RESUMEN
PURPOSE: The ability to assess the extracellular pH (pHe) is an important issue in oncology, because extracellular acidification is associated with tumor aggressiveness and resistance to cytotoxic therapies. In this study, a stable triphosphonated triarylmethyl (TPTAM) radical was qualified as a pHe electron paramagnetic resonance (EPR) molecular reporter. METHODS: Calibration of hyperfine splitting as a function of pH was performed using a 1.2-GHz EPR spectrometer. Gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) was used as an extracellular paramagnetic broadening agent to assess the localization of TPTAM when incubated with cells. In vivo EPR pH-metry was performed in MDA, SiHa, and TLT tumor models and in muscle. Bicarbonate therapy was used to modulate the tumor pHe. EPR measurements were compared with microelectrode readouts. RESULTS: The hyperfine splitting of TPTAM was strongly pH-dependent around the pKa of the probe (pKa = 6.99). Experiments with Gd-DTPA demonstrated that TPTAM remained in the extracellular compartment. pHe was found to be more acidic in the MDA, SiHa, and TLT tumor models compared with muscle. Treatment of animals by bicarbonate induced an increase in pHe in tumors: similar variations in pHe were found when using in vivo EPR or invasive microelectrodes measurements. CONCLUSION: This study demonstrates the potential usefulness of TPTAM for monitoring pHe in tumors. Magn Reson Med 77:2438-2443, 2017. © 2016 International Society for Magnetic Resonance in Medicine.
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Espectroscopía de Resonancia por Spin del Electrón/métodos , Radicales Libres/química , Concentración de Iones de Hidrógeno , Técnicas de Sonda Molecular , Sondas Moleculares/química , Neoplasias Experimentales/química , Neoplasias Experimentales/diagnóstico , Algoritmos , Animales , Humanos , Células K562 , Masculino , Ratones , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The fast development of nanomedicines requires more and more reliable chemical tools in order to accurately design materials and control the surface properties of the nano-objects used in biomedical applications. In this study we describe a smooth and simple photografting technique, i.e., the clip photochemistry, that allows the introduction of molecules of interest in inert polymers or on stealth nanoparticles directly in aqueous solution. First we developed the methodology on polyethylene glycol (PEG) and looked for critical parameters of the process (irradiation times, concentrations, washings) by using several molecular probes and adapted analytical techniques ((19)F qNMR, EA, LSC). We found that the clip photochemistry in water is a robust and efficient method to functionalize PEG. Second we applied it on PEGylated USPIO (USPIO-PEG) magnetic resonance imaging agent and succeeded in introducing RGD peptide and homemade peptidomimetics on their PEG segments. The magnetic abilities of the conjugated nanoparticles were unchanged by the derivatization process as evidenced by their relaxometric properties and their NMRD profile. When tested on Jurkat lymphocyte T Cells, which express αvß3 integrins, the USPIO conjugated with RGD ligands leads to an increase of the transverse relaxation rate (R2) by a factor 10 to 14 as compared to USPIO-PEG. Consequently, it makes them good candidates for targeted imaging technology in cancer therapy.
Asunto(s)
Compuestos Férricos/química , Imagen por Resonancia Magnética/métodos , Nanopartículas/química , Oligopéptidos/química , Procesos Fotoquímicos , Polietilenglicoles/química , Agua/química , Medios de Contraste/química , Humanos , Células Jurkat , Ligandos , Fenómenos MagnéticosRESUMEN
Self-assembled prodrugs forming nanoaggregates are a promising approach to enhance the antitumor efficacy and to reduce the toxicity of anticancer drugs. To achieve this goal, doxorubicin was chemically conjugated to d-α-tocopherol succinate through an amide bond to form N-doxorubicin-α-d-tocopherol succinate (N-DOX-TOS). The prodrug self-assembled in water into 250 nm nanostructures when stabilized with d-α-tocopherol poly(ethylene glycol) 2000 succinate. Cryo-TEM analysis revealed the formation of nanoparticles with a highly ordered lamellar inner structure. NMR spectra of the N-DOX-TOS nanoparticles indicated that N-DOX-TOS is located in the core of the nanoparticles while PEG chains and part of the tocopherol are in the corona. High drug loading (34% w/w) and low in vitro drug release were achieved. In vitro biological assessment showed significant anticancer activity and temperature-dependent cellular uptake of N-DOX-TOS nanoparticles. In vivo, these nanoparticles showed a greater antitumor efficacy than free DOX. N-DOX-TOS nanoparticles might have the potential to improve DOX-based chemotherapy.
