RESUMEN
Microplastic pollution was studied in surface waters of Isfjorden, Svalbard in July 2021 as a part of an international regional harmonisation exercise. Surface microplastics (0.5-5 mm) were sampled with a neuston net in triplicate per study site in several branches of Isfjorden, covering populated and unpopulated fjords. High spatial variability of microplastic abundance (0-32,700 items/km2) was observed within a single fjord resulting from the hydrodynamic pattern formed through the interaction of surface currents, freshwater runoff, and wind conditions. Maximum microplastic abundance was not correlated with the distance from the local source and was instead defined by local small-scale hydrodynamics. Future recommendations for correct assessment of surface microplastics concentration in estuarine environments are presented.
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Monitoreo del Ambiente , Estuarios , Microplásticos , Contaminantes Químicos del Agua , Monitoreo del Ambiente/métodos , Microplásticos/análisis , Contaminantes Químicos del Agua/análisis , SvalbardRESUMEN
Reference materials (RMs) are vital tools in the validation of methods used to detect environmental pollutants. Microplastics, a relatively new environmental pollutant, require a variety of complex approaches to address their presence in environmental samples. Both interlaboratory comparison (ILC) studies and RMs are essential to support the validation of methods used in microplastic analysis. Presented here are results of quality assurance and quality control (QA/QC) performed on two types of candidate microplastic RMs: dissolvable gelatin capsules and soda tablets. These RMs have been used to support numerous international ILC studies in recent years (2019-2022). Dissolvable capsules containing polyethylene terephthalate (PET), polyvinyl chloride (PVC), polyethylene (PE), and polystyrene (PS), in different size fractions from 50 to 1000 µm, were produced for one ILC study, obtaining relative standard deviation (RSD) from 0 to 24%. The larger size fraction allowed for manual addition of particles to the capsules, yielding 0% error and 100% recovery during QA/QC. Dissolvable capsules were replaced by soda tablets in subsequent ILC studies and recovery test exercises because they were found to be a more reliable carrier for microplastic RMs. Batches of soda tablets were produced containing different single and multiple polymer mixtures, i.e., PE, PET, PS, PVC, polypropylene (PP), and polycarbonate (PC), with RSD ranging from 8 to 21%. Lastly, soda tablets consisting of a mixture of PE, PVC, and PS (125-355 µm) were produced and used for recovery testing during pretreatment of environmental samples. These had an RSD of 9%. Results showed that soda tablets and capsules containing microplastics >50 µm could be produced with sufficient precision for internal recovery tests and external ILC studies. Further work is required to optimize this method for smaller microplastics (< 50 µm) because variation was found to be too large during QA/QC. Nevertheless, this approach represents a valuable solution addressing many of the challenges associated with validating microplastic analytical methods.
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Northern Dvina River is one of the largest rivers in the European Arctic flowing into the White Sea through the populated regions with developed industry. Floating plastics include microplastics (0.5-5 mm) and mesoplastics (5-25 mm) were observed on seasonal variations in the Northern Dvina River mouth. The samples were collected every month from September to November 2019 and from May to October 2020 with a Neuston net that was togged 3 nautical miles in the Korbel'nyy Branch of the River delta. Chemical composition of the plastic particles was determined using a Fourier transmission infrared spectrometer. The majority of the microplastics were identified as polyethylene 52.6%, followed by polypropylene 36.8%. After estimating the export fluxes of microplastics from the Northern Dvina River to the Arctic, there is no significant seasonal variation of the river export of microplastics. The microplastics export rate during the spring flood period in May turned out to be maximum, 58 items/s, while the minimum discharge was in September with a value of 9 items/s. The average weight concentration of microplastics was 18.5 µg/m3, which is higher than it was found in the Barents Sea - 12.5 µg/m3 and several times higher than in the Eurasian Arctic on average - 3.7 µg/m3. These results indicate that the Northern Dvina River is being one of the main sources of microplastic pollution of the White and the Barents Seas.
