synthesis, Characterization, and Sensing Study

A dinuclear organometallic acceptor 4,4'-bis[trans-Pt(PEt(3))(2)(O(3)SCF(3))(ethynyl)]biphenyl (1) containing Pt-ethynyl functionality is synthesized. Multinuclear NMR ((1)H, (31)P, and (13)C), infrared (IR), and electrospray ionization mass spectrometry (ESI-MS) including single-crystal X-ray diffraction analysis established the formation of 1. Equimolar treatment of acceptor 1 separately with three different ``clip'' type ditopic donors (L(a)-L(c)) yielded [2 + 2] self-assembled three metallamacrocycles 2a-2c, respectively. These macrocycles were characterized by various spectroscopic techniques, and their sizes/shapes were obtained through geometry optimization using molecular mechanics universal force field (MMUFF) simulations. Attachment of unsaturated ethynyl functionality to biphenyl building unit helped to make the macrocycles (2a-2c) pi-electron rich and thereby fluorescent in nature. Furthermore, 2c in solution has been examined to be suitable for sensing electron-deficient nitroaromatic like picric acid, which is often considered as a secondary chemical explosive. The fluorescence study of 2c showed a marked quenching of initial emission intensity upon titrating with picric acid (PA), and it exhibited the largest fluorescence quenching response with high selectivity among various other electron deficient aromatic compounds tested.

Coordination-Driven Self-Assembly of 2D-Metallamacrocycles Using a New Carbazole-Based Dipyridyl Donor: Synthesis, Characterization, and C-60 Binding Study

A new carbazole-based 90 degrees dipyridyl donor 3,6-di(4-pyridylethynyl)carbazole (L) containing carbazole-ethynyl functionality is synthesized in reasonable yield using the Sonagashira coupling reaction. Multinuclear NMR, electrospray ionization-mass spectrometry (ESI-MS), including single crystal X-ray diffraction analysis characterized this 90 degrees building unit. The stoichiometry combination of L with several Pd(II)/Pt(II)-based 90 degrees acceptors (1a-1d) yielded 2 + 2] self-assembled metallacycles (2a-2d) under mild conditions in quantitative yields 1a = cis-(dppf)Pd(OTf)(2); 1b = cis-(dppf)Pt(OTf)(2); 1c = cis-(tmen)Pd(NO3)(2); 1d = 3,6-bis{trans-Pt(C C) (PEt3)(2)(NO3))carbazole]. All these macrocycles were characterized by various spectroscopic techniques, and the molecular structure of 2a was unambiguously determined by single crystal X-ray diffraction analysis. Incorporation of ethynyl functionality to the carbazole backbone causes the resulted macrocycles (2a-2d) to be pi-electron rich and thereby exhibit strong emission characteristics. The macrocycle 2a has a large internal concave aromatic surface. The fluorescence quenching study suggests that 2a forms a similar to 1:1 complex with C-60 with a high association constant of K-sv = 1.0 X 10(5) M-1.

High precision Mg isotope measurements of meteoritic samples by secondary ion mass spectrometry

The possibility of establishing an accurate relative chronology of the early solar system events based on the decay of short-lived Al-26 to Mg-26 (half-life of 0.72 Myr) depends on the level of homogeneity (or heterogeneity) of Al-26 and Mg isotopes. However, this level is difficult. to constrain precisely because of the very high precision needed for the determination of isotopic ratios, typically of +/- 5 ppm. In this study, we report for the first time a detailed analytical protocol developed for high precision in situ Mg isotopic measurements ((25)mg/(24)mg and (26)mg/Mg-24 ratios, as well as Mg-26 excess) by MC-SIMS. As the data reduction process is critical for both accuracy and precision of the final isotopic results, factors such as the Faraday cup (FC) background drift and matrix effects on instrumental fractionation have been investigated. Indeed these instrumental effects impacting the measured Mg-isotope ratios can be as large or larger than the variations we are looking for to constrain the initial distribution of Al-26 and Mg isotopes in the early solar system. Our results show that they definitely are limiting factors regarding the precision of Mg isotopic compositions, and that an under- or over-correction of both FC background instabilities and instrumental isotopic fractionation leads to important bias on delta Mg-25, delta(26)mg and Delta Mg-26 values (for example, olivines not corrected for FC background drifts display Delta Mg-26 values that can differ by as much as 10 ppm from the truly corrected value). The new data reduction process described here can then be applied to meteoritic samples (components of chondritic meteorites for instance) to accurately establish their relative chronology of formation.

Self-assembly of discrete metallamacrocycles employing half-sandwich octahedral diruthenium(II) building units and imidazole-based ligands