Asunto(s)
Antineoplásicos/farmacología , Doxorrubicina/farmacología , Sistemas de Liberación de Medicamentos , Neoplasias Experimentales/tratamiento farmacológico , Profármacos/farmacología , alfa-Tocoferol/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Doxorrubicina/química , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Nanoestructuras/química , Neoplasias Experimentales/patología , Profármacos/síntesis química , Profármacos/química , Relación Estructura-Actividad , alfa-Tocoferol/químicaRESUMEN
A series of lipophilic ester derivatives (2a-g) of (S)-1-(pent-4'-enoyl)-4-(hydroxymethyl)-azetidin-2-one has been synthesised in three steps from (S)-4-(benzyloxycarbonyl)-azetidin-2-one and evaluated as novel, reversible, ß-lactamic inhibitors of endocannabinoid-degrading enzymes (human fatty acid amide hydrolase (hFAAH) and monoacylglycerol lipase (hMAGL)). The compounds showed IC50 values in the micromolar range and selectivity for hFAAH versus hMAGL. The unexpected 1000-fold decrease in activity of 2a comparatively to the known regioisomeric structure 1a (i.e. lipophilic chains placed on N1 and C3 positions of the ß-lactam core) could be explained on the basis of docking studies into a revisited model of hFAAH active site, considering one or two water molecules in interaction with the catalytic triad.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Azetidinas/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Modelos Moleculares , Amidohidrolasas/metabolismo , Azetidinas/síntesis química , Azetidinas/química , Dominio Catalítico/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Humanos , Estructura Molecular , Monoacilglicerol Lipasas/antagonistas & inhibidores , Monoacilglicerol Lipasas/metabolismo , Relación Estructura-ActividadRESUMEN
New host-guest materials have been prepared by incorporation of a home-made organic probe displaying a pyrene motif and a phosphonate function into a regular amphiphilic copolymer. Using powder X-Ray diffraction, photoluminescence and FT-IR spectroscopy, we have been able to study the non-covalent interactions between the host matrix and the guest molecule in the solid state. Interestingly, we have shown that the matrix directs the guest spatial localization and alters its properties. Thanks to the comparison of pyrene vs. N-pyrenylmaleimide derivatives, the influence of the chemical nature of the guest molecules on the non-covalent interactions with the host have been studied. In addition, using polyethylene glycol as a reference host, we have been able to evidence a true matrix effect within our new insertion materials. The phosphonated guest molecule appears to be a novel probe targeting the hydrophilic domain of the host copolymer.