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Microplásticos , Ríos , Estaciones del Año , Contaminantes Químicos del Agua , Regiones Árticas , Monitoreo del Ambiente/métodos , Microplásticos/análisis , Océanos y Mares , Ríos/química , Contaminantes Químicos del Agua/análisisRESUMEN
Comparative investigations of microplastic (MP) occurrence in the global ocean are often hampered by the application of different methods. In this study, the same sampling and analytical approach was applied during five different cruises to investigate MP covering a route from the East-Siberian Sea in the Arctic, through the Atlantic, and into the Antarctic Peninsula. A total of 121 subsurface water samples were collected using underway pump-through system on two different vessels. This approach allowed subsurface MP (100 µm-5 mm) to be evaluated in five regions of the World Ocean (Antarctic, Central Atlantic, North Atlantic, Barents Sea and Siberian Arctic) and to assess regional differences in MP characteristics. The average abundance of MP for whole studied area was 0.7 ± 0.6 items/m3 (ranging from 0 to 2.6 items/m3), with an equal average abundance for both fragments and fibers (0.34 items/m3). Although no statistical difference was found for MP abundance between the studied regions. Differences were found between the size, morphology, polymer types and weight concentrations. The Central Atlantic and Barents Sea appeared to have more MP in terms of weight concentration (7-7.5 µg/m3) than the North Atlantic and Siberian Arctic (0.6 µg/m3). A comparison of MP characteristics between the two Hemispheres appears to indicate that MP in the Northern Hemisphere mostly originate from terrestrial input, while offshore industries play an important role as a source of MP in the Southern Hemisphere. The waters of the Northern Hemisphere were found to be more polluted by fibers than those of the Southern Hemisphere. The results presented here suggest that fibers can be transported by air and water over long distances from the source, while distribution of fragments is limited mainly to the water mass where the source is located.
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Microplásticos , Contaminantes Químicos del Agua , Regiones Antárticas , Regiones Árticas , Monitoreo del Ambiente , Plásticos , Agua , Contaminantes Químicos del Agua/análisisRESUMEN
A major challenge for new antibiotic discovery is predicting the physicochemical properties that enable small molecules to permeate Gram-negative bacterial membranes. We have applied physicochemical lessons from previous work to redesign and improve the antibacterial potency of pyridopyrimidine inhibitors of biotin carboxylase (BC) by up to 64-fold and 16-fold against Escherichia coli and Pseudomonas aeruginosa, respectively. Antibacterial and enzyme potency assessments in the presence of an outer membrane-permeabilizing agent or in efflux-compromised strains indicate that penetration and efflux properties of many redesigned BC inhibitors could be improved to various extents. Spontaneous resistance to the improved pyridopyrimidine inhibitors in P. aeruginosa occurs at very low frequencies between 10-8 and 10-9. However, resistant isolates had alarmingly high minimum inhibitory concentration shifts (16- to >128-fold) compared to the parent strain. Whole-genome sequencing of resistant isolates revealed that either BC target mutations or efflux pump overexpression can lead to the development of high-level resistance.
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Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Ligasas de Carbono-Nitrógeno/antagonistas & inhibidores , Escherichia coli/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Antibacterianos/síntesis química , Antibacterianos/química , Membrana Externa Bacteriana/efectos de los fármacos , Membrana Externa Bacteriana/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/metabolismo , Fenómenos Químicos , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/enzimología , Escherichia coli/genética , Pruebas de Sensibilidad Microbiana , Modelos Químicos , Estructura Molecular , Mutación , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genéticaRESUMEN
The regio- and stereo-specific oxygenation of polyunsaturated fatty acids is catalyzed by lipoxygenases (LOX); both Fe and Mn forms of the enzyme have been described. Structural elements of the Fe and Mn coordination spheres and the helical catalytic domain in which the metal center resides are highly conserved. However, animal, plant, and microbial LOX each have distinct features. We report five crystal structures of a LOX from the fungal plant pathogen Fusarium graminearum. This LOX displays a novel amino terminal extension that provides a wrapping domain for dimerization. Moreover, this extension appears to interfere with the iron coordination sphere, as the typical LOX configuration is not observed at the catalytic metal when the enzyme is dimeric. Instead novel tetra-, penta-, and hexa-coordinate Fe2+ ligations are apparent. In contrast, a monomeric structure indicates that with repositioning of the amino terminal segment, the enzyme can assume a productive conformation with the canonical Fe2+ coordination sphere.