Equimolar combination of a series of binuclear half-sandwich p-cymene ruthenium(II) building units Ru-2(mu-eta(4)-C2O4)(MeOH)(2)(eta(6)-p-cymene)(2)](OTf)(2) 1a](OTf)(2), Ru-2(mu-eta(4)-N,N'-diphenyloxamidato)( MeOH)(2)(eta(6)-p-cymene)(2)](OTf)(2) 1b](OTf)(2) and Ru-2(mu-eta(4)-C6H2O4)(MeOH)(2)(eta(6)-p-cymene)(2)](OTf)(2) 1c](OTf)(2) separately with imidazole-based ditopic ligands (L-1-L-2) in methanol yielded a series of tetranuclear metallamacrocycles 2-7](OTf)(4), respectively L-1 = 1,4-bis(imidazole-1-yl)benzene; L-2 = 4,4'-bis(imidazole-1-yl)biphenyl; OTf- = O3SCF3-]. Similarly, the reaction of Ru-2(mu-eta(4)-C2O4)(MeOH)(2)(eta(6)-p-cymene)2](OTf)(2) 1a](OTf)(2) with a triazine-based tritopic ligand 1,3,5-tris(imidazole-1-yl) triazine (L3) in 3: 2 M ratio afforded an unexpected tetranuclear macrocycle 8](OTf)(4) instead of an expected trigonal prismatic cage 8a](OTf)(6). All the self-assembled macrocycles 2-8](OTf)(4) were isolated in moderate to high yields and were fully characterized by multinuclear H-1, F-19] NMR, IR and electrospray ionization mass spectrometry (ESI-MS). In addition, X-ray diffraction study on the single crystals of 3](OTf)(4) and 8](OTf)(4) also indicated the formation 2 + 2] self-assembled macrocycles. Despite the possibility of formation of different conformational isomeric macrocycles (syn-and anti) and polymeric product due to free rotation of ligand sites of imidazole linkers, the selective formation of single conformational isomer (anti) as the only product is quite interesting. Furthermore, the photo-and electrochemical properties of these assemblies have been studied using UV/Vis absorption and cyclic voltammetry analysis. (c) 2013 Elsevier B.V. All rights reserved.

Evaluation of hexane and ethyl acetate extracts of the sponge Jaspis diastra collected from Mauritius Waters on HeLa cells

Objectives Based on previous screening results, the cytotoxic effect of the hexane (JDH) and ethyl acetate extracts (JDE) of the marine sponge Jaspis diastra were evaluated on HeLa cells and the present study aimed at determining their possible mechanism of cell death. Methods Nuclear staining, membrane potential change, flow cytometry analysis of cell cycle distribution and annexin V staining were undertaken to investigate the effects of JDE and JDH. Electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance were used to characterize an isolated bioactive molecule. Key findings JDE displayed an IC50 25 times more significant than the JDH. Flow cytometry analysis revealed JDE induced apoptosis in HeLa cells accompanied by the collapse of mitochondrial membrane potential. Fractionation of JDE resulted in the isolation of the known cytotoxic cyclodepsipeptide, Jaspamide. Conclusions Taking our results together suggest that JDE can be valuable for the development of anticancer drugs, especially for cervical cancer. Further investigations are currently in progress with the aim to determine and isolate other bioactive compounds from this extract.

Mass spectrometry based characterization of Hb Beckman variant in a falsely elevated HbA(1c) sample

Glycated hemoglobin (HbA(1c)) is a `gold standard' biomarker for assessing the glycemic index of an individual. HbA(1c) is formed due to nonenzymatic glycosylation at N-terminal valine residue of the P-globin chain. Cation exchange based high performance liquid chromatography (CE HPLC) is mostly used to quantify HbA(1c), in blood sample. A few genetic variants of hemoglobin and post-translationally modified variants of hemoglobin interfere with CE HPLC-based quantification,. resulting in its false positive estimation. Using mass spectrometry, we analyzed a blood sample with abnormally high HbA(1c) (52.1%) in the CE HPLC method. The observed HbA(1c) did not corroborate the blood glucose level of the patient. A mass spectrometry based bottom up proteomics approach, intact globin chain mass analysis, and chemical modification of the proteolytic peptides identified the presence of Hb Beckman, a genetic variant of hemoglobin, in the experimental sample. A similar surface area to charge ratio between HbA(1c) and Hb Beckman might have resulted in the coelution of the variant with HbA(1c) in CE HPLC. Therefore, in the screening of diabetes mellitus through the estimation of HbA(1c), it is important to look for genetic variants of hemoglobin in samples that show abnormally high glycemic index, and HbA(1c) must be estimated using an alternative method. (C) 2015 Elsevier Inc. All rights reserved.

Self-assembly of Metallamacrocycles Employing a New Benzil-based Organometallic Bisplatinum(II) Acceptor

A benzil-based semi-rigid dinuclear organometallic acceptor 4,4'-bistrans-Pt(PEt3)(2)(NO3)(ethynyl)]benzil (bisPt-NO3) containing a Pt-ethynyl functionality was synthesized in good yield and characterized by multinuclear NMR (H-1, P-31, and C-13), electrospray ionization mass spectrometry (ESI-MS), and single-crystal X-ray diffraction analysis of the iodide analogue bisPt-I. The stoichiometric (1:1) combination of the acceptor bisPt-NO3 separately with four different ditopic donors (L-1-L-4; L-1 = 9-ethyl-3,6-di(1H-imidazol-1-yl)-9H-carbazole, L-2 = 1,4-bis((1H-imidazol-1-yl)methyl)benzene, L-3 = 1,3-bis((1H-imidazol-1-yl)methyl)benzene and L-4 = 9,10-bis((1H-imidazol-1-yl) methyl)anthracene) yielded four 2 + 2] self-assembled metallacycles M-1-M-4 in quantitative yields, respectively. All these newly synthesized assemblies were characterized by various spectroscopic techniques (NMR, IR, ESI-MS) and their sizes/shapes were predicted through geometry optimization employing the PM6 semi-empirical method. The benzil moiety was introduced in the backbone of the acceptor bisPt-NO3 due to the interesting structural feature of long carbonyl C-C bond (similar to 1.54 angstrom), which enabled us to probe the role of conformational flexibility on size and shapes of the resulting coordination ensembles.