Asunto(s)
Colorantes Fluorescentes/química , Organofosfonatos/química , Polímeros/química , Pirenos/química , Polietilenglicoles/química , Polímeros/síntesis química , Espectrofotometría Infrarroja , Difracción de Rayos XRESUMEN
Poly(lactide-co-glycolide) (PLGA) is extensively used in pharmaceutical applications, for example, in targeted drug delivery, because of biocompatibility and degradation rate, which is easily tuned by the copolymer composition. Nevertheless, synthesis of sugar-labeled amphiphilic copolymers with a PLGA backbone is quite a challenge because of high sensitivity to hydrolytic degradation. This Article reports on the synthesis of a new amphiphilic copolymer of PLGA grafted by mannosylated poly(ethylene oxide) (PEO). A novel building block, that is, α-methoxy-ω-alkyne PEO-clip-N-hydroxysuccinimide (NHS) ester, was prepared on purpose by photoreaction of a diazirine containing molecular clip. This PEO block was mannosylated by reaction of the NHS ester groups with an aminated sugar, that is, 2-aminoethyl-α-d-mannopyroside. Then, the alkyne ω-end-group of PEO was involved in a copper alkyne- azide coupling (CuAAC) with the pendent azides of the aliphatic copolyester. The targeted mannose-labeled poly(lactide-co-glycolide-co-ε-caprolactone)-graft-poly(ethylene oxide) copolymer was accordingly formed. Copolymerization of d,l-lactide and glycolide with α-chloro-ε-caprolactone, followed by substitution of chlorides by azides provided the azido-functional PLGA backbone. Finally, micelles of the amphiphilic mannosylated graft copolymer were prepared in water, and their interaction with Concanavalin A (ConA), a glyco-receptor protein, was studied by quartz crystal microbalance. This study concluded to the prospect of using this novel bioconjugate in targeted drug delivery.
Asunto(s)
Materiales Biocompatibles/síntesis química , Manosa/metabolismo , Poliésteres/química , Polietilenglicoles/química , Polímeros/síntesis química , Concanavalina A/metabolismo , Espectroscopía de Resonancia Magnética , Micelas , Estructura Molecular , Cuarzo/químicaRESUMEN
The tripeptide Leu-Asp-Val (LDV) is known to bind α(4)ß(1) integrin in leukemia cells. Here we have synthesized a LDV peptidomimetic equipped with a biotin-conjugated spacer-arm. Compound 9 acts as an inhibitor of the α(4)ß(1) integrin in an adhesion assay using fluorescently labeled, α(4)ß(1) integrin-expressing leukemia CCRF-CEM cells. Furthermore, when bound to neutravidin-coated plates, compound 9 could capture CCRF-CEM cells. Such biotin-conjugated LDV peptidomimetic may thus represent a novel tool for biotechnological applications using avidin interaction for leukapheresis or leukemia cell targeting.
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Química Farmacéutica/métodos , Oligopéptidos/química , Peptidomiméticos/química , Antineoplásicos/farmacología , Biotina/química , Adhesión Celular , Línea Celular , Línea Celular Tumoral , Diseño de Fármacos , Regulación Leucémica de la Expresión Génica , Humanos , Concentración 50 Inhibidora , Integrina alfa4beta1/metabolismo , Cinética , Leucemia/metabolismo , Modelos Químicos , Péptidos/químicaRESUMEN
Oxidative cross-coupling reactions of substituted o-aminophenols were catalyzed by a commercial laccase to produce non-symmetrically substituted phenoxazinones for the first time. Identification by (1)H-, (13)C- and (31)P-NMR, and by HPLC-PDA and HPLC-MS/MS of exclusively two kinds of substituted phenoxazinones out of four potential heterocyclic frameworks was confirmed by a DFT study. The redox-properties of the substrates, their relative rates of conversion and the rigid docking of selected substrates led to a revisited mechanistic pathway for phenoxazinones biosynthesis. Our suggestions concern both the first formal two-electron oxidation by laccase and the first intermolecular 1,4-conjugated addition which secures the observed regioselectivity.
Asunto(s)
Aminofenoles/química , Lacasa/metabolismo , Oxazinas/síntesis química , Trametes/enzimología , Aminofenoles/metabolismo , Productos Biológicos/química , Modelos Moleculares , Estructura Molecular , Oxidación-Reducción , Estereoisomerismo , Especificidad por SustratoRESUMEN
The absolute configuration of a relatively large and conformationally flexible chiral compound, 3-(1'-hydroxyethyl)-1-(3'-phenylpropanoyl)-azetidin-2-one, is determined using Vibrational Circular Dichroism (VCD) spectroscopy, Optical Rotation Dispersion (ORD) and Electronic Circular Dichroism (ECD). To that end a state of the art experimental VCD spectrum is compared to a theoretical spectrum and the absolute configuration is assigned. ORD and ECD are also used in the assignment to investigate the complementarity of the three techniques. VCD spectroscopy is found to have important advantages over ORD and ECD for diastereomers. The concept of robust modes is applied to this conformationally flexible molecule, showing that its use is limited for such large and flexible molecules.