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Fusarium/enzimología , Hierro/metabolismo , Lipooxigenasas/química , Lipooxigenasas/metabolismo , Manganeso/metabolismo , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Fusarium/química , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , Especificidad por SustratoRESUMEN
NMR and X-ray diffraction studies were conducted on Pt(II)LCl2 complexes prepared with the new N-donor ligands N(SO2R)Me ndpa (R = Me, Tol; n = 2, 4). These ligands differ from N(H)dpa (di-2-picolylamine) in having the central N within a tertiary sulfonamide group instead of a secondary amine group and having Me groups at the 6,6'-positions ( n = 2) or 3,3',5,5'-positions ( n = 4) of the pyridyl rings. The N(SO2R)3,3',5,5'-Me4dpa ligands are coordinated in a bidentate fashion in Pt( N(SO2R)3,3',5,5'-Me4dpa)Cl2 complexes, forming a rare eight-membered chelate ring. The sulfonamide N atom did not bind to Pt(II), consistent with indications in the literature that tertiary sulfonamides are unlikely to anchor two meridionally coordinated five-membered chelate rings in solutions of coordinating solvents. The N(SO2R)6,6'-Me2dpa ligands coordinate in a monodentate fashion to form the binuclear complexes [ trans-Pt(DMSO)Cl2]2( N(SO2R)6,6'-Me2dpa). The monodentate instead of bidentate N(SO2R)6,6'-Me2dpa coordination is attributed to 6,6'-Me steric bulk. These binuclear complexes are indefinitely stable in DMF- d7, but in DMSO- d6 the N(SO2R)6,6'-Me2dpa ligands dissociate completely. In DMSO- d6, the bidentate ligands in Pt( N(SO2R)3,3',5,5'-Me4dpa)Cl2 complexes also dissociate, but incompletely; these complexes provide rare examples of association-dissociation equilibria of N,N bidentate ligands in Pt(II) chemistry. Like typical cis-PtLCl2 complexes, the Pt( N(SO2R)3,3',5,5'-Me4dpa)Cl2 complexes undergo monosolvolysis in DMSO- d6 to form the [Pt( N(SO2R)3,3',5,5'-Me4dpa)(DMSO- d6)Cl]+ cations. However, unlike typical cis-PtLCl2 complexes, the Pt( N(SO2R)3,3',5,5'-Me4dpa)Cl2 complexes surprisingly do not react readily with the excellent N-donor bioligand guanosine. A comparison of the structural features of over 50 known relevant Pt(II) complexes having smaller chelate rings with those of the very few relevant Pt(II) complexes having eight-membered chelate rings indicates that the pyridyl rings in Pt( N(SO2R)3,3',5,5'-Me4dpa)Cl2 complexes are well positioned to form strong Pt-N bonds. Therefore, the dissociation of the bidentate ligand and the poor biomolecule reactivity of the Pt( N(SO2R)3,3',5,5'-Me4dpa)Cl2 complexes arise from steric consequences imposed by the -CH2-N(SO2R)-CH2- chain in the eight-membered chelate ring.
RESUMEN
Multi-target inhibitors have become increasing popular as a means to leverage the advantages of poly-pharmacology while simplifying drug delivery. Here, we describe dual inhibitors for soluble epoxide hydrolase (sEH) and fatty acid amide hydrolase (FAAH), two targets known to synergize when treating inflammatory and neuropathic pain. The structure activity relationship (SAR) study described herein initially started with t-TUCB (trans-4-[4-(3-trifluoromethoxyphenyl-l-ureido)-cyclohexyloxy]-benzoic acid), a potent sEH inhibitor that was previously shown to weakly inhibit FAAH. Inhibitors with a 6-fold increase of FAAH potency while maintaining high sEH potency were developed by optimization. Interestingly, compared to most FAAH inhibitors that inhibit through time-dependent covalent modification, t-TUCB and related compounds appear to inhibit FAAH through a time-independent, competitive mechanism. These inhibitors are selective for FAAH over other serine hydrolases. In addition, FAAH inhibition by t-TUCB appears to be higher in human FAAH over other species; however, the new dual sEH/FAAH inhibitors have improved cross-species potency. These dual inhibitors may be useful for future studies in understanding the therapeutic application of dual sEH/FAAH inhibition.
