RESUMEN
Noncovalent or supramolecular PEGylation, in combination with the site of administration, has great potential to increase the half-life of therapeutic proteins. To date, a variety of noncovalent PEGylation strategies have been devised. However, questions remain concerning the nature of the protein-PEG interaction. Here, we report structural analyses of a model system that comprised the lysine-rich cytochrome c and two PEGylated variants of sulfonatocalix[4]arene. Complex formation was characterized in solution by NMR spectroscopy. It was found that mono- or di-PEGylated sulfonatocalix[4]arene bound the protein similar to the parent calixarene. X-ray crystal structures at <2.7 Å resolution of the PEGylated derivatives in complex with cytochrome c revealed that the PEG chains were mostly disordered or encapsulated within the calixarene cavity. These results suggest that there was minimal interaction between the PEG and the protein surface, providing further evidence in favor of PEG maintaining a random coil conformation.
Asunto(s)
Calixarenos/química , Cristalografía por Rayos X/métodos , Polietilenglicoles/química , Ácidos Sulfónicos/química , Sitios de Unión , Estructura Molecular , Proteínas/química , Análisis Espectral/métodosRESUMEN
Lower rim amide linked 8-amino quinoline and 8-amino naphthalene moiety 1,3,5-triderivatives of calix[6]arene L1 and L2 have been synthesized and characterized. While the L1 acts as a receptor molecule, the L2 acts as a control molecule. The complexation between L1 and Cu(2+) or Zn(2+) was delineated by the absorption and electrospray ionization (ESI) MS spectra. The binding ability of these molecules toward biologically important metal ions was studied by fluorescence and absorption spectroscopy. The derivative L1 detects Zn(2+) by bringing ratiometric change in the fluorescence signals at 390 and 490 nm, but in the case of Cu(2+), it is only the fluorescence quenching of 390 nm band that is observed, while no new band is observed at 390 nm. The stoichiometry of both the complexes is 1:1 and was confirmed in both the cases by measuring the ESI mass spectra. The isotopic peak pattern observed in the ESI MS confirmed the presence of Zn(2+) or Cu(2+) present in the corresponding complex formed with L1. Among these two ions, the Cu(2+) exhibits higher sensitivity. The density-functional theory (DFT) studies revealed the conformational changes in the arms and also revealed the coordination features in the case of the metal complexes. The arm conformational changes upon Zn(2+) binding were supported by nuclear Overhauser effect spectrometry (NOESY) studies. The stronger binding of Cu(2+) over that of Zn(2+) observed from the absorption study was further supported by the complexational energies computed from the computational data. While the L1 exhibited spherical particles, upon complexation with Cu(2+), it exhibits chain like morphological features in scanning electron microscopy (SEM) but only small aggregates in the case of Zn(2+). Thus, even the microscopy data can differentiate the complex formed between L1 and Cu(2+) from that formed with Zn(2+).
Asunto(s)
Calixarenos/química , Cobre/análisis , Fenoles/química , Quinolinas/química , Zinc/análisis , Sitios de Unión , Iones/análisis , Sustancias Macromoleculares/síntesis química , Sustancias Macromoleculares/química , Modelos Moleculares , Estructura Molecular , Tamaño de la Partícula , Teoría Cuántica , Espectrometría de Fluorescencia , Espectrometría de Masa por Ionización de Electrospray , Propiedades de SuperficieRESUMEN
Herein we report the synthesis and characterization of 7-oxanorbornadiene (OND)-appended 1,3,5-tris conjugate of calix[6]arene (L2). L2 has been shown to exhibit selective reactivity toward cysteine (Cys) over homocysteine (Hcy) and glutathione (GSH) under stoichiometric conditions. The selectivity of L2 is attributed to the steric crowding of three Diels-Alder centers possessing OND units present on the calix[6]arene platform, while a control molecular system possessing only one such unit without the calix[6]arene platform (L1) does not show any selectivity toward Cys. While L2 exhibited spherical particles, its reactivity with Cys resulted in flowerlike morphological features, as revealed by scanning electron microscopy. However, the reaction with GSH did not result in any such morphological features, a result that is in agreement with that observed from fluorescence studies in solution. L2 has been shown to react with Cys present in HeLa and Jurkat E6 cells by fluorescence microscopy.
Asunto(s)
Calixarenos/química , Calixarenos/síntesis química , Cisteína/química , Glutatión/química , Homocisteína/química , Norbornanos/química , Norbornanos/síntesis química , Fenoles/química , Fenoles/síntesis química , Reacción de Cicloadición , Células HeLa , Humanos , Espectroscopía de Resonancia Magnética , Microscopía/métodos , Microscopía Fluorescente/métodosRESUMEN
A new triazole-linked anthracenyl-appended calix[4]arene-1,3-diconjugate (L) has been synthesized and characterized, and its single crystal XRD structure has been established. Binding properties of L toward different biologically relevant metal ions have been studied by fluorescence and absorption spectroscopy in ethanol. L exhibits selective recognition of Co(2+) and can detect down to a concentration of 55 ppb (0.92 µM). The roles of the calix[4]arene platform as well as the preorganized binding core in L's selective recognition have been demonstrated by studying appropriate control molecules. The mode of binding of L with Co(2+) has been modeled both by DFT and MD computational calculations. L and its Co(2+) complex could be differentiated on the basis of the nanostructural features observed in AFM and TEM.
Asunto(s)
Antracenos/química , Calixarenos/química , Calixarenos/síntesis química , Cobalto/química , Nanoestructuras/química , Triazoles/química , Triazoles/síntesis química , Cristalografía por Rayos X , Fluorescencia , Espectroscopía de Resonancia Magnética , Microscopía , Modelos MolecularesRESUMEN
A new 1,3,5-tris-triazole linked picolylimine conjugate of calix[6]arene (L) has been shown to be selective toward La(3+) by turn on fluorescence with â¼70-fold enhancement and emits blue, with a minimal detection limit of 65 ± 5 ppb (490 nM). The species of recognition has been modeled computationally to have a monocapped twisted square antiprism with a N(6)O(3) binding core about La(3+). The in situ complex of L with La(3+) recognizes F(-) via fluorescence quenching. The reversible response of sensing La(3+) and F(-) sequentially by L has been demonstrated.
Asunto(s)
Calixarenos/química , Colorantes Fluorescentes/química , Flúor/química , Iminas/química , Lantano/química , Fenoles/química , Triazoles/química , Colorantes Fluorescentes/análisis , Flúor/análisis , Iones/química , Lantano/análisis , Modelos Moleculares , Estructura MolecularRESUMEN
Carboxamidoquinoline appended calix[4]arene-1,3-di-conjugate (L) has been synthesized and characterized and its single crystal XRD structure has been established. L has been shown to act as selective ratiometric turn-on fluorescence sensor for Zn(2+) up to a lowest concentration of 183 ± 18 ppb (2.82 µM) with a nine-fold enhancement by exhibiting blue-green emission. The coordination features of the species of recognition have been computationally evaluated by DFT methods and found to have distorted tetrahedral Zn(2+) center in an N(4) core. The spherical nano-structural features observed for L in TEM are being transformed into the Koosh nano-flower like structure when complexed with Zn(2+) and hence these two can be easily differentiated. Even the features observed in AFM can distinctly differentiate L from its Zn(2+) complex.