RESUMEN
An endophytic whorl-forming Streptomyces sp. designated as TS3RO having antifungal activity against a large number of fungal pathogens, including Sclerotinia sclerotiorum, Rhizoctonia solani, Colletotrichum gloeosporioides, Cryphonectria parasitica, Fusarium oxysporum, Pyrenophora tritici-repentis, Epidermophyton floccosum, and Trichophyton rubrum, was isolated from surface-sterilized Catharanthus roseus stems. Preliminary identification showed that Streptomyces cinnamoneus subsp. sparsus was its closest related species. However, strain TS3RO could readily be distinguished from this species using a combination of phenotypic properties, 16S rDNA sequence similarity, and phylogenetic analyses. Thus, the whorl-forming Streptomyces sp. strain TS3RO is likely a new subspecies within the Streptomyces cinnamoneus group. Direct bioautography on a thin-layer chromatography plate with Cladosporium cucumerinum was conducted throughout the purification steps for bioassay-guided isolation of the active antifungal compounds from the crude extract. Structural elucidation of the isolated bioactive compound was obtained via LC-MS spectrometry, UV-visible spectra, and nuclear magnetic resonance data. It revealed that fungichromin, a known methylpentaene macrolide antibiotic, was the main antifungal component of TS3RO strain, as shown by thin-layer chromatography bioautography. This is the first report of an endophytic whorl-forming Streptomyces isolated from the medically important plant Catharanthus roseus.
Asunto(s)
Antifúngicos/aislamiento & purificación , Catharanthus/microbiología , Macrólidos/aislamiento & purificación , Streptomyces/química , Antifúngicos/metabolismo , Cromatografía en Capa Delgada , ADN Bacteriano/genética , Fermentación , Macrólidos/metabolismo , Filogenia , Polienos/aislamiento & purificación , Polienos/metabolismo , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Streptomyces/genética , Streptomyces/aislamiento & purificaciónRESUMEN
The complexation of calcium and zinc cations by pyrroglutamate analogs has been studied in the gas phase by means of electrospray ionization mass spectrometry (ESI-MS). Complexes were obtained from the solutions of calcium perchlorate and zinc perchlorate in acetonitrile. The complexes with calcium are singly and doubly charged with various stoichiometries while zinc complexes are singly charged except for one ligand. Solvation with acetonitrile and presence of perchlorate counter-ions are observed when the complexes are in the gas phase. The complexes formed with both metals are mainly L(2)M and LM species. All tested compounds are better complexing agents for calcium than for zinc.
Asunto(s)
Calcio/química , Ácido Pirrolidona Carboxílico/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Zinc/químicaRESUMEN
The regulation of 2-arachidonoylglycerol (2-AG) levels is a major issue as 2-AG has been proven to participate in numerous physiopathological phenomena such as neuroprotection or analgesia. Octhilinone, a cysteine-reagent compound, has recently been shown to inhibit in the nanomolar range monoacylglycerol lipase (MAGL), the major enzyme responsible for the degradation of 2-AG. Here, we further investigate the mechanism by which octhilinone and its benzisothiazolinone analog inhibit human MAGL. We also provide new information on the structural requirements for MAGL inhibition by these compounds. Finally, we describe for N-octylbenzisothiazolinone a mode of inhibition which is partially different from that described for octhilinone, especially with regard to the targeted cysteine residues in the vicinity of the catalytic site.