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Amidohidrolasas/antagonistas & inhibidores , Benzoatos/farmacología , Inhibidores Enzimáticos/farmacología , Epóxido Hidrolasas/antagonistas & inhibidores , Compuestos de Fenilurea/farmacología , Animales , Benzoatos/síntesis química , Benzoatos/química , Dominio Catalítico , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Ratones , Microsomas/efectos de los fármacos , Simulación del Acoplamiento Molecular , Estructura Molecular , Compuestos de Fenilurea/síntesis química , Compuestos de Fenilurea/química , Ratas , Relación Estructura-ActividadRESUMEN
Antifreeze proteins (AFPs) enhance the survival of organisms inhabiting cold environments by affecting the formation and/or structure of ice. We report the crystal structure of the first multi-domain AFP that has been characterized. The two ice binding domains are structurally similar. Each consists of an irregular ß-helix with a triangular cross-section and a long α-helix that runs parallel on one side of the ß-helix. Both domains are stabilized by hydrophobic interactions. A flat plane on the same face of each domain's ß-helix was identified as the ice binding site. Mutating any of the smaller residues on the ice binding site to bulkier ones decreased the antifreeze activity. The bulky side chain of Leu174 in domain A sterically hinders the binding of water molecules to the protein backbone, partially explaining why antifreeze activity by domain A is inferior to that of domain B. Our data provide a molecular basis for understanding differences in antifreeze activity between the two domains of this protein and general insight on how structural differences in the ice-binding sites affect the activity of AFPs.
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Proteínas Anticongelantes/química , Proteínas Bacterianas/química , Cristalografía por Rayos X , Disulfuros/química , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformación ProteicaRESUMEN
Our goal is to develop convenient methods for obtaining trans-[PtII(4-Xpy)2Cl2] complexes applicable to 4-substituted pyridines (4-Xpy) with limited volatility and water solubility, properties typical of 4-Xpy, with X being a moiety targeting drug delivery. Treatment of cis-[PtII(DMSO)2Cl2] (DMSO = dimethyl sulfoxide) with 4-Xpy in acetonitrile allowed isolation of a new series of simple trans-[PtII(4-Xpy)2Cl2] complexes. A side product with very downfield H2/6 signals led to our synthesis of a series of new [PtII(4-Xpy)4]Cl2 salts. For both series in CDCl3, the size of the H2/6 Δδ [coordinated minus "free" 4-Xpy H2/6 shift] decreased as 4-Xpy donor ability increased from 4-CNpy to 4-Me2Npy. This finding can be attributed to the greater synergistic reduction in the inductive effect of the Pt(II) center with increased 4-Xpy donor ability. The high solubility of [PtII(4-Xpy)4]Cl2 salts in CDCl3 (a solvent with low polarity) and the very downfield shift of the [PtII(4-Xpy)4]Cl2 H2/6 signals for the solutions provide evidence for the presence of strong {[PtII(4-Xpy)4]2+,2Cl-} ion pairs that are stabilized by multiple CH···Cl contacts. This conclusion gains considerable support from [PtII(4-Xpy)4]Cl2 crystal structures revealing that a chloride anion occupies a pseudoaxial position with nonbonding (py)C-H···Cl contacts (2.4-3.0 Å). Evidence for (py)C-H···Y contacts was obtained in NMR studies of [PtII(4-Xpy)4]Y2 salts with Y counterions less capable of forming H-bonds than chloride ion. Our synthetic approaches and spectroscopic analysis are clearly applicable to other nonvolatile ligands.
RESUMEN
[Pt(N(R)-1,1'-Me2dma)Cl]Cl complexes with tridentate ligands (bis(1-methyl-2-methylimidazolyl)amine, R = H; N-(methyl)bis(1-methyl-2-methylimidazolyl)amine, R = Me) were prepared in order to investigate Pt(N(R)-1,1'-Me2dma)G adducts (G = monodentate N9-substituted guanine or hypoxanthine derivative). Solution NMR spectroscopy is the primary tool for studying metal complexes of nucleosides and nucleotides because such adducts rarely crystallize. However, [Pt(N(H)-1,1'-Me2dma)(3'-GMPH)]NO3·5H2O (5) was crystallized, allowing, to our knowledge, the first crystallographic molecular structure determination for a 3'-GMP platinum complex. The structure is one of only a very few structures of a 3'-GMP complex with any metal. Complex 5 has the syn rotamer conformation, with 3'-GMP bound by N7. All Pt(N(R)-1,1'-Me2dma)G adducts exhibit two new downfield-shifted G H8 signals, consistent with G bound to platinum by N7 and a syn/anti rotamer mixture. Anticancer-active monofunctional platinum(II) complexes have bulky carrier ligands that cause DNA adducts to be distorted. Hence, understanding carrier-ligand steric effects is key in designing new platinum drugs. Ligand bulk can be correlated with the degree of impeded rotation of the G nucleobase about the Pt-N7 bond, as assessed by the observation of rotamers. The signals of syn and anti rotamers are connected by EXSY cross-peaks in 2D ROESY spectra of Pt(N(H)-1,1'-Me2dma)G adducts but not in spectra of Pt(N(H)dpa)G adducts (N(H)dpa = bis(2-picolyl)amine), indicating that rotamer interchange is more facile and carrier-ligand bulk is lower in Pt(N(H)-1,1'-Me2dma)G than in Pt(N(H)dpa)G adducts. The lower steric hindrance is a direct consequence of the greater distance of the G nucleobase from the H4/4' protons in the N(R)-1,1'-Me2dma carrier ligand in comparison to that from the H6/6' protons in the N(H)dpa carrier ligand. Although in 5 the nucleotide is 3'-GMP (not the usual 5'-GMP) and the N(H)-1,1'-Me2dma carrier ligand is very different from those typically present in structurally characterized Pt(II) G complexes, the rocking and canting angles in 5 adhere to long-recognized trends.
RESUMEN
A novel ligand bearing a central piperidinyl sulfonamide group, N(SO2pip)dpa, and its corresponding Re tricarbonyl complex, [Re(CO)3(N(SO2pip)dpa)]+, have been synthesized in good yield. The methylene CH2 signal seen as a singlet (4.54 ppm) in a 1H NMR spectrum of the ligand in DMSO-d6 appears as two doublets (5.39, 5.01 ppm) in a spectrum of the [Re(CO)3(N(SO2pip)dpa)]+ complex and confirms the presence of magnetically nonequivalent protons upon coordination to Re. Structural results revealed that the Re-N bond lengths fall within the normal range establishing coordination of ligand to metal. The presence of intraligand π â πâ and n â πâ transitions is indicated by the absorption peaks around 200-250 nm in UV-visible spectra. Absorption peaks in UV-visible spectra around 300 nm for metal complexes were identified as MLCT transitions. The S-N stretch observed as a strong peak at 923 cm-1 for N(SO2pip)dpa appeared at a shorter frequency, at 830 cm-1 in an FTIR spectrum of the [Re(CO)3(N(SO2pip)dpa)]+. The intense fluorescence displayed by the N(SO2pip)dpa ligand has quenched upon coordination to Re. Relatively low IC50 values given by human breast cancer cells, MCF-7, (N(SO2pip)dpa = 139 µM, [Re(CO)3(N(SO2pip)dpa)]+ = 360 µM) indicate that N(SO2pip)dpa and [Re(CO)3(N(SO2pip)dpa)]+ are promising novel compounds that can be further investigated on their usage as potential anticancer agents.