Asunto(s)
Inhibidores Enzimáticos/química , Monoacilglicerol Lipasas/antagonistas & inhibidores , Tiazoles/química , Tiazolidinas/química , Diseño de Fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Humanos , Monoacilglicerol Lipasas/genética , Monoacilglicerol Lipasas/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiazoles/síntesis química , Tiazoles/farmacología , Tiazolidinas/farmacologíaRESUMEN
α-Methoxy-ω-alkyne poly(ethylene glycol) (PEG) was tagged with pendent N-hydroxy-succinimidyl activated esters by photografting of a molecular clip. This easily synthesized heterofunctional PEG was found to be a versatile building block for (i) conjugation with an amino derivative and (ii) grafting to azido functional aliphatic polyesters backbone by Huisgen's 1,3-dipolar cycloaddition. This original combination of "clip" and "click" reactions provides a versatile and straightforward pathway for the synthesis of functional amphiphilic and degradable copolymers valuable for biomedical applications such as in drug-delivery.
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Alquinos/síntesis química , Poliésteres/síntesis química , Polietilenglicoles/síntesis química , Química Clic , Polietilenglicoles/químicaRESUMEN
The discrimination between cyclomonomers and various oligomers formed during a ring-closing metathesis (RCM) process is not an easy task. Their (1)H NMR patterns are often very similar, and the use of mass spectrometry techniques is usually recommended. Here, we show that the DOSY-NMR method is a reliable tool to help in the identification of cyclomonomers versus cyclodimers by comparing the translational diffusion coefficient of the compounds issued from RCM reactions with the diffusion coefficient of their respective precursors.
RESUMEN
Aminophenoxazinone dyes with variable water solubilities were assayed for the first time in a live-cell imaging application. Among a library of ten sulfonylated chromophores, one compound gave excellent results as an endocytic marker, showing a precise subcellular distribution. The compound was compared to four commercial vital tracers, including Lucifer Yellow. The first laccase-mediated regioselective synthesis of a diphosphorylated 2-aminophenoxazinone dye was also described. This compound, water-soluble at 10(-2) M, displayed modest fluorescence properties and the ability to complex Mg(2+) and Ca(2+) cations, therefore giving fluorescence quenching.
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Colorantes Fluorescentes/metabolismo , Lacasa/metabolismo , Oxazinas/metabolismo , Animales , Biocatálisis , Células CHO , Calcio/química , Cricetinae , Cricetulus , Colorantes Fluorescentes/química , Magnesio/química , Microscopía Fluorescente , Oxazinas/química , Agua/químicaRESUMEN
The hetero-Diels-Alder (HDA) reaction of 1-(diethoxyphosphonyl)-1,3-butadiene, 1-(dibenzyloxyphosphonyl)-1,3-butadiene, and 1-(diethoxyphosphonyl)-3-tert-butyldimethylsilyloxy-1,3-butadiene with various nitroso heterodienophiles has been investigated as a new synthetic route for aminophosphonic derivatives. The HDA cycloadditions regioselectively led to the proximal isomers, i.e., presenting the NR(3) group in the meta position regarding the phosphonate substituent. From the resulting 6-phosphono-3,6-dihydro-1,2-oxazine cycloadducts, a limited number of chemical steps were allowed to obtain a significant variety of aminophosphonic compounds of potential interest in medicinal chemistry. This has been illustrated through the synthesis of (Z)-4-(o-tolylamino)-1-hydroxybut-2-enylphosphonic acid, diethyl 3,4-dihydroxy-1-o-tolylpyrrolidin-2-yl-2-phosphonate, 4-(o-tolylamino)-1,2,3-trihydroxybutylphosphonic acid, diethyl 3-(2-(o-tolylamino)-1-hydroxyethyl)oxiran-2-yl-2-phosphonate, and diethyl 4,5-dihydroxymorpholin-6-yl-6-phosphonate.