RESUMEN
The dramatic increase in the prevalence of antibiotic-resistant bacteria has necessitated a search for new antibacterial agents against novel targets. Moiramide B is a natural product, broad-spectrum antibiotic that inhibits the carboxyltransferase component of acetyl-CoA carboxylase, which catalyzes the first committed step in fatty acid synthesis. Herein, we report the 2.6 Å resolution crystal structure of moiramide B bound to carboxyltransferase. An unanticipated but significant finding was that moiramide B bound as the enol/enolate. Crystallographic studies demonstrate that the (4S)-methyl succinimide moiety interacts with the oxyanion holes of the enzyme, supporting the notion that an anionic enolate is the active form of the antibacterial agent. Structure-activity studies demonstrate that the unsaturated fatty acid tail of moiramide B is needed only for entry into the bacterial cell. These results will allow the design of new antibacterial agents against the bacterial form of carboxyltransferase.
Asunto(s)
Amidas/metabolismo , Antibacterianos/metabolismo , Transferasas de Carboxilo y Carbamoilo/química , Staphylococcus aureus/enzimología , Succinimidas/metabolismo , Transferasas de Carboxilo y Carbamoilo/metabolismo , Cristalografía por Rayos X , Conformación ProteicaRESUMEN
Five functionalized bis(ß-diketones) and their Cu(II) molecular squares are described. The new bis(ß-diketones), m-pbhxH2 (3), 5-MeO-m-pbaH2 (4), 5-BuO-m-pbaH2 (5), 2-MeO-m-pbaH2 (6), and 2-MeO-m-pbprH2 (7), were prepared by reaction of the corresponding aldehydes with phospholenes, as we previously reported for m-pbaH2 (1) and m-pbprH2 (2). Ligand 3 has long alkyl chains in its ß-diketone moieties, while ligands 4-7 have alkoxy substituents on their aromatic rings. When treated with Cu(2+), the new bis(ß-diketones) 3, 4, 5, and 7 afford molecular squares, Cu4(m-pbhx)4 (10), Cu4(5-MeO-m-pba)4 (11), Cu4(5-BuO-m-pba)4 (12), and Cu4(2-MeO-m-pbpr)4 (13), respectively. Two of the new molecular squares, 10 and 12, contain longer-chain substituents and are soluble in a wider range of organic solvents. The other squares, 11 and 13, contain external and internal methoxy groups, respectively, and they show smaller changes in solubility. Single-crystal X-ray analyses are reported for three of the molecular squares without guest molecules, and for five adducts of the squares with σ- (polypyridine) and π-bonded (fullerene) guests. The Cu···Cu distances in the "empty" squares range from 14.047 to 14.904 Å; those in the adducts vary over a wider range depending on the guest molecule involved.
RESUMEN
Monofunctional Pt(II) complexes bind to G residues in DNA and, if the carrier ligands are bulky, cause DNA structural distortions that lead to anticancer activity. We assessed the steric effects of the tridentate carrier ligand, N(H)6-Medpa (N-(6-methyl-2-picolyl)-N-(2-picolyl)amine), bearing a 6-methyl group and a 6'-proton projecting toward the nucleobase in Pt(N(H)6-Medpa)G adducts (G=9-ethylguanine, 3'-GMP, 5'-GMP, 5'-GTP). Pt(N(H)6-Medpa)G adducts form syn and anti rotamers with the guanine O6 and the central N-H of N(H)6-Medpa on the same or opposite side of the coordination plane, respectively. Pt(N(H)6-Medpa)G adducts have some properties (ease of rotamer interchange and extent of conversion to bis adducts, Pt(N(H)6-Medpa)G2) intermediate to properties reported for analogs having a tridentate ligand with zero or two methyl groups. However, in comparison, the syn rotamer of Pt(N(H)6-Medpa)G adducts has an unexpectedly high abundance. This result is attributable to guanine base canting, such that the 6-membered guanine ring is positioned away from the bulky 6-Me group. This canting both relieves electrostatic repulsion between the partially positive H6' and the guanine H8 protons and creates a favorable electrostatic attraction between the H6' proton and the partially negative guanine O6. This combined information provides insight useful for designing monofunctional anticancer agents.
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Antineoplásicos/síntesis química , Aductos de ADN/química , Guanina/química , Compuestos Organoplatinos/síntesis química , Antineoplásicos/química , Guanina/análogos & derivados , Compuestos Organoplatinos/químicaRESUMEN
Acetyl-CoA carboxylase catalyzes the first and regulated step in fatty acid synthesis. In most Gram-negative and Gram-positive bacteria, the enzyme is composed of three proteins: biotin carboxylase, a biotin carboxyl carrier protein (BCCP), and carboxyltransferase. The reaction mechanism involves two half-reactions with biotin carboxylase catalyzing the ATP-dependent carboxylation of biotin-BCCP in the first reaction. In the second reaction, carboxyltransferase catalyzes the transfer of the carboxyl group from biotin-BCCP to acetyl-CoA to form malonyl-CoA. In this report, high-resolution crystal structures of biotin carboxylase from Haemophilus influenzae were determined with bicarbonate, the ATP analogue AMPPCP; the carboxyphosphate intermediate analogues, phosphonoacetamide and phosphonoformate; the products ADP and phosphate; and the carboxybiotin analogue N1'-methoxycarbonyl biotin methyl ester. The structures have a common theme in that bicarbonate, phosphate, and the methyl ester of the carboxyl group of N1'-methoxycarbonyl biotin methyl ester all bound in the same pocket in the active site of biotin carboxylase and as such utilize the same set of amino acids for binding. This finding suggests a catalytic mechanism for biotin carboxylase in which the binding pocket that binds tetrahedral phosphate also accommodates and stabilizes a tetrahedral dianionic transition state resulting from direct transfer of CO2 from the carboxyphosphate intermediate to biotin.
Asunto(s)
Adenosina Difosfato/química , Adenosina Trifosfato/química , Proteínas Bacterianas/química , Biotina/química , Ligasas de Carbono-Nitrógeno/química , Haemophilus influenzae/enzimología , Modelos Moleculares , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Bicarbonatos/química , Bicarbonatos/metabolismo , Biocatálisis , Biotina/análogos & derivados , Biotina/metabolismo , Ligasas de Carbono-Nitrógeno/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Bases de Datos de Proteínas , Foscarnet/química , Foscarnet/metabolismo , Conformación Molecular , Compuestos Organofosforados/química , Compuestos Organofosforados/metabolismo , Fosfatos/química , Fosfatos/metabolismo , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismoRESUMEN
Anticancer-active monofunctional Pt(II) complexes have bulky carrier ligands and bind to G residues in DNA, causing structural distortions. To gain fundamental chemical information on such monofunctional adducts, we assessed the 9-ethylguanine (9-EtG) adducts formed by [Pt(N(H)6,6'-Me2dpa)Cl]Cl (N(H)6,6'-Me2dpa = di-(6-methyl-2-picolyl)amine). 9-EtG added to [Pt(N(H)6,6'-Me2dpa)Cl]Cl to form not only the expected [Pt(N(H)6,6'-Me2dpa)(9-EtG)](2+) monoadduct having syn and anti conformers but also a [Pt(N(H)6,6'-Me2dpa)(9-EtG)2](2+) bisadduct consisting of ΛHT and ΔHT conformers (HT = head-to-tail). For both adducts, the two conformers exist as a dynamic equilibrium mixture. Concomitant with formation of the bisadduct, the binding mode of the N(H)6,6'-Me2dpa ligand converts from tridentate to bidentate. A Pt(II)-bound 6-methyl-2-picolyl chain and the secondary amine constitute the bidentate chelate ring. The other 6-methyl-2-picolyl chain is dangling. The secondary nitrogen is an asymmetric center, and each conformer exists as a racemic mixture of two enantiomers. For a given configuration at the secondary amine of the [Pt(N(H)6,6'-Me2dpa)(9-EtG)2](2+) adduct, the more abundant HT conformer can form a hydrogen bond between the NH of the bidentate ligand and the cis 9-EtG O6. [Pt(N(H)6,6'-Me2dpa)Cl]Cl forms the monoadduct in â¼1/20 the time for its parent, [Pt(N(H)dpa)Cl]Cl (N(H)dpa = di(2-picolyl)amine), which exhibited typical behavior in forming only a monoadduct. We attribute the unusual new findings for [Pt(N(H)6,6'-Me2dpa)Cl]Cl to Pt-N bond weakening induced by the steric bulk of 6/6'-Me groups. We hypothesize that undetectable intermediates with a dangling 6-methyl-2-picolyl chain facilitate both rapid monoadduct formation and also bisadduct formation. Consistent with the intermediacy of such species with a dangling chain, addition of HCl to a [Pt(N(H)6,6'-Me2dpa)Cl]Cl solution readily produced a dichloro complex with the N(H)6,6'-Me2dpa chelate ligand in the bidentate mode, whereas HCl addition had no effect on [Pt(N(H)dpa)Cl]Cl.