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Butadienos/química , Compuestos Nitrosos/química , Organofosfonatos/síntesis química , Ciclización , Estructura Molecular , Organofosfonatos/química , EstereoisomerismoRESUMEN
Ultrasmall particles of iron oxide (USPIOs) coated with 3,3'-bis(phosphonate)propionic acid were covalently coupled to a home-made Arg-Gly-Asp (RGD) peptidomimetic molecule via a short oligoethylene-glycol (OEG) spacer. The conjugation rate was measured by X-ray photoelectron spectroscopy (XPS). The particle size and magnetic characteristics were kept. Our novel conjugate targeted efficiently Jurkat cells (increase of 229% vs the control).
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Compuestos Férricos/química , Imitación Molecular , Oligopéptidos/química , Tamaño de la Partícula , Análisis Espectral/métodos , Rayos XRESUMEN
Laccases are members of the blue copper oxidases family found in nature. They commonly oxidise a wide range of phenol and aniline derivatives, which in turn are involved in oxidative coupling reactions. Yet, laccases remain rarely described as biocatalysts in organic synthesis. This paper describes the chemical preparation of original sulfonated aminophenol substrates and their enzyme-mediated dimerisation into phenoxazine chromophores that feature tuneable water solubility as a function of the sulfonyl substituent. The scope and limitations of the biocatalysed synthetic process are outlined. Kinetic data were collected to evaluate the influence of physicochemical parameters. The structure of the novel phenoxazine dyes ("head-to-head" or "head-to-tail" dimer) was assessed by NMR spectroscopic analysis. Two crystalline compounds were analysed by X-ray diffraction. Such laccase-mediated synthesis (a green chemistry process) was proven to be more efficient than the chemical oxidation of o-aminophenols with silver oxide.
Asunto(s)
Compuestos Heterocíclicos/química , Lacasa/química , Oxazinas/metabolismo , Biotransformación , Cromatografía , Cristalografía por Rayos X , Lacasa/metabolismo , Estructura Molecular , Oxazinas/química , Oxidación-Reducción , Espectrofotometría Ultravioleta , Sulfonamidas/químicaRESUMEN
Functionalized poly-epsilon-caprolactone-block-polyethyleneglycol (PCL-PEG) amphiphilic copolymers were prepared to be constituents of nanocarriers used for the targeting of specific cells. Hence, we conceived a smooth and simple photografting methodology on these copolymers using a bifunctional molecular clip (O-succinimidyl-4-(p-azido-phenyl)butanoate). We prepared PCL-PEGs with pendent N-hydroxysuccinimide esters and studied the grafting with 3H-lysine, which radioactivity was counted by LSC. Several parameters were investigated, such as behavior of homopolymers, initial concentrations, irradiation, and incubation durations. Evidences of a "PEG directed photografting" are discussed and this selectivity could be improved by a selective solvent technique. The photografting on different PCL-PEGs revealed a dependency of the rates to the crystallinity of the copolymers. Several controls by SEC, DLS, and TEM of the treated copolymers were realized. Lastly, the coupling of alpha-D-mannopyranoside ligand was performed, reaching amounts of 5400 nmol/g of PCL-PEG. This derivatized PCL-PEG enters in the preparation of nanocarriers used for the targeting of antigen presenting cells.
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Portadores de Fármacos , Óxido de Etileno/química , Óxido de Etileno/metabolismo , Lactonas/química , Lactonas/metabolismo , Polímeros/química , Polímeros/metabolismo , Tritio , Materiales Biocompatibles , Rastreo Diferencial de Calorimetría , Lisina/química , Lisina/metabolismo , Ensayo de Materiales , Micelas , Microscopía Electrónica de Rastreo , Succinimidas/químicaRESUMEN
Aminocitrate (and homolog) derivatives have been prepared by bis-alkylation of glycinate Schiff bases with bromoacetates (and ethyl acrylate), followed by N-acylation and esters (partial or complete) deprotection. Aminoisocitrate was similarly obtained by mono-alkylation with diethyl fumarate. Evaluation against representative beta-lactamases revealed that the free acid derivatives are modest inhibitors of class A enzymes, whilst their benzyl esters showed a good inhibition of OXA-10 (class D enzyme). A docking experiment featured hydrophobic interactions in the active site.