Asunto(s)
Antineoplásicos/química , Complejos de Coordinación/química , Guanina/análogos & derivados , Compuestos Organoplatinos/química , Picolinas/química , Antineoplásicos/síntesis química , Sitios de Unión , Complejos de Coordinación/síntesis química , ADN/química , Guanina/química , Humanos , Enlace de Hidrógeno , Conformación Molecular , Compuestos Organoplatinos/síntesis química , Soluciones , EstereoisomerismoRESUMEN
A series of five mono-styryl and their corresponding symmetric di-styryl-2,6-diiodo-BODIPYs containing indolyl, pyrrolyl, thienyl or tri(ethylene glycol)phenyl groups were synthesized using Knoevenagel condensations. The yields for the condensation reactions were improved up to 40% using microwave irradiation (90°C for 1h at 400W) due to lower decomposition of BODIPYs upon prolonged heating. The spectroscopic, structural (including the X-ray of a di-styryl-2,6-diiodo-BODIPY) and in vitro properties of the BODIPYs were investigated. The extension of π-conjugation through the 3,5-dimethyls of the known phototoxic 2,6-diiodo-BODIPY 1 produced bathochromic shifts in the absorption and emission spectra, in the order of 63-125nm for the mono-styryl- and 128-220nm for the di-styryl-BODIPYs in DMSO. The largest red-shifts were observed for the indolyl-containing BODIPYs while the largest fluorescence quantum yields were observed for the tri(ethyleneglycol)phenylstyryl-BODIPYs. Among this series, only the mono-styryl-BODIPYs were phototoxic (IC50=2-15µM at 1.5J/cm(2)), and were observed to localize preferentially in the cell ER and mitochondria. On the other hand, the di-styryl-BODIPYs were found to have low or no phototoxicity (IC50>100µM at 1.5J/cm(2)). Among this series of compounds BODIPY 2a shows the most promise for application as photosensitizer in PDT.
Asunto(s)
Compuestos de Boro/química , Compuestos de Boro/metabolismo , Compuestos de Boro/toxicidad , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Cristalografía por Rayos X , Retículo Endoplásmico/química , Retículo Endoplásmico/metabolismo , Humanos , Luz , Microscopía Fluorescente , Microondas , Mitocondrias/química , Mitocondrias/metabolismo , Conformación Molecular , Neoplasias/tratamiento farmacológico , Fotoquimioterapia , Fármacos Fotosensibilizantes/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Oxígeno Singlete/metabolismo , Espectrometría de FluorescenciaRESUMEN
Diabetes is affecting the life of millions of people. A large proportion of diabetic patients suffer from severe complications such as neuropathic pain, and current treatments for these complications have deleterious side effects. Thus, alternate therapeutic strategies are needed. Recently, the elevation of epoxy-fatty acids through inhibition of soluble epoxide hydrolase (sEH) was shown to reduce diabetic neuropathic pain in rodents. In this report, we describe a series of newly synthesized sEH inhibitors with at least 5-fold higher potency and doubled residence time inside both the human and rodent sEH enzyme than previously reported inhibitors. These inhibitors also have better physical properties and optimized pharmacokinetic profiles. The optimized inhibitor selected from this new series displayed improved efficacy of almost 10-fold in relieving pain perception in diabetic neuropathic rats as compared to the approved drug, gabapentin, and previously published sEH inhibitors. Therefore, these new sEH inhibitors could be an attractive alternative to treat diabetic neuropathy in